Enhancing Thermostability of Bacillus licheniformis Lipase with LEA Peptide Co-expression System.

Enhancing Thermostability of Bacillus licheniformis Lipase with LEA Peptide Co-expression System. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Enhancing Thermostability of Bacillus licheniformis Lipase with LEA Peptide Co-expression System. Ammar Khazaal Kadhim Almansoori, Kang Siang Yu, Faisal Mohamed, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4160767/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Heterozygous protein expression in E. coli facilitates high yield and quality. However, the challenges of protein instability due to environmental stress are still an issue that affects the activity of the protein produced. In this study, the improvement of protein thermostability was done using a peptide co-expression system. The developed system exploited the usefulness of Late Abundant Embryogenesis (LEA) proteins to protect proteins from damage. Recombinant lipase from Bacillus licheniformis was expressed along with the LEA-like peptide, whose design was inspired by the 11 repetitive amino acid sequences of the LEA protein. In total, four LEA-like peptide co-expression systems were assessed. The evaluation of improvements in protein thermostability was conducted using a standard lipase assay. The purified lipase was challenged at 45 °C, a higher temperature than its optimal temperature. Two-fold lipase activity was recorded from the protein co-expressed with the LEA-II-like peptide. Based on amino acid sequence comparison, LEA-II has the advantage of containing more polar residues with several aliphatic amino acids, which may improve LipA B.licheniformis -LEA II complex stability at higher temperatures. Next, molecular docking and molecular dynamic simulation were employed to analyze the stability of the lipase in the presence and absence of LEA II. The findings of the RMSD, MM-GBSA and related analyses showed that the LipA B.licheniformis -LEA II complexes have better stability than the LipA B.licheniformis alone, thus supporting the lipase assay. These findings successfully unravel the potential of the LEA-like peptide co-expression system as a novel approach to improve enzyme thermostability. LipAB.licheniformis Late Embryogenesis Abundant (LEA) Thermostability Co-expression Molecular docking and Molecular dynamic simulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Key points LipA B.licheniformis thermostability was improved through the novel implementation of the LEA peptide co-expression system. LipA B.licheniformis properties have been improved after interacting with the LEA II peptide. The LEA II peptide aids in enhancing the stability and affinity of the LipA B.licheniformis . INTRODUCTION Lipases are a group of enzymes responsible for the hydrolysis of triglycerides into free fatty acids and glycerol ( 1 ) as well as esterification by using glycerol and fatty acids as substrates under low water conditions ( 2 ). Lipase can be divided into several groups based on substrate specificity. However, lipase generally shared common properties such as cofactor independent, active in organic solvent, highly efficient and stable as well as regioselective, chemoselective and enantioselective ( 3 ). The usefulness of lipase makes it widely utilized in various sectors, including detergent, food, biodiesel, medical, and pharmaceutical industries ( 4 , 5 ). Microbial lipases are preferable for commercial uses since the production using microorganisms is scalable and cost-efficient. Nevertheless, the number of strains used for commercial purposes is still low ( 6 , 7 ). One of the limiting factors that contribute to low number of microbial lipases is because of heat sensitive. The producing, storing, or using protein, exposure to heat can significantly reduce the enzyme half-life due to stress. Furthermore, when used for esterification without water, lipase often requires higher temperatures to melt the fat ( 8 ). While there are many heat-resistant lipases available on the market, their numbers are decreasing because obtaining thermostable lipases is challenging. Gupta et al., 2004 reviewed that most of the microbial lipases used in the industries come from mesophilic bacteria which commonly produced from Genus of Pseudomonas, Burkholderia, Chromobacterium. Of the high demand on thermostable enzymes, researchers usually apply rationale design to overcome heat sensitive limitation. The approach integrates bioinformatic tools and mutagenesis to create specific protein with desired attribute. This strategy requires high skill designer, time consuming and specifically design for protein candidate which are the drawback ( 9 ). In this study, the target lipase used was isolated from Bacillus licheniformis IBRL-CHS2 and is called LipA B.licheniformis . It is a small enzyme with a size of 18.6 kDa, functioning optimally at a temperature of 37°C and pH 7 while hydrolyzing the C12 triglyceride substrate, laurate ( 10 ). The catalytic function of LipA B.licheniformis is modulated by the presence of its active site, in contrast to being governed by a lid. This is attributed to the absence of a lid in LipA B.licheniformis , leading to the formation of a pre-existing oxyanion hole at the active site. This structural feature facilitates substrate binding without requiring interfacial activation. Consequently, LipA B.licheniformis demonstrates efficient performance at low substrate concentrations, exhibiting high enzymatic activity. ( 11 ). However, similar to many enzymes, LipA B.licheniformis is sensitive to temperature fluctuations, leading to the unfolding of the enzyme and a substantial decrease in activity at 45 ºC ( 11 ). Late embryogenesis abundant (LEA) proteins, initially recognized in plants and later found in various organisms, play a pivotal role in responding to stress conditions ( 12 ). With the exception of Group 5 LEA proteins, all LEA proteins usually have a molecular weight between 10–30 kDa, a hydrophilic nature, and stability, marked by repetitive hydrophilic amino acids ( 13 ). These LEA proteins contain an intrinsically disordered structure, contributing to a lack of proper folding in a hydrated state; however, this structure transforms into a well-defined secondary structure when water is absent ( 14 ). In a significant discovery by Liu and colleagues (2010), it was revealed that a Group 3 LEA protein, specifically the PM2 protein isolated from soybean, effectively enhances the heat tolerance of Escherichia coli when over-expressed in the cell ( 15 ). Interestingly, Ikeno and Haruyama (2013) have successfully demonstrated that the modified 11-mers repetitive motif found in Group 3 LEA from the sleeping chironomid, Polypedilum vanderplanki , is helpful in improving protein production in Escherichia coli through the LEA-like peptide co-expression system ( 16 ). Moreover, more success stories involving the use of LEA-like peptides to enhance cellular tolerance to UV radiation, acidic stress, temperature variations, and changes in salinity have been reported in recent years ( 17 – 19 ). However, no study has delved further into the impact of LEA co-expression in enhancing protein properties. This research, using lipase (LipA B.licheniformis ) as a protein of interest and heat stress as one of the limiting factors in protein stability, represents the first attempt to explore the utility of the LEA-like co-expression system, which could potentially be applied to produce enzymes with stress tolerance. Additionally, utilized molecular docking and molecular dynamic simulation to comprehend how the interaction of LEA-like peptides can contribute to protein stability. MATERIALS AND METHODS Construction of pCold- LipA B.licheniformis -T7LEA The pCLEA-LipA B.licheniformis series was constructed by cloning LEA-like peptide; I, II, E, and K into pCOLD-LipA B.licheniformis , respectively at Bam HI and Hind III sites. The pCOLD-LipA B.licheniformis was derived from pCOLD-I (Takara, Clontech, Japan), reserving lipase gene at Nde I and Bam HI sites ( 11 ). The plasmid DNA template to amplify LEA-like peptides ( 16 ). To ensure LEA-like peptide co-expressed with LipA B.licheniformis , T7 promoter was introduced to upstream of LEA-like DNA sequence, while the expression of LipA B.licheniformis was driven by the original promoter of the pCold™ I vector. The sequence of LEA peptides, origin of the plasmid along with primers and the constructed map, can be found in S1. Co-expression and purification of LipA B.licheniformis and LEA-like peptides Protein expression was carried out by inducing 0.2 mM IPTG prechilled bacterial culture; E. coli BL21 (DE3), pCLEA-LipA B.licheniformis for 30 minutes after reaching OD 600 ~ 0.5. Cultures were shaken-incubated at 150rpm and 15 ºC for 16 hours. Protein purification was conducted using His-tagged immobilized metal affinity chromatography (IMAC) with the HisTALON™ Gravity Column Purification Kit (Takara, Clontech, Japan) as suggested by manufacturer. Following purification, the lipase was subjected to SDS-PAGE, and the concentration was determined using the Bio-Rad Protein Assay (Biorad, USA), based on the Bradford assay ( 20 ). Lipase assay The lipase's enzymatic activity was assessed using the p -nitrophenyl laurate ( p NPL) colorimetric method, following a slight modification of the lipase assay outlined by Ch’ng and Sudesh (2013) ( 21 ). The assay was chosen and optimized in our lab by Reddy (2016) ( 11 ). Subsequently, lipase activity, relative activity, and residual activity were calculated using the equations provided in S2. Prediction of interactions between LipA B.licheniformis and LEA II peptide by in silico approach Tertiary structure prediction The Phyre2 webserver ( http://www.sbg.bio.ic.ac.uk/phyre2 ) ( 22 ) was employed to predict the tertiary structure of LipA B.licheniformis as for secondary structure of was assigned using Stride webserver ( http://webclu.bio.wzw.tum.de/stride/ ) ( 23 ). Simultaneously, the tertiary structure prediction for the LEA II peptide was conducted using the PEP-FOLD 3.5 webserver ( http://bioserv.rpbs.univ-paris-diderot.fr/PEP-FOLD ) ( 24 ). The model of the LEA II peptide with the highest Optimized Potential for Efficient structure Prediction (sOPEP) score was chosen for subsequent docking with LipA B.licheniformis as shown in S3. Molecular Docking Protein-protein docking of LipA B.licheniformis and LEAII was carried out using ClusPro web server ( https://cluspro.org ) ( 25 ). Post-analysis of protein-protein interaction was performed using PDBSum web server ( https://www.ebi.ac.uk/thornton-srv/databases/pdbsum/ ). The binding propensities of p NPL with LipA B.licheniformis and LipA B.licheniformis +LEAII proteins was also investigated through molecular docking studies which were performed on Autodock Vina Version 1.2.3 ( 26 ), where p NPL was docked into the binding site of LipA B.licheniformis or LipA B.licheniformis +LEAII. The binding site residues of LipA B.licheniformis have been determined using DoG Site Scorer ( https://proteins.plus/ last accessed on 28/08/2023). The molecular structure of p NPL was constructed and optimized using Avogadro program Version 1.2.0 ( 27 ). Subsequently the enzyme structure and p NPL structure was converted and saved into pdbqt format. Molecular docking was performed within a grid box dimension 30 x 30 x 30 Å with center of box along X, Y, and Z at 28.34, 16.65, and 151.78, respectively. The exhaustiveness value was set to 100 for efficient search. All other parameters were set at their default values. The best docked conformations were chosen from amongst the best cluster that had the lowest binding free energies. Molecular dynamics simulation (MDS) The docked complexes of LipA B.licheniformis + p NPL and LipA B.licheniformis + p NPL + LEAII were subjected to 500 ns extended molecular dynamics simulations using the Gromacs Version 2023 program ( 28 ). The topologies of LipA B.licheniformis and LipA B.licheniformis +LEAII were generated using the CHARMM-36 force field parameters ( 29 , 30 ); while the topology of p NPL was generated from the CGenFF server ( 29 , 31 ). The complexes of LipA B.licheniformis + p NPL and LipA B.licheniformis + p NPL_LEAII were placed in a dodecahedron unit cell such as that the edges of systems remained 1 nm away from the edges of box. The systems were solvated by adding the water using the TIP3P water model ( 32 ) and neutralized by adding appropriate number of sodium and chloride counter-ions so as to achieve the molar concentration of 0.15. The system was relieved of the steric strain by energy minimization with the steepest descent algorithm until the force-constant reaches the threshold of 100 kJ mol-1 nm-1. Two step equilibration was performed on resulting system, where the initial equilibration was performed at constant temperature condition (NVT) of 318. K temperature and later at constant pressure conditions (NPT) at 1 atm pressure. During equilibration steps, the constant temperature conditions were achieved using a modified Berendsen thermostat ( 33 ) and the constant pressure conditions were achieved using the Berendsen barostat ( 34 ), for 1 ns each. The final 500 ns production phase MD simulation were