Cosolvent-induced Spontaneous Refolding of Lipase

Cosolvent-induced Spontaneous Refolding of Lipase | 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 Cosolvent-induced Spontaneous Refolding of Lipase Cheng Cheng, Yongqin Su, Lupeng Cui, Yumeng Qiu, Jialing Wang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6737700/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Jan, 2026 Read the published version in Applied Microbiology and Biotechnology → Version 1 posted You are reading this latest preprint version Abstract The overexpression of proteins in Escherichia coli often results in the formation of inclusion bodies, which are biologically inactive, especially for proteins with exposed hydrophobic surfaces. Solubilization of IBs and subsequent refolding is essential for obtaining correctly folded and active protein. However, protein refolding involves multiple steps—namely isolation, solubilization, and refolding—which is a labor-intensive process. In this study, we developed a strategy for soluble production and protein refolding. A fusion tag was applied to Burkholderia ambifaria lipase YCJ01, enabling abundant soluble expression in E. coli . Despite this, the soluble protein exhibited only partial enzymatic activity, suggesting an unfolded state of soluble lipase YCJ01. Lipase activity increased significantly after incubation with cosolvents, reaching 1,003 U/mL, 754 U/mL, and 501 U/mL in 25% (v/w) glycerol, 15% (v/w) DMSO, and 4M trimethylamine N-oxide (TMAO) solutions, respectively. Correctly folded and highly active lipase YCJ01 with a natural N-terminus was obtained. Moreover, the cosolvent-induced refolding mechanism was elucidated through molecular dynamics simulations. Glycerol and DMSO were found to aggregate around hydrophobic regions of lipase, directly stabilizing structure by displacing water molecules and weakening water–protein hydrogen (H) bonds within the hydration shell. Conversely, TMAO molecules indirectly influenced the lipase structure by strengthening water–water H bonds. Lipase YCJ01 soluble expression spontaneous refolding cosolvents MD simulations Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Key Points Cosolvents enhance lipase activity, with glycerol showing the highest improvement MD simulations show glycerol and DMSO directly interact with hydrophobic regions Glycerol and DMSO stabilize lipase directly, while TMAO enhances stability indirectly Introduction Natural hydrophobic proteins, such as lipases, hydrophobins, and membrane proteins, have significant potential for diverse applications in biocatalysis and biomedical fields (Berger and Sallada 2019 ; Kaur et al. 2017 ; Mouritsen et al. 2006 ; Reis et al. 2009 ; Sackmann and Tanaka 2000 ). However, their widespread use is substantially hindered by the challenges in preparing functionally folded proteins, which often exhibit reduced activity and poor stability, limiting their practical utility (Baneyx and Mujacic 2004 ; Berger and Sallada 2019 ; Cheng et al. 2018 ; Cui et al. 2021 ; Kaur et al. 2017 ; Mouritsen et al. 2006 ; Reis et al. 2009 ; Sackmann and Tanaka 2000 ). Overexpression of proteins in Escherichia coli typically results in the formation of inclusion bodies (IBs), which are generally biologically inactive and require processing to yield an active final product (Baneyx and Mujacic 2004 ). The strong hydrophobicity of hydrophobic proteins increases their tendency to form IBs, which compete with correct folding, particularly at high concentrations. This has been identified as a key challenge limiting their efficient application in the bioengineering industry. Conversely, the hydrophobic nature of these proteins often enhances their activity or stability in oil–water biphasic systems. For example, lipase exhibits biocatalytic activity at the interface of aqueous and oil phases, facilitating an optimal site for lipolysis (Mouritsen et al. 2006 ). The localized hydrophobic nature of lipases allows them to become active at interfaces (Cheng et al. 2018 ). Hydrophobins typically self-assemble at the air–water interface (Berger and Sallada 2019 ; Cui et al. 2021 ). Membrane proteins require a specific environment containing lipids and water to facilitate the transport of various molecules across the cell membrane to maintain homeostasis (Junge et al. 2008 ). Consequently, self-folding in nonaqueous media presents a promising approach for the industrial production of bulk and/or fine bioactive proteins with high hydrophobicity. Therefore, a novel strategy for soluble production and hydrophobic protein refolding by leveraging nonaqueous media is highly sought after. Fusion tags can be used to reduce prokaryotic IB formation and enhance protein solubility expression (Cheng et al. 2017 ; Ma et al. 2024 ). A large variety of fusion tags are available for solubility enhancement, including the maltose-binding protein (MBP), N-utilization substance (NusA), thioredoxin (TrxA), Ffu and glutathione S-transferase (GST) (Costa et al. 2014 ). However, soluble expression does not always guarantee high enzymatic activity due to the formation of unfolding intermediates. Subsequent folding is essential to achieve correctly folded and active proteins. The equilibrium of the protein refolding process can be influenced by adjusting the thermodynamic state of the system, such as pH, pressure, temperature, or cosolvents (Agam et al. 2024 ; Beck and Daggett 2004 ; Schuler et al. 2002 ). Cosolvents can shift the equilibrium toward the folded state of proteins (Canchi and García 2013 ). For example, the cosolvent glycerol-assisted refolding of denatured Xfpal derived from Xylella fastidiosa resulted in a correctly structured protein (Santos et al. 2012 ). The intrinsic mechanism of cosolvents in inducing protein refolding has been effectively demonstrated using atomistic molecular dynamics (MD) simulations. Two potential mechanisms, “direct interaction” (Bruzdziak et al. 2015 ) and “indirect interaction” (Bennion and Daggett 2003 ; Shiraga et al. 2018 ), have been proposed in independent systems. For example, as a cosolvent, urea directly interacts with N-methylacetamide and dimethyl sulfoxide by weakening hydrogen (H) bonds within overlapping hydration shells (Stasiulewicz et al. 2020 ). Tobi et al. ( 2003 ) illustrated cosolvent urea directly interacts with the peptide backbone during denaturation by selectively binding to the expanding surface of unfolded proteins, shifting the equilibrium toward the unfolded state. Conversely, protective cosolvents, such as trimethylamine N-oxide (TMAO) and glycerol, were preferentially excluded from the protein surface, favoring the folded state through indirect interaction. Bennion and Valerie ( 2004 ) observed that TMAO enhanced water–water H bonding in both binary TMAO–water mixtures and ternary urea–TMAO–water solutions, while also strengthening water–urea interactions to reduce urea–protein H bonding. Moreover, the protective role of TMAO in countering the denaturing effects of urea was demonstrated by analyzing the hydration and conformational dynamics of wild-type villin headpiece protein HP35 and a doubly norleucine-substituent mutant (Lys24/29Nle) HP35NN in pure urea and urea–TMAO mixed solutions (Yang et al. 2016 ). TMAO preferentially protects the hydrophobic core of proteins from urea-induced denaturation by reducing urea accumulation around hydrophobic residues. Meanwhile, the thermodynamics of protein and cosolvent mixtures have been studied, where denaturing cosolvents lower the free energy of the unfolded state, thereby favoring the unfolded population, whereas protecting cosolvents raise the free energy of the unfolded state, favoring the folded population (Auton and Bolen 2005 ; Bolen and Baskakov 2001 ). However, most MD simulations of cosolvent action focus on specific protein models, such as short peptides or small truncated proteins. Practical applications of cosolvent-induced refolding systems for assisting highly hydrophobic proteins with high molecular weight to correctly fold are still limited. Burkholderia ambifaria lipase YCJ01 is a well-characterized enzyme known for its high thermal stability, organic-solvent tolerance, and significant potential in various important applications, particularly in organic synthesis and the production of pharmaceuticals, flavors, and fragrances, as demonstrated in our previous studies (Cheng et al. 2018 ; Yao et al. 2013a ). In this study, we developed a new method for soluble production in E. coli and self-refolding enhancement for lipase YCJ01 in cosolvent systems. To the best of our knowledge, this is the first comprehensive attempt to study, at the molecular level, the effects of glycerol, DMSO, and TMAO as cosolvents on the self-refolding of the entire lipase YCJ01 protein. Additionally, we addressed the following two scientific questions using MD simulation: (1) How do cosolvents affect enzyme structure and dynamics? (2) What significant structural and dynamic observables can be applied to the refolding of other proteins? Materials and Methods Materials E. coli DH5α (Invitrogen) was used for subcloning and plasmid amplification, while E. coli BL21 (DE3) (Novagen) was employed for protein expression. The pET-28a (+) (Novagen) plasmid served as the backbone for the expression. Restriction enzymes Xho I, Nco I, and Hind III were purchased from Takara Bio. All DNA ligations were conducted using T4 ligase (TaKaRa). Construction and expression of lipase YCJ01 and NusA-lipase YCJ01 The construction of pNusA vectors was performed as follows: primers F1&R1 (Table S1 ) were used to amplify NusA fragments from the commercial vector pET43a. The purified PCR products and the pET28a (+) plasmid were digested with Nco I and Hind III restriction enzymes. The pNusA vector was obtained after ligating the digested DNAs using T4 ligase. Recombinant plasmids were then transformed into E. coli BL21 (DE3). The resulting clones containing recombinant plasmids of pNusA vectors were confirmed through sequencing. To construct the lipase YCJ01 and NusA-lipase YCJ01 expression vector, Burkholderia ambifaria YCJ01 (containing a full-length lipase YCJ01) was used as the template to obtain lipase YCJ01. Primers F2&R2 (Table S1 ) were synthesized to generate the gene fragment of lipase YCJ01 without its natural signal peptide, with the restriction sites underlined. After litigating the digested PCR products with pET28a and pNusA plasmids, the target genes were inserted into the constructed vectors. These constructions were then transformed into E. coli BL21 (DE3), and pET28a-lipaseYCJ01 and pNusA-lipase YCJ01 was verified through restriction digestion and sequencing. The expression of lipase YCJ01 fused with the NusA fusion tag was conducted as follows: cells containing the pET28a-lipaseYCJ01 and pNusA-lipase YCJ01 vectors were grown to mid-log phase (OD 600 = 0.6) at 37℃ in LB-broth supplemented with 50 mg/L kanamycin. The lipase YCJ01 and fusion protein NusA-lipase YCJ01 overproduction were induced with IPTG at a final concentration of 1 mM at 20℃. The cells were harvested once the OD 600 value reached 3.0. After centrifugation for 10 mins (4℃, 12000rpm), the supernatants were discarded, and the pelleted cells were resuspended in distilled deionized water and lysed through ultrasonication. The