Immobilization of Thermomyces lanuginosus lipase in a novel polysaccharide-based hydrogel by a two-step crosslinking method and its use in the lauroylation of α-arbutin

Immobilization of Thermomyces lanuginosus lipase in a novel polysaccharide-based hydrogel by a two-step crosslinking method and its use in the lauroylation of α-arbutin | 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 Immobilization of Thermomyces lanuginosus lipase in a novel polysaccharide-based hydrogel by a two-step crosslinking method and its use in the lauroylation of α-arbutin Ming Chen, Wei na She, Xin Zhao, Cheng Chen, Ben wei Zhu, Yun Sun, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3445915/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Jan, 2024 Read the published version in Bioresources and Bioprocessing → Version 1 posted 4 You are reading this latest preprint version Abstract The Thermomyces lanuginosus lipase (TLLs) was successfully immobilized within a novel hydrogel matrix through a two-step crosslinking method. TLLs was initially crosslinked through the Schiff-base reaction by oxidized carboxymethyl cellulose (OCMC). The water-soluble OCMC@TLLs complex was subsequently crosslinked by carboxymethyl chitosan (CMCSH) in a microfluidic apparatus to form the CMCHS/OCMC@TLLs microspheres. The CD (Circular Dichroism, CD) and FTIR (Fourier Transform infrared spectroscopy, FTIR) spectra demonstrated that the crosslinking of TLLs with OCMC resulted in a less significant impact on their structure compared to that with glutaraldehyde. CMCHS/OCMC@TLLs showed decreased catalytic performance due to the mass transfer resistance, while its thermal stability was greatly improved. The CMCHS/OCMC@TLLs were used to catalyze the lauroylation of arbutinin tetrahydrofuran. After 12 h of reaction under optimal conditions, the yield of 6′-O-laurylarbutin reached an impressive 92.12%. The prepared 6′-O-laurylarbutin has high lipophilicity and exhibits similar tyrosinase inhibitory activity and higher antioxidant activity compared to its parent compound. Arbutin oxidized carboxymethyl cellulose carboxymethyl chitosan microfluidic covalent cross-linked polymer lipase acylation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Arbutin, hydroquinone O-(α/β)-D-glucopyranoside, is a natural compound found int the leaves of Ericaceae plants (Bearberry). As a member of phenolic glycosides, arbutin possesses unique biological activities of antioxidant, anti-inflammatory, and anti-tumor (Shang et al. 2020 ; Lee and Kim 2012 ; Dong-Seok Kim et al. 2011 ; Takebayashi et al. 2010 ), assuming its clinical application in the treatment of urinary tract infections, kidney stones, cystitis (Schindler et al. 2002 ), and respiratory diseases (Zhou et al. 2019 ). Moreover, arbutin, particularly its α-isomer, serves as a potent tyrosinase inhibitor that blocks the production of melanin in vivo. Thus, it is now widely used in the production of whitening cosmetics. From a structural perspective, arbutin consists of two components, namely a glucoside and a phenolic group. The active Ph-OH in the phenolic group plays a crucial role in scavenging free radicals and protecting cellular constituents from oxidative harm (Watanabe et al. 2009 ), thus providing multiple physiological activities for arbutin. On the other hand, glucoside makes arbutin highly water soluble, but it hinders its transportation through cell membranes, resulting in a low oral bioavailability. In an effort to solve the aforementioned issue, researchers have attempted to graft hydrophobic groups, such as long-chain fatty acyls onto arbutin by chemical (Wang and Kong 2021 ) and enzymatic methods (NAGAI et al. 2009 ). However, there are two active reaction sites in the arbutin structure, namely the primary hydroxyl in glycoside and the phenolic hydroxyl in a phenolic group. It is difficult to achieve selective acylation through traditional chemical modification. Compared to chemical methods, biocatalysis offers several advantages including high regioselectivity, short reaction routes, high yields, and cost-saving benefits. Thus, the application of biosynthesis in the selective acylation of phenolic glycosides has garnered increasing attention. Tokiwaetal. ( 2007 ) synthesized arbutin undecylenic acid ester using alkaline protease from Bacillus subtilis as a catalyst. The acylated arbutin showed 100-fold higher inhibitory activity against tyrosinase than arbutin (Tokiwa et al. 2007 ). Watanabe et al. ( 2009 ) studied the lipase-catalyzed acylation of phenol glycosides (i.e., arbutin, naringenin, and phlorizin) using lauric acid as an acyl donor. Lipase CALB showed a broad substrate spectrum and the yields of the resulting esters were all up to 50%. The lauroylphenol glycosides were more active against linoleic acid oxidation than its parent compound; probably resulting from its increased solubility and improved membrane penetration in oil-based systems (Watanabe et al. 2009 ). Liu et al. (2021) developed a novel one-step enzymatic method for the synthesis of hydrophobic arbutin esters in supercritical carbon dioxide (SC-CO 2 ), using CALB as the biocatalyst and ethyl palmitate as the acyl donor (Liu 2021 ). The stability and solubility of the resulting esters were greatly improved, making them more useful in cosmetic and pharmaceutical applications. Based on prior studies, immobilized lipases are preferred in the selective acylation of phenolic glycosides due to their exceptional characteristics, including abroad range of substrates, extensive source, excellent solvent tolerance, and cost-effectiveness (Milivojević et al. 2017 ). In recent years, there has been a significant increase in the research and development of immobilized lipase, with a primary focus on optimizing the supports (Parnianchi et al. 2018 ; Harris et al. 2021 ; Zahirinejad et al. 2021 ). As a host material, the supports for enzyme immobilization should possess not only high enzyme capacity but also excellent stability and mechanical strength to match different applications. In this regard, polysaccharide-based hydrogels are highly appealing due to their porous structure, non-toxicity, exceptional biocompatibility, easy modification, and biodegradability (Okura et al. 2020 ). The 3D mesh structure of hydrogels has the ability to both trap enzymes and allow the passage of substrates and products. Moreover, due to their highly hydrophilic nature and water-rich structure, hydrogels provide an optimal microenvironment for enzymes, thereby promoting the preservation of their conformation (Dai et al. 2017 ). Recently, researchers have investigated the hydrogel-based immobilization technology of lipase in detail. Park et al. ( 2015 ) immobilized Candida rugosa lipase in cellulose/lignin composite hydrogel beads. The immobilized lipase showed higher activity and stability than those embedded in pure cellulose beads (Park et al. 2015 ). Kim et al. ( 2017 ) prepared bacterial cellulose (BC)-chitosan composite hydrogel beads and used to immobilization of Candida lipase by physical adsorption and covalent cross-linking. As compared to microcrystalline cellulose (MCC)-chitosan hydrogel beads, BC-chitosan hydrogel exhibits higher adsorption capacity and activity recovery(Kim et al. 2017 ). Despite achieving some positive results in previous work, the application of polysaccharide-based hydrogels in enzyme immobilization still faces challenges. Natural polysaccharides, for example, chitosan, cellulose, and alginate are polyhydroxy polymers lacking highly reactive groups. During the formation of hydrogels, polysaccharide chains are commonly crosslinked through non-covalent interactions, such as hydrogen bonds and electrostatic forces. The weak interactions that exist between the chains are inadequate to ensure the structural stability of hydrogel. When exposed to solvents for an extended period, hydrogels are susceptible to swelling or even disintegration, leading to the leakage of enzymes. To stabilize the structure of hydrogel, bifunctional reagents such as glutaraldehyde and divalent metal ions Ca 2+ are frequently used as crosslinkers to enhance the interactions between polymer chains and enzymes (Turner et al. 2005 ). However, it is possible that this small reagent scan infiltrates the enzyme structure and causes excessive intermolecular and/or intramolecular crosslinking, resulting in irreversible changes in the conformation and a reduction in enzyme activity. In this study, we propose a novel method for the immobilization of lipase TLLs that involves the covalent cross-linking of oxidized carboxymethyl cellulose (OCMC) and carboxymethyl chitosan (CMCHS). First, OCMC with high aldehyde content was used to establish a chemical link through Schiff base’s reaction between the free TLLs; the OCMC@TLLs complex was subsequently crosslinked with CMCHS to yield the TLLs-containing hydrogel (OCMC/CMCHS@TLLs). To prepare hydrogel beads of uniform size, a microfluidic apparatus was utilized in this step (see Fig. 1 for the preparation route of OCMC/CMCHS@TLLs microspheres). The structure, catalytic properties, and stability of OCMC/CMCHS@TLLs were investigated in detail. In the end, we perfected a high-yield process for synthesizing 6'-O-lauroyl arbutin with OCMC/CMCHS@TLLs as a catalyst. The oil-water partition coefficient, inhibitory activity of tyrosinase, and DPPH scavenging capacity of 6'-O-lauroyl arbutin were also assessed. 2. Results 2.1. Preparation of OCMC@TLLs To improve the immobilization of TLLs, free TLLs were first linked by linear OCMC through the Schiff-base reaction. The changes in activity and conformation of the OCMC@TLLs complex were investigated and compared to those of GA@TLLs, the complex formed by using GA as a crosslinker. As described in 3.2.2, TLLs solution was treated with OCMC and glutaraldehyde (GA) solution, both of which had the same aldehyde concentration (approximately 0.7 mol/L), for a duration of one hour, respectively. The changes in the activity of reactants were monitored at 10-minute intervals, while the activity of untreated TLLs was determined and used as a control. The initial activity of free TLLs was set at 100%. As illustrated in Fig. 2 , the impact of GA on the activity of TLLs could be classified into two stages. In the first stage, the relative activity of TLLs decreased immediately to 82.5% in contact with GA, indicating that a rapid reaction occurred between GA and TLLs. As time progressed, the relative activity of GA@TLLs remained constant. However, a second decrease in the activity of GA@TLLs was observed when the reaction time exceeded 30 minutes. This phenomenon might be attributed to changes in the conformation of TLLs due to GA-induced intramolecular crosslinking. On the contrary, the relative activity of OCMC@TLLs descended slowly during the crosslinking reaction and remained higher than that of GA@TLLs and even the free TLLs. At the end of the reaction, the relative activity of OCMC@TLLs was as high as 93.85%, whereas that of GA@TLLs was only 62.82%. This phenomenon might be explained by the special structure of OCMC. OCMC is a carboxymethylated derivative of cellulose, having a linear and flexible structure. It makes a high steric hindrance effect against the Schiff-base reaction between OCMC and TLLs, which reduces the intensity of the crosslinking reaction and aids in preserving