performed where the temperature conditions of 45°C were achieved with a modified Berendensen thermostat and pressure conditions of 1 atm were achieved with the Parrinello-Rahman barostat ( 35 ). The restrain on covalent bonds was achieved with LINCS algorithm ( 36 ). The long-range electrostatic energies were computed with Particle Mesh Ewald (PME) method ( 37 ) with a cut-off of 1.2 nm. The output trajectories were treated for periodic boundary conditions before MD analysis. The root mean square deviations (RMSD) in the backbone atoms, the root mean square fluctuation (RMSF) in the side chain atoms, radius of gyration (Rg), and the analysis of solvent accessible solvent area (SASA) of both the system were analyzed and compared for the stability aspects. The hydrogen bonds formatted between the p NPL and LipA B.licheniformis as well as the hydrogen bonds formed between the LipA B.licheniformis and LEAII were analyzed and the representative trajectories at the beginning of simulation and at 100, 200, 300, 400, and 500 ns were investigated. The major path of motions in each complex was studied through the principal component analysis (PCA) ( 38 ), where the covariance matrix for the C-α atom was constructed and diagonalized to obtain the eigenvectors and eigenvalues. The eigenvectors represent the motion path, while eigenvalues represent the mean square fluctuation. The first two eigenvectors, principal components (PC1 and PC2) were further used as a reaction coordinate in the Gibb’s free energy landscape ( 39 ) analysis. Molecular mechanics energies combined with Poisson Boltzmann surface area continuum solvation (MM-PBSA) and Molecular mechanics with generalised Born and surface area solvation (MM-GBSA) calculations were performed with gmx_MMPBSA tool ( 40 ) on the trajectories sampled at 1 ns each from the 450 ns to 500 ns simulation period. RESULTS Co-expression and purification of LipA B.licheniformis and LEA-like peptides The lipases produced from LEA co-expression system were successfully expressed and purified as shown in SDS- polyacrylamide gel (S3). Temperature study of LipA B.licheniformis co-expressed with LEA-like peptides The lipase activity was assessed using the p NPL lipase assay at 37°C and 45°C. At 37°C, no significant difference was observed between LipA B.licheniformis alone and LipA B.licheniformis co-expressed with the LEA series peptides in both relative and residual activities. At 45°C, the resulting relative activities were calculated and visually represented in Figure 1A. Subsequently, a one-way ANOVA analysis was performed, followed by multiple comparisons utilizing the Tukey test at a significance level of 5%. The findings revealed that LipA B.licheniformis exhibited the highest relative activity when co-expressed with the LEA II peptide at 45°C. As shown in Figure 1A, the statistical analysis demonstrated a significant difference compared to LipA B.licheniformis alone (****p < 0.05). Conversely, no significant difference was observed after co-expression with LEA I, LEA E, and LEA K peptides. Furthermore, the lipase activity after incubation at 45°C for an hour showed slightly higher residual activity after co-expression with the LEA II peptide, while for LipA B.licheniformis alone and co-expressed with LEA I, E, and K peptides, the residual activities showed no significant difference as shown in Figure 1B. Prediction of LipA B.licheniformis -LEA interaction The Phyre2-predicted tertiary structure of LipA B.licheniformis exhibited a 100% confidence level and 99% identity with the template lipase lip_vut1 structure from the metagenome of goat rumen (PDB: 6nkc.1). Illustrated in Figure 2A, this lipase adopted an α/β hydrolase fold, with the six β-strands (β3-β8) forming a parallel β-sheet acting as the core. It featured nine α-helices, comprising four α-helices (A, B, C, and F) and five 310-helices. The catalytic triad Ser-78 was situated at the nucleophilic elbow between the β5 strand and helix C, while Asp-130 and His-153 were located in the loop after the β7 and β8 strands, respectively. Since LEA II peptide showed the best improvement in the relative and residual activities of LipA B.licheniformis , the tertiary structure of LEA II peptide was predicted by PEP-FOLD webserver for further in silico investigation. As shown in Figure 2B, the LEA II peptide was predicted to be made up of loops with an α-helix at the center of the structure. The LipA B.licheniformis 2D structure, LEA II structures and the validation of the 3D structures are shown in S4. Protein-protein docking Protein-protein docking (PPI) between LipA B.licheniformis and LEAII results are shown in Figure 3. The binding energy score calculated from the ClusPro server for LipA B.licheniformis -LEAII was -450, where the LEAII made 57 non-bonded interactions and three hydrogen bond interactions between the Glu9 of LEAII and Met79 and Ser78 of LipA B.licheniformis and one hydrogen bond between Lys10 of LEAII and Ile132 of LipA B.licheniformis . A total of 13 residues from the LipA B.licheniformis and 7 residues from LEAII were involved in these non-bonded interactions. Of these 7 residues, 5 residues are conserved in all LEA-like peptides while 2 consecutive residues; Gly6 and Leu7 are unique to LEA II. Molecular Docking studies The evaluation of binding energy was done to look at the effect of LipA B.licheniformis -LEA II interaction with ligand. The control, LipA B.licheniformis with p NPL has a lower binding energy ΔG = -3.914 kcal/mol, as compared to the LipA B.licheniformis -LEAII with p NPL binding energy which is ΔG = -6.0091 kcal/mol. The p NPL showed hydrogen bonds with His77, Ser78, Gly12, and Ile13, while the hydrophobic alkyl part of p NPL showed the hydrophobic alkyl interactions with Met79, Ala106, Val133, Leu109, and Leu137 residues. The p NPL formed five conventional hydrogen bonds with the residues His77, Ser78, Gly12, Ile13, and Met79. Hydrophobic interactions were found with similar residues at the binding site viz. Ala106, Val133, Leu109, and Leu137. The docked poses of p NPL in LipA B.licheniformis and LipA B.licheniformis -LEAII the complexes were almost similar as shown in Figure 4. Molecular Dynamics Simulation Molecular dynamics simulations extended to 500 ns were carried out in order to determine the stability and convergence of LipA B.licheniformis - p NPL-LEAII complex and LipA B.licheniformis - p NPL complex at 45 °C . The RMSD of backbone atoms of LipA B.licheniformis - p NPL and LipA B.licheniformis - p NPL-LEAII exhibited reasonable convergence and stability (Fig. 5 A). The noteworthy difference arose from 200 ns onwards where the RMSD in backbone atoms of LipA B.licheniformis - p NPL-LEAII was found reasonably lower than the LipA B.licheniformis - p NPL complex. The average RMSD for LipA B.licheniformis - p NPL-LEAII complex was around 0.125 nm, while for LipA B.licheniformis - p NPL was around 0.15 nm. The LEAII being a separate chain in LipA B.licheniformis - p NPL-LEAII complex the RMSD in backbone atoms of LEAII was separately investigated. It was observed that the RMSD in LEAII was comparatively high reaching a maximum of 0.65 nm and average around 0.45 nm (Fig. 5 B). The RMSD in p NPL atoms in both the systems remained almost similar with an average of around 0.3 nm (Fig. 5 C). It is evident from the results of RMSD in backbone atoms of LipA B.licheniformis and atoms of p NPL that the LipA B.licheniformis - p NPL-LEAII complex is quite stable compared to LipA B.licheniformis - p NPL complex. The intermolecular non-bonded interactions between LipA B.licheniformis and LEAII might have stabilized the LipA B.licheniformis . The fluctuations inside chain atoms of LipA B.licheniformis was further assessed through RMSF analysis which showed that the LipA B.licheniformis - p NPL has higher magnitude of fluctuations, except the residues in the range 50-60, compared to LipA B.licheniformis - p NPL-LEAII (Fig. 5 D). The residues in the range 50-60 showed slightly higher fluctuations in LipA B.licheniformis - p NPL-LEAII complex and most of the residues in this range are interface residues between LipA B.licheniformis and LEAII. However, in both the complexes the RMSF is below 0.35 which indicates reasonable stability of the complexes. Radius of gyration (Rg) is the measure of compactness of the protein. During the first 50 ns simulation period LipA B.licheniformis - p NPL-LEAII displayed slightly higher Rg compared to the Rg of LipA B.licheniformis - p NPL (Figure 5 E). Thereafter, LipA B.licheniformis - p NPL-LEAII complex showed lower Rg than LipA B.licheniformis - p NPL until the end of simulation with an average of around 1.47 nm, while the average for LipA B.licheniformis - p NPL was around 1.48 nm. The solvent accessible surface area (SASA) provides insights of disordered regions of protein and smaller the SASA more is the compactness of protein and better is the stability. LipA B.licheniformis - p NPL-LEAII complex has significantly lower SASA with an average of around 20 nm 2 , while LipA B.licheniformis - p NPL has an average of around 80 nm 2 (Fig. 5 F). The lower SASA values in LipA B.licheniformis - p NPL-LEAII might be due to interactions of LEAII with LipA B.licheniformis and proportionally lower surface area available for solvent access. The binding affinity of p NPL was assessed in terms of the hydrogen bonds formed between p NPL and LipA B.licheniformis . The LipA B.licheniformis - p NPL complex showed that around 2 consistent hydrogen bonds formed between p NPL and LipA B.licheniformis , while in LipA B.licheniformis - p NPL-LEAII around 3 consistent hydrogen bonds were formed between p NPL and LipA B.licheniformis . LEAII might have caused the conformational change in LipA B.licheniformis so that p NPL binds with more affinity and with more number of hydrogen bonds at the binding cavities. The LEAII also formed around 6 hydrogen bonds with the LipA B.licheniformis in LipA B.licheniformis - p NPL-LEAII complex. The hydrogen bonds formed in different trajectories extracted at various time intervals showed that the initial equilibrated trajectory of LipA B.licheniformis - p NPL complex has hydrogen bonds between p NPL and Ile13 and Gly12. The 100 ns trajectory showed a hydrogen bond with Ile43, which suggested that p NPL occupied a different binding cavity. Similarly, the 200 ns trajectory showed a hydrogen bond with Ala16 in this binding cavity. None of 300, 400, and 500 ns trajectories showed any hydrogen bonds and at 400 ns p NPL moved out of binding cavity (Fig. 6 A). On the other hand, the initial equilibrated trajectory of LipA B.licheniformis - p NPL-LEAII complex also showed a hydrogen bond between p NPL and Gly12 and Ile13. p NPL occupied a different binding cavity in the 100 ns without any hydrogen bond. The trajectory at 200 ns showed hydrogen bonds with the residues Asn48 and Asn49. No hydrogen bonds were seen in the 300 ns and 500 ns trajectories, while the trajectory at 400 ns showed a hydrogen bond with Thr38, Glu36, and His4 residues (Fig. 6 B). After presenting the analysis of RMSD, RMSF, RG, and H-bond, Figure 7 (a and b) illustrates the dynamic behavior of the pNPL substrate in the absence and presence of LEA II. In the absence of LEA II, the pNPL substrate undergoes multiple conformational changes during the simulation, particularly notable at 400 ns when the substrate deviates from the binding site (Fig. 7a). Conversely, in the presence of LEA II, the LipA B.licheniformis -pNPL complex demonstrates stability, with the substrate consistently positioned inside the binding site and displaying fewer conformational changes. This suggests that the presence of LEA II contributes to a more stable and well-oriented interaction between the pNPL and LipA B.licheniformis enzyme (Fig. 7b). Molecular Mechanics Poisson Boltzman Surface Area Continuum solvation (MM-PBSA) calculations The trajectories from 450 ns to 500 ns extracted at 1 ns each were utilized in the MM-PBSA calculations. The binding free energy along with other contributing energy in form of MM-GBSA is determined for LipA B.licheniformis - p NPL-LEAII and LipA B.licheniformis - p NPL complexes. The results are shown in Table 1. The relative binding free energies (ΔTOTAL) for LipA B.licheniformis - p NPL and LipA B.licheniformis - p NPL-LEAII complexes were -20.52 and -24.41 kcal/mol, respectively. The ΔG binding energies for LipA B.licheniformis - p NPL and LipA B.licheniformis - p NPL-LEAII complexes after taking into account the entropy were -2.31 and -11.68 kcal/mol, respectively. Notably, in the case of p NPL in LipA B.licheniformis - p NPL-LEAII complex has significantly lower van der Waals energy (ΔVDWAALS) energy of -23.15 kcal/mol, and significantly higher polar solvation free energy (ΔEGB) of -0.91 kcal/mol and non-polar solvation free energy (ΔESURF) of -3.72, compared to p NPL in LipA B.licheniformis - p NPL complex. Further, the entropy in p NPL in LipA B.licheniformis - p NPL-LEAII complex was significantly lower compared to LipA B.licheniformis - p NPL complex. The resultant ΔG binding for p NPL in LipA B.licheniformis - p NPL-LEAII complex was significantly lower compared to the LipA B.licheniformis - p NPL complex. These results suggest that the p