supernatant of the total lysate (soluble fractions) was obtained through centrifugation. The pellet (insoluble fraction) was resuspended in distilled deionized water and subjected to further analysis. The construction and expression of Ffu209-lipaseYCJ01, Ffu217-lipaseYCJ01, Ffu312-lipaseYCJ01, GST-lipaseYCJ01, and MBP-lipaseYCJ01 were performed using the same method as NusA-lipase YCJ01. The primers used for plasmid construction are listed in Table S1 in the Additional files. To analyze the supernatant of the total lysate (soluble fractions) and pellet (insoluble fractions), 12% SDS-PAGE was performed. Coomassie-stained protein bands were then scanned and analyzed using densitometry (Duoscan T1200, Bio-Rad, Hercules, CA). Refolding of cosolvents on lipase YCJ01 The refolding process of lipase using chemical chaperone (25% glycerol, 15% DMSO, 4 M TMAO, and 8M urea with 4 M TMAO) was investigated. The cells were harvested through centrifugation, resuspended in distilled deionized water, and lysed through ultrasonication. The supernatants of the total lysate were collected for further analysis. The total protein content in the supernatant of cell lysate was monitored using the Bradford assay. The solvent was not removed after the incubation with the cosolvent. The lipase needs diluted 5 times before the activity assay. The NusA-lipase YCJ01 solution was diluted to 50 g/ml. Different concentrations of cosolvents were added to the solution in sealed glass vial. The mixtures were incubated at 4℃, 17℃, and 28℃, respectively. The activity of lipase was measured every 24 h. Assay of lipase activity and protein concentration Lipase activity was measured using a modified spectrophotometric method with p-nitrophenyl palmitate (p-NPP) as a substrate (Yao et al. 2013a ). p-NPP (3 mg), dissolved in 1 mL of isopropanol to a final concentration of 0.3 mg/mL, was mixed with 9 mL of 50 mM sodium phosphate buffer (pH 8.0) containing gum arabic (0.1%) and Triton X-100 (0.6%). The reaction mixture consisted of 240 µL of substrate solution and 10 µL of appropriately diluted enzyme solution, incubated at 40℃ for 10 min. The amount of p-nitrophenol (p-NP) produced in the reaction mixture was quantified spectrophotometrically at 410 nm. One unit of enzyme activity was defined as the amount of enzyme that liberated 1 mol of p-NP per min under standard assay conditions. The activity measurements were performed three times on each sample to ensure reproducibility. MD simulation Atomistic MD simulations were conducted using GROMACS 5.0. The GROMOS 54A7 force fields (Schmid et al. 2011 ) were employed to model protein atoms, while the TIP3P explicit solvent model (Agam et al. 2024 ) was used to represent water molecules. The three-dimensional structure of lipase YCJ01 was generated using the SWISS-MODEL, with the template PDB-ID 4LIP (Zhu et al. 2016 ) (PAL lipase, sequence identity 95.6%, crystal resolution 1.75 Å) and optimized through energy minimization. Additionally, the force field parameters for DMSO, glycerol, and TMAO were sourced from A.W. Schuettelkopf (Schüttelkopf and van Aalten 2010 ). For each simulation system, a relatively extended structure of lipase YCJ01, lacking native structural elements, was placed in a cubic box containing plenty of water and DMSO, glycerol, or TMAO molecules. Suitable counterions were added to each system to balance the charge of the protein. The detailed simulation parameters, including the number of solvent molecules, box size, and simulation time, for all simulation systems, are provided in Table S2 . For each system, the replica exchange MD simulations (Sugita and Okamoto 1999 ) were conducted in the NVT ensemble. MD simulations were performed at 300 K and 1 atm for 100 ns. The SHAKE algorithm (Ryckaert et al. 1977 ) was applied to constrain bonds involving H atoms, enabling a time step of 2.0 fs, with coordinates saved every 1 ps. Particle mesh Ewald was employed to manage long-range electrostatic interactions, and a nonbonded cutoff of 10 Å was used (Darden et al. 1992 ). Langevin dynamics with a collision frequency of 3.0 ps − 1 was utilized to maintain the temperature of the system. The trajectories were analyzed using GROMACS tools. Root means square deviations (RMSD) values were calculated using g_rms, while the number of H bonds, defined by a cutoff distance of 0.35 nm and an angle cutoff of 30 o , was determined using g_hbond. The radius of gyration (Rg) was calculated using g_gyrate to evaluate protein compactness, and the radial distribution function was computed using g_rdf. Images of the molecular graphics were generated and visualized using the VMD package (Humphrey et al. 1996 ). Results Soluble expression of highly hydrophobic lipase YCJ01 As reported in our previous studies, the lipase from B. ambifaria YCJ01 exhibits exceptional thermal stability, distinct tolerance to organic solvents, and significant potential for biocatalysis and the resolution of pharmaceutical intermediates (Bennion and Valerie 2004 ; Yang et al. 2016 ). The complete sequence of the lipase consists of a native signal peptide, a propeptide, and a mature peptide. Due to the high hydrophobic nature of lipase YCJ01, both the complete sequence and the mature peptide were expressed as IBs in E. coli . To enhance lipase YCJ01 solubility and circumvent the need for solubilizing IBs, fusion tags (Ffu209, Ffu217, Ffu312, GST, MBP, and NusA (Cheng et al. 2017 )) were employed to confer stability and high solubility to lipase YCJ01. NusA tag (55kDa) performed preferably when compared with other fusion tags as shown in Fig. S1 . As shown in Fig. 1 , direct expression of lipase YCJ01 resulted in high yields, but the majority of the protein was sequestered in IBs, with only 3% solubility observed, even at an induction temperature of 20℃. Fortunately, the fusion protein NusA-lipase YCJ01 achieved significantly improved solubility (67.8%). The molecular weight of lipase YCJ01, as determined by SDS-PAGE, is 34 kDa, aligning with previous reports (Yao et al. 2013b ), the molecular weight of the NusA-lipase YCJ01 fusion protein, comprising NusA (55 kDa) and lipase YCJ01 (34 kDa), exhibits a molecular weight of approximately 89 kDa. The molecular weight difference between NusA-lipase YCJ01 and lipase YCJ01 specifically corresponds to the molecular weight of NusA (55 kDa). However, the soluble NusA-lipase YCJ01 exhibited weak enzymatic activity. Cosolvent-induced refolding of NusA-lipase YCJ01 Due to the misconception that soluble protein expression guarantees proper protein folding into its correct structure, limited studies have focused on the refolding of soluble protein to enhance bioactivity. Lipase biocatalysis occurs at the interface of aqueous and oil phases to create an optimal site for lipolysis, and the enzyme demonstrates excellent organic-solvent tolerance. Therefore, cosolvents, such as glycerol, DMSO, and TMAO, were chosen to facilitate the refolding of fusion protein NusA-lipase YCJ01 into its correct structure. Among these, an organic solvent, DMSO, was initially used as a cosolvent to promote positive effects on protein refolding. Meanwhile, to better examine the cosolvent-induced refolding process, conditions involving water and a mixed solution of TMAO and urea were considered. Additionally, the impacts of different concentrations and inducing temperatures of cosolvents on lipase YCJ01 refolding were systematically investigated. As shown in Fig. 2 A, lipase YCJ01 activity peaked at 210 U/ml after incubation with 25% (v/w) glycerol for 24 h. The activity rose to 125 U/ml with a 15% (v/w) DMSO concentration, reaching its maximum at 115 U/ml with a 4 mol/L TMAO concentration. However, excessive concentrations of cosolvent did not promote lipase YCJ01 refolding. Protein samples were analyzed using SDS-PAGE and stained with Coomassie blue. Lane 1: Soluble fraction of lipase YCJ01 with molecular weight of 34 kDa (Baneyx and Mujacic 2004 ). Lane 2: Inclusion bodies (IBs) of lipase YCJ01. Lane 3: Soluble fraction of NusA-lipase YCJ01 with molecular weight of 89 kDa. Lane 4: IBs of NusA-lipase YCJ01. Temperature plays a significant role in the refolding process. The enzymatic activities of lipase YCJ01 were evaluated at different temperatures (4℃, 17℃, and 28℃) using the optimal concentrations of each cosolvent solution (25% glycerol, 15% DMSO, and 4 mol/L TMAO). As shown in Fig. 2 B, a refolding temperature of 17℃ was more favorable than 28℃ and 4℃. The lowest enzymatic activity of lipase was observed at 28℃. Additionally, the refolding of NusA-lipase YCJ01 in water was found to be slow (Fig. 2 C), with enzymatic activity increasing from 1.5 U/ml to 69 U/ml. Conversely, the presence of cosolvent molecules significantly facilitated lipase YCJ01 refolding. Particularly, the enzymatic activities of lipase increased to 1,003 U/ml in 25% glycerol, 754 U/ml in 15% DMSO, and 501 U/ml in 4 M TMAO. The refolding efficiency of the cosolvents followed the order: TMAO < DMSO < glycerol. However, the combination of urea and TMAO in a 1:2 ratio did not improve the enzymatic activity of lipase (Fig. 2 C). Lipase YCJ01 has an open reading frame of 1095 bp that encodes 364 amino acids (Yao et al. 2013b ). Analysis by the SignalP 3.0 Server indicates it contains a signal peptide of 40 amino acids and a propeptide of 12 amino acids in Fig. S2 . The N-terminal sequence analysis (Fig. S3) demonstrates that the N-terminal sequence of the refolded lipase YCJ01 is YPIILVHGLTGTDK, which is entirely consistent with the reported N-terminal of the mature peptide of lipase YCJ01. Replica exchange MD analysis of cosolvent-induced refolding mechanism In this study, we employed MD simulations to explore the microcosmic mechanisms by which different cosolvent molecules—glycerol, DMSO, and TMAO—affect the refolding of a highly hydrophobic lipase with a high molecular weight. To assess the stability and overall structural changes of lipase YCJ01 in water, 15% DMSO, 25% glycerol, and 4 M TMAO solutions, the backbone RMSDs averaged over four trajectories for each solution were calculated. As shown in Fig. 3 A, all RMSD values reached a plateau, indicating that the protein backbone maintained a stable structure across these solutions. Compared to the RMSD values for the overall conformation, the changing RMSD value in water (0.445 nm) was significantly higher than in 15% DMSO, 25% glycerol, and 4 M TMAO solutions (0.272 nm, 0.273 nm, and 0.287nm, respectively). Moreover, the growth rates of RMSD varied across different solutions during the first 25 ns. The RMSD value for lipase YCJ01 rose significantly in water, 15% DMSO, and 25% glycerol, while RMSD in 4 M TMAO remained relatively stable. Furthermore, the Rg of the lipase backbone in water, 15% DMSO, 25% glycerol, and 4 M TMAO solutions was calculated to assess structural compactness. As shown in Fig. 3 B, slight increases in the Rg value were observed, specifically 0.072 nm in 15% DMSO and 0.073 nm in 25% glycerol. However, the Rg value of lipase in water and 4 M TMAO showed a contrasting trend, decreasing from 1.93 nm to 1.88 nm (0.05 nm) in water, and from 1.93 nm to 1.91 nm (0.02 nm) in 4 M TMAO. This indicates that the overall conformation of lipase in 15% DMSO and 25% glycerol becomes more loosened, whereas lipase tends to