the activity of TLLs. In addition, the larger size of OCMC molecules makes it difficult to penetrate the interior of TLL. The aldehyde groups in OCMC can only react with the exposed -NH2 on the surface of TLLs molecules, thereby reducing the effect of crosslinking on enzyme conformation. To clarify the impact of OCMC and GA on the activity of TLLs, the secondary structure of free TLLs, OCMC@TLLs, and GA@TLLs was analyzed by CD and FT-IR spectra. As shown in Fig. 3 a, the CD spectrum of free TLLs presented a positive peak at 197 nm and two broad negative peaks at 210 and 221 nm in the far-UV region, which were assigned to the structures of β-sheet and α-helix, respectively (Li et al. 2021 ). Compared to the free enzyme, the absorbance at 210 and 221 nm of GA@TLLs both decreased, but that at 197 nm increased simultaneously. It could be inferred that the proportion of α-helix decreased and that of β-sheet increased in the structure of GA@TLLs (Chen et al. 2021 ). The CD spectrum of OCMC@TLLs exhibited different changes from that of GA@TLLs, in which the positive peak at 197 nm was greatly weakened, suggesting a decreased proportion of β-sheet in the OCMC@TLLs structure. FT-IR Analysis of free TLLs, GA@TLLs, and OCMC@TLLs was done, and the curves of spectrum in the amide I band (1600 ~ 1700 cm -1 ) were fitted into their respective secondary components by means of deconvolution (Fig. 3 B ~ Fig. 3 D), respectively. The starting values for the center position and height of each amide I subpeak were manually fixed by inspecting each spectrum, while the width was initially fixed at 15 cm − 1 . As listed in Table 1 , the proportion of α-helix and β-sheet in GA@TLLs was about 15.37% and 44.19%, which were 5.65% lower and 21.64% higher than those in free TLLs, respectively. In OCMC@TLLs, however, although the proportion of α-helix and β-sheet showed a slight decrease, the changes were not significant (< 3%). These findings were consistent with the result from the CD spectrum, confirming the distinct influences of GA and OCMC on the secondary structure of TLLs.2.2. Fabrication and Characterization of OCMC/CMCS@TLLs Table 1 Results of deconvolution of amide I bands corresponding to TLLs, GA@TLLs and OCMC@TLLs. Wavenumber, cm -1 1624 ~ 1640 1674 ~ 1695 1648 ~ 1660 1640 ~ 1650 1662 ~ 1686 Samples Secondary structure, % β-sheet α-helix Random coil Turn Other TLLs 36.33 16.29 20.66 15.76 10.96 OCMC@TLLs 35.31 15.99 19.26 19.51 9.93 GA@TLLs 44.19 15.37 22.62 8.02 9.80 The OCMC/CMCS@TLLs microspheres were manufactured using the water-in-oil method with the aid of microfluidic devices (Yang et al. 2016 ). The size of microspheres was regulated by altering the flow rates of disperse phase and oil phase. Upon microscopic observation, the prepared microspheres were in a spherical shape with a mean diameter of 193.28 ± 18.35µm (Fig. 4 A, B). The cross-section of freeze-dried OCMC/CMCS@TLLs revealed a developed porous structure (Fig. 4 D), providing a vast area for the binding of enzymes. In addition, some regular particles were observed on the surface of support from the high-resolution image (Fig. 4 C), which demonstrated the successful immobilization of TLLs. The FT-IR spectra of TLLs, OCMC/CMCHS, and OCMC/CMCHS@TLLs were showed in Fig. 5 . The absorption peak at 3420 cm -1 is attributed to the stretching vibration of -NH 2 and -OH groups. In the spectrum of OCMC/CMCHS@TLLs, the peak at 1053 cm -1 and 2926 cm -1 correspond to the bending vibration of C-O and the stretching vibration of C-H; 1417 cm -1 is the stretching vibration of -CH 2 , and the strong absorptions at 1600 cm -1 and 1327 cm -1 correspond to the characteristic peaks of C = N and C-N, respectively, confirming the generation of Schiff-based OCMC/CMCHS@TLLs. 2.3. Enzymatic properties of OCMC/CMCHS@TLLs 2.3.1. Subsubsection The effects of pH on the activity and stability of OCMC/CMCHS@TLLs were examined and contrasted with those of free TLLs. In the experimental condition, the optimum pH of free TLLs and OCMC/CMCHS@TLLs was found to be approximately 8.0 (Fig. 6 A, B). To assess the pH stability, OCMC/CMCHS@TLLs and free TLLs were separately mixed with a buffer solution of varying pH values (from 4.0 ~ 11.0). The samples were stood at 4℃ for 12 h, followed by assaying the activity of the samples and dividing it by its initial activity to obtain the residual activity (%). The residual activity with the highest value was established at 100%. As presented in (Fig. 6 C, 6 D), OCMC/CMCHS@TLLs and free TLLs were more stable in alkaline solutions (pH > 8); the highest stability for both was obtained at pH 8.0. Within the pH range of 8.0 ~ 11.0, the stability of OCMC/CMCHS@TLLs was slightly higher than that of the free form. The impact of temperature on the activity of OCMC/CMCHS@TLLs and free TLLs were depicted in Fig. 6 E. The optimal temperature for both was 50°C. Under moderate temperature conditions (20–40°C), the relative activity of OCMC/CMCHS@TLLs was noticeably lower than that of the free ones, which might be attributed to high mass transfer resistance in hydrogels. As the temperature increased further, the fluidity of the polymer chains in hydrogels correspondingly increased, reducing the resistance of mass transfer. In addition, the deformation of hydrogels can also mitigate the thermal denaturation of TLLs (Xu et al. 2020 ). Therefore, OCMC/CMCHS@TLLs displayed higher relative activity than free TLLs within a high temperature range of 50 to 80°C. The thermal stability of OCMC/CMCHS@TLLs was assessed by monitoring the residual activity after incubation at 60°C for 1 h. The results presented in Fig. 8 B demonstrated that the activity of TLLs remained constant within the temperature range of 20°C ~ 40°C, but it experienced a dramatic drop in stability as the temperature exceeded 40°C. In comparison to TLLs, OCMC/CMCHS@TLLs exhibited a high level of stability across a broader temperature range of 20–60°C. After being incubated at 60°C for one hour, the residual activity of OCMC/CMCHS@TLLs was as high as 94.26%, confirming the protective effect of the hydrogel on TLLs activity. 3.3.1. Kinetics The kinetics of free TLLs, OCMC@TLLs complex, and OCMC/CMCS@TLLs were evaluated by the Michaelis-Menten equation, using p -NPP as the substrate. By performing linear regression on \(\frac{1}{{\text{r}}_{\text{s}}}\) and \(\frac{1}{{\text{C}}_{\text{s}}}\) , the K m values of TLLs in three forms were calculated and listed in Table 2 . Table 2 Km value of free TLLs, OCMC@TLLs and OCMC/CMCHS@TLLs. Enzyme TLLs OCMC@TLLs OCMC/CMCHS@TLLs Km, mmol/L 1.396 2.917 9.938 The Km value of OCMC@TLLs complex was 2.917 mmol/L, whereas that of free TLLs was 1.396 mmol/L, indicating a slight alteration in the conformation of TLLs. The Km of OCMC/CMCS@TLLs was calculated to be 9.938 mmol/L, about 7-fold higher than that of free TLLs, revealing a significant decrease in its affinity for the substrate. The aforementioned results could be attributed to the high mass transfer resistance in hydrogel. In contrast to free TLLs, the TLLs in OCMC/CMCS@TLLs were restricted within the hydrogels. During the reaction, the substrates in the solution need to be diffused through the mesh of hydrogels to the reaction site. This process resulted in high mass transfer resistance, ultimately leading to a great increase in the Km value of OCMC/CMCS@TLLs. 2.4. Preparation of 6'-O-lauroyl arbutin 2.4.1 Screening of the reaction media As previously reported, the specific physicochemical properties of the reaction medium greatly influence lipase-catalyzed esterification (GARCIA-MOLINA et al. 2010). In this study, seven organic solvents with varying solubility (Log P) and dielectric constant (ε) were tested for their suitability as the reaction medium, while OCMC/CMCS@TLLs were used as the catalyst. After a reaction at 50°C for 12 h, the conversion rate of 6'-O-lauroyl arbutin in different reaction media was found to be in descending order, i.e., THF > tert-amyl alcohol > acetone > cyclohexane > acetonitrile > methanol (Table 3 ). This result could be attributed to the difference in the solubility and polarity of solvents. In the arbutin esterification system, the acyl donor and acceptor have opposite solubility. An excessively hydrophobic or hydrophilic solvent promotes the dissolution of one substrate while limiting the dissolution of the other, resulting in a decrease in the conversion rate. In addition, the polar solvents with high ε values, such as methanol and acetonitrile may damage the structural stability of TLLs and cause the denaturation of enzymes. Table 3 Conversion rate of 6'-O-lauroylarbutinin different medium Medium Log P Dielectric constant, ε Conversion rate, % Methanol -0.76 32.6 1.11 ± 0.37 Acetonitrile -0.33 37.5 9.83 ± 1.91 Acetone -0.23 20.5 15.92 ± 0.99 THF 0.49 7.39 90.13 ± 1.55 tert-Amyl alcohol 1.3 13.35 66.31 ± 0.89 Cyclohexane 3.2 1.18 15.03 ± 1.04 * Reaction conditions: arbutin, 0.1 mmol; vinyl laurate, 0.6 mmol; solvent volume, 5 mL; temperature, 50°C; enzyme dosage, 50 mg. 2.4.2. Donor/acceptor ratio In order to optimize the reaction conditions, a set of experiments were conducted with a fixed arbutin concentration and varying concentrations of vinyl laurate, resulting in the final molar ratios of 1:1, 1:2, 1:4, 1:6, 1:8, and 1:10; OCMC/CMCHS@TLLs was used as the catalyst with a dosage of 50 mg, and the reaction temperature was set at 50°C. Figure 7 showed that the reaction rate and the conversion of 6'-O-lauroyl arbutin both increased with the molar ratio of arbutin and vinyl laurate. While the molar ratio was 1:6, the final conversion rate was as high as 90.79% after a 12 h reaction. Increasing the donor/acceptor ratio further had little improvement in the conversion rate and reaction time. In comparison with other relevant literature, this result was advantageous in terms of conversion rate and reaction time (Table 4 ). Table 4 Comparison of conversion rate of arbutin with literatures Source of lipase Acyl donor Medium Reaction time, h Conversion rate,% Ref. Candida antarctica B Ferulic acid tert-butanol 168 62 (Ishihara et al. 2010 ) Candida sp. 99 − 1 Vinyl acetate THF 24 91.42 (Cai et al. 2015a ) Candida parapsilosis Vinyl laurate Pyridine-isooctane 24 71.1 (Li et al. 2018 ) Penicillium expanse Vinyl vanillic acid tetrahydrofuran 62 93 (Yang et al. 2020 ) Candida antarctica B Ethyl palmitate SC − CO 2 20 85.21 (Liu 2021 ) Thermomyceslanuginosus Vinyl laurate THF 12 90.79 This work 2.4.3 Operating stability of OCMC/CMCHS@TLLs The operating stability of OCMC/CMCHS@TLLs was evaluated by subjecting them to a reusability test in the production of 6′-O-laurylarbutin at optimal conditions. The same operations were conducted using TILM, commercially immobilized TLLs, instead of OCMC/CMCHS@TLLs. Figure 8 revealed that when using OCMC/CMCHS@TLLs or TILM as the catalyst, the conversion rate of 6′-O-laurylarbutin gradually decreased with each reaction batch, indicating a decrease in enzyme performance. After seven repetitions, the performance of OCMC/CMCHS@TLLs and TLIM dropped to 53.52% and 60.77%, respectively. Assuming the deactivation of both OCMC/CMCHS@TLLs and TLIM in THF followed the first-order reaction kinetics. The deactivation constant (k d ) can be calculated by linear regression of the following equation: $$\text{ln}\left(\frac{{C}_{i}}{{C}_{0}}\right)=-{k}_{d}\bullet t+b$$ 1 