NPL has better affinity to LipA B.licheniformis in the LipA B.licheniformis -LEAII bound complex than the LipA B.licheniformis alone. Table 1: MM-PBSA calculations for LipA B.licheniformis - p NPL and LipA B.licheniformis - p NPL-LEAII complexes. Energy component (kcal/mol) Averages for p NPL LipA B.licheniformis - p NPL complex LipA B.licheniformis - p NPL -LEAII complex ΔVDWAALS -19.62 -23.15 ΔEEL -1.83 -0.91 ΔEGB 3.95 3.37 ΔESURF -3.01 -3.72 ΔGGAS -21.45 -24.06 ΔGSOLV 0.93 -0.35 ΔTOTAL -20.52 -24.41 -TΔS (Entropy) 18.21 12.74 ΔG binding -2.31 -11.68 ΔVDWAALS: van der Waals energy; ΔEEL: Electrostatic energies; ΔEGB: Polar solvation free energy; ΔESURF: Non-polar solvation free energy; ΔGGAS = ΔVDWAALS+ΔEEL; ΔGSOLV = ΔEGB + ΔESURF; ΔTOTAL = ΔGSOLV +ΔGGAS; ΔG binding = ΔTOTAL - TΔS. Principal Component Analysis and Gibb’s free energy analysis Principal component analysis (PCA) of the MD simulation trajectories for LipA B.licheniformis - p NPL and LipA B.licheniformis - p NPL-LEAII were analyzed. The first two principal components (PC1 and PC2) were used as reaction coordinates in Gibb’s free energy analysis. The lowest energy metastable conformations were identified from Gibb’s free energy analysis. The LipA B.licheniformis - p NPL complex showed unique metastable conformations occupied in three low energy basins with energy below 2.5 kJ/mol. While the LipA B.licheniformis - p NPL-LEAII complex showed a larger and single unique energy basin where the metastable conformations were clustered having ΔG below 2.5 kJ/mol. The results clearly indicate that the bound LEAII holds the LipA B.licheniformis in stable and unique conformational state. While the LipA B.licheniformis - p NPL complex showed that multiple metastable conformational states are possible due to freedom in global motions in LipA B.licheniformis conformations as shown in Figure 8. DISCUSSION Over the past two decades, there are many attentions and studies were conducted focusing on protein thermostability. Realizing it could be beneficial for industrial application, the number of related studies is growing as there is a demand for higher thermostable protein in which it could help helpful to reduce the production cost due to a better protein stability under thermal stress. Improvement in protein thermostability could open more progress in scientific research by discovering, understanding and exploiting the potentials of novel heat-sensitive enzymes to be used in the future. However, while most studies focus on creating a specific thermostable enzyme candidate, the current approach requires a long process as it involves several complex experimental designs starting from computational analyses to site-targeted mutagenesis. This study is an attempt to explore a new horizon of enhancing protein thermostability through a general, applicable approach for different types of enzymes. The adopted experimental design applied a simple heterologous expression of desired protein, lipase from Bacillus licheniformis (LipA B.licheniformis ) using LEA co-expression system as a novel strategy to improve its thermostability. Lipase was chosen as it is one of the most common and important enzymes in industry. From the previous research findings, it was observed that expressing LEA-like peptides has successfully improved protein and cellular tolerances towards different stresses like UV radiation, salinity, heat stress and pH ( 17 – 19 ). In this work, the usefulness of LEA-like peptides was evaluated further to see the rescue effect of LEA-like peptide to protect desired recombinant protein against heat stress using co-expression system as a strategy. The expression of desired protein, LipA B.licheniformis and LEA-like peptide was induced by IPTG in E. coli BL21 (DE3) as microbial factory. As the expression of LipA B.licheniformis and LEA-like peptide were driven by cold shock prooter and T7 promoter respectively, it was assumed that the supply of LEA peptide is adequate to interact with LipA B.licheniformis produced. Ideally, the strong interaction between LipA B.licheniformis -LEA peptide was undisturbed by sonication and remained intact during protein purification. As a result, the purified LipA B.licheniformis -LEA complex was eluted and subjected for lipase activity at 45˚C. The lipase assay was conducted at its challenging temperature to see the impact of LEA-like peptide interaction in protecting lipase thermostability which reflected through the result of enzyme activity. Comparing the LipA B.licheniformis relative activity and LipA B.licheniformis residual activity among different LEA-like peptide- LipA B.licheniformis complexes, LipA B.licheniformis -LEA II complex has the best improvement of lipase activity. Notably, the LipA B.licheniformis -LEA II complex has a significant increment in lipase relative activity with the reading almost double as compared to the control while the lipase residual activity was also shown slight improvement. The improvement of lipase activity at higher temperature indicates the structure of lipase was protected from denatured. Denaturation of protein by the strong heat stress affects the LipA B.licheniformis folding and bonding that cause the deformation of active site and functional structure. Thus, by forming a strong interaction with LEA-like peptide, the outer surface of LipA B.licheniformis is protected by the peptide, leaving the LipA B.licheniformis structure intact as close as its native structure even with the exposure of a higher temperature. However, it is important to note that the interaction of LipA B.licheniformis with LEA-like peptide should not hinder the active site region. Else, the lipase activity can still be low despite having a thermostable lipase structure. To evaluate the possibilities, a series of bioinformatic analyses starting from molecular docking to molecular dynamic simulation was conducted to understand the peptide-protein interaction and its molecular properties changes during heat challenge. The molecular docking outcome suggested that the p NPL has better binding free energy in the case of LipA B.licheniformis -LEAII complex than the LipA B.licheniformis alone. The overall lower energy state of LipA B.licheniformis -LEAII complex is responsible for more favorable binding affinity of p NPL than the LipA B.licheniformis alone. The MD studies also corroborate the results of molecular docking where the RMSD in LipA B.licheniformis in LipA B.licheniformis - p NPL-LEAII complex clearly indicate the significant stability. Further, the fluctuations inside chain atoms in LipA B.licheniformis and LipA B.licheniformis - p NPL-LEAII complex and the radius of gyration around the center of mass of LipA B.licheniformis in LipA B.licheniformis - p NPL-LEAII complex confirms better stability compared to the LipA B.licheniformis - p NPL complex. Substantially lower solvent accessible surface area in LipA B.licheniformis - p NPL-LEAII complex is suggestive of compact structure. Slightly better propensity of hydrogen bonds between LipA B.licheniformis and p NPL in LipA B.licheniformis - p NPL-LEAII complex further confirms better stability of LipA B.licheniformis - p NPL-LEAII complex. The MM-GBSA calculations confirmed that the p NPL has significantly better binding free energy in LipA B.licheniformis - p NPL-LEAII complex than the LipA B.licheniformis - p NPL complex. Since the interaction of LEA-like peptide- LipA B.licheniformis plays a major role for the successful thermostable improvement, a detailed observation on LEA-like peptide sequences was done. As Fig. 9 shows, there are 13 amino acid residues for each peptide. The differences of LEA-like peptides can be seen on the 6th, 7th, and 12th amino acid. LEA I and LEA II have a subtle difference at amino acid number 7. LEA II replaced threonine, a polar and non-charged amino acid, with leucine, an aliphatic amino acid from the nonpolar group. Changing this amino acid in LEA II made the peptide to be more hydrophobic due to non-polar residue. Meanwhile, the differences between LEA II with LEA E and LEA K are found at amino acid position number 6 and 12. LEA E and LEA K peptides substitute nonpolar, aliphatic amino acid of glycine with glutamic acid, a negative charge amino acid and lysine, a positive charged nonpolar amino acid, respectively. Overall, LEA II has more non-polar and aliphatic amino acids compared to other LEA peptides. The less sensitive attribute shown by lipase-LEA II complex by the heat temperature most likely due to having extra non-polar aliphatic acid. ( 41 ) demonstrated the importance of aliphatic residue in enhancing protein thermostability. Meanwhile, increment of hydrophobic properties in LEA II helps for a better protein-protein interaction with LipA B.licheniformis . A similar observation was mentioned by ( 42 ). Thus, although all LEA-like peptides have the ability to bind with LipA B.licheniformis , the interaction can be weakened by heat energy that could dissociate the complexes. Nevertheless, the thermostability and hydrophobicity properties owned by LEA II promote for the LEA II-LipA B.licheniformis stronger interaction despite challenged with higher temperature. As a result, LEA II promotes better lipase structure stability which was proven by having a good lipase activity at higher temperature. Possessing charged amino acid in LEA E and LEA K did not give good results. Again, the possible reason is because the peptides are losing its hydrophobicity and thermostability properties at these specific, crucial amino acids. Another possibility of lower performance of charged LEA-like peptide is also due to its specific surface preference while establishing interaction with the LipA B.licheniformis . For example, LEA-K has more tendency to bind at negative charge region on LipA B.licheniformis surface and vice versa. This preference probably is not the best to protect LipA B.licheniformis structure because it has more selective binding as compared to LEA II. In conclusion, this study highlights the significant enhancement of LipA B.licheniformis thermostability at 45°C through the incorporation of LEA-II peptide, showcasing its efficacy in bolstering heat tolerance. The observed impact underscores the critical role of specific amino acid position. Minor alterations with different groups of amino acid even with a single amino acid make a big difference by comparing lipase activity between lipase-LEA I and lipase-LEA II. Furthermore, the study suggests a useful direction to explore by testing hypotheses, specifically in changing the 6th and 12th amino acids to non-charged hydrophobic counterparts, offering valuable insights for optimizing LipA B.licheniformis thermostability. Although the precise mechanism of improved stress tolerance in protein using LEA-like peptides remains elusive, this approach can be employed to extend its broader applicability for other proteins. Indeed, if this approach is applicable for different proteins, it reduces complexity, saves time to produce other useful protein with higher stress tolerance properties. Declarations Author Contribution Statement: Ammar Khazaal Kadhim Almansoori, Kang Siang Yu and Rashidah Abdul Rahim conceptualized, designed the research idea and analyzed data. Ammar Khazaal Kadhim Almansoori, Faisal Mohamed and Kang Siang Yu conducted the experiments, wrote the main manuscript, reviewing and editing. Shinya Ikeno contributed with LEA peptides and provided the analytical tools of in vitro experiments. Ammar Khazaal Kadhim Almansoori, Rajesh B. Patil and Ropón-Palacios G provided the analytical tools of in silico experiments, analyzed data, reviewing and editing. Rashidah Abdul Rahim supervision, resources, reviewing and editing. Acknowledgments : The authors would like to thank the Malaysian Ministry of Higher Education, Fundamental Research Grant Scheme (FRGS) (203.PBiologi.6712194) and Universiti Sains Malaysia, Research University (RU) grant (No: 1001/PBIOLOGI/8011009) for supporting this research. Data Availability Statement: All data are present in the manuscript and it’s Supplement. Statement and Declarations: The authors declare that we do not have any conflict of interest to the content of this article. Funding: This research was supported by the Malaysian Ministry of Higher Education, Fundamental Research Grant Scheme (FRGS) (203.PBiologi.6712194) and Universiti Sains Malaysia, Research University (RU) grant (No: 1001/PBIOLOGI/8011009). Ethical approval statement: This article does not contain any studies with human participants or animals performed by any of the authors. References Guncheva M, Zhiryakova D. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4160767","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":287733174,"identity":"f1ba7cd2-c7d7-4253-9f51-3411a6161e74","order_by":0,"name":"Ammar Khazaal Kadhim Almansoori","email":"data:image/png;base64,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","orcid":"","institution":"Universiti Sains Malaysia","correspondingAuthor":true,"prefix":"","firstName":"Ammar","middleName":"Khazaal Kadhim","lastName":"Almansoori","suffix":""},{"id":287733175,"identity":"d711dd94-5bcd-4154-92c6-5419f5c7029d","order_by":1,"name":"Kang Siang Yu","email":"","orcid":"","institution":"Universiti Sains Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Kang","middleName":"Siang","lastName":"Yu","suffix":""},{"id":287733176,"identity":"b9571bd5-d603-4783-bb82-3c9563d22d93","order_by":2,"name":"Faisal Mohamed","email":"","orcid":"","institution":"Universiti Sains Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Faisal","middleName":"","lastName":"Mohamed","suffix":""},{"id":287733177,"identity":"ef12ae7a-ba48-4f85-bc32-c6fc126e3059","order_by":3,"name":"Shinya Ikeno","email":"","orcid":"","institution":"Kyushu Institute of Technology: Kyushu Kogyo Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Shinya","middleName":"","lastName":"Ikeno","suffix":""},{"id":287733178,"identity":"de945ed7-ccb5-4bb2-83ad-6eb1869031ef","order_by":4,"name":"Rajesh B. Patil","email":"","orcid":"","institution":"Sinhgad College of Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Rajesh","middleName":"B.","lastName":"Patil","suffix":""},{"id":287733179,"identity":"d06669b7-153b-4272-a05f-74d12a2626af","order_by":5,"name":"Ropón-Palacios G","email":"","orcid":"","institution":"Universidade Federal de Alfenas","correspondingAuthor":false,"prefix":"","firstName":"Ropón-Palacios","middleName":"","lastName":"G","suffix":""},{"id":287733180,"identity":"e6580ea0-0e3c-405b-9c61-4fe7da2029ac","order_by":6,"name":"Rashidah Abdul Rahim","email":"","orcid":"","institution":"Universiti Sains Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Rashidah","middleName":"Abdul","lastName":"Rahim","suffix":""}],"badges":[],"createdAt":"2024-03-25 05:09:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4160767/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4160767/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54365675,"identity":"89a63182-0321-4fdd-bdd7-d964ef076ce1","added_by":"auto","created_at":"2024-04-09 12:28:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":71575,"visible":true,"origin":"","legend":"\u003cp\u003eRelative activity (A) and residual activity (B) of LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e and LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e co-expressed with LEA I, II, E and K peptides at 45 °C using pCold expression system.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4160767/v1/b5ac875ca03388d75e744414.png"},{"id":54365677,"identity":"87358ab4-d70c-48d7-be15-276733dd8025","added_by":"auto","created_at":"2024-04-09 12:28:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":299366,"visible":true,"origin":"","legend":"\u003cp\u003eThe 3D tertiary structure of (A). LEA II peptide using PEP-FOLD webservers, and C-terminus are labelled as N and C, respectively. The α-helix and the loop are coloured green. (B). LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e obtained using Phyre2 webserver. The catalytic triad, Ser78, Asp129 and His152 are labelled and shown in blue stick form. The α-helices are red in colour, β-strands are yellow in colour and loops are green in colour.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4160767/v1/b4fab8f797ffb46adea2f674.png"},{"id":54365673,"identity":"ca25da8e-6e87-49a0-a472-ce294e0a8c24","added_by":"auto","created_at":"2024-04-09 12:28:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":469884,"visible":true,"origin":"","legend":"\u003cp\u003eStructures of protein-protein docking best pose between LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e (Chain A) and LEAII (Chain B, right panel). The left panel explaining the residual interactions between LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e and LEAII. The middle panel exhibits interface statistics.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4160767/v1/4430be0c694923931e81d03b.png"},{"id":54365678,"identity":"165d3dd5-68b2-4826-bb75-b021a8905cb6","added_by":"auto","created_at":"2024-04-09 12:28:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":816843,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular surface view of (A) LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e-\u003cem\u003ep\u003c/em\u003eNPL (B) LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e-\u003cem\u003ep\u003c/em\u003eNPL-LEAII binding core accommodating the ligand (Left panel) (the surface on LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e is shown in light blue color while surface on LEAII is shown in light pink color. The \u003cem\u003ep\u003c/em\u003eNPL is shown in pink stick representation). 3D and 2D interaction with hydrogen bond and other non-bonded interactions (Right panel).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4160767/v1/50e7824473227f1e7ffb5802.png"},{"id":54365680,"identity":"9924df00-3d1d-4a09-920a-f74a54fd9521","added_by":"auto","created_at":"2024-04-09 12:28:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":303093,"visible":true,"origin":"","legend":"\u003cp\u003eMD analysis. (A) RMSD plots for backbone atoms of LipA\u003csub\u003eB.licheniformis\u003c/sub\u003e in LipA\u003csub\u003eB.licheniformis\u003c/sub\u003e-pNPL complex (black) and LipA\u003csub\u003eB.licheniformis\u003c/sub\u003e in LipA\u003csub\u003eB.licheniformis\u003c/sub\u003e-pNPL-LEAII complex(red), (B) RMSD in LEAII backbone atoms in LipA\u003csub\u003eB.licheniformis\u003c/sub\u003e-pNPL-LEAII complex, (C) RMSD in pNPL atoms in the respective complexes, (D)RMSF of side chain atoms of LipA\u003csub\u003eB.licheniformis\u003c/sub\u003e in both the complexes, (E) Radius of gyration (Rg), and (F) Solvent accessible surface area of LipA\u003csub\u003eB.licheniformis\u003c/sub\u003e-pNPL and LipA\u003csub\u003eB.licheniformis\u003c/sub\u003e-pNPL-LEAII complex.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4160767/v1/2e8a3ab3e1f2d63c3e64838a.png"},{"id":54365679,"identity":"7180ca3c-d0ed-4226-9e8c-a930238ec5dc","added_by":"auto","created_at":"2024-04-09 12:28:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":65083,"visible":true,"origin":"","legend":"\u003cp\u003eHydrogen bond analysis. Hydrogen bonds between (A) LipA\u003csub\u003eB.licheniformis\u003c/sub\u003e and pNPL in LipA\u003csub\u003eB.licheniformis\u003c/sub\u003e-pNPL complex, (B) LipA\u003csub\u003eB.licheniformis\u003c/sub\u003e and pNPL in LipA\u003csub\u003eB.licheniformis\u003c/sub\u003e-pNPL-LEAII complex.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4160767/v1/3aa85b3a3904cf448c1cbe65.png"},{"id":54365672,"identity":"78688dd8-4daf-466e-a470-abecde5f8f45","added_by":"auto","created_at":"2024-04-09 12:28:47","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2636520,"visible":true,"origin":"","legend":"\u003cp\u003ea\u003cstrong\u003e:\u003c/strong\u003e Snapshots of MD trajectories of LipA\u003csub\u003eB.licheniformis\u003c/sub\u003e-pNPL complex extracted at (A) equilibrium state (0 ns), (B) 100 ns, (C) 200 ns, (D) 300 ns, (E) 400 ns, and (F) 500 ns. (The hydrogen bonds formed are appropriatly shown in zoom in panel).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eb\u003cstrong\u003e:\u003c/strong\u003e Snapshots of MD trajectories of LipA\u003csub\u003eB.licheniformis\u003c/sub\u003e-pNPL-LEAII complex extracted at (A) equilibrium state (0 ns), (B) 100 ns, (C) 200 ns, (D) 300 ns, (E) 400 ns, and (F) 500 ns.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4160767/v1/5c21a11c6cedc34df42a093f.png"},{"id":54365676,"identity":"3234eeb4-1eb5-4ad7-a7d7-695557a90c5b","added_by":"auto","created_at":"2024-04-09 12:28:48","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":585079,"visible":true,"origin":"","legend":"\u003cp\u003eGibb’s free energy analysis for (A) LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e-\u003cem\u003ep\u003c/em\u003eNPL complex and (B) LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e-\u003cem\u003ep\u003c/em\u003eNPL-LEAII complex.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4160767/v1/6b7c6e5e95ee168bbf28139c.png"},{"id":54365671,"identity":"7ed1dd5c-f5bc-49b5-9041-6dfb94b0af53","added_by":"auto","created_at":"2024-04-09 12:28:47","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":104683,"visible":true,"origin":"","legend":"\u003cp\u003eLEA peptide series amino acid residues.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-4160767/v1/03b03272eaaef256166fb380.png"},{"id":55296707,"identity":"af5da6fd-7248-4a5e-9940-afb3993cf0ab","added_by":"auto","created_at":"2024-04-25 10:50:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3885754,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4160767/v1/fbc6404a-f91d-4bf8-abf6-64cec1114a51.pdf"},{"id":54365670,"identity":"0a48c272-94e1-4508-8535-d4f9067fbf73","added_by":"auto","created_at":"2024-04-09 12:28:47","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2360491,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-4160767/v1/b2aee8a7a74bccf408bf30f3.docx"},{"id":54365669,"identity":"095f62c2-1c3e-488b-ba86-27923b4c716f","added_by":"auto","created_at":"2024-04-09 12:28:47","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1436896,"visible":true,"origin":"","legend":"","description":"","filename":"graphicalabstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-4160767/v1/b1d9f130603029168d437f5e.tif"}],"financialInterests":"","formattedTitle":"Enhancing Thermostability of Bacillus licheniformis Lipase with LEA Peptide Co-expression System.","fulltext":[{"header":"Key points","content":"\u003cul\u003e\n \u003cli\u003eLipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e thermostability was improved through the novel implementation of the LEA peptide co-expression system.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eLipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e properties have been improved after interacting with the LEA II peptide.\u003c/li\u003e\n \u003cli\u003eThe LEA II peptide aids in enhancing the stability and affinity of the LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"INTRODUCTION","content":"\u003cp\u003eLipases are a group of enzymes responsible for the hydrolysis of triglycerides into free fatty acids and glycerol (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) as well as esterification by using glycerol and fatty acids as substrates under low water conditions (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Lipase can be divided into several groups based on substrate specificity. However, lipase generally shared common properties such as cofactor independent, active in organic solvent, highly efficient and stable as well as regioselective, chemoselective and enantioselective (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). The usefulness of lipase makes it widely utilized in various sectors, including detergent, food, biodiesel, medical, and pharmaceutical industries (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Microbial lipases are preferable for commercial uses since the production using microorganisms is scalable and cost-efficient. Nevertheless, the number of strains used for commercial purposes is still low (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOne of the limiting factors that contribute to low number of microbial lipases is because of heat sensitive. The producing, storing, or using protein, exposure to heat can significantly reduce the enzyme half-life due to stress. Furthermore, when used for esterification without water, lipase often requires higher temperatures to melt the fat (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). While there are many heat-resistant lipases available on the market, their numbers are decreasing because obtaining thermostable lipases is challenging. Gupta et al., 2004 reviewed that most of the microbial lipases used in the industries come from mesophilic bacteria which commonly produced from Genus of \u003cem\u003ePseudomonas, Burkholderia, Chromobacterium.\u003c/em\u003e Of the high demand on thermostable enzymes, researchers usually apply rationale design to overcome heat sensitive limitation. The approach integrates bioinformatic tools and mutagenesis to create specific protein with desired attribute. This strategy requires high skill designer, time consuming and specifically design for protein candidate which are the drawback (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, the target lipase used was isolated from \u003cem\u003eBacillus licheniformis\u003c/em\u003e IBRL-CHS2 and is called LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e. It is a small enzyme with a size of 18.6 kDa, functioning optimally at a temperature of 37\u0026deg;C and pH 7 while hydrolyzing the C12 triglyceride substrate, laurate (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). The catalytic function of LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e is modulated by the presence of its active site, in contrast to being governed by a lid. This is attributed to the absence of a lid in LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e, leading to the formation of a pre-existing oxyanion hole at the active site. This structural feature facilitates substrate binding without requiring interfacial activation. Consequently, LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e demonstrates efficient performance at low substrate concentrations, exhibiting high enzymatic activity. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). However, similar to many enzymes, LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e is sensitive to temperature fluctuations, leading to the unfolding of the enzyme and a substantial decrease in activity at 45 \u0026ordm;C (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLate embryogenesis abundant (LEA) proteins, initially recognized in plants and later found in various organisms, play a pivotal role in responding to stress conditions (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). With the exception of Group 5 LEA proteins, all LEA proteins usually have a molecular weight between 10\u0026ndash;30 kDa, a hydrophilic nature, and stability, marked by repetitive hydrophilic amino acids (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). These LEA proteins contain an intrinsically disordered structure, contributing to a lack of proper folding in a hydrated state; however, this structure transforms into a well-defined secondary structure when water is absent (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). In a significant discovery by Liu and colleagues (2010), it was revealed that a Group 3 LEA protein, specifically the PM2 protein isolated from soybean, effectively enhances the heat tolerance of \u003cem\u003eEscherichia coli\u003c/em\u003e when over-expressed in the cell (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Interestingly, Ikeno and Haruyama (2013) have successfully demonstrated that the modified 11-mers repetitive motif found in Group 3 LEA from the sleeping chironomid, \u003cem\u003ePolypedilum vanderplanki\u003c/em\u003e, is helpful in improving protein production in \u003cem\u003eEscherichia coli\u003c/em\u003e through the LEA-like peptide co-expression system (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMoreover, more success stories involving the use of LEA-like peptides to enhance cellular tolerance to UV radiation, acidic stress, temperature variations, and changes in salinity have been reported in recent years (\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). However, no study has delved further into the impact of LEA co-expression in enhancing protein properties. This research, using lipase (LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e) as a protein of interest and heat stress as one of the limiting factors in protein stability, represents the first attempt to explore the utility of the LEA-like co-expression system, which could potentially be applied to produce enzymes with stress tolerance. Additionally, utilized molecular docking and molecular dynamic simulation to comprehend how the interaction of LEA-like peptides can contribute to protein stability.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e \u003cb\u003eConstruction of pCold- LipA\u003c/b\u003e \u003csub\u003e \u003cb\u003eB.licheniformis\u003c/b\u003e \u003c/sub\u003e \u003cb\u003e-T7LEA\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe pCLEA-LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e series was constructed by cloning LEA-like peptide; I, II, E, and K into pCOLD-LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e, respectively at \u003cem\u003eBam\u003c/em\u003eHI and \u003cem\u003eHind\u003c/em\u003eIII sites. The pCOLD-LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e was derived from pCOLD-I (Takara, Clontech, Japan), reserving lipase gene at \u003cem\u003eNde\u003c/em\u003eI and \u003cem\u003eBam\u003c/em\u003eHI sites (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). The plasmid DNA template to amplify LEA-like peptides (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). To ensure LEA-like peptide co-expressed with LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e, T7 promoter was introduced to upstream of LEA-like DNA sequence, while the expression of LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e was driven by the original promoter of the pCold\u0026trade; I vector. The sequence of LEA peptides, origin of the plasmid along with primers and the constructed map, can be found in S1.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCo-expression and purification of LipA\u003c/b\u003e \u003csub\u003e \u003cb\u003eB.licheniformis\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eand LEA-like peptides\u003c/b\u003e\u003c/p\u003e \u003cp\u003eProtein expression was carried out by inducing 0.2 mM IPTG prechilled bacterial culture; \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3), pCLEA-LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e for 30 minutes after reaching OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;~\u0026thinsp;0.5. Cultures were shaken-incubated at 150rpm and 15 \u0026ordm;C for 16 hours. Protein purification was conducted using His-tagged immobilized metal affinity chromatography (IMAC) with the HisTALON\u0026trade; Gravity Column Purification Kit (Takara, Clontech, Japan) as suggested by manufacturer. Following purification, the lipase was subjected to SDS-PAGE, and the concentration was determined using the Bio-Rad Protein Assay (Biorad, USA), based on the Bradford assay (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eLipase assay\u003c/h2\u003e \u003cp\u003eThe lipase's enzymatic activity was assessed using the \u003cem\u003ep\u003c/em\u003e-nitrophenyl laurate (\u003cem\u003ep\u003c/em\u003eNPL) colorimetric method, following a slight modification of the lipase assay outlined by Ch\u0026rsquo;ng and Sudesh (2013) (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). The assay was chosen and optimized in our lab by Reddy (2016) (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Subsequently, lipase activity, relative activity, and residual activity were calculated using the equations provided in S2.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePrediction of interactions between LipA\u003c/b\u003e \u003csub\u003e \u003cb\u003eB.licheniformis\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eand LEA II peptide by\u003c/b\u003e \u003cb\u003ein silico\u003c/b\u003e \u003cb\u003eapproach\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eTertiary structure prediction\u003c/h2\u003e \u003cp\u003eThe Phyre2 webserver (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.sbg.bio.ic.ac.uk/phyre2\u003c/span\u003e\u003cspan address=\"http://www.sbg.bio.ic.ac.uk/phyre2\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) was employed to predict the tertiary structure of LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e as for secondary structure of was assigned using Stride webserver (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://webclu.bio.wzw.tum.de/stride/\u003c/span\u003e\u003cspan address=\"http://webclu.bio.wzw.tum.de/stride/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Simultaneously, the tertiary structure prediction for the LEA II peptide was conducted using the PEP-FOLD 3.5 webserver (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioserv.rpbs.univ-paris-diderot.fr/PEP-FOLD\u003c/span\u003e\u003cspan address=\"http://bioserv.rpbs.univ-paris-diderot.fr/PEP-FOLD\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). The model of the LEA II peptide with the highest Optimized Potential for Efficient structure Prediction (sOPEP) score was chosen for subsequent docking with LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e as shown in S3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMolecular Docking\u003c/h2\u003e \u003cp\u003eProtein-protein docking of LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e and LEAII was carried out using ClusPro web server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cluspro.org\u003c/span\u003e\u003cspan address=\"https://cluspro.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Post-analysis of protein-protein interaction was performed using PDBSum web server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/thornton-srv/databases/pdbsum/\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/thornton-srv/databases/pdbsum/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The binding propensities of \u003cem\u003ep\u003c/em\u003eNPL with LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e and LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e+LEAII proteins was also investigated through molecular docking studies which were performed on Autodock Vina Version 1.2.3 (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e), where \u003cem\u003ep\u003c/em\u003eNPL was docked into the binding site of LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e or LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e+LEAII. The binding site residues of LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e have been determined using DoG Site Scorer (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://proteins.plus/\u003c/span\u003e\u003cspan address=\"https://proteins.plus/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e last accessed on 28/08/2023). The molecular structure of \u003cem\u003ep\u003c/em\u003eNPL was constructed and optimized using Avogadro program Version 1.2.0 (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Subsequently the enzyme structure and \u003cem\u003ep\u003c/em\u003eNPL structure was converted and saved into pdbqt format. Molecular docking was performed within a grid box dimension 30 x 30 x 30 \u0026Aring; with center of box along X, Y, and Z at 28.34, 16.65, and 151.78, respectively. The exhaustiveness value was set to 100 for efficient search. All other parameters were set at their default values. The best docked conformations were chosen from amongst the best cluster that had the lowest binding free energies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMolecular dynamics simulation (MDS)\u003c/h2\u003e \u003cp\u003eThe docked complexes of LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e+\u003cem\u003ep\u003c/em\u003eNPL and LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e+\u003cem\u003ep\u003c/em\u003eNPL\u0026thinsp;+\u0026thinsp;LEAII were subjected to 500 ns extended molecular dynamics simulations using the Gromacs Version 2023 program (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). The topologies of LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e and LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e+LEAII were generated using the CHARMM-36 force field parameters (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e); while the topology of \u003cem\u003ep\u003c/em\u003eNPL was generated from the CGenFF server (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). The complexes of LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e+\u003cem\u003ep\u003c/em\u003eNPL and LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e+\u003cem\u003ep\u003c/em\u003eNPL_LEAII were placed in a dodecahedron unit cell such as that the edges of systems remained 1 nm away from the edges of box. The systems were solvated by adding the water using the TIP3P water model (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e) and neutralized by adding appropriate number of sodium and chloride counter-ions so as to achieve the molar concentration of 0.15. The system was relieved of the steric strain by energy minimization with the steepest descent algorithm until the force-constant reaches the threshold of 100 kJ mol-1 nm-1. Two step equilibration was performed on resulting system, where the initial equilibration was performed at constant temperature condition (NVT) of 318. K temperature and later at constant pressure conditions (NPT) at 1 atm pressure. During equilibration steps, the constant temperature conditions were achieved using a modified Berendsen thermostat (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e) and the constant pressure conditions were achieved using the Berendsen barostat (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e), for 1 ns each. The final 500 ns production phase MD simulation were performed where the temperature conditions of 45\u0026deg;C were achieved with a modified Berendensen thermostat and pressure conditions of 1 atm were achieved with the Parrinello-Rahman barostat (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). The restrain on covalent bonds was achieved with LINCS algorithm (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). The long-range electrostatic energies were computed with Particle Mesh Ewald (PME) method (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) with a cut-off of 1.2 nm.