become more compact in water and 4 M TMAO. Moreover, more residues are exposed to the solvent in 15% DMSO and 25% glycerol. To evaluate the interactions between cosolvent molecules and lipase, a hydrophobic analysis of lipase YCJ01 was conducted. Most hydrophobic residues were found on the surface of the lipase and clustered into two core regions, R1 and R2. The R1 region comprises residues Val130, Leu138, Val142, Ile143, Val149, and Leu165, while the R2 region includes residues Ile236, Leu238, Ile239, Leu245, Leu247, Leu250, Val258, Ile260, Leu270, Val271, Val281, Leu298, and Val300. Snapshots from MD simulations at 1 ns, 30 ns, and 100 ns intervals for the 15% DMSO, 25% glycerol, and 4 M TMAO solution were captured (Fig. 4 ). The cosolvent molecules were consistently positioned at least 3.5 Å away from the protein surface. During the simulation, glycerol and DMSO molecules gradually accumulated around the hydrophobic core regions of the lipase, while TMAO molecules remained evenly distributed on the protein surface. Additionally, the time evolution of RMSF for each residue in the different simulation systems was monitored (Fig. 5 ). The overall residues in water exhibited higher RMSF values than those in 25% (w/v) glycerol, 15% (w/v) DMSO, and 4 M TMAO. Notably, residues in the R1 and R2 hydrophobic regions of lipase became unstable after approximately 20 ns in water. Conversely, these regions were stabilized in the glycerol and DMSO systems, where the molecules effectively stabilized the hydrophobic core regions of the lipase, preventing aggregation and unfolding. However, the instability in the R1 region was observed after 40 ns in the 4 M TMAO solution, unlike in the DMSO or glycerol systems. This structural variation may explain the reduced Rg value of the protein in the 4 M TMAO solution. These findings align with the RMSD and Rg observations. To better illustrate the flexibility/rigidity of the regions in the absence and presence of cosolvent molecules, the RMSF in water and 25% (w/v) glycerol were further analyzed (Fig. 6 ) , in the water, the residue region (Ser150-Ala160) has a high RMSF, corresponding to the loop region of lipase YCJ01, which is significantly disturbed. In the 25% (w/v) glycerol, the disturbance in this region is markedly reduced, which is consistent with the results in Fig. 5 . In the 25% (w/v) glycerol, the residue region (Gly126-Thr150) has a higher RMSF value than in water. This region corresponds to the α5 region of lipase YCJ01, namely, the “lid” of lipaseYCJ01 (Cheng et al. 2018 ). Moreover, the numbers of cosolvent and water molecules within 3.5 Å of the overall conformation of lipase and its hydrophobic regions (R1 and R2) were calculated to evaluate the distribution behaviors of the cosolvent molecules on the hydrophobic surface of the lipase (Table 1 ). The presence of glycerol and DMSO molecules reduced the number of water molecules in the hydration shell of lipase, primarily in the hydrophobic regions. Conversely, the number of water molecules in the TMAO system showed minimal change compared to the water system. Furthermore, the number of cosolvent molecules within the hydration shell of the lipase was quantified. As shown in Table 1 , the percentage of DMSO and glycerol molecules surrounding the hydrophobic regions (R1 and R2) relative to the total in the hydration shell were 94.9% and 74.7%, respectively, while the percentage of TMAO was only 37.4%. DMSO and glycerol molecules aggregated at the hydrophobic regions of the lipase due to hydrophobic interactions, stabilizing the structure of these regions. Conversely, TMAO molecules were distributed on the surface lipase without apparent direct interaction with the hydrophobic regions. Additionally, the numbers of H bonds in the final nanosecond of the MD simulations were calculated. The average number of protein–water H bonds decreased in cosolvent solutions: from 498 in water to 414 in 15% DMSO, 407 in 25% glycerol, and 443 in 4 M TMAO solutions. Meanwhile, the average numbers of H bonds formed between lipase and cosolvent molecules were 19 for DMSO, 7 for glycerol, and 5 for TMAO. Protein stability is significantly affected by water properties, as cosolvent molecules can intrinsically affect water structure in various ways, thereby affecting proteins through indirect mechanisms. To explore the protective roles of these cosolvents indirectly, their impact on water structure was analyzed. The oxygen–oxygen (O–O) water radial distribution functions in the simulations of water, 15% DMSO, 25% glycerol, and 4 M TMAO systems were calculated. As shown in Fig. 7 A, a distinct peak at 0.275 nm was observed, and adding glycerol, DMSO, and TMAO caused a significant increase in the first peak. The O–O water radial distribution increased from 3.269 in the water system to 4.901 in the 25% glycerol system, indicating that cosolvents, particularly glycerol, enhanced the order of water structure. Furthermore, DMSO showed a greater effect on water structure than TMAO. These findings align with the superior ability of glycerol, demonstrating that glycerol and DMSO enhance lipase refolding more effectively than TMAO, primarily due to indirect interactions. The impact of cosolvents on water–water H bonds in water, 15% DMSO, 25% glycerol, and 4 M TMAO solutions was also examined. Figure 7 B illustrates the distribution of H-bond distances. The strength of water–water H bonds is significantly affected by the solvent environment. Discussion In this study, an E. coli heterologous expression system and a self-refolding strategy were established to enhance enzyme activity using cosolvents. A large, highly hydrophobic lipase YCJ01 was successfully expressed in abundance with the aid of the fusion tag NusA. The intrinsically solubility and biological activity of NusA in E. coli contribute to its ability to improve the soluble production of fusion proteins. NusA slows down translation at the transcription pauses, offering more time for protein folding (Costa et al. 2014 ). It is likely that the transient intermediates formed during expression engaged in nonspecific intermolecular interactions due to the exposed hydrophobic surfaces, resulting in the unfolded state of lipase YCJ01. This suggests that the NusA fusion tag effectively enhanced the solubility of passenger lipase YCJ01 but failed to facilitate the correct folding of lipase YCJ01. Additionally, optimal refolding conditions using cosolvents for high enzymatic activity were explored. Spontaneous refolding occurred when NusA-lipase YCJ01 was in 25% (v/w) glycerol, 15% (v/w) DMSO, and 4 M TMAO solutions in vitro , achieving activities of 1,003 U/mL, 754 U/mL and 501 U/mL, respectively. Remarkably, the enzymatic activity of lipase YCJ01 significantly increased, and cosolvent molecules facilitated the refolding process of lipase YCJ01, this phenomenon that the improvement of lipase activity in the presence of cosolvents is also observed for other lipases (Kamal et al. 2013 ; Mangiagalli et al. 2020 ; Tsuzuki et al. 2001 ; TSUZUKI et al. 2003 ). The refolding efficiency of the cosolvents suggests that glycerol and DMSO are more effective than TMAO for refolding large, highly hydrophobic proteins such as lipase YCJ01. The concentration optimization results suggest that excessive concentrations of cosolvent did not promote lipase YCJ01 refolding. Instead, the excessive concentrations of cosolvent molecules caused steric hindrance, making it difficult for the stretched peptide chains to fold correctly, ultimately suppressing the protein refolding process. Moreover, the temperature optimization results show that NusA-lipase YCJ01 refolding at 17°C exhibited better activity than at 4°C, due to the slower refolding rate at lower temperatures, which also decelerates the cleavage of the propeptide. The lowest enzymatic activity of lipase at 28℃ indicates that high refolding temperature hinders proper protein refolding. This effect was likely due to increased intermolecular interactions, leading to a higher probability of protein aggregation caused by excessive molecular collisions. The N-terminal sequence analysis suggests that the signal peptide and propeptide of lipase YCJ01 have been successfully cleaved during the cosolvent-induced refolding process. This process also allowed the removal of the fusion tag NusA as shown in Fig. 2 D. The pro-peptides act as intramolecular chaperones to facilitate lipase folding and are subsequently cleaved to release the mature protein, which is essential for obtaining lipase with the native N-terminus, as previously reported (Luo et al. 2018 ). MD simulations at atomic resolution are widely used to describe the mechanisms by which cosolvent molecules influence protein folding equilibria, offering valuable insights into their effects on proteins and peptides (Daggett 2006 ). However, most studies have focused on short-model peptides or truncated proteins, with limited research available on the folding mechanisms of integral proteins. Using MD simulations, the effect of cosolvents on such a large, highly hydrophobic protein was studied for the first time. Compared to the RMSD values for the overall conformation, the changing RMSD value in water was significantly higher than in 15% DMSO, 25% glycerol, and 4 M TMAO solutions. This suggests that lipase YCJ01 exhibited more conformational changes in water, whereas it maintained a relatively stable spatial structure in the three cosolvent solutions. The RMSD finding also implies that the refolding process in TMAO differs from that in other cosolvents, such as glycerol and DMSO. The Rg results showed that the conformation of lipase YCJ01 in water and 4 M TMAO was more compact than that in 15% DMSO and 25% glycerol, which hindered the refolding process of lipase YCJ01. This aligns with the enzymatic activity of lipase YCJ01 in different solutions, with higher activity observed in 15% DMSO and 25% glycerol than in 4 M TMAO (Fig. 2 B ) . Surface hydrophobicity is a key factor in the accumulation of highly hydrophobic proteins (Kaur et al. 2017 ). The clustering of DMSO and glycerol molecules at the hydrophobic regions effectively reduced protein–protein interactions, thereby promoting the refolding process. Conversely, the uniform distribution of TMAO on the protein surface prevented aggregation but did so indirectly. Unlike DMSO and glycerol molecules, which directly interacted with the hydrophobic regions of lipase YCJ01, TMAO molecules likely influenced the hydrophobic lipase YCJ01 indirectly. Cosolvents, glycerol and DMSO, aggregated around the hydrophobic regions of the lipase, while TMAO primarily enhanced the water structure to reduce water attack on lipase. The RMSF results indicate that the lid region of lipase YCJ01 has a high degree of structural flexibility. In the 25% (w/v) glycerol, the conformation of the “lid” is more prone to change, suggesting that it is closely related to the activation mechanism of lipase YCJ01, which makes the active site accessible. In addition, the decrease in H bonds between lipase and water molecules occurred due to the competition of cosolvents with water molecules on the surface of lipase. It has been reported that water molecules play a significant role in protein denaturation by first solvating the hydrophobic core