Where C 0 and C i represented the conversion rate of 6′-O-laurylarbutin in the first and batch, respectively. The calculated k d value of OCMC/CMCHS@TLLs was 0.1104, which was slightly higher than that of TLIM, suggesting its high operating stability in THF. 2.5. Properties of 6′-O-laurylarbutin The logarithmic value of distribution coefficient ratio, also known as the oil-water partition coefficient (Log P), is frequently utilized to determine the hydrophilicity and lipophilicity of organics. This coefficient is specific for a given compound in n -octanol and water. The Log P values of arbutin and 6'-O-lauroyl arbutin were determined in an octanol/water solution with a phase ratio of 1:1. As illustrated in Fig. 9 A, the Log P value of α-arbutin was − 1.51, indicating its potent hydrophilicity; however, the Log P value of 6'-O-lauroylarbutin was as high as 3.18, demonstrating that the lipophilicity of arbutin was greatly enhanced by introducing a long-chain fatty acyl group. In order to examine the impacts of acylation modification on the activity of arbutin, the capabilities for inhibiting tyrosinase and scavenging DPPH radicals of both 6′-O-lauroyl arbutin and arbutin were assessed by using Vitamin E as the control. As presented in Fig. 9 B, the activity of tyrosinase decreased rapidly with the concentration of inhibitors in the low dose range (< 0.2 mmol/L), but then leveled off. In the experimental range, the maximal inhibition activity of V E on tyrosinase was only 50.93%, which was lower than that of α-arbutin and 6′-O-lauroylarbutin. The half-inhibitory concentration (IC 50 ) of 6′-O-lauroylarbutin was 0.035 mmol/L, slightly lower than that of α-arbutin (0.041 mmol/L). This result suggested that the acylation modification on the C'-6 position of α-arbutin has a minimal impact on the tyrosinase inhibition activity. The activity of α-arbutin and 6′-O-lauroylarbutin as anti-oxidants was assessed by the DPPH method. Figure 9 C demonstrates that the scavenging rate of all three samples against DPPH radical gradually increases as their concentrations rise, indicating a distinct dose-response relationship. Compared to the control, α-arbutin showed superior radical scavenging abilities, particularly in the low dose range of < 0.35 mmol/L. Moreover, the DPPH radical scavenging ability of 6'-O-lauroyl arbutin was found to be greater than that of its parent compound. The IC 50 value of 6'-O-lauroyl arbutin was about 0.12 mmol/L, obviously lower than that of α-arbutin. This result can be explained by the fact that the alkane chain in lauroyl is electron-donating, resulting in an increase in the electron density in the oxygen of C = O. The electron-rich oxygen is likely to release electrons, thereby improving the scavenging ability of α-arbutin against DPPH radicals. 3. Conclusion Polysaccharide-based hydrogels possess a well-established grid structure, excellent biocompatibility, and biodegradability. However, its stability and mechanical strength are generally poor due to the weak interactions between polymer chains as well as between polymers and enzymes, thereby limiting its application in enzyme immobilization. In this study, we propose a novel two-step crosslinking method for the preparation of OCMC/CMCHS@TLLs hydrogel. The merits of this methodology are that, firstly, the free TLLs are initially crosslinked with a low-toxic OCMC through a Schiff base reaction, which strengthens the connection between enzymes and the carrier, thereby preventing enzyme leakage in use. Then the water-soluble OCMC@TLLs complexes are further crosslinked with CMCHS through imine bridges, created by the Schiff base reaction between the excess aldehyde groups in OCMC and the amine groups in CMCHS. This stabilizes the structural stability of hydrogels and facilitates improving the performance of OCMC/CMCHS@TLLs. The OCMC/CMCHS@TLLs microspheres were prepared with the aid of a microfluidic apparatus, which had similar enzymatic properties to free enzymes, as well as superior pH and thermal stability. Unfortunately, the catalytic performance of OCMC/CMCHS@TLLs was greatly lower than that of free enzymes due to the limitation of high mass transfer resistance in hydrogels. Moreover, OCMC/CMCHS@TLLs demonstrated remarkable performance and stability in the synthesis of 6′-O-lauroyl arbutin using THF as the reaction medium. After reacting for 12 hours, the maximal yield of 6'-O-lauroyl arbutin was 92.12%, which was comparably more efficient than previous reports. The as-prepared 6′-O-lauroyl arbutin was highly lipophilic and showed nearly equivalent inhibition activity against tyrosinase as α-arbutin. In addition, the DPPH radical scavenging ability of 6'-O-lauroyl arbutin was greatly higher than its parent compound due to the electron-donating effect of lauroyls, which gives stronger physiological activity to α-arbutin. 4. Materials and Methods 4.1. Materials TLLs were purchased from Novozymes (Denmark). Tetrahydrofuran (THF) and CMC with a viscosity of 800–1200 mPa・s were provided by Sinopharm Chemical Reagent Co. Bovine serum albumin (BSA), Triton X-100, 4-nitrophenyl palmitate ( p -NPP), and p-nitrophenol ( p -NP) were sourced from Sigma. CMCHS, vinyl ester, α-arbutin, n -hexane, and methyl silicone oil were purchased from Aladdin. All other chemicals were acquired at analytical grade. 4.2. Methods 4.2.1. Preparation of oxidized carboxymethyl cellulose The oxidation of CMC was conducted in accordance with prior studies (Yi et al. 2020 ). 5.0 g of CMC and 4.28 g of sodium periodate were separately dissolved in 25 mL of anhydrous ethanol and deionized water separately. Both sample solutions were mixed up and stirred magnetically for 12 hours in the absence of light. The reaction was terminated by adding 1.3 mL of ethylene glycol and continued to stir for 0.5 h. A four-fold volume of anhydrous ethanol was added to the mixture, followed by centrifugation. The precipitates were re-dissolved in deionized water and subsequently subjected to dialysis in water for 24 hours. The dialyzed solution was freeze-dried to yield OCMC powder. According to Benghanem's method (Benghanem et al. 2017 ), the molar fraction of -CHO in OCMC was determined to be 97.2%. 4.2.2. Immobilization of TLLs The lipase TLLs were diluted with deionized water to a final concentration of 2.14 mg/mL, and mixed with OCMC aqueous solution (8% w/v). The mixture was stirred at 4°C for 30 min to facilitate the formation of OCMC@TLLs complex. The aqueous solution of CMCHS (5% w/v) and the obtained OCMC@TLLs complexes were separately pumped as the dispersed phase into a Y-shaped microchip (with an inner channel diameter of 200 µm); the OCMC@TLLs-CMCHS mixture was then sheared into droplets by the continuous phase (methyl silicone oil). The flow rates of the dispersed phase were both set at 1.0 mL/min, while that of the continuous phase were 5 mL/min. The droplets flowed forward along a 0.3-m-long microchannel together with the continuous phase and solidified simultaneously. The as-prepared microspheres were collected and kept at -30°C for 30 min. Subsequently, they were washed with N -hexane and water in sequence to remove the residual methyl silicone oil and free TLLs on the surface. 4.2.3. Characterization The morphology of microspheres was observed using an optical microscope (Nikon, ECLIPSE TS2, Japan) and a Scanning Electron Microscope (SEM, Zeiss Ultra 55, German), respectively. Fourier-transform infrared spectra (FT-IR) of free TLLs, OCMC@TLLs, and CMCHS/OCMC@TLLs were obtained by a Nicolet 380 spectrometer (Thermo Fisher Scientific Co., Ltd.) at a resolution of 4 cm -1 in the range of 400–4000 cm -1 . The secondary structures of free TLLs and OCMC@TLLs were analyzed by circular dichroism (BRIGHTTIME Chirascan, UK) within a scanning range of 190 ~ 400 nm. 4.2.4. Analytical methods The concentration of TLLs was determined using BCA protein assay kits (Biyuntian Biotechnology Company Limited, China). The p-NPP method was used to assess the activity of both free and immobilized TLLs (Akhlaghi and Najafpour-Darzi 2022 ) (The specific procedure is shown in supplementary S1). One unit (U) of enzyme activity was defined as the amount of enzyme required to catalyze the hydrolysis of p -NPP ( p -nitrophenyl palmitate) to produce 1 µmol p-nitrophenol within one minute. Quantitative analysis of α-arbutin and 6′-O-laurylarbutin was performed by an HPLC device (Ultimate3000, the U.S.) equipped with a Venusil XBP C 18 column (4.6 mm×250 mm). The detection wavelength was set at 280nm. For arbutin analysis, the mobile phase consisted of (water: methanol) was 80:20, while for 6'-O-lauryl arbutin, it was 20:80. The retention time of α-arbutin and 6′-O-laurylarbutin was 4.79 min and 17.17 min, respectively. 4.2.5. NMR data of arbutin mono-esters Highly purified arbutin esters were obtained by FPLC (GE Health, the U.S.) equipped with a RP-preparative column (GP-C 18 , 21.2×150 mm). The chemical structure of the purified monoester was determined by 1 H and 13 C NMR spectroscopic analysis in dimethyl sulfoxide (DMSO)- d 6 on a Bruker 400 MHz spectrometer (Bruker, Kurtabaugh, Germany). The detailed assignment of 1 H NMR and 13 C NMR signals was provided in the supplementary information (The spectra of 1 H NMR and 13 C NMR were supplied in Chapters S1, S2, and S3). 4.2.6. Enzymaticreaction The enzymatic reaction was carried out in 5.0 mL of dried tetrahydrofuran, which contained α-arbutin and vinyl laurate in varying ratio. After adding 20 mg of CMCHS/OCMC@TLLs, the reaction mixture was stirred at 50°C and 200 rpm for 12 h, and the reaction was monitored by detecting the concentration of 6′-O-laurylarbutin in reactant at two-hour intervals of two hours. Each Experiment was performed in triplicate, and all data were calculated based on the mean values. 4.2.7. Inhibitory activity of tyrosinase The inhibitory activities of α-arbutin and 6′-O-lauroylarbutin against tyrosinase were evaluated in a 96-microtiter plate (Ai et al. 2022 ). α-arbutin,6′-O-lauroyl arbutin and vitamin E (positive control) were dissolved in 20%methanol aqueous solution. L-tyrosine (0.1 mg/mL) and tyrosinase (100 U/mL) solution were prepared using PBS buffer, respectively. Each well contained 80 µL of PBS buffer, 40 µL of sample solution, 40 µL of L-tyrosine solution and 40µL of tyrosinase solution. The plate was allowed to stand at 37°C for 30 min. During the incubation, the absorbance at 492 nm was measured at fixed intervals. The inhibition activity was calculated using the following equation (Cai et al. 2015a ). The blank was composed of PBS buffer without any inhibitors. $$\text{I}\text{n}\text{h}\text{i}\text{b}\text{i}\text{t}\text{i}\text{o}\text{n} \text{r}\text{a}\text{t}\text{e} \left(\text{%}\right)=\frac{[\left(A-B\right)-\left(C-D\right)]}{(A-B)}$$ 2 Where A and B represent the absorbance value of the blank after and before incubation; C and D are the absorbance values of the sample solution after and before incubation. 