\u003c/p\u003e \u003cp\u003eThe output trajectories were treated for periodic boundary conditions before MD analysis. The root mean square deviations (RMSD) in the backbone atoms, the root mean square fluctuation (RMSF) in the side chain atoms, radius of gyration (Rg), and the analysis of solvent accessible solvent area (SASA) of both the system were analyzed and compared for the stability aspects. The hydrogen bonds formatted between the \u003cem\u003ep\u003c/em\u003eNPL and LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e as well as the hydrogen bonds formed between the LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e and LEAII were analyzed and the representative trajectories at the beginning of simulation and at 100, 200, 300, 400, and 500 ns were investigated. The major path of motions in each complex was studied through the principal component analysis (PCA) (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e), where the covariance matrix for the C-α atom was constructed and diagonalized to obtain the eigenvectors and eigenvalues. The eigenvectors represent the motion path, while eigenvalues represent the mean square fluctuation. The first two eigenvectors, principal components (PC1 and PC2) were further used as a reaction coordinate in the Gibb\u0026rsquo;s free energy landscape (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e) analysis. Molecular mechanics energies combined with Poisson Boltzmann surface area continuum solvation (MM-PBSA) and Molecular mechanics with generalised Born and surface area solvation (MM-GBSA) calculations were performed with gmx_MMPBSA tool (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e) on the trajectories sampled at 1 ns each from the 450 ns to 500 ns simulation period.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eCo-expression and purification of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eLipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and LEA-like peptides\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe lipases produced from LEA co-expression system were successfully expressed and purified as shown in SDS- polyacrylamide gel (S3). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTemperature study of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eLipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;co-expressed with LEA-like peptides\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe lipase activity was assessed using the \u003cem\u003ep\u003c/em\u003eNPL lipase assay at 37\u0026deg;C and 45\u0026deg;C. At 37\u0026deg;C, no significant difference was observed between LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e alone and LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e co-expressed with the LEA series peptides in both relative and residual activities.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAt 45\u0026deg;C, the resulting relative activities were calculated and visually represented in Figure 1A. Subsequently, a one-way ANOVA analysis was performed, followed by multiple comparisons utilizing the Tukey test at a significance level of 5%. The findings revealed that LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e exhibited the highest relative activity when co-expressed with the LEA II peptide at 45\u0026deg;C. As shown in Figure 1A, the statistical analysis demonstrated a significant difference compared to LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e alone (****p \u0026lt; 0.05). Conversely, no significant difference was observed after co-expression with LEA I, LEA E, and LEA K peptides.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurthermore, the lipase activity after incubation at 45\u0026deg;C for an hour showed slightly higher residual activity after co-expression with the LEA II peptide, while for LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e alone and co-expressed with LEA I, E, and K peptides, the residual activities showed no significant difference as shown in Figure 1B.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrediction of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eLipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e-LEA interaction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Phyre2-predicted tertiary structure of\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e exhibited a 100% confidence level and 99% identity with the template lipase lip_vut1 structure from the metagenome of goat rumen (PDB: 6nkc.1). Illustrated in Figure 2A, this lipase adopted an \u0026alpha;/\u0026beta; hydrolase fold, with the six \u0026beta;-strands (\u0026beta;3-\u0026beta;8) forming a parallel \u0026beta;-sheet acting as the core. It featured nine \u0026alpha;-helices, comprising four \u0026alpha;-helices (A, B, C, and F) and five 310-helices. The catalytic triad Ser-78 was situated at the nucleophilic elbow between the \u0026beta;5 strand and helix C, while Asp-130 and His-153 were located in the loop after the \u0026beta;7 and \u0026beta;8 strands, respectively.\u003c/p\u003e\n\u003cp\u003eSince LEA II peptide showed the best improvement in the relative and residual activities of\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e, the tertiary structure of LEA II peptide was predicted by PEP-FOLD webserver for further \u003cem\u003ein silico\u003c/em\u003e investigation. As shown in Figure 2B, the LEA II peptide was predicted to be made up of loops with an \u0026alpha;-helix at the center of the structure. The LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e 2D structure, LEA II structures and the validation of the 3D structures are shown in S4.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein-protein docking\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein-protein docking (PPI) between LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e and LEAII results are shown in Figure 3. The binding energy score calculated from the ClusPro server for LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-LEAII was -450, where the LEAII made 57 non-bonded interactions and three hydrogen bond interactions between the Glu9 of LEAII and Met79 and Ser78 of\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e and one hydrogen bond between Lys10 of LEAII and Ile132 of LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e. A total of 13 residues from the\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e and 7 residues from LEAII were involved in these non-bonded interactions. Of these 7 residues, 5 residues are conserved in all LEA-like peptides while 2 consecutive residues; Gly6 and Leu7 are unique to LEA II.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular Docking studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe evaluation of binding energy was done to look at the effect of\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-LEA II interaction with ligand. The control,\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e with \u003cem\u003ep\u003c/em\u003eNPL has a lower binding energy \u0026Delta;G = -3.914 kcal/mol, as compared to the\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-LEAII with \u003cem\u003ep\u003c/em\u003eNPL binding energy which is \u0026Delta;G = -6.0091 kcal/mol. The \u003cem\u003ep\u003c/em\u003eNPL showed hydrogen bonds with His77, Ser78, Gly12, and Ile13, while the hydrophobic alkyl part of \u003cem\u003ep\u003c/em\u003eNPL showed the hydrophobic alkyl interactions with Met79, Ala106, Val133, Leu109, and Leu137 residues. The \u003cem\u003ep\u003c/em\u003eNPL formed five conventional hydrogen bonds with the residues His77, Ser78, Gly12, Ile13, and Met79. Hydrophobic interactions were found with similar residues at the binding site \u003cem\u003eviz.\u0026nbsp;\u003c/em\u003eAla106, Val133, Leu109, and Leu137. The docked poses of \u003cem\u003ep\u003c/em\u003eNPL in\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e and LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-LEAII the complexes were almost similar as shown in Figure 4. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular Dynamics Simulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMolecular dynamics simulations extended to 500 ns were carried out in order to determine the stability and convergence of\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL-LEAII\u0026nbsp;complex and\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL complex at\u0026nbsp;45 \u0026deg;C\u0026nbsp;. The RMSD of backbone atoms of\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL and\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL-LEAII exhibited reasonable convergence and stability (Fig. 5 A). The noteworthy difference arose from 200 ns onwards where the RMSD in backbone atoms of\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL-LEAII was found reasonably lower than the\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL complex. The average RMSD for\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL-LEAII complex was around 0.125 nm, while for\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL was around 0.15 nm. The LEAII being a separate chain in\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL-LEAII complex the RMSD in backbone atoms of LEAII was separately investigated. It was observed that the RMSD in LEAII was comparatively high reaching a maximum of 0.65 nm and average around 0.45 nm (Fig. 5 B). The RMSD in \u003cem\u003ep\u003c/em\u003eNPL atoms in both the systems remained almost similar with an average of around 0.3 nm (Fig. 5 C). It is evident from the results of RMSD in backbone atoms of\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e and atoms of \u003cem\u003ep\u003c/em\u003eNPL that the\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL-LEAII complex is quite stable compared to\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL complex. The intermolecular non-bonded interactions between\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e and LEAII might have stabilized the LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e. The fluctuations inside chain atoms of\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e was further assessed through RMSF analysis which showed that the LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL has higher magnitude of fluctuations, except the residues in the range 50-60, compared to\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL-LEAII (Fig. 5 D). The residues in the range 50-60 showed slightly higher fluctuations in\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL-LEAII complex and most of the residues in this range are interface residues between\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e and LEAII. However, in both the complexes the RMSF is below 0.35 which indicates reasonable stability of the complexes. Radius of gyration (Rg) is the measure of compactness of the protein. During the first 50 ns simulation period LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL-LEAII displayed slightly higher Rg compared to the Rg of\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL (Figure 5 E). Thereafter,\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL-LEAII complex showed lower Rg than\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL until the end of simulation with an average of around 1.47 nm, while the average for\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL was around 1.48 nm. The solvent accessible surface area (SASA) provides insights of disordered regions of protein and smaller the SASA more is the compactness of protein and better is the stability.\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL-LEAII complex has significantly lower SASA with an average of around 20 nm\u003csup\u003e2\u003c/sup\u003e, while\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL has an average of around 80 nm\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e(Fig. 5 F). The lower SASA values in\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL-LEAII might be due to interactions of LEAII with\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e and proportionally lower surface area available for solvent access. The binding affinity of \u003cem\u003ep\u003c/em\u003eNPL was assessed in terms of the hydrogen bonds formed between \u003cem\u003ep\u003c/em\u003eNPL and\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e. The\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL complex showed that around 2 consistent hydrogen bonds formed between \u003cem\u003ep\u003c/em\u003eNPL and\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e, while in\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL-LEAII around 3 consistent hydrogen bonds were formed between \u003cem\u003ep\u003c/em\u003eNPL and\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e. LEAII might have caused the conformational change in\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e so that \u003cem\u003ep\u003c/em\u003eNPL binds with more affinity and with more number of hydrogen bonds at the binding cavities. The LEAII also formed around 6 hydrogen bonds with the\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e in LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL-LEAII complex.