of the protein, followed by its overall structure (Bennion and Daggett 2003 ). Under these conditions, cosolvent molecules enhanced the refolding process of highly hydrophobic proteins by reducing the formation of water–protein H bonds, thereby mitigating water attack. The relatively few H bonds formed between lipase and cosolvent molecules, particularly TMAO, support an indirect interaction mechanism underlying the protective role of TMAO (Paul and Patey 2007 ; Yang et al. 2016 ). In this study, the presence of cosolvents resulted in shorter and more robust H-bond lengths than those in the water system. Compared to the water system, water–water interactions became more pronounced in the presence of cosolvent molecules, particularly TMAO, which hindered water–protein interactions. This is beneficial for the formation of hydrophobic cavities in lipase, facilitating its refolding. These observations indicate that glycerol and DMSO affect hydrophobic protein folding both directly and indirectly, whereas TMAO primarily relies on indirect interactions. This aligns with the observation that the RMSD of glycerol and DMSO increases more rapidly than TMAO within the first 25 ns (Fig. 3 A). This is consistent with the fact that lipase YCJ01 activity increased more quickly in DMSO and glycerol solutions than in the TMAO solution (Fig. 2 C). The effect of cosolvents on lipase YCJ01 analysized by MD simulation reveals that cosolvents can accelerate the refolding of lipaseYCJ01. Meanwhile, the addition of glycerol opens the “lid” region, boosting the solvent accessibility of the active site of lipaseYCJ01. These cosolvent molecules stabilized the water structure and enhanced water–water H bonds. Therefore, we concluded that glycerol and DMSO influenced hydrophobic proteins both directly and indirectly, while TMAO indirectly stabilized the hydrophobic lipase YCJ01. Furthermore, adding cosolvent molecules effectively reduced hydrophobic interactions between lipase molecules, thereby inhibiting aggregation. This approach in this study could be applied to the soluble production and self-refolding of other hydrophobic proteins, as well as the stabilization of other industrial enzymes in nonaqueous environments. Declarations Author contributions C. C.,Y.q. S., L.p. C.: Conceptualization, Methodology, Software. Y.m. Q., J.l. W.: Data curation, Writing-Original draft preparation. T.y. J.: Visualization, Investigation. C. C., B.f. H.: Writing-Reviewing and Editing.All authors reviewed the manuscript. Acknowledgement This study was supported by grants from the National Natural Science Foundation Youth Fund (22308159), the Natural Science Foundation of Jiangsu Province (BK20220335), the Jiangsu Provincial Association for Science and Technology Youth Talent Support Project (TJ-2023-021). In addition, we are grateful to the High Performance Computing Center of Nanjing Tech University for supporting the computational resources. Funding This study was supported by grants from the National Natural Science Foundation Youth Fund (22308159), the Natural Science Foundation of Jiangsu Province (BK20220335), the Jiangsu Provincial Association for Science and Technology Youth Talent Support Project (TJ-2023-021). Availability of data and materials All data generated or analyzed during this study are included in this published article. Ethics approval and consent to participate Not applicable. 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Supplementary Files rawimage.docx supplementarymaterial1.docx Cite Share Download PDF Status: Published Journal Publication published 06 Jan, 2026 Read the published version in Applied Microbiology and Biotechnology → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6737700","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":471410249,"identity":"ce8be35d-2ec2-4143-b5a6-e1a157033885","order_by":0,"name":"Cheng Cheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIie3RMQrCMBSA4VcKmQJdU6J4hUqhLuI5HC2FuogKLo6VQkfnOnmFeoNIQRfRVXCxS2ehIrqoie5pR8H8w0uGfCQQAJXqBzNiMadt0AIAnW/FKo8cxdz5oEVVidUVx6IUAFUmNNxkV3QY6stwXYyhXU+Ynp+lpLbp2zV8mmgR8mgMvp0w1LKkhAwcSsjJnUXY0TGkbsIwInIyulFi7Tkxbpy8KpDuAJmXHhO3IE5YOSFH36HAPE58m2LLsxcpcqTEiL3cfDw77ipMswJPO/X5NsylRMTfA9AMPu+E7++Upd35aFQ4qFKpVH/aG4TKRoB8kIKhAAAAAElFTkSuQmCC","orcid":"","institution":"Nanjing Tech University","correspondingAuthor":true,"prefix":"","firstName":"Cheng","middleName":"","lastName":"Cheng","suffix":""},{"id":471410250,"identity":"f510997c-5c54-4e2d-9fd7-fb33b74a4bb8","order_by":1,"name":"Yongqin Su","email":"","orcid":"","institution":"Nanjing Tech University","correspondingAuthor":false,"prefix":"","firstName":"Yongqin","middleName":"","lastName":"Su","suffix":""},{"id":471410252,"identity":"ab9a6c33-e89b-420d-9e7a-61235d2fc4b0","order_by":2,"name":"Lupeng Cui","email":"","orcid":"","institution":"Nanjing Tech University","correspondingAuthor":false,"prefix":"","firstName":"Lupeng","middleName":"","lastName":"Cui","suffix":""},{"id":471410254,"identity":"2b303b0e-edbf-46f6-9516-7bd6b9ac5464","order_by":3,"name":"Yumeng Qiu","email":"","orcid":"","institution":"Nanjing Tech University","correspondingAuthor":false,"prefix":"","firstName":"Yumeng","middleName":"","lastName":"Qiu","suffix":""},{"id":471410258,"identity":"92f1b70a-d9c2-438e-a79d-1d6ec9bd228e","order_by":4,"name":"Jialing Wang","email":"","orcid":"","institution":"Nanjing Tech University","correspondingAuthor":false,"prefix":"","firstName":"Jialing","middleName":"","lastName":"Wang","suffix":""},{"id":471410260,"identity":"e60b8453-bf13-4974-b4ba-836c5039e317","order_by":5,"name":"Tianyue Jiang","email":"","orcid":"","institution":"Nanjing Tech University","correspondingAuthor":false,"prefix":"","firstName":"Tianyue","middleName":"","lastName":"Jiang","suffix":""},{"id":471410262,"identity":"37a8d6da-f701-4cd5-bc2d-7c8dab89dc59","order_by":6,"name":"Bingfang He","email":"","orcid":"","institution":"Nanjing Tech University","correspondingAuthor":false,"prefix":"","firstName":"Bingfang","middleName":"","lastName":"He","suffix":""}],"badges":[],"createdAt":"2025-05-24 08:08:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6737700/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6737700/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00253-025-13660-6","type":"published","date":"2026-01-06T15:58:14+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84754095,"identity":"b73d66db-a276-440f-8428-8f233d298681","added_by":"auto","created_at":"2025-06-17 03:50:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":115949,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of lipase YCJ01 in \u003cem\u003eE. coli\u003cbr\u003e\n \u003c/em\u003eProtein samples were analyzed using SDS-PAGE and stained with Coomassie blue. Lane 1: Soluble fraction of lipase YCJ01 with molecular weight of 34 kDa (Baneyx and Mujacic 2004). Lane 2: Inclusion bodies (IBs) of lipase YCJ01. Lane 3: Soluble fraction of NusA-lipase YCJ01 with molecular weight of 89 kDa. Lane 4: IBs of NusA-lipase YCJ01.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6737700/v1/e7cc492f96667590ed88c44f.png"},{"id":84754086,"identity":"74a43837-a615-476e-b4da-5f49a4d9e9f2","added_by":"auto","created_at":"2025-06-17 03:50:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":295045,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Effect of different concentrations of osmolyte (TMAO, DMSO, and glycerol) on the folding and stability of Lipase YCJ01. (B) Enzymatic activities of lipase YCJ01 were measured after incubation with various chemical chaperones for 7 days at 4℃, 17℃, and 28℃. (C) Time curve showing the enzyme activity of recombinant NusA-lipaseYCJ01 refolding in water, 15% DMSO, 20% glycerol, and 4 M TMAO at 17℃. (D) SDS-PAGE analysis of recombinant NusA-lipase YCJ01 refolding in osmolyte solutions at 17℃. Lane 1: Control; Lane 2: Refolding in 20% glycerol; Lane 3: Refolding in 4 M TMAO; Lane 4: Refolding in 15% DMSO\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6737700/v1/23da50739ec9c0ee719934c4.png"},{"id":84754883,"identity":"9a9da911-1f50-40d7-b548-49e566b47771","added_by":"auto","created_at":"2025-06-17 03:58:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":294511,"visible":true,"origin":"","legend":"\u003cp\u003eRMSD values (A) of the backbone of lipase YCJ01 and the radius of gyration (B) for the backbone atoms of lipase YCJ01 in water, 15% DMSO, 25% glycerol, and 4 M TMAO\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6737700/v1/207952793f84a33a47af63e6.png"},{"id":84754099,"identity":"8f28fdbf-e0af-4f8b-a4b2-8576d0a33405","added_by":"auto","created_at":"2025-06-17 03:50:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":479368,"visible":true,"origin":"","legend":"\u003cp\u003eSnapshots captured at 1 ns, 30 ns, and 100 ns intervals during the simulation of 15% DMSO (A), 25% glycerol (B), and 4 M TMAO solution. Key: The protein is displayed in a surface style, with the hydrophobic residues shown in green. DMSO, glycerol, and TMAO molecules are depicted as yellow, orange, and cyan spheres, respectively\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6737700/v1/5666caf97cb6294804ead5f2.png"},{"id":84754101,"identity":"529d4422-2498-470c-ae6d-407ce3d8f236","added_by":"auto","created_at":"2025-06-17 03:50:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":246803,"visible":true,"origin":"","legend":"\u003cp\u003eTime evolution of the root mean squared fluctuation (RMSF) for the residues of lipase YCJ01 in water, 25% glycerol, and 15% DMSO system\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6737700/v1/9e5ed54220f65a99f3bdf4d7.png"},{"id":84755153,"identity":"6579b242-6bcf-497d-83e1-f971cf8aeef7","added_by":"auto","created_at":"2025-06-17 04:06:58","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":143199,"visible":true,"origin":"","legend":"\u003cp\u003eThe RMSF values of lipaseYCJ01 in water and 25% glycerol system\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6737700/v1/ceb410091be68cdccb56627e.png"},{"id":84754885,"identity":"fed7ee47-c513-4d64-95f8-b104e8df7caa","added_by":"auto","created_at":"2025-06-17 03:58:58","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":233624,"visible":true,"origin":"","legend":"\u003cp\u003eO–O water radial distribution functions (A) and H bond length distributions (B) for pure water, 15% DMSO, 25% Glycerol, and 4 M TMAO simulations\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-6737700/v1/98ef8c02bef5c0161c601f9b.png"},{"id":100069271,"identity":"a64e1d8b-6bbd-45ad-9f6d-3c6bd3aa05b2","added_by":"auto","created_at":"2026-01-12 16:12:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2415635,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6737700/v1/f77fa627-d19b-4ac2-8ce0-dd7994458a2c.pdf"},{"id":84754087,"identity":"58f24d5c-5ee1-4d45-ba31-b839432b00f2","added_by":"auto","created_at":"2025-06-17 03:50:58","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1528972,"visible":true,"origin":"","legend":"","description":"","filename":"rawimage.docx","url":"https://assets-eu.researchsquare.com/files/rs-6737700/v1/3d5d25bb05ca957c65b715a8.docx"},{"id":84754091,"identity":"a624a46a-2076-4194-9341-bd8f777a6639","added_by":"auto","created_at":"2025-06-17 03:50:58","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":300333,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarymaterial1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6737700/v1/85a92c0692e6541fe41b5913.