4.2.8. DPPH radical scavenging The DPPH radical scavenging ability of arbutin and 6'-O-lauroyl arbutin was evaluated using an enhanced approach inspired by Cai's study (Cai et al. 2015b ). The reaction system consisted of 195 µL of DPPH ethanol solution (6×10 − 5 mol/L) and 5 µL of the tested sample with concentrations ranging from 0 to 1.0mM. The absorbance of the reactant was continually monitored using a microplate reader at a wavelength of 517 nm. Once the absorbance value stabilized, it was recorded. The clearance rate of DPPH radicals was calculated using the following equation: $$\text{C}\text{l}\text{e}\text{a}\text{r}\text{a}\text{n}\text{c}\text{e} \text{r}\text{a}\text{t}\text{e} \left(\text{%}\right)=\frac{{\text{A}}_{0}-\text{A}}{{\text{A}}_{\text{o}}}$$ 3 Where A 0 and A denoted the absorbance of the blank and the sample, respectively, at the wavelength of 517 nm. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials All data generated or analysed during this study are included in this published article and its supplementary information files. Competing interests The authors declare that they have no competing interests. Author Contributions: M.C. and W.S. conducted the main experiments, analyzed the data, and wrote the manuscript; C.C. and X.Z. helped with the experimental procedures; Z.Y. 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Molecules 24:. https://doi.org/10.3390/molecules24183303 Supplementary Files SupplementaryMaterials.docx Supplementary Materials: The spectra of 1 H NMR, 13 C NMR was supplied in Fig. S1, S2, S3. The 13 C NMR、 1 H NMR signals and structure of 6′-O-laurylarbutin is shown in Fig. S1. Positive and negative ion mode mass spectra of purified arbutin monolauroyl derivatives is shown in Fig. S2. The 6′- O-lauroylgeniposide structure and nuclear magnetic spectrum assignment is shown in Fig. S3. graphicabstract.tif Cite Share Download PDF Status: Published Journal Publication published 04 Jan, 2024 Read the published version in Bioresources and Bioprocessing → Version 1 posted Reviewers agreed at journal 18 Oct, 2023 Reviewers invited by journal 18 Oct, 2023 Editor assigned by journal 16 Oct, 2023 First submitted to journal 14 Oct, 2023 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3445915","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":241241085,"identity":"6bd12b44-dff2-46af-8705-95786c510db7","order_by":0,"name":"Ming 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20:31:11","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":114936,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in the relative activity of free TLLs, GA@TLLs and OCMC@TLLs during reaction.\u003c/p\u003e","description":"","filename":"floatimage2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3445915/v1/d80d7a1dce4778b5037da9e7.jpg"},{"id":45109441,"identity":"734b1dec-8f94-4824-b34c-0a083bb9543b","added_by":"auto","created_at":"2023-10-23 20:31:12","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":643518,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e)Circular dichroism spectrum and deconvolution fitting of amide I region of (\u003cstrong\u003eB\u003c/strong\u003e) TLLs (\u003cstrong\u003eC\u003c/strong\u003e) GA@TLLs and (\u003cstrong\u003eD\u003c/strong\u003e) 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stability of TLLs and OCMC/CMCHS@TLLs\u003c/p\u003e","description":"","filename":"floatimage6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3445915/v1/92f6bc5426dbf96f978c2b04.jpg"},{"id":45110017,"identity":"903e61d4-20c5-42c9-90a2-a5d7513247a5","added_by":"auto","created_at":"2023-10-23 20:39:11","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":38527,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of donor concentration on the conversion rate of 6'-O-lauroyl arbutin\u003c/p\u003e","description":"","filename":"floatimage7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3445915/v1/35006133f13a094662200d93.jpg"},{"id":45109440,"identity":"54f19acf-d2fe-4eb7-8bcb-9790343a157d","added_by":"auto","created_at":"2023-10-23 20:31:12","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":44320,"visible":true,"origin":"","legend":"\u003cp\u003eOperating stability of OCMC/CMCHS@TLLs in the reusability test\u003c/p\u003e","description":"","filename":"floatimage8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3445915/v1/e5dac3deb229f52a07a59054.jpg"},{"id":45109444,"identity":"16cac86c-477d-41ac-886f-ecb2850a1ddd","added_by":"auto","created_at":"2023-10-23 20:31:12","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":821647,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eThe Log P values of arbutin and 6'-O-lauroylarbutin.\u003cstrong\u003e (B)\u003c/strong\u003eTyrosinase inhibitory activity of 6'-O-lauroylarbutin, α-arbutin and vitamin E.\u003cstrong\u003e(C) \u003c/strong\u003eDPPH radicals scavenging ability of 6′-O-lauroylarbutin, α-arbutin and vitamin E.\u003c/p\u003e","description":"","filename":"floatimage9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3445915/v1/7a61f59e2476c86659776c3e.jpg"},{"id":49315428,"identity":"770b2961-4d42-4b55-a832-8d7f194a705f","added_by":"auto","created_at":"2024-01-08 15:07:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1189207,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3445915/v1/3f257df8-282d-481d-8618-1a06223091fb.pdf"},{"id":45110018,"identity":"759410b6-3f46-412c-a489-e3523f5b58d6","added_by":"auto","created_at":"2023-10-23 20:39:12","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":298070,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Materials:\u0026nbsp;\u003c/strong\u003eThe spectra of\u0026nbsp;\u003csup\u003e1\u003c/sup\u003eH NMR,\u0026nbsp;\u003csup\u003e13\u003c/sup\u003eC NMR was supplied in Fig. S1, S2, S3. The\u0026nbsp;\u003csup\u003e13\u003c/sup\u003eC NMR、\u003csup\u003e1\u003c/sup\u003eH NMR signals and structure of 6′-O-laurylarbutin is shown in Fig. S1. Positive and negative ion mode mass spectra of purified arbutin monolauroyl derivatives is shown in Fig. S2. The 6′- O-lauroylgeniposide structure and nuclear magnetic spectrum assignment is shown in Fig. S3.\u003c/p\u003e","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-3445915/v1/4d680db8ad7a9ac6f18af31c.docx"},{"id":45109445,"identity":"87350226-28a4-4997-a622-1bb08dd00920","added_by":"auto","created_at":"2023-10-23 20:31:12","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1683080,"visible":true,"origin":"","legend":"","description":"","filename":"graphicabstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-3445915/v1/63c53b9fcbb4845a0c29cbeb.tif"}],"financialInterests":"","formattedTitle":"Immobilization of Thermomyces lanuginosus lipase in a novel polysaccharide-based hydrogel by a two-step crosslinking method and its use in the lauroylation of α-arbutin","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eArbutin, hydroquinone O-(α/β)-D-glucopyranoside, is a natural compound found int the leaves of Ericaceae plants (Bearberry). As a member of phenolic glycosides, arbutin possesses unique biological activities of antioxidant, anti-inflammatory, and anti-tumor (Shang et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Lee and Kim \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Dong-Seok Kim et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Takebayashi et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), assuming its clinical application in the treatment of urinary tract infections, kidney stones, cystitis (Schindler et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), and respiratory diseases (Zhou et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Moreover, arbutin, particularly its α-isomer, serves as a potent tyrosinase inhibitor that blocks the production of melanin in vivo. Thus, it is now widely used in the production of whitening cosmetics.\u003c/p\u003e \u003cp\u003eFrom a structural perspective, arbutin consists of two components, namely a glucoside and a phenolic group. The active Ph-OH in the phenolic group plays a crucial role in scavenging free radicals and protecting cellular constituents from oxidative harm (Watanabe et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), thus providing multiple physiological activities for arbutin. On the other hand, glucoside makes arbutin highly water soluble, but it hinders its transportation through cell membranes, resulting in a low oral bioavailability.\u003c/p\u003e \u003cp\u003eIn an effort to solve the aforementioned issue, researchers have attempted to graft hydrophobic groups, such as long-chain fatty acyls onto arbutin by chemical (Wang and Kong \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and enzymatic methods (NAGAI et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). However, there are two active reaction sites in the arbutin structure, namely the primary hydroxyl in glycoside and the phenolic hydroxyl in a phenolic group. It is difficult to achieve selective acylation through traditional chemical modification. Compared to chemical methods, biocatalysis offers several advantages including high regioselectivity, short reaction routes, high yields, and cost-saving benefits. Thus, the application of biosynthesis in the selective acylation of phenolic glycosides has garnered increasing attention. Tokiwaetal. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) synthesized arbutin undecylenic acid ester using alkaline protease from \u003cem\u003eBacillus subtilis\u003c/em\u003e as a catalyst. The acylated arbutin showed 100-fold higher inhibitory activity against tyrosinase than arbutin (Tokiwa et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Watanabe et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) studied the lipase-catalyzed acylation of phenol glycosides (i.e., arbutin, naringenin, and phlorizin) using lauric acid as an acyl donor. Lipase CALB showed a broad substrate spectrum and the yields of the resulting esters were all up to 50%. The lauroylphenol glycosides were more active against linoleic acid oxidation than its parent compound; probably resulting from its increased solubility and improved membrane penetration in oil-based systems (Watanabe et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Liu et al. (2021) developed a novel one-step enzymatic method for the synthesis of hydrophobic arbutin esters in supercritical carbon dioxide (SC-CO\u003csub\u003e2\u003c/sub\u003e), using CALB as the biocatalyst and ethyl palmitate as the acyl donor (Liu \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The stability and solubility of the resulting esters were greatly improved, making them more useful in cosmetic and pharmaceutical applications.