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe hydrogen bonds formed in different trajectories extracted at various time intervals showed that the initial equilibrated trajectory of LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL complex has hydrogen bonds between \u003cem\u003ep\u003c/em\u003eNPL and Ile13 and Gly12. The 100 ns trajectory showed a hydrogen bond with Ile43, which suggested that \u003cem\u003ep\u003c/em\u003eNPL occupied a different binding cavity. Similarly, the 200 ns trajectory showed a hydrogen bond with Ala16 in this binding cavity. None of 300, 400, and 500 ns trajectories showed any hydrogen bonds and at 400 ns \u003cem\u003ep\u003c/em\u003eNPL moved out of binding cavity (Fig. 6 A). On the other hand, the initial equilibrated trajectory of\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL-LEAII complex also showed a hydrogen bond between \u003cem\u003ep\u003c/em\u003eNPL and Gly12 and Ile13. \u003cem\u003ep\u003c/em\u003eNPL occupied a different binding cavity in the 100 ns without any hydrogen bond. The trajectory at 200 ns showed hydrogen bonds with the residues Asn48 and Asn49. No hydrogen bonds were seen in the 300 ns and 500 ns trajectories, while the trajectory at 400 ns showed a hydrogen bond with Thr38, Glu36, and His4 residues (Fig. 6 B). \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter presenting the analysis of RMSD, RMSF, RG, and H-bond, Figure 7 (a and b) illustrates the dynamic behavior of the pNPL substrate in the absence and presence of LEA II. In the absence of LEA II, the pNPL substrate undergoes multiple conformational changes during the simulation, particularly notable at 400 ns when the substrate deviates from the binding site (Fig. 7a). Conversely, in the presence of LEA II, the LipA\u003csub\u003eB.licheniformis\u003c/sub\u003e-pNPL complex demonstrates stability, with the substrate consistently positioned inside the binding site and displaying fewer conformational changes. This suggests that the presence of LEA II contributes to a more stable and well-oriented interaction between the pNPL and \u0026nbsp;LipA\u003csub\u003eB.licheniformis\u003c/sub\u003e enzyme (Fig. 7b).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular Mechanics Poisson Boltzman Surface Area Continuum solvation (MM-PBSA) calculations\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe trajectories from 450 ns to 500 ns extracted at 1 ns each were utilized in the MM-PBSA calculations. The binding free energy along with other contributing energy in form of MM-GBSA is determined for\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL-LEAII and\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL complexes. The results are shown in Table 1. The relative binding free energies (\u0026Delta;TOTAL) for\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL and\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL-LEAII complexes were -20.52 and -24.41 kcal/mol, respectively. The \u0026Delta;G binding energies for\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL and\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL-LEAII complexes after taking into account the entropy were -2.31 and -11.68 kcal/mol, respectively. Notably, in the case of \u003cem\u003ep\u003c/em\u003eNPL in\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL-LEAII complex has significantly lower van der Waals energy (\u0026Delta;VDWAALS) energy of -23.15 kcal/mol, and significantly higher polar solvation free energy (\u0026Delta;EGB) of -0.91 kcal/mol and non-polar solvation free energy (\u0026Delta;ESURF) of -3.72, compared to \u003cem\u003ep\u003c/em\u003eNPL in\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL complex. Further, the entropy in \u003cem\u003ep\u003c/em\u003eNPL in\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL-LEAII complex was significantly lower compared to\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL complex. The resultant \u0026Delta;G binding for \u003cem\u003ep\u003c/em\u003eNPL in\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL-LEAII complex was significantly lower compared to the\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL complex. These results suggest that the \u003cem\u003ep\u003c/em\u003eNPL has better affinity to\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e in the LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-LEAII bound complex than the\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e alone. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 1:\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eMM-PBSA calculations for\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL and\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL-LEAII complexes.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"623\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.879614767255216%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eEnergy component (kcal/mol)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"75.12038523274478%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAverages for \u003cem\u003ep\u003c/em\u003e\u003c/strong\u003eNPL\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.54797441364605%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eLipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e-\u003cem\u003ep\u003c/em\u003e\u003c/strong\u003eNPL\u003cstrong\u003e\u0026nbsp;complex\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"52.45202558635395%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eLipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e-\u003cem\u003ep\u003c/em\u003e\u003c/strong\u003eNPL\u003cstrong\u003e-LEAII complex\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.83974358974359%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026Delta;VDWAALS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.73717948717949%\" valign=\"top\"\u003e\n \u003cp\u003e-19.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.42307692307692%\" valign=\"top\"\u003e\n \u003cp\u003e-23.15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.83974358974359%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026Delta;EEL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.73717948717949%\" valign=\"top\"\u003e\n \u003cp\u003e-1.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.42307692307692%\" valign=\"top\"\u003e\n \u003cp\u003e-0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.83974358974359%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026Delta;EGB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.73717948717949%\" valign=\"top\"\u003e\n \u003cp\u003e3.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.42307692307692%\" valign=\"top\"\u003e\n \u003cp\u003e3.37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.83974358974359%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026Delta;ESURF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.73717948717949%\" valign=\"top\"\u003e\n \u003cp\u003e-3.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.42307692307692%\" valign=\"top\"\u003e\n \u003cp\u003e-3.72\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.83974358974359%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026Delta;GGAS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.73717948717949%\" valign=\"top\"\u003e\n \u003cp\u003e-21.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.42307692307692%\" valign=\"top\"\u003e\n \u003cp\u003e-24.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.83974358974359%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026Delta;GSOLV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.73717948717949%\" valign=\"top\"\u003e\n \u003cp\u003e0.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.42307692307692%\" valign=\"top\"\u003e\n \u003cp\u003e-0.35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.83974358974359%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026Delta;TOTAL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.73717948717949%\" valign=\"top\"\u003e\n \u003cp\u003e-20.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.42307692307692%\" valign=\"top\"\u003e\n \u003cp\u003e-24.41\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.83974358974359%\" valign=\"top\"\u003e\n \u003cp\u003e-T\u0026Delta;S (Entropy)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.73717948717949%\" valign=\"top\"\u003e\n \u003cp\u003e18.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.42307692307692%\" valign=\"top\"\u003e\n \u003cp\u003e12.74\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.83974358974359%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026Delta;G binding\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.73717948717949%\" valign=\"top\"\u003e\n \u003cp\u003e-2.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.42307692307692%\" valign=\"top\"\u003e\n \u003cp\u003e-11.68\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026Delta;VDWAALS: van der Waals energy; \u0026Delta;EEL: Electrostatic energies; \u0026Delta;EGB: Polar solvation free energy; \u0026Delta;ESURF: Non-polar solvation free energy; \u0026Delta;GGAS = \u0026Delta;VDWAALS+\u0026Delta;EEL; \u0026Delta;GSOLV = \u0026Delta;EGB + \u0026Delta;ESURF; \u0026Delta;TOTAL = \u0026Delta;GSOLV +\u0026Delta;GGAS; \u0026Delta;G binding = \u0026Delta;TOTAL - T\u0026Delta;S.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrincipal Component Analysis and Gibb\u0026rsquo;s free energy analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrincipal component analysis (PCA) of the MD simulation trajectories for\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL and\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL-LEAII were\u0026nbsp;analyzed. The first two principal components (PC1 and PC2) were used as reaction coordinates in Gibb\u0026rsquo;s free energy analysis. The lowest energy metastable conformations were identified from Gibb\u0026rsquo;s free energy analysis. The\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL complex showed unique metastable conformations occupied in three low energy basins with energy below 2.5 kJ/mol. While the\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL-LEAII complex showed a larger and single unique energy basin where the metastable conformations were clustered having\u0026nbsp;\u0026Delta;G below 2.5 kJ/mol. The results clearly indicate that the bound LEAII holds the\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e in stable and unique conformational state. While the LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ep\u003c/em\u003eNPL complex showed that multiple metastable conformational states are possible due to freedom in global motions in\u0026nbsp;LipA\u003cem\u003e\u003csub\u003eB.licheniformis\u003c/sub\u003e\u003c/em\u003e conformations as shown in Figure 8. \u0026nbsp; \u0026nbsp;\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOver the past two decades, there are many attentions and studies were conducted focusing on protein thermostability. Realizing it could be beneficial for industrial application, the number of related studies is growing as there is a demand for higher thermostable protein in which it could help helpful to reduce the production cost due to a better protein stability under thermal stress. Improvement in protein thermostability could open more progress in scientific research by discovering, understanding and exploiting the potentials of novel heat-sensitive enzymes to be used in the future. However, while most studies focus on creating a specific thermostable enzyme candidate, the current approach requires a long process as it involves several complex experimental designs starting from computational analyses to site-targeted mutagenesis. This study is an attempt to explore a new horizon of enhancing protein thermostability through a general, applicable approach for different types of enzymes. The adopted experimental design applied a simple heterologous expression of desired protein, lipase from \u003cem\u003eBacillus licheniformis\u003c/em\u003e (LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e) using LEA co-expression system as a novel strategy to improve its thermostability. Lipase was chosen as it is one of the most common and important enzymes in industry.\u003c/p\u003e \u003cp\u003eFrom the previous research findings, it was observed that expressing LEA-like peptides has successfully improved protein and cellular tolerances towards different stresses like UV radiation, salinity, heat stress and pH (\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). In this work, the usefulness of LEA-like peptides was evaluated further to see the rescue effect of LEA-like peptide to protect desired recombinant protein against heat stress using co-expression system as a strategy. The expression of desired protein, LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e and LEA-like peptide was induced by IPTG in \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) as microbial factory. As the expression of LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e and LEA-like peptide were driven by cold shock prooter and T7 promoter respectively, it was assumed that the supply of LEA peptide is adequate to interact with LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e produced. Ideally, the strong interaction between LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e-LEA peptide was undisturbed by sonication and remained intact during protein purification. As a result, the purified LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e-LEA complex was eluted and subjected for lipase activity at 45˚C. The lipase assay was conducted at its challenging temperature to see the impact of LEA-like peptide interaction in protecting lipase thermostability which reflected through the result of enzyme activity.