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cosolvent-induced Spontaneous Refolding of Lipase","fulltext":[{"header":"Key Points","content":"\u003cul start=\"50\"\u003e\n \u003cli\u003e\u003cem\u003eCosolvents enhance lipase activity, with glycerol showing the highest improvement\u003c/em\u003e\u003c/li\u003e\n \u003cli\u003e\u003cem\u003eMD simulations show glycerol and DMSO directly interact with hydrophobic regions\u003c/em\u003e\u003c/li\u003e\n \u003cli\u003e\u003cem\u003eGlycerol and DMSO stabilize lipase directly, while TMAO enhances stability indirectly\u003c/em\u003e\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eNatural hydrophobic proteins, such as lipases, hydrophobins, and membrane proteins, have significant potential for diverse applications in biocatalysis and biomedical fields (Berger and Sallada \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Kaur et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Mouritsen et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Reis et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Sackmann and Tanaka \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). However, their widespread use is substantially hindered by the challenges in preparing functionally folded proteins, which often exhibit reduced activity and poor stability, limiting their practical utility (Baneyx and Mujacic \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Berger and Sallada \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Cheng et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Cui et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kaur et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Mouritsen et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Reis et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Sackmann and Tanaka \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOverexpression of proteins in \u003cem\u003eEscherichia coli\u003c/em\u003e typically results in the formation of inclusion bodies (IBs), which are generally biologically inactive and require processing to yield an active final product (Baneyx and Mujacic \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The strong hydrophobicity of hydrophobic proteins increases their tendency to form IBs, which compete with correct folding, particularly at high concentrations. This has been identified as a key challenge limiting their efficient application in the bioengineering industry. Conversely, the hydrophobic nature of these proteins often enhances their activity or stability in oil\u0026ndash;water biphasic systems. For example, lipase exhibits biocatalytic activity at the interface of aqueous and oil phases, facilitating an optimal site for lipolysis (Mouritsen et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The localized hydrophobic nature of lipases allows them to become active at interfaces (Cheng et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Hydrophobins typically self-assemble at the air\u0026ndash;water interface (Berger and Sallada \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Cui et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Membrane proteins require a specific environment containing lipids and water to facilitate the transport of various molecules across the cell membrane to maintain homeostasis (Junge et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Consequently, self-folding in nonaqueous media presents a promising approach for the industrial production of bulk and/or fine bioactive proteins with high hydrophobicity. Therefore, a novel strategy for soluble production and hydrophobic protein refolding by leveraging nonaqueous media is highly sought after.\u003c/p\u003e \u003cp\u003eFusion tags can be used to reduce prokaryotic IB formation and enhance protein solubility expression (Cheng et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Ma et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). A large variety of fusion tags are available for solubility enhancement, including the maltose-binding protein (MBP), N-utilization substance (NusA), thioredoxin (TrxA), Ffu and glutathione S-transferase (GST) (Costa et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). However, soluble expression does not always guarantee high enzymatic activity due to the formation of unfolding intermediates. Subsequent folding is essential to achieve correctly folded and active proteins. The equilibrium of the protein refolding process can be influenced by adjusting the thermodynamic state of the system, such as pH, pressure, temperature, or cosolvents (Agam et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Beck and Daggett \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Schuler et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Cosolvents can shift the equilibrium toward the folded state of proteins (Canchi and Garc\u0026iacute;a \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). For example, the cosolvent glycerol-assisted refolding of denatured Xfpal derived from \u003cem\u003eXylella fastidiosa\u003c/em\u003e resulted in a correctly structured protein (Santos et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The intrinsic mechanism of cosolvents in inducing protein refolding has been effectively demonstrated using atomistic molecular dynamics (MD) simulations. Two potential mechanisms, \u0026ldquo;direct interaction\u0026rdquo; (Bruzdziak et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and \u0026ldquo;indirect interaction\u0026rdquo; (Bennion and Daggett \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Shiraga et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), have been proposed in independent systems. For example, as a cosolvent, urea directly interacts with N-methylacetamide and dimethyl sulfoxide by weakening hydrogen (H) bonds within overlapping hydration shells (Stasiulewicz et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Tobi et al. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) illustrated cosolvent urea directly interacts with the peptide backbone during denaturation by selectively binding to the expanding surface of unfolded proteins, shifting the equilibrium toward the unfolded state. Conversely, protective cosolvents, such as trimethylamine N-oxide (TMAO) and glycerol, were preferentially excluded from the protein surface, favoring the folded state through indirect interaction. Bennion and Valerie (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) observed that TMAO enhanced water\u0026ndash;water H bonding in both binary TMAO\u0026ndash;water mixtures and ternary urea\u0026ndash;TMAO\u0026ndash;water solutions, while also strengthening water\u0026ndash;urea interactions to reduce urea\u0026ndash;protein H bonding. Moreover, the protective role of TMAO in countering the denaturing effects of urea was demonstrated by analyzing the hydration and conformational dynamics of wild-type villin headpiece protein HP35 and a doubly norleucine-substituent mutant (Lys24/29Nle) HP35NN in pure urea and urea\u0026ndash;TMAO mixed solutions (Yang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). TMAO preferentially protects the hydrophobic core of proteins from urea-induced denaturation by reducing urea accumulation around hydrophobic residues. Meanwhile, the thermodynamics of protein and cosolvent mixtures have been studied, where denaturing cosolvents lower the free energy of the unfolded state, thereby favoring the unfolded population, whereas protecting cosolvents raise the free energy of the unfolded state, favoring the folded population (Auton and Bolen \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Bolen and Baskakov \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). However, most MD simulations of cosolvent action focus on specific protein models, such as short peptides or small truncated proteins. Practical applications of cosolvent-induced refolding systems for assisting highly hydrophobic proteins with high molecular weight to correctly fold are still limited.\u003c/p\u003e \u003cp\u003e \u003cem\u003eBurkholderia ambifaria\u003c/em\u003e lipase YCJ01 is a well-characterized enzyme known for its high thermal stability, organic-solvent tolerance, and significant potential in various important applications, particularly in organic synthesis and the production of pharmaceuticals, flavors, and fragrances, as demonstrated in our previous studies (Cheng et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Yao et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013a\u003c/span\u003e). In this study, we developed a new method for soluble production in \u003cem\u003eE. coli and\u003c/em\u003e self-refolding enhancement for lipase YCJ01 in cosolvent systems. To the best of our knowledge, this is the first comprehensive attempt to study, at the molecular level, the effects of glycerol, DMSO, and TMAO as cosolvents on the self-refolding of the entire lipase YCJ01 protein. Additionally, we addressed the following two scientific questions using MD simulation: (1) How do cosolvents affect enzyme structure and dynamics? (2) What significant structural and dynamic observables can be applied to the refolding of other proteins?\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003e \u003cem\u003eE. coli\u003c/em\u003e DH5α (Invitrogen) was used for subcloning and plasmid amplification, while \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) (Novagen) was employed for protein expression. The pET-28a (+) (Novagen) plasmid served as the backbone for the expression. Restriction enzymes \u003cem\u003eXho\u003c/em\u003eI, \u003cem\u003eNco\u003c/em\u003eI, and \u003cem\u003eHind\u003c/em\u003eIII were purchased from Takara Bio. All DNA ligations were conducted using T4 ligase (TaKaRa).