\u003c/p\u003e \u003cp\u003eBased on prior studies, immobilized lipases are preferred in the selective acylation of phenolic glycosides due to their exceptional characteristics, including abroad range of substrates, extensive source, excellent solvent tolerance, and cost-effectiveness (Milivojević et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In recent years, there has been a significant increase in the research and development of immobilized lipase, with a primary focus on optimizing the supports (Parnianchi et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Harris et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zahirinejad et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). As a host material, the supports for enzyme immobilization should possess not only high enzyme capacity but also excellent stability and mechanical strength to match different applications. In this regard, polysaccharide-based hydrogels are highly appealing due to their porous structure, non-toxicity, exceptional biocompatibility, easy modification, and biodegradability (Okura et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The 3D mesh structure of hydrogels has the ability to both trap enzymes and allow the passage of substrates and products. Moreover, due to their highly hydrophilic nature and water-rich structure, hydrogels provide an optimal microenvironment for enzymes, thereby promoting the preservation of their conformation (Dai et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Recently, researchers have investigated the hydrogel-based immobilization technology of lipase in detail. Park et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) immobilized \u003cem\u003eCandida rugosa\u003c/em\u003e lipase in cellulose/lignin composite hydrogel beads. The immobilized lipase showed higher activity and stability than those embedded in pure cellulose beads (Park et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Kim et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) prepared bacterial cellulose (BC)-chitosan composite hydrogel beads and used to immobilization of \u003cem\u003eCandida\u003c/em\u003e lipase by physical adsorption and covalent cross-linking. As compared to microcrystalline cellulose (MCC)-chitosan hydrogel beads, BC-chitosan hydrogel exhibits higher adsorption capacity and activity recovery(Kim et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite achieving some positive results in previous work, the application of polysaccharide-based hydrogels in enzyme immobilization still faces challenges. Natural polysaccharides, for example, chitosan, cellulose, and alginate are polyhydroxy polymers lacking highly reactive groups. During the formation of hydrogels, polysaccharide chains are commonly crosslinked through non-covalent interactions, such as hydrogen bonds and electrostatic forces. The weak interactions that exist between the chains are inadequate to ensure the structural stability of hydrogel. When exposed to solvents for an extended period, hydrogels are susceptible to swelling or even disintegration, leading to the leakage of enzymes. To stabilize the structure of hydrogel, bifunctional reagents such as glutaraldehyde and divalent metal ions Ca\u003csup\u003e2+\u003c/sup\u003e are frequently used as crosslinkers to enhance the interactions between polymer chains and enzymes (Turner et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). However, it is possible that this small reagent scan infiltrates the enzyme structure and causes excessive intermolecular and/or intramolecular crosslinking, resulting in irreversible changes in the conformation and a reduction in enzyme activity.\u003c/p\u003e \u003cp\u003eIn this study, we propose a novel method for the immobilization of lipase TLLs that involves the covalent cross-linking of oxidized carboxymethyl cellulose (OCMC) and carboxymethyl chitosan (CMCHS). First, OCMC with high aldehyde content was used to establish a chemical link through Schiff base\u0026rsquo;s reaction between the free TLLs; the OCMC@TLLs complex was subsequently crosslinked with CMCHS to yield the TLLs-containing hydrogel (OCMC/CMCHS@TLLs). To prepare hydrogel beads of uniform size, a microfluidic apparatus was utilized in this step (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e for the preparation route of OCMC/CMCHS@TLLs microspheres). The structure, catalytic properties, and stability of OCMC/CMCHS@TLLs were investigated in detail. In the end, we perfected a high-yield process for synthesizing 6'-O-lauroyl arbutin with OCMC/CMCHS@TLLs as a catalyst. The oil-water partition coefficient, inhibitory activity of tyrosinase, and DPPH scavenging capacity of 6'-O-lauroyl arbutin were also assessed.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Preparation of OCMC@TLLs\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo improve the immobilization of TLLs, free TLLs were first linked by linear OCMC through the Schiff-base reaction. The changes in activity and conformation of the OCMC@TLLs complex were investigated and compared to those of GA@TLLs, the complex formed by using GA as a crosslinker.\u003c/p\u003e \u003cp\u003eAs described in 3.2.2, TLLs solution was treated with OCMC and glutaraldehyde (GA) solution, both of which had the same aldehyde concentration (approximately 0.7 mol/L), for a duration of one hour, respectively. The changes in the activity of reactants were monitored at 10-minute intervals, while the activity of untreated TLLs was determined and used as a control. The initial activity of free TLLs was set at 100%.\u003c/p\u003e \u003cp\u003eAs illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the impact of GA on the activity of TLLs could be classified into two stages. In the first stage, the relative activity of TLLs decreased immediately to 82.5% in contact with GA, indicating that a rapid reaction occurred between GA and TLLs. As time progressed, the relative activity of GA@TLLs remained constant. However, a second decrease in the activity of GA@TLLs was observed when the reaction time exceeded 30 minutes. This phenomenon might be attributed to changes in the conformation of TLLs due to GA-induced intramolecular crosslinking.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eOn the contrary, the relative activity of OCMC@TLLs descended slowly during the crosslinking reaction and remained higher than that of GA@TLLs and even the free TLLs. At the end of the reaction, the relative activity of OCMC@TLLs was as high as 93.85%, whereas that of GA@TLLs was only 62.82%. This phenomenon might be explained by the special structure of OCMC. OCMC is a carboxymethylated derivative of cellulose, having a linear and flexible structure. It makes a high steric hindrance effect against the Schiff-base reaction between OCMC and TLLs, which reduces the intensity of the crosslinking reaction and aids in preserving the activity of TLLs. In addition, the larger size of OCMC molecules makes it difficult to penetrate the interior of TLL. The aldehyde groups in OCMC can only react with the exposed -NH2 on the surface of TLLs molecules, thereby reducing the effect of crosslinking on enzyme conformation.\u003c/p\u003e \u003cp\u003eTo clarify the impact of OCMC and GA on the activity of TLLs, the secondary structure of free TLLs, OCMC@TLLs, and GA@TLLs was analyzed by CD and FT-IR spectra. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, the CD spectrum of free TLLs presented a positive peak at 197 nm and two broad negative peaks at 210 and 221 nm in the far-UV region, which were assigned to the structures of β-sheet and α-helix, respectively (Li et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCompared to the free enzyme, the absorbance at 210 and 221 nm of GA@TLLs both decreased, but that at 197 nm increased simultaneously. It could be inferred that the proportion of α-helix decreased and that of β-sheet increased in the structure of GA@TLLs (Chen et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The CD spectrum of OCMC@TLLs exhibited different changes from that of GA@TLLs, in which the positive peak at 197 nm was greatly weakened, suggesting a decreased proportion of β-sheet in the OCMC@TLLs structure.\u003c/p\u003e \u003cp\u003eFT-IR Analysis of free TLLs, GA@TLLs, and OCMC@TLLs was done, and the curves of spectrum in the amide I band (1600\u0026thinsp;~\u0026thinsp;1700 cm\u003csup\u003e-1\u003c/sup\u003e) were fitted into their respective secondary components by means of deconvolution (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u0026thinsp;~\u0026thinsp;Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), respectively. The starting values for the center position and height of each amide I subpeak were manually fixed by inspecting each spectrum, while the width was initially fixed at 15 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. As listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the proportion of α-helix and β-sheet in GA@TLLs was about 15.37% and 44.19%, which were 5.65% lower and 21.64% higher than those in free TLLs, respectively. In OCMC@TLLs, however, although the proportion of α-helix and β-sheet showed a slight decrease, the changes were not significant (\u0026lt;\u0026thinsp;3%). These findings were consistent with the result from the CD spectrum, confirming the distinct influences of GA and OCMC on the secondary structure of TLLs.2.2. Fabrication and Characterization of OCMC/CMCS@TLLs\u003c/p\u003e \u003ctable id=\"Tab1\" border=\"1\" style=\"margin-right: calc(19%); width: 81%;\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eResults of deconvolution of amide I bands corresponding to TLLs, GA@TLLs and OCMC@TLLs.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" style=\"width: 11.6628%;\"\u003e\n \u003cp\u003eWavenumber, cm\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\" style=\"width: 18.6704%;\"\u003e\n \u003cp\u003e1624\u0026thinsp;~\u0026thinsp;1640\u003c/p\u003e\n \u003cp\u003e1674\u0026thinsp;~\u0026thinsp;1695\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\" style=\"width: 27.0086%;\"\u003e\n \u003cp\u003e1648\u0026thinsp;~\u0026thinsp;1660\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\" style=\"width: 18.1954%;\"\u003e\n \u003cp\u003e1640\u0026thinsp;~\u0026thinsp;1650\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\" style=\"width: 14.0673%;\"\u003e\n \u003cp\u003e1662\u0026thinsp;~\u0026thinsp;1686\u003c/p\u003e\n \u003c/th\u003e\n \u003cth style=\"width: 6.3537%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\" style=\"width: 10.3938%;\"\u003e\n \u003cp\u003eSamples\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"9\" style=\"width: 28.7038%;\"\u003e\n \u003cp\u003eSecondary structure, %\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 18.6704%;\"\u003e\n \u003cp\u003e\u0026beta;-sheet\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 27.0086%;\"\u003e\n \u003cp\u003e\u0026alpha;-helix\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 18.1954%;\"\u003e\n \u003cp\u003eRandom coil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 14.0673%;\"\u003e\n \u003cp\u003eTurn\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.3537%;\"\u003e\n \u003cp\u003eOther\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 10.3938%;\"\u003e\n \u003cp\u003eTLLs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 18.6704%;\"\u003e\n \u003cp\u003e36.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 27.0086%;\"\u003e\n \u003cp\u003e16.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 18.1954%;\"\u003e\n \u003cp\u003e20.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 14.0673%;\"\u003e\n \u003cp\u003e15.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.3537%;\"\u003e\n \u003cp\u003e10.96\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 10.3938%;\"\u003e\n \u003cp\u003eOCMC@TLLs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 18.6704%;\"\u003e\n \u003cp\u003e35.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 27.0086%;\"\u003e\n \u003cp\u003e15.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 18.1954%;\"\u003e\n \u003cp\u003e19.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 14.0673%;\"\u003e\n \u003cp\u003e19.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.3537%;\"\u003e\n \u003cp\u003e9.93\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 10.3938%;\"\u003e\n \u003cp\u003eGA@TLLs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 18.6704%;\"\u003e\n \u003cp\u003e44.