\u003c/p\u003e \u003cp\u003eComparing the LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e relative activity and LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e residual activity among different LEA-like peptide- LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e complexes, LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e-LEA II complex has the best improvement of lipase activity. Notably, the LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e-LEA II complex has a significant increment in lipase relative activity with the reading almost double as compared to the control while the lipase residual activity was also shown slight improvement. The improvement of lipase activity at higher temperature indicates the structure of lipase was protected from denatured. Denaturation of protein by the strong heat stress affects the LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e folding and bonding that cause the deformation of active site and functional structure. Thus, by forming a strong interaction with LEA-like peptide, the outer surface of LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e is protected by the peptide, leaving the LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e structure intact as close as its native structure even with the exposure of a higher temperature. However, it is important to note that the interaction of LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e with LEA-like peptide should not hinder the active site region. Else, the lipase activity can still be low despite having a thermostable lipase structure. To evaluate the possibilities, a series of bioinformatic analyses starting from molecular docking to molecular dynamic simulation was conducted to understand the peptide-protein interaction and its molecular properties changes during heat challenge.\u003c/p\u003e \u003cp\u003eThe molecular docking outcome suggested that the \u003cem\u003ep\u003c/em\u003eNPL has better binding free energy in the case of LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e-LEAII complex than the LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e alone. The overall lower energy state of LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e-LEAII complex is responsible for more favorable binding affinity of \u003cem\u003ep\u003c/em\u003eNPL than the LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e alone. The MD studies also corroborate the results of molecular docking where the RMSD in LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e in LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e-\u003cem\u003ep\u003c/em\u003eNPL-LEAII complex clearly indicate the significant stability. Further, the fluctuations inside chain atoms in LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e and LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e-\u003cem\u003ep\u003c/em\u003eNPL-LEAII complex and the radius of gyration around the center of mass of LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e in LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e-\u003cem\u003ep\u003c/em\u003eNPL-LEAII complex confirms better stability compared to the LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e-\u003cem\u003ep\u003c/em\u003eNPL complex. Substantially lower solvent accessible surface area in LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e-\u003cem\u003ep\u003c/em\u003eNPL-LEAII complex is suggestive of compact structure. Slightly better propensity of hydrogen bonds between LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003ep\u003c/em\u003eNPL in LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e-\u003cem\u003ep\u003c/em\u003eNPL-LEAII complex further confirms better stability of LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e-\u003cem\u003ep\u003c/em\u003eNPL-LEAII complex. The MM-GBSA calculations confirmed that the \u003cem\u003ep\u003c/em\u003eNPL has significantly better binding free energy in LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e-\u003cem\u003ep\u003c/em\u003eNPL-LEAII complex than the LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e-\u003cem\u003ep\u003c/em\u003eNPL complex.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSince the interaction of LEA-like peptide- LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e plays a major role for the successful thermostable improvement, a detailed observation on LEA-like peptide sequences was done. As Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e shows, there are 13 amino acid residues for each peptide. The differences of LEA-like peptides can be seen on the 6th, 7th, and 12th amino acid. LEA I and LEA II have a subtle difference at amino acid number 7. LEA II replaced threonine, a polar and non-charged amino acid, with leucine, an aliphatic amino acid from the nonpolar group. Changing this amino acid in LEA II made the peptide to be more hydrophobic due to non-polar residue. Meanwhile, the differences between LEA II with LEA E and LEA K are found at amino acid position number 6 and 12. LEA E and LEA K peptides substitute nonpolar, aliphatic amino acid of glycine with glutamic acid, a negative charge amino acid and lysine, a positive charged nonpolar amino acid, respectively. Overall, LEA II has more non-polar and aliphatic amino acids compared to other LEA peptides. The less sensitive attribute shown by lipase-LEA II complex by the heat temperature most likely due to having extra non-polar aliphatic acid. (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e) demonstrated the importance of aliphatic residue in enhancing protein thermostability. Meanwhile, increment of hydrophobic properties in LEA II helps for a better protein-protein interaction with LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e. A similar observation was mentioned by (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Thus, although all LEA-like peptides have the ability to bind with LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e, the interaction can be weakened by heat energy that could dissociate the complexes. Nevertheless, the thermostability and hydrophobicity properties owned by LEA II promote for the LEA II-LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e stronger interaction despite challenged with higher temperature. As a result, LEA II promotes better lipase structure stability which was proven by having a good lipase activity at higher temperature. Possessing charged amino acid in LEA E and LEA K did not give good results. Again, the possible reason is because the peptides are losing its hydrophobicity and thermostability properties at these specific, crucial amino acids. Another possibility of lower performance of charged LEA-like peptide is also due to its specific surface preference while establishing interaction with the LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e. For example, LEA-K has more tendency to bind at negative charge region on LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e surface and vice versa. This preference probably is not the best to protect LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e structure because it has more selective binding as compared to LEA II.\u003c/p\u003e \u003cp\u003eIn conclusion, this study highlights the significant enhancement of LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e thermostability at 45\u0026deg;C through the incorporation of LEA-II peptide, showcasing its efficacy in bolstering heat tolerance. The observed impact underscores the critical role of specific amino acid position. Minor alterations with different groups of amino acid even with a single amino acid make a big difference by comparing lipase activity between lipase-LEA I and lipase-LEA II. Furthermore, the study suggests a useful direction to explore by testing hypotheses, specifically in changing the 6th and 12th amino acids to non-charged hydrophobic counterparts, offering valuable insights for optimizing LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e thermostability.\u003c/p\u003e \u003cp\u003eAlthough the precise mechanism of improved stress tolerance in protein using LEA-like peptides remains elusive, this approach can be employed to extend its broader applicability for other proteins. Indeed, if this approach is applicable for different proteins, it reduces complexity, saves time to produce other useful protein with higher stress tolerance properties.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contribution Statement:\u0026nbsp;\u003c/strong\u003eAmmar Khazaal Kadhim Almansoori, Kang Siang Yu and Rashidah Abdul Rahim conceptualized, designed the research idea and analyzed data. Ammar Khazaal Kadhim Almansoori, Faisal Mohamed and Kang Siang Yu conducted the experiments, wrote the main manuscript, reviewing and editing. Shinya Ikeno contributed with LEA peptides and provided the analytical tools of \u003cem\u003ein vitro\u003c/em\u003e experiments. \u0026nbsp;Ammar Khazaal Kadhim Almansoori, Rajesh B. Patil and Rop\u0026oacute;n-Palacios G provided the analytical tools of \u003cem\u003ein silico\u003c/em\u003e experiments, analyzed data, reviewing and editing. Rashidah Abdul Rahim supervision, resources, reviewing and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eThe authors would like to thank\u0026nbsp;the Malaysian Ministry of Higher Education, Fundamental Research Grant Scheme (FRGS) (203.PBiologi.6712194) and Universiti Sains Malaysia, Research University (RU) grant (No: 1001/PBIOLOGI/8011009)\u0026nbsp;for supporting this research.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e All data are present in the manuscript and it\u0026rsquo;s Supplement.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatement and Declarations:\u0026nbsp;\u003c/strong\u003eThe authors declare that we do not have any conflict of interest to the content of this article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis research was supported by the Malaysian Ministry of Higher Education, Fundamental Research Grant Scheme (FRGS) (203.PBiologi.6712194) and Universiti Sains Malaysia, Research University (RU) grant (No: 1001/PBIOLOGI/8011009).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval statement:\u0026nbsp;\u003c/strong\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGuncheva M, Zhiryakova D. 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Proceedings of the National Academy of Sciences. 2021;118(6):e2018234118.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"LipAB.licheniformis, Late Embryogenesis Abundant (LEA), Thermostability, Co-expression, Molecular docking and Molecular dynamic simulation","lastPublishedDoi":"10.21203/rs.3.rs-4160767/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4160767/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHeterozygous protein expression in \u003cem\u003eE. coli\u003c/em\u003e facilitates high yield and quality. However, the challenges of protein instability due to environmental stress are still an issue that affects the activity of the protein produced. In this study, the improvement of protein thermostability was done using a peptide co-expression system. The developed system exploited the usefulness of Late Abundant Embryogenesis (LEA) proteins to protect proteins from damage. Recombinant lipase from \u003cem\u003eBacillus licheniformis\u003c/em\u003e was expressed along with the LEA-like peptide, whose design was inspired by the 11 repetitive amino acid sequences of the LEA protein. In total, four LEA-like peptide co-expression systems were assessed. The evaluation of improvements in protein thermostability was conducted using a standard lipase assay. The purified lipase was challenged at 45 °C, a higher temperature than its optimal temperature. Two-fold lipase activity was recorded from the protein co-expressed with the LEA-II-like peptide. Based on amino acid sequence comparison, LEA-II has the advantage of containing more polar residues with several aliphatic amino acids, which may improve LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e-LEA II complex stability at higher temperatures. Next, molecular docking and molecular dynamic simulation were employed to analyze the stability of the lipase in the presence and absence of LEA II. The findings of the RMSD, MM-GBSA and related analyses showed that the LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e-LEA II complexes have better stability than the LipA\u003csub\u003e\u003cem\u003eB.licheniformis\u003c/em\u003e\u003c/sub\u003e alone, thus supporting the lipase assay. These findings successfully unravel the potential of the LEA-like peptide co-expression system as a novel approach to improve enzyme thermostability.\u003c/p\u003e","manuscriptTitle":"Enhancing Thermostability of Bacillus licheniformis Lipase with LEA Peptide Co-expression System.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-09 12:28:42","doi":"10.21203/rs.3.rs-4160767/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c82f6859-0125-4344-9f9d-c5f49cbb6197","owner":[],"postedDate":"April 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-25T10:42:13+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-09 12:28:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4160767","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4160767","identity":"rs-4160767","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}