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eConstruction and expression of lipase YCJ01 and NusA-lipase YCJ01\u003c/h3\u003e\n\u003cp\u003eThe construction of pNusA vectors was performed as follows: primers F1\u0026amp;R1 (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) were used to amplify NusA fragments from the commercial vector pET43a. The purified PCR products and the pET28a (+) plasmid were digested with \u003cem\u003eNco\u003c/em\u003eI and \u003cem\u003eHind\u003c/em\u003eIII restriction enzymes. The pNusA vector was obtained after ligating the digested DNAs using T4 ligase. Recombinant plasmids were then transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3). The resulting clones containing recombinant plasmids of pNusA vectors were confirmed through sequencing. To construct the lipase YCJ01 and NusA-lipase YCJ01 expression vector, \u003cem\u003eBurkholderia ambifaria\u003c/em\u003e YCJ01 (containing a full-length lipase YCJ01) was used as the template to obtain lipase YCJ01. Primers F2\u0026amp;R2 (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) were synthesized to generate the gene fragment of lipase YCJ01 without its natural signal peptide, with the restriction sites underlined. After litigating the digested PCR products with pET28a and pNusA plasmids, the target genes were inserted into the constructed vectors. These constructions were then transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3), and pET28a-lipaseYCJ01 and pNusA-lipase YCJ01 was verified through restriction digestion and sequencing. The expression of lipase YCJ01 fused with the NusA fusion tag was conducted as follows: cells containing the pET28a-lipaseYCJ01 and pNusA-lipase YCJ01 vectors were grown to mid-log phase (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.6) at 37℃ in LB-broth supplemented with 50 mg/L kanamycin. The lipase YCJ01 and fusion protein NusA-lipase YCJ01 overproduction were induced with IPTG at a final concentration of 1 mM at 20℃. The cells were harvested once the OD\u003csub\u003e600\u003c/sub\u003e value reached 3.0. After centrifugation for 10 mins (4℃, 12000rpm), the supernatants were discarded, and the pelleted cells were resuspended in distilled deionized water and lysed through ultrasonication. The supernatant of the total lysate (soluble fractions) was obtained through centrifugation. The pellet (insoluble fraction) was resuspended in distilled deionized water and subjected to further analysis. The construction and expression of Ffu209-lipaseYCJ01, Ffu217-lipaseYCJ01, Ffu312-lipaseYCJ01, GST-lipaseYCJ01, and MBP-lipaseYCJ01 were performed using the same method as NusA-lipase YCJ01. The primers used for plasmid construction are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e in the Additional files. To analyze the supernatant of the total lysate (soluble fractions) and pellet (insoluble fractions), 12% SDS-PAGE was performed. Coomassie-stained protein bands were then scanned and analyzed using densitometry (Duoscan T1200, Bio-Rad, Hercules, CA).\u003c/p\u003e\n\u003ch3\u003eRefolding of cosolvents on lipase YCJ01\u003c/h3\u003e\n\u003cp\u003eThe refolding process of lipase using chemical chaperone (25% glycerol, 15% DMSO, 4 M TMAO, and 8M urea with 4 M TMAO) was investigated. The cells were harvested through centrifugation, resuspended in distilled deionized water, and lysed through ultrasonication. The supernatants of the total lysate were collected for further analysis. The total protein content in the supernatant of cell lysate was monitored using the Bradford assay. The solvent was not removed after the incubation with the cosolvent. The lipase needs diluted 5 times before the activity assay. The NusA-lipase YCJ01 solution was diluted to 50 g/ml. Different concentrations of cosolvents were added to the solution in sealed glass vial. The mixtures were incubated at 4℃, 17℃, and 28℃, respectively. The activity of lipase was measured every 24 h.\u003c/p\u003e\n\u003ch3\u003eAssay of lipase activity and protein concentration\u003c/h3\u003e\n\u003cp\u003eLipase activity was measured using a modified spectrophotometric method with p-nitrophenyl palmitate (p-NPP) as a substrate (Yao et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013a\u003c/span\u003e). p-NPP (3 mg), dissolved in 1 mL of isopropanol to a final concentration of 0.3 mg/mL, was mixed with 9 mL of 50 mM sodium phosphate buffer (pH 8.0) containing gum arabic (0.1%) and Triton X-100 (0.6%). The reaction mixture consisted of 240 \u0026micro;L of substrate solution and 10 \u0026micro;L of appropriately diluted enzyme solution, incubated at 40℃ for 10 min. The amount of p-nitrophenol (p-NP) produced in the reaction mixture was quantified spectrophotometrically at 410 nm. One unit of enzyme activity was defined as the amount of enzyme that liberated 1 mol of p-NP per min under standard assay conditions. The activity measurements were performed three times on each sample to ensure reproducibility.\u003c/p\u003e\n\u003ch3\u003eMD simulation\u003c/h3\u003e\n\u003cp\u003eAtomistic MD simulations were conducted using GROMACS 5.0. The GROMOS 54A7 force fields (Schmid et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) were employed to model protein atoms, while the TIP3P explicit solvent model (Agam et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) was used to represent water molecules. The three-dimensional structure of lipase YCJ01 was generated using the SWISS-MODEL, with the template PDB-ID 4LIP (Zhu et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) (PAL lipase, sequence identity 95.6%, crystal resolution 1.75 \u0026Aring;) and optimized through energy minimization. Additionally, the force field parameters for DMSO, glycerol, and TMAO were sourced from A.W. Schuettelkopf (Sch\u0026uuml;ttelkopf and van Aalten \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). For each simulation system, a relatively extended structure of lipase YCJ01, lacking native structural elements, was placed in a cubic box containing plenty of water and DMSO, glycerol, or TMAO molecules. Suitable counterions were added to each system to balance the charge of the protein. The detailed simulation parameters, including the number of solvent molecules, box size, and simulation time, for all simulation systems, are provided in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e. For each system, the replica exchange MD simulations (Sugita and Okamoto \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) were conducted in the NVT ensemble. MD simulations were performed at 300 K and 1 atm for 100 ns. The SHAKE algorithm (Ryckaert et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1977\u003c/span\u003e) was applied to constrain bonds involving H atoms, enabling a time step of 2.0 fs, with coordinates saved every 1 ps. Particle mesh Ewald was employed to manage long-range electrostatic interactions, and a nonbonded cutoff of 10 \u0026Aring; was used (Darden et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Langevin dynamics with a collision frequency of 3.0 ps\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was utilized to maintain the temperature of the system.\u003c/p\u003e \u003cp\u003eThe trajectories were analyzed using GROMACS tools. Root means square deviations (RMSD) values were calculated using g_rms, while the number of H bonds, defined by a cutoff distance of 0.35 nm and an angle cutoff of 30\u003csup\u003eo\u003c/sup\u003e, was determined using g_hbond. The radius of gyration (Rg) was calculated using g_gyrate to evaluate protein compactness, and the radial distribution function was computed using g_rdf. Images of the molecular graphics were generated and visualized using the VMD package (Humphrey et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1996\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003eSoluble expression of highly hydrophobic lipase YCJ01\u003c/h2\u003e\n \u003cp\u003eAs reported in our previous studies, the lipase from \u003cem\u003eB. ambifaria\u003c/em\u003e YCJ01 exhibits exceptional thermal stability, distinct tolerance to organic solvents, and significant potential for biocatalysis and the resolution of pharmaceutical intermediates (Bennion and Valerie \u003cspan class=\"CitationRef\"\u003e2004\u003c/span\u003e; Yang et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). The complete sequence of the lipase consists of a native signal peptide, a propeptide, and a mature peptide. Due to the high hydrophobic nature of lipase YCJ01, both the complete sequence and the mature peptide were expressed as IBs in \u003cem\u003eE. coli\u003c/em\u003e. To enhance lipase YCJ01 solubility and circumvent the need for solubilizing IBs, fusion tags (Ffu209, Ffu217, Ffu312, GST, MBP, and NusA (Cheng et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e)) were employed to confer stability and high solubility to lipase YCJ01. NusA tag (55kDa) performed preferably when compared with other fusion tags as shown in \u003cstrong\u003eFig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/strong\u003e. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, direct expression of lipase YCJ01 resulted in high yields, but the majority of the protein was sequestered in IBs, with only 3% solubility observed, even at an induction temperature of 20℃. Fortunately, the fusion protein NusA-lipase YCJ01 achieved significantly improved solubility (67.8%). The molecular weight of lipase YCJ01, as determined by SDS-PAGE, is 34 kDa, aligning with previous reports (Yao et al. \u003cspan class=\"CitationRef\"\u003e2013b\u003c/span\u003e), the molecular weight of the NusA-lipase YCJ01 fusion protein, comprising NusA (55 kDa) and lipase YCJ01 (34 kDa), exhibits a molecular weight of approximately 89 kDa. The molecular weight difference between NusA-lipase YCJ01 and lipase YCJ01 specifically corresponds to the molecular weight of NusA (55 kDa). However, the soluble NusA-lipase YCJ01 exhibited weak enzymatic activity.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eCosolvent-induced refolding of NusA-lipase YCJ01\u003c/h3\u003e\n\u003cp\u003eDue to the misconception that soluble protein expression guarantees proper protein folding into its correct structure, limited studies have focused on the refolding of soluble protein to enhance bioactivity. Lipase biocatalysis occurs at the interface of aqueous and oil phases to create an optimal site for lipolysis, and the enzyme demonstrates excellent organic-solvent tolerance. Therefore, cosolvents, such as glycerol, DMSO, and TMAO, were chosen to facilitate the refolding of fusion protein NusA-lipase YCJ01 into its correct structure. Among these, an organic solvent, DMSO, was initially used as a cosolvent to promote positive effects on protein refolding. Meanwhile, to better examine the cosolvent-induced refolding process, conditions involving water and a mixed solution of TMAO and urea were considered. Additionally, the impacts of different concentrations and inducing temperatures of cosolvents on lipase YCJ01 refolding were systematically investigated. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, lipase YCJ01 activity peaked at 210 U/ml after incubation with 25% (v/w) glycerol for 24 h. The activity rose to 125 U/ml with a 15% (v/w) DMSO concentration, reaching its maximum at 115 U/ml with a 4 mol/L TMAO concentration. However, excessive concentrations of cosolvent did not promote lipase YCJ01 refolding.\u003c/p\u003e\n\u003cp\u003eProtein samples were analyzed using SDS-PAGE and stained with Coomassie blue. Lane 1: Soluble fraction of lipase YCJ01 with molecular weight of 34 kDa (Baneyx and Mujacic \u003cspan class=\"CitationRef\"\u003e2004\u003c/span\u003e). Lane 2: Inclusion bodies (IBs) of lipase YCJ01. Lane 3: Soluble fraction of NusA-lipase YCJ01 with molecular weight of 89 kDa. Lane 4: IBs of NusA-lipase YCJ01.