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 27.0086%;\"\u003e\n \u003cp\u003e15.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 18.1954%;\"\u003e\n \u003cp\u003e22.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 14.0673%;\"\u003e\n \u003cp\u003e8.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.3537%;\"\u003e\n \u003cp\u003e9.80\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e \u003cp\u003eThe OCMC/CMCS@TLLs microspheres were manufactured using the water-in-oil method with the aid of microfluidic devices (Yang et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The size of microspheres was regulated by altering the flow rates of disperse phase and oil phase. Upon microscopic observation, the prepared microspheres were in a spherical shape with a mean diameter of 193.28\u0026thinsp;\u0026plusmn;\u0026thinsp;18.35\u0026micro;m (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). The cross-section of freeze-dried OCMC/CMCS@TLLs revealed a developed porous structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), providing a vast area for the binding of enzymes. In addition, some regular particles were observed on the surface of support from the high-resolution image (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), which demonstrated the successful immobilization of TLLs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe FT-IR spectra of TLLs, OCMC/CMCHS, and OCMC/CMCHS@TLLs were showed in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The absorption peak at 3420 cm\u003csup\u003e-1\u003c/sup\u003e is attributed to the stretching vibration of -NH\u003csub\u003e2\u003c/sub\u003e and -OH groups. In the spectrum of OCMC/CMCHS@TLLs, the peak at 1053 cm\u003csup\u003e-1\u003c/sup\u003eand 2926 cm\u003csup\u003e-1\u003c/sup\u003ecorrespond to the bending vibration of C-O and the stretching vibration of C-H; 1417 cm\u003csup\u003e-1\u003c/sup\u003e is the stretching vibration of -CH\u003csub\u003e2\u003c/sub\u003e, and the strong absorptions at 1600 cm\u003csup\u003e-1\u003c/sup\u003e and 1327 cm\u003csup\u003e-1\u003c/sup\u003e correspond to the characteristic peaks of C\u0026thinsp;=\u0026thinsp;N and C-N, respectively, confirming the generation of Schiff-based OCMC/CMCHS@TLLs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Enzymatic properties of OCMC/CMCHS@TLLs\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1. Subsubsection\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe effects of pH on the activity and stability of OCMC/CMCHS@TLLs were examined and contrasted with those of free TLLs. In the experimental condition, the optimum pH of free TLLs and OCMC/CMCHS@TLLs was found to be approximately 8.0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). To assess the pH stability, OCMC/CMCHS@TLLs and free TLLs were separately mixed with a buffer solution of varying pH values (from 4.0\u0026thinsp;~\u0026thinsp;11.0). The samples were stood at 4℃ for 12 h, followed by assaying the activity of the samples and dividing it by its initial activity to obtain the residual activity (%). The residual activity with the highest value was established at 100%. As presented in (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), OCMC/CMCHS@TLLs and free TLLs were more stable in alkaline solutions (pH\u0026thinsp;\u0026gt;\u0026thinsp;8); the highest stability for both was obtained at pH 8.0. Within the pH range of 8.0\u0026thinsp;~\u0026thinsp;11.0, the stability of OCMC/CMCHS@TLLs was slightly higher than that of the free form.\u003c/p\u003e \u003cp\u003eThe impact of temperature on the activity of OCMC/CMCHS@TLLs and free TLLs were depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE. The optimal temperature for both was 50\u0026deg;C. Under moderate temperature conditions (20\u0026ndash;40\u0026deg;C), the relative activity of OCMC/CMCHS@TLLs was noticeably lower than that of the free ones, which might be attributed to high mass transfer resistance in hydrogels. As the temperature increased further, the fluidity of the polymer chains in hydrogels correspondingly increased, reducing the resistance of mass transfer. In addition, the deformation of hydrogels can also mitigate the thermal denaturation of TLLs (Xu et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Therefore, OCMC/CMCHS@TLLs displayed higher relative activity than free TLLs within a high temperature range of 50 to 80\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe thermal stability of OCMC/CMCHS@TLLs was assessed by monitoring the residual activity after incubation at 60\u0026deg;C for 1 h. The results presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eB demonstrated that the activity of TLLs remained constant within the temperature range of 20\u0026deg;C\u0026thinsp;~\u0026thinsp;40\u0026deg;C, but it experienced a dramatic drop in stability as the temperature exceeded 40\u0026deg;C. In comparison to TLLs, OCMC/CMCHS@TLLs exhibited a high level of stability across a broader temperature range of 20\u0026ndash;60\u0026deg;C. After being incubated at 60\u0026deg;C for one hour, the residual activity of OCMC/CMCHS@TLLs was as high as 94.26%, confirming the protective effect of the hydrogel on TLLs activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1. Kinetics\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe kinetics of free TLLs, OCMC@TLLs complex, and OCMC/CMCS@TLLs were evaluated by the Michaelis-Menten equation, using \u003cem\u003ep\u003c/em\u003e-NPP as the substrate. By performing linear regression on\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\frac{1}{{\\text{r}}_{\\text{s}}}\\)\u003c/span\u003e\u003c/span\u003eand\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\frac{1}{{\\text{C}}_{\\text{s}}}\\)\u003c/span\u003e\u003c/span\u003e, the \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e values of TLLs in three forms were calculated and listed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eKm value of free TLLs, OCMC@TLLs and OCMC/CMCHS@TLLs.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEnzyme\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTLLs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOCMC@TLLs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOCMC/CMCHS@TLLs\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKm, mmol/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.396\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.917\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.938\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe Km value of OCMC@TLLs complex was 2.917 mmol/L, whereas that of free TLLs was 1.396 mmol/L, indicating a slight alteration in the conformation of TLLs. The Km of OCMC/CMCS@TLLs was calculated to be 9.938 mmol/L, about 7-fold higher than that of free TLLs, revealing a significant decrease in its affinity for the substrate. The aforementioned results could be attributed to the high mass transfer resistance in hydrogel. In contrast to free TLLs, the TLLs in OCMC/CMCS@TLLs were restricted within the hydrogels. During the reaction, the substrates in the solution need to be diffused through the mesh of hydrogels to the reaction site. This process resulted in high mass transfer resistance, ultimately leading to a great increase in the Km value\u003c/p\u003e \u003cp\u003eof OCMC/CMCS@TLLs. 2.4. Preparation of 6'-O-lauroyl arbutin\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 Screening of the reaction media\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAs previously reported, the specific physicochemical properties of the reaction medium greatly influence lipase-catalyzed esterification (GARCIA-MOLINA et al. 2010). In this study, seven organic solvents with varying solubility (Log P) and dielectric constant (ε) were tested for their suitability as the reaction medium, while OCMC/CMCS@TLLs were used as the catalyst. After a reaction at 50\u0026deg;C for 12 h, the conversion rate of 6'-O-lauroyl arbutin in different reaction media was found to be in descending order, i.e., THF\u0026thinsp;\u0026gt;\u0026thinsp;tert-amyl alcohol\u0026thinsp;\u0026gt;\u0026thinsp;acetone\u0026thinsp;\u0026gt;\u0026thinsp;cyclohexane\u0026thinsp;\u0026gt;\u0026thinsp;acetonitrile\u0026thinsp;\u0026gt;\u0026thinsp;methanol (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This result could be attributed to the difference in the solubility and polarity of solvents. In the arbutin esterification system, the acyl donor and acceptor have opposite solubility. An excessively hydrophobic or hydrophilic solvent promotes the dissolution of one substrate while limiting the dissolution of the other, resulting in a decrease in the conversion rate. In addition, the polar solvents with high ε values, such as methanol and acetonitrile may damage the structural stability of TLLs and cause the denaturation of enzymes.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eConversion rate of 6'-O-lauroylarbutinin different medium\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMedium\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLog P\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDielectric constant, \u003cem\u003eε\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eConversion rate, %\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethanol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-0.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcetonitrile\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.91\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcetone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTHF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003etert-Amyl alcohol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e66.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCyclohexane\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.03\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e* Reaction conditions: arbutin, 0.1 mmol; vinyl laurate, 0.6 mmol; solvent volume, 5 mL; temperature, 50\u0026deg;C; enzyme dosage, 50 mg.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2. Donor/acceptor ratio\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIn order to optimize the reaction conditions, a set of experiments were conducted with a fixed arbutin concentration and varying concentrations of vinyl laurate, resulting in the final molar ratios of 1:1, 1:2, 1:4, 1:6, 1:8, and 1:10; OCMC/CMCHS@TLLs was used as the catalyst with a dosage of 50 mg, and the reaction temperature was set at 50\u0026deg;C. Figure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e showed that the reaction rate and the conversion of 6'-O-lauroyl arbutin both increased with the molar ratio of arbutin and vinyl laurate. While the molar ratio was 1:6, the final conversion rate was as high as 90.79% after a 12 h reaction. Increasing the donor/acceptor ratio further had little improvement in the conversion rate and reaction time. In comparison with other relevant literature, this result was advantageous in terms of conversion rate and reaction time (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of conversion rate of arbutin with literatures\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSource of lipase\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAcyl donor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMedium\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eReaction time, h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eConversion rate,%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRef.\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCandida antarctica B\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFerulic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003etert-butanol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e168\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e(Ishihara et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2010\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCandida sp.\u003c/em\u003e 99\u0026thinsp;\u0026minus;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVinyl acetate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTHF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e91.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e(Cai et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015a\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCandida parapsilosis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVinyl laurate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePyridine-isooctane\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e71.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e(Li et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePenicillium expanse\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVinyl vanillic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003etetrahydrofuran\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e(Yang et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCandida antarctica B\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEthyl palmitate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSC\u0026thinsp;\u0026minus;\u0026thinsp;CO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e85.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e(Liu \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eThermomyceslanuginosus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVinyl laurate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTHF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e90.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eThis work\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3 Operating stability of OCMC/CMCHS@TLLs\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe operating stability of OCMC/CMCHS@TLLs was evaluated by subjecting them to a reusability test in the production of 6\u0026prime;-O-laurylarbutin at optimal conditions. The same operations were conducted using TILM, commercially immobilized TLLs, instead of OCMC/CMCHS@TLLs.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003e revealed that when using OCMC/CMCHS@TLLs or TILM as the catalyst, the conversion rate of 6\u0026prime;-O-laurylarbutin gradually decreased with each reaction batch, indicating a decrease in enzyme performance. After seven repetitions, the performance of OCMC/CMCHS@TLLs and TLIM dropped to 53.52% and 60.77%, respectively. Assuming the deactivation of both OCMC/CMCHS@TLLs and TLIM in THF followed the first-order reaction kinetics. The deactivation constant (k\u003csub\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sub\u003e) can be calculated by linear regression of the following equation:\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Equ1\" class=\"Equation\"\u003e \u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\text{ln}\\left(\\frac{{C}_{i}}{{C}_{0}}\\right)=-{k}_{d}\\bullet t+b$$\u003c/div\u003e \u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eWhere C\u003csub\u003e0\u003c/sub\u003e and C\u003csub\u003ei\u003c/sub\u003e represented the conversion rate of 6\u0026prime;-O-laurylarbutin in the first and batch, respectively. The calculated k\u003cem\u003ed\u003c/em\u003e value of OCMC/CMCHS@TLLs was 0.1104, which was slightly higher than that of TLIM, suggesting its high operating stability in THF.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Properties of 6\u0026prime;-O-laurylarbutin\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe logarithmic value of distribution coefficient ratio, also known as the oil-water partition coefficient (Log P), is frequently utilized to determine the hydrophilicity and lipophilicity of organics. This coefficient is specific for a given compound in \u003cem\u003en\u003c/em\u003e-octanol and water. The Log P values of arbutin and 6'-O-lauroyl arbutin were determined in an octanol/water solution with a phase ratio of 1:1.\u003c/p\u003e \u003cp\u003eAs illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA, the Log P value of α-arbutin was \u0026minus;\u0026thinsp;1.51, indicating its potent hydrophilicity; however, the Log P value of 6'-O-lauroylarbutin was as high as 3.18, demonstrating that the lipophilicity of arbutin was greatly enhanced by introducing a long-chain fatty acyl group.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn order to examine the impacts of acylation modification on the activity of arbutin, the capabilities for inhibiting tyrosinase and scavenging DPPH radicals of both 6\u0026prime;-O-lauroyl arbutin and arbutin were assessed by using Vitamin E as the control. As presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB, the activity of tyrosinase decreased rapidly with the concentration of inhibitors in the low dose range (\u0026lt;\u0026thinsp;0.2 mmol/L), but then leveled off. In the experimental range, the maximal inhibition activity of V\u003csub\u003eE\u003c/sub\u003e on tyrosinase was only 50.93%, which was lower than that of α-arbutin and 6\u0026prime;-O-lauroylarbutin. The half-inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) of 6\u0026prime;-O-lauroylarbutin was 0.035 mmol/L, slightly lower than that of α-arbutin (0.041 mmol/L). This result suggested that the acylation modification on the C'-6 position of α-arbutin has a minimal impact on the tyrosinase inhibition activity.\u003c/p\u003e \u003cp\u003eThe activity of α-arbutin and 6\u0026prime;-O-lauroylarbutin as anti-oxidants was assessed by the DPPH method. Figure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC demonstrates that the scavenging rate of all three samples against DPPH radical gradually increases as their concentrations rise, indicating a distinct dose-response relationship. Compared to the control, α-arbutin showed superior radical scavenging abilities, particularly in the low dose range of \u0026lt;\u0026thinsp;0.35 mmol/L. Moreover, the DPPH radical scavenging ability of 6'-O-lauroyl arbutin was found to be greater than that of its parent compound. The IC\u003csub\u003e50\u003c/sub\u003e value of 6'-O-lauroyl arbutin was about 0.12 mmol/L, obviously lower than that of α-arbutin. This result can be explained by the fact that the alkane chain in lauroyl is electron-donating, resulting in an increase in the electron density in the oxygen of C\u0026thinsp;=\u0026thinsp;O. The electron-rich oxygen is likely to release electrons, thereby improving the scavenging ability of α-arbutin against DPPH radicals.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Conclusion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003ePolysaccharide-based hydrogels possess a well-established grid structure, excellent biocompatibility, and biodegradability. However, its stability and mechanical strength are generally poor due to the weak interactions between polymer chains as well as between polymers and enzymes, thereby limiting its application in enzyme immobilization. In this study, we propose a novel two-step crosslinking method for the preparation of OCMC/CMCHS@TLLs hydrogel. The merits of this methodology are that, firstly, the free TLLs are initially crosslinked with a low-toxic OCMC through a Schiff base reaction, which strengthens the connection between enzymes and the carrier, thereby preventing enzyme leakage in use. Then the water-soluble OCMC@TLLs complexes are further crosslinked with CMCHS through imine bridges, created by the Schiff base reaction between the excess aldehyde groups in OCMC and the amine groups in CMCHS. This stabilizes the structural stability of hydrogels and facilitates improving the performance of OCMC/CMCHS@TLLs. The OCMC/CMCHS@TLLs microspheres were prepared with the aid of a microfluidic apparatus, which had similar enzymatic properties to free enzymes, as well as superior pH and thermal stability. Unfortunately, the catalytic performance of OCMC/CMCHS@TLLs was greatly lower than that of free enzymes due to the limitation of high mass transfer resistance in hydrogels. Moreover, OCMC/CMCHS@TLLs demonstrated remarkable performance and stability in the synthesis of 6\u0026prime;-O-lauroyl arbutin using THF as the reaction medium. After reacting for 12 hours, the maximal yield of 6'-O-lauroyl arbutin was 92.12%, which was comparably more efficient than previous reports. The as-prepared 6\u0026prime;-O-lauroyl arbutin was highly lipophilic and showed nearly equivalent inhibition activity against tyrosinase as α-arbutin. In addition, the DPPH radical scavenging ability of 6'-O-lauroyl arbutin was greatly higher than its parent compound due to the electron-donating effect of lauroyls, which gives stronger physiological activity to α-arbutin.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"4. Materials and Methods","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Materials\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTLLs were purchased from Novozymes (Denmark). Tetrahydrofuran (THF) and CMC with a viscosity of 800\u0026ndash;1200 mPa・s were provided by Sinopharm Chemical Reagent Co. Bovine serum albumin (BSA), Triton X-100, 4-nitrophenyl palmitate (\u003cem\u003ep\u003c/em\u003e-NPP), and p-nitrophenol (\u003cem\u003ep\u003c/em\u003e-NP) were sourced from Sigma. CMCHS, vinyl ester, α-arbutin, \u003cem\u003en\u003c/em\u003e-hexane, and methyl silicone oil were purchased from Aladdin. All other chemicals were acquired at analytical grade.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.2. Methods\u003c/h2\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e4.2.1. Preparation of oxidized carboxymethyl cellulose\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe oxidation of CMC was conducted in accordance with prior studies (Yi et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). 5.0 g of CMC and 4.28 g of sodium periodate were separately dissolved in 25 mL of anhydrous ethanol and deionized water separately. Both sample solutions were mixed up and stirred magnetically for 12 hours in the absence of light. The reaction was terminated by adding 1.3 mL of ethylene glycol and continued to stir for 0.5 h. A four-fold volume of anhydrous ethanol was added to the mixture, followed by centrifugation. The precipitates were re-dissolved in deionized water and subsequently subjected to dialysis in water for 24 hours. The dialyzed solution was freeze-dried to yield OCMC powder. According to Benghanem's method (Benghanem et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), the molar fraction of -CHO in OCMC was determined to be 97.2%.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e4.2.2. Immobilization of TLLs\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe lipase TLLs were diluted with deionized water to a final concentration of 2.14 mg/mL, and mixed with OCMC aqueous solution (8% w/v). The mixture was stirred at 4\u0026deg;C for 30 min to facilitate the formation of OCMC@TLLs complex. The aqueous solution of CMCHS (5% w/v) and the obtained OCMC@TLLs complexes were separately pumped as the dispersed phase into a Y-shaped microchip (with an inner channel diameter of 200 \u0026micro;m); the OCMC@TLLs-CMCHS mixture was then sheared into droplets by the continuous phase (methyl silicone oil). The flow rates of the dispersed phase were both set at 1.0 mL/min, while that of the continuous phase were 5 mL/min. The droplets flowed forward along a 0.3-m-long microchannel together with the continuous phase and solidified simultaneously. The as-prepared microspheres were collected and kept at -30\u0026deg;C for 30 min. Subsequently, they were washed with \u003cem\u003eN\u003c/em\u003e-hexane and water in sequence to remove the residual methyl silicone oil and free TLLs on the surface.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e4.2.3. Characterization\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe morphology of microspheres was observed using an optical microscope (Nikon, ECLIPSE TS2, Japan) and a Scanning Electron Microscope (SEM, Zeiss Ultra 55, German), respectively. Fourier-transform infrared spectra (FT-IR) of free TLLs, OCMC@TLLs, and CMCHS/OCMC@TLLs were obtained by a Nicolet 380 spectrometer (Thermo Fisher Scientific Co., Ltd.) at a resolution of 4 cm\u003csup\u003e-1\u003c/sup\u003e in the range of 400\u0026ndash;4000 cm\u003csup\u003e-1\u003c/sup\u003e. The secondary structures of free TLLs and OCMC@TLLs were analyzed by circular dichroism (BRIGHTTIME Chirascan, UK) within a scanning range of 190\u0026thinsp;~\u0026thinsp;400 nm.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e4.2.4. Analytical methods\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe concentration of TLLs was determined using BCA protein assay kits (Biyuntian Biotechnology Company Limited, China). The p-NPP method was used to assess the activity of both free and immobilized TLLs (Akhlaghi and Najafpour-Darzi \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) (The specific procedure is shown in supplementary S1). One unit (U) of enzyme activity was defined as the amount of enzyme required to catalyze the hydrolysis of \u003cem\u003ep\u003c/em\u003e-NPP (\u003cem\u003ep\u003c/em\u003e-nitrophenyl palmitate) to produce 1 \u0026micro;mol p-nitrophenol within one minute. Quantitative analysis of α-arbutin and 6\u0026prime;-O-laurylarbutin was performed by an HPLC device (Ultimate3000, the U.S.) equipped with a Venusil XBP C\u003csub\u003e18\u003c/sub\u003e column (4.6 mm\u0026times;250 mm). The detection wavelength was set at 280nm. For arbutin analysis, the mobile phase consisted of (water: methanol) was 80:20, while for 6'-O-lauryl arbutin, it was 20:80. The retention time of α-arbutin and 6\u0026prime;-O-laurylarbutin was 4.79 min and 17.17 min, respectively.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e4.2.5. NMR data of arbutin mono-esters\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eHighly purified arbutin esters were obtained by FPLC (GE Health, the U.S.) equipped with a RP-preparative column (GP-C\u003csub\u003e18\u003c/sub\u003e, 21.2\u0026times;150 mm). The chemical structure of the purified monoester was determined by \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC NMR spectroscopic analysis in dimethyl sulfoxide (DMSO)-\u003cem\u003ed\u003c/em\u003e6 on a Bruker 400 MHz spectrometer (Bruker, Kurtabaugh, Germany). The detailed assignment of \u003csup\u003e1\u003c/sup\u003eH NMR and \u003csup\u003e13\u003c/sup\u003eC NMR signals was provided in the supplementary information (The spectra of \u003csup\u003e1\u003c/sup\u003eH NMR and \u003csup\u003e13\u003c/sup\u003eC NMR were supplied in Chapters S1, S2, and S3).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e4.2.6. Enzymaticreaction\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe enzymatic reaction was carried out in 5.0 mL of dried tetrahydrofuran, which contained α-arbutin and vinyl laurate in varying ratio. After adding 20 mg of CMCHS/OCMC@TLLs, the reaction mixture was stirred at 50\u0026deg;C and 200 rpm for 12 h, and the reaction was monitored by detecting the concentration of 6\u0026prime;-O-laurylarbutin in reactant at two-hour intervals of two hours. Each Experiment was performed in triplicate, and all data were calculated based on the mean values.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e4.2.7. Inhibitory activity of tyrosinase\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe inhibitory activities of α-arbutin and 6\u0026prime;-O-lauroylarbutin against tyrosinase were evaluated in a 96-microtiter plate (Ai et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). α-arbutin,6\u0026prime;-O-lauroyl arbutin and vitamin E (positive control) were dissolved in 20%methanol aqueous solution. L-tyrosine (0.1 mg/mL) and tyrosinase (100 U/mL) solution were prepared using PBS buffer, respectively. Each well contained 80 \u0026micro;L of PBS buffer, 40 \u0026micro;L of sample solution, 40 \u0026micro;L of L-tyrosine solution and 40\u0026micro;L of tyrosinase solution. The plate was allowed to stand at 37\u0026deg;C for 30 min. During the incubation, the absorbance at 492 nm was measured at fixed intervals. The inhibition activity was calculated using the following equation (Cai et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015a\u003c/span\u003e). The blank was composed of PBS buffer without any inhibitors.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Equ2\" class=\"Equation\"\u003e \u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\text{I}\\text{n}\\text{h}\\text{i}\\text{b}\\text{i}\\text{t}\\text{i}\\text{o}\\text{n} \\text{r}\\text{a}\\text{t}\\text{e} \\left(\\text{%}\\right)=\\frac{[\\left(A-B\\right)-\\left(C-D\\right)]}{(A-B)}$$\u003c/div\u003e \u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eWhere A and B represent the absorbance value of the blank after and before incubation; C and D are the absorbance values of the sample solution after and before incubation.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e4.2.8. DPPH radical scavenging\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe DPPH radical scavenging ability of arbutin and 6'-O-lauroyl arbutin was evaluated using an enhanced approach inspired by Cai's study (Cai et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015b\u003c/span\u003e). The reaction system consisted of 195 \u0026micro;L of DPPH ethanol solution (6\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e mol/L) and 5 \u0026micro;L of the tested sample with concentrations ranging from 0 to 1.0mM. The absorbance of the reactant was continually monitored using a microplate reader at a wavelength of 517 nm. Once the absorbance value stabilized, it was recorded. The clearance rate of DPPH radicals was calculated using the following equation:\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Equ3\" class=\"Equation\"\u003e \u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ3\" name=\"EquationSource\"\u003e\n$$\\text{C}\\text{l}\\text{e}\\text{a}\\text{r}\\text{a}\\text{n}\\text{c}\\text{e} \\text{r}\\text{a}\\text{t}\\text{e} \\left(\\text{%}\\right)=\\frac{{\\text{A}}_{0}-\\text{A}}{{\\text{A}}_{\\text{o}}}$$\u003c/div\u003e \u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eWhere A\u003csub\u003e0\u003c/sub\u003e and A denoted the absorbance of the blank and the sample, respectively, at the wavelength of 517 nm.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article and its supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eM.C. and W.S. conducted the main experiments, analyzed the data, and wrote the manuscript; C.C. and X.Z. helped with the experimental procedures; Z.Y. (Zhong Yao) was the supervisor for this work and revised the manuscript; Benwei Zhu and Y.S. revised the manuscript. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAi Y-F, Dong S-H, Lin B, et al (2022) Acylated sucroses and butenolide analog from the leaves of Tripterygium wilfordii Hook. f. and their potential anti-tyrosinase effects. Fitoterapia 161:105250. https://doi.org/10.1016/j.fitote.2022.105250\u003c/li\u003e\n\u003cli\u003eAkhlaghi N, Najafpour-Darzi G (2022) Preparation of immobilized lipase on Co2+-chelated carboxymethyl cellulose based MnFe2O4 magnetic nanocomposite particles. Molecular Catalysis 519:112118. https://doi.org/10.1016/j.mcat.2022.112118\u003c/li\u003e\n\u003cli\u003eBenghanem S, Chetouani A, Elkolli M, et al (2017) Grafting of oxidized carboxymethyl cellulose with hydrogen peroxide in presence of Cu(II) to chitosan and biological elucidation. 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Molecules 24:. https://doi.org/10.3390/molecules24183303\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bioresources-and-bioprocessing","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biob","sideBox":"Learn more about [Bioresources and Bioprocessing](http://bioresourcesbioprocessing.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/biob/default.aspx","title":"Bioresources and Bioprocessing","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Arbutin, oxidized carboxymethyl cellulose, carboxymethyl chitosan, microfluidic, covalent cross-linked polymer, lipase, acylation","lastPublishedDoi":"10.21203/rs.3.rs-3445915/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3445915/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe \u003cem\u003eThermomyces lanuginosus\u003c/em\u003e lipase (TLLs) was successfully immobilized within a novel hydrogel matrix through a two-step crosslinking method. TLLs was initially crosslinked through the Schiff-base reaction by oxidized carboxymethyl cellulose (OCMC). The water-soluble OCMC@TLLs complex was subsequently crosslinked by carboxymethyl chitosan (CMCSH) in a microfluidic apparatus to form the CMCHS/OCMC@TLLs microspheres. The CD (Circular Dichroism, CD) and FTIR (Fourier Transform infrared spectroscopy, FTIR) spectra demonstrated that the crosslinking of TLLs with OCMC resulted in a less significant impact on their structure compared to that with glutaraldehyde. CMCHS/OCMC@TLLs showed decreased catalytic performance due to the mass transfer resistance, while its thermal stability was greatly improved. The CMCHS/OCMC@TLLs were used to catalyze the lauroylation of arbutinin tetrahydrofuran. After 12 h of reaction under optimal conditions, the yield of 6\u0026prime;-O-laurylarbutin reached an impressive 92.12%. The prepared 6\u0026prime;-O-laurylarbutin has high lipophilicity and exhibits similar tyrosinase inhibitory activity and higher antioxidant activity compared to its parent compound.\u003c/p\u003e","manuscriptTitle":"Immobilization of Thermomyces lanuginosus lipase in a novel polysaccharide-based hydrogel by a two-step crosslinking method and its use in the lauroylation of α-arbutin","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-10-23 20:31:07","doi":"10.21203/rs.3.rs-3445915/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2023-10-18T13:40:42+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-10-18T13:17:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-10-16T07:27:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"Bioresources and Bioprocessing","date":"2023-10