\u003c/p\u003e\n\u003cp\u003eTemperature plays a significant role in the refolding process. The enzymatic activities of lipase YCJ01 were evaluated at different temperatures (4℃, 17℃, and 28℃) using the optimal concentrations of each cosolvent solution (25% glycerol, 15% DMSO, and 4 mol/L TMAO). As shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB, a refolding temperature of 17℃ was more favorable than 28℃ and 4℃. The lowest enzymatic activity of lipase was observed at 28℃. Additionally, the refolding of NusA-lipase YCJ01 in water was found to be slow (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC), with enzymatic activity increasing from 1.5 U/ml to 69 U/ml. Conversely, the presence of cosolvent molecules significantly facilitated lipase YCJ01 refolding. Particularly, the enzymatic activities of lipase increased to 1,003 U/ml in 25% glycerol, 754 U/ml in 15% DMSO, and 501 U/ml in 4 M TMAO. The refolding efficiency of the cosolvents followed the order: TMAO\u0026thinsp;\u0026lt;\u0026thinsp;DMSO\u0026thinsp;\u0026lt;\u0026thinsp;glycerol. However, the combination of urea and TMAO in a 1:2 ratio did not improve the enzymatic activity of lipase (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\n\u003cp\u003eLipase YCJ01 has an open reading frame of 1095 bp that encodes 364 amino acids (Yao et al. \u003cspan class=\"CitationRef\"\u003e2013b\u003c/span\u003e). Analysis by the SignalP 3.0 Server indicates it contains a signal peptide of 40 amino acids and a propeptide of 12 amino acids in \u003cstrong\u003eFig. \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/strong\u003e. The N-terminal sequence analysis \u003cstrong\u003e(Fig. S3)\u003c/strong\u003e demonstrates that the N-terminal sequence of the refolded lipase YCJ01 is YPIILVHGLTGTDK, which is entirely consistent with the reported N-terminal of the mature peptide of lipase YCJ01.\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eReplica exchange MD analysis of cosolvent-induced refolding mechanism\u003c/h2\u003e\n \u003cp\u003eIn this study, we employed MD simulations to explore the microcosmic mechanisms by which different cosolvent molecules\u0026mdash;glycerol, DMSO, and TMAO\u0026mdash;affect the refolding of a highly hydrophobic lipase with a high molecular weight. To assess the stability and overall structural changes of lipase YCJ01 in water, 15% DMSO, 25% glycerol, and 4 M TMAO solutions, the backbone RMSDs averaged over four trajectories for each solution were calculated. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA, all RMSD values reached a plateau, indicating that the protein backbone maintained a stable structure across these solutions. Compared to the RMSD values for the overall conformation, the changing RMSD value in water (0.445 nm) was significantly higher than in 15% DMSO, 25% glycerol, and 4 M TMAO solutions (0.272 nm, 0.273 nm, and 0.287nm, respectively). Moreover, the growth rates of RMSD varied across different solutions during the first 25 ns. The RMSD value for lipase YCJ01 rose significantly in water, 15% DMSO, and 25% glycerol, while RMSD in 4 M TMAO remained relatively stable.\u003c/p\u003e\n \u003cp\u003eFurthermore, the Rg of the lipase backbone in water, 15% DMSO, 25% glycerol, and 4 M TMAO solutions was calculated to assess structural compactness. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB, slight increases in the Rg value were observed, specifically 0.072 nm in 15% DMSO and 0.073 nm in 25% glycerol. However, the Rg value of lipase in water and 4 M TMAO showed a contrasting trend, decreasing from 1.93 nm to 1.88 nm (0.05 nm) in water, and from 1.93 nm to 1.91 nm (0.02 nm) in 4 M TMAO. This indicates that the overall conformation of lipase in 15% DMSO and 25% glycerol becomes more loosened, whereas lipase tends to become more compact in water and 4 M TMAO. Moreover, more residues are exposed to the solvent in 15% DMSO and 25% glycerol.\u003c/p\u003e\n \u003cp\u003eTo evaluate the interactions between cosolvent molecules and lipase, a hydrophobic analysis of lipase YCJ01 was conducted. Most hydrophobic residues were found on the surface of the lipase and clustered into two core regions, R1 and R2. The R1 region comprises residues Val130, Leu138, Val142, Ile143, Val149, and Leu165, while the R2 region includes residues Ile236, Leu238, Ile239, Leu245, Leu247, Leu250, Val258, Ile260, Leu270, Val271, Val281, Leu298, and Val300. Snapshots from MD simulations at 1 ns, 30 ns, and 100 ns intervals for the 15% DMSO, 25% glycerol, and 4 M TMAO solution were captured (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). The cosolvent molecules were consistently positioned at least 3.5 \u0026Aring; away from the protein surface. During the simulation, glycerol and DMSO molecules gradually accumulated around the hydrophobic core regions of the lipase, while TMAO molecules remained evenly distributed on the protein surface.\u003c/p\u003e\n \u003cp\u003eAdditionally, the time evolution of RMSF for each residue in the different simulation systems was monitored (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). The overall residues in water exhibited higher RMSF values than those in 25% (w/v) glycerol, 15% (w/v) DMSO, and 4 M TMAO. Notably, residues in the R1 and R2 hydrophobic regions of lipase became unstable after approximately 20 ns in water. Conversely, these regions were stabilized in the glycerol and DMSO systems, where the molecules effectively stabilized the hydrophobic core regions of the lipase, preventing aggregation and unfolding. However, the instability in the R1 region was observed after 40 ns in the 4 M TMAO solution, unlike in the DMSO or glycerol systems. This structural variation may explain the reduced Rg value of the protein in the 4 M TMAO solution. These findings align with the RMSD and Rg observations. To better illustrate the flexibility/rigidity of the regions in the absence and presence of cosolvent molecules, the RMSF in water and 25% (w/v) glycerol were further analyzed (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e\u003cstrong\u003e)\u003c/strong\u003e, in the water, the residue region (Ser150-Ala160) has a high RMSF, corresponding to the loop region of lipase YCJ01, which is significantly disturbed. In the 25% (w/v) glycerol, the disturbance in this region is markedly reduced, which is consistent with the results in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e. In the 25% (w/v) glycerol, the residue region (Gly126-Thr150) has a higher RMSF value than in water. This region corresponds to the \u0026alpha;5 region of lipase YCJ01, namely, the \u0026ldquo;lid\u0026rdquo; of lipaseYCJ01 (Cheng et al. \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eMoreover, the numbers of cosolvent and water molecules within 3.5 \u0026Aring; of the overall conformation of lipase and its hydrophobic regions (R1 and R2) were calculated to evaluate the distribution behaviors of the cosolvent molecules on the hydrophobic surface of the lipase (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The presence of glycerol and DMSO molecules reduced the number of water molecules in the hydration shell of lipase, primarily in the hydrophobic regions. Conversely, the number of water molecules in the TMAO system showed minimal change compared to the water system. Furthermore, the number of cosolvent molecules within the hydration shell of the lipase was quantified. As shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, the percentage of DMSO and glycerol molecules surrounding the hydrophobic regions (R1 and R2) relative to the total in the hydration shell were 94.9% and 74.7%, respectively, while the percentage of TMAO was only 37.4%. DMSO and glycerol molecules aggregated at the hydrophobic regions of the lipase due to hydrophobic interactions, stabilizing the structure of these regions. Conversely, TMAO molecules were distributed on the surface lipase without apparent direct interaction with the hydrophobic regions. Additionally, the numbers of H bonds in the final nanosecond of the MD simulations were calculated. The average number of protein\u0026ndash;water H bonds decreased in cosolvent solutions: from 498 in water to 414 in 15% DMSO, 407 in 25% glycerol, and 443 in 4 M TMAO solutions. Meanwhile, the average numbers of H bonds formed between lipase and cosolvent molecules were 19 for DMSO, 7 for glycerol, and 5 for TMAO.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cimg 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\" width=\"584\" height=\"324\"\u003e\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003eProtein stability is significantly affected by water properties, as cosolvent molecules can intrinsically affect water structure in various ways, thereby affecting proteins through indirect mechanisms. To explore the protective roles of these cosolvents indirectly, their impact on water structure was analyzed. The oxygen\u0026ndash;oxygen (O\u0026ndash;O) water radial distribution functions in the simulations of water, 15% DMSO, 25% glycerol, and 4 M TMAO systems were calculated. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA, a distinct peak at 0.275 nm was observed, and adding glycerol, DMSO, and TMAO caused a significant increase in the first peak. The O\u0026ndash;O water radial distribution increased from 3.269 in the water system to 4.901 in the 25% glycerol system, indicating that cosolvents, particularly glycerol, enhanced the order of water structure. Furthermore, DMSO showed a greater effect on water structure than TMAO. These findings align with the superior ability of glycerol, demonstrating that glycerol and DMSO enhance lipase refolding more effectively than TMAO, primarily due to indirect interactions. The impact of cosolvents on water\u0026ndash;water H bonds in water, 15% DMSO, 25% glycerol, and 4 M TMAO solutions was also examined. Figure \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB illustrates the distribution of H-bond distances. The strength of water\u0026ndash;water H bonds is significantly affected by the solvent environment.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, an \u003cem\u003eE. coli\u003c/em\u003e heterologous expression system and a self-refolding strategy were established to enhance enzyme activity using cosolvents. A large, highly hydrophobic lipase YCJ01 was successfully expressed in abundance with the aid of the fusion tag NusA. The intrinsically solubility and biological activity of NusA in \u003cem\u003eE. coli\u003c/em\u003e contribute to its ability to improve the soluble production of fusion proteins. NusA slows down translation at the transcription pauses, offering more time for protein folding (Costa et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). It is likely that the transient intermediates formed during expression engaged in nonspecific intermolecular interactions due to the exposed hydrophobic surfaces, resulting in the unfolded state of lipase YCJ01. This suggests that the NusA fusion tag effectively enhanced the solubility of passenger lipase YCJ01 but failed to facilitate the correct folding of lipase YCJ01.\u003c/p\u003e \u003cp\u003eAdditionally, optimal refolding conditions using cosolvents for high enzymatic activity were explored. Spontaneous refolding occurred when NusA-lipase YCJ01 was in 25% (v/w) glycerol, 15% (v/w) DMSO, and 4 M TMAO solutions \u003cem\u003ein vitro\u003c/em\u003e, achieving activities of 1,003 U/mL, 754 U/mL and 501 U/mL, respectively. Remarkably, the enzymatic activity of lipase YCJ01 significantly increased, and cosolvent molecules facilitated the refolding process of lipase YCJ01, this phenomenon that the improvement of lipase activity in the presence of cosolvents is also observed for other lipases (Kamal et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Mangiagalli et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Tsuzuki et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; TSUZUKI et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The refolding efficiency of the cosolvents suggests that glycerol and DMSO are more effective than TMAO for refolding large, highly hydrophobic proteins such as lipase YCJ01. The concentration optimization results suggest that excessive concentrations of cosolvent did not promote lipase YCJ01 refolding. Instead, the excessive concentrations of cosolvent molecules caused steric hindrance, making it difficult for the stretched peptide chains to fold correctly, ultimately suppressing the protein refolding process. Moreover, the temperature optimization results show that NusA-lipase YCJ01 refolding at 17\u0026deg;C exhibited better activity than at 4\u0026deg;C, due to the slower refolding rate at lower temperatures, which also decelerates the cleavage of the propeptide. The lowest enzymatic activity of lipase at 28℃ indicates that high refolding temperature hinders proper protein refolding. This effect was likely due to increased intermolecular interactions, leading to a higher probability of protein aggregation caused by excessive molecular collisions. The N-terminal sequence analysis suggests that the signal peptide and propeptide of lipase YCJ01 have been successfully cleaved during the cosolvent-induced refolding process. This process also allowed the removal of the fusion tag NusA as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD. The pro-peptides act as intramolecular chaperones to facilitate lipase folding and are subsequently cleaved to release the mature protein, which is essential for obtaining lipase with the native N-terminus, as previously reported (Luo et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMD simulations at atomic resolution are widely used to describe the mechanisms by which cosolvent molecules influence protein folding equilibria, offering valuable insights into their effects on proteins and peptides (Daggett \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). However, most studies have focused on short-model peptides or truncated proteins, with limited research available on the folding mechanisms of integral proteins. Using MD simulations, the effect of cosolvents on such a large, highly hydrophobic protein was studied for the first time.\u003c/p\u003e \u003cp\u003eCompared to the RMSD values for the overall conformation, the changing RMSD value in water was significantly higher than in 15% DMSO, 25% glycerol, and 4 M TMAO solutions. This suggests that lipase YCJ01 exhibited more conformational changes in water, whereas it maintained a relatively stable spatial structure in the three cosolvent solutions. The RMSD finding also implies that the refolding process in TMAO differs from that in other cosolvents, such as glycerol and DMSO. The Rg results showed that the conformation of lipase YCJ01 in water and 4 M TMAO was more compact than that in 15% DMSO and 25% glycerol, which hindered the refolding process of lipase YCJ01. This aligns with the enzymatic activity of lipase YCJ01 in different solutions, with higher activity observed in 15% DMSO and 25% glycerol than in 4 M TMAO (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Surface hydrophobicity is a key factor in the accumulation of highly hydrophobic proteins (Kaur et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The clustering of DMSO and glycerol molecules at the hydrophobic regions effectively reduced protein\u0026ndash;protein interactions, thereby promoting the refolding process. Conversely, the uniform distribution of TMAO on the protein surface prevented aggregation but did so indirectly. Unlike DMSO and glycerol molecules, which directly interacted with the hydrophobic regions of lipase YCJ01, TMAO molecules likely influenced the hydrophobic lipase YCJ01 indirectly. Cosolvents, glycerol and DMSO, aggregated around the hydrophobic regions of the lipase, while TMAO primarily enhanced the water structure to reduce water attack on lipase.\u003c/p\u003e \u003cp\u003eThe RMSF results indicate that the lid region of lipase YCJ01 has a high degree of structural flexibility. In the 25% (w/v) glycerol, the conformation of the \u0026ldquo;lid\u0026rdquo; is more prone to change, suggesting that it is closely related to the activation mechanism of lipase YCJ01, which makes the active site accessible. In addition, the decrease in H bonds between lipase and water molecules occurred due to the competition of cosolvents with water molecules on the surface of lipase. It has been reported that water molecules play a significant role in protein denaturation by first solvating the hydrophobic core of the protein, followed by its overall structure (Bennion and Daggett \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Under these conditions, cosolvent molecules enhanced the refolding process of highly hydrophobic proteins by reducing the formation of water\u0026ndash;protein H bonds, thereby mitigating water attack. The relatively few H bonds formed between lipase and cosolvent molecules, particularly TMAO, support an indirect interaction mechanism underlying the protective role of TMAO (Paul and Patey \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, the presence of cosolvents resulted in shorter and more robust H-bond lengths than those in the water system. Compared to the water system, water\u0026ndash;water interactions became more pronounced in the presence of cosolvent molecules, particularly TMAO, which hindered water\u0026ndash;protein interactions. This is beneficial for the formation of hydrophobic cavities in lipase, facilitating its refolding. These observations indicate that glycerol and DMSO affect hydrophobic protein folding both directly and indirectly, whereas TMAO primarily relies on indirect interactions. This aligns with the observation that the RMSD of glycerol and DMSO increases more rapidly than TMAO within the first 25 ns (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). This is consistent with the fact that lipase YCJ01 activity increased more quickly in DMSO and glycerol solutions than in the TMAO solution (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The effect of cosolvents on lipase YCJ01 analysized by MD simulation reveals that cosolvents can accelerate the refolding of lipaseYCJ01. Meanwhile, the addition of glycerol opens the \u0026ldquo;lid\u0026rdquo; region, boosting the solvent accessibility of the active site of lipaseYCJ01. These cosolvent molecules stabilized the water structure and enhanced water\u0026ndash;water H bonds. Therefore, we concluded that glycerol and DMSO influenced hydrophobic proteins both directly and indirectly, while TMAO indirectly stabilized the hydrophobic lipase YCJ01. Furthermore, adding cosolvent molecules effectively reduced hydrophobic interactions between lipase molecules, thereby inhibiting aggregation. This approach in this study could be applied to the soluble production and self-refolding of other hydrophobic proteins, as well as the stabilization of other industrial enzymes in nonaqueous environments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eC. C.,Y.q. S., L.p. C.: Conceptualization, Methodology, Software. Y.m. Q., J.l. W.: Data curation, Writing-Original draft preparation. T.y. J.: Visualization, Investigation. C. C., B.f. H.: Writing-Reviewing and Editing.All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003eAcknowledgement\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study was supported by grants from the National Natural Science Foundation Youth Fund (22308159), the Natural Science Foundation of Jiangsu Province (BK20220335), the Jiangsu Provincial Association for Science and Technology Youth Talent Support Project (TJ-2023-021). In addition, we are grateful to the High Performance Computing Center of Nanjing Tech University for supporting the computational resources.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis study was supported by grants from the National Natural Science Foundation Youth Fund (22308159), the Natural Science Foundation of Jiangsu Province (BK20220335), the Jiangsu Provincial Association for Science and Technology Youth Talent Support Project (TJ-2023-021).\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAgam G, Barth A, Lamb DC (2024) Folding pathway of a discontinuous two-domain protein. 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J Mol Catal B Enzym 133:S150-S156. doi:10.1016/j.molcatb.2016.12.009\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":true,"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":"Lipase YCJ01, soluble expression, spontaneous refolding, cosolvents, MD simulations","lastPublishedDoi":"10.21203/rs.3.rs-6737700/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6737700/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe overexpression of proteins in \u003cem\u003eEscherichia coli\u003c/em\u003e often results in the formation of inclusion bodies, which are biologically inactive, especially for proteins with exposed hydrophobic surfaces. Solubilization of IBs and subsequent refolding is essential for obtaining correctly folded and active protein. However, protein refolding involves multiple steps\u0026mdash;namely isolation, solubilization, and refolding\u0026mdash;which is a labor-intensive process. In this study, we developed a strategy for soluble production and protein refolding. A fusion tag was applied to \u003cem\u003eBurkholderia ambifaria\u003c/em\u003e lipase YCJ01, enabling abundant soluble expression in \u003cem\u003eE. coli\u003c/em\u003e. Despite this, the soluble protein exhibited only partial enzymatic activity, suggesting an unfolded state of soluble lipase YCJ01. Lipase activity increased significantly after incubation with cosolvents, reaching 1,003 U/mL, 754 U/mL, and 501 U/mL in 25% (v/w) glycerol, 15% (v/w) DMSO, and 4M trimethylamine N-oxide (TMAO) solutions, respectively. Correctly folded and highly active lipase YCJ01 with a natural N-terminus was obtained. Moreover, the cosolvent-induced refolding mechanism was elucidated through molecular dynamics simulations. Glycerol and DMSO were found to aggregate around hydrophobic regions of lipase, directly stabilizing structure by displacing water molecules and weakening water\u0026ndash;protein hydrogen (H) bonds within the hydration shell. Conversely, TMAO molecules indirectly influenced the lipase structure by strengthening water\u0026ndash;water H bonds.\u003c/p\u003e","manuscriptTitle":"Cosolvent-induced Spontaneous Refolding of Lipase","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-17 03:50:53","doi":"10.21203/rs.3.rs-6737700/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":"e165823b-86f8-4204-b7f8-399308c82acd","owner":[],"postedDate":"June 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-12T16:03:50+00:00","versionOfRecord":{"articleIdentity":"rs-6737700","link":"https://doi.org/10.1007/s00253-025-13660-6","journal":{"identity":"applied-microbiology-and-biotechnology","isVorOnly":false,"title":"Applied Microbiology and Biotechnology"},"publishedOn":"2026-01-06 15:58:14","publishedOnDateReadable":"January 6th, 2026"},"versionCreatedAt":"2025-06-17 03:50:53","video":"","vorDoi":"10.1007/s00253-025-13660-6","vorDoiUrl":"https://doi.org/10.1007/s00253-025-13660-6","workflowStages":[]},"version":"v1","identity":"rs-6737700","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6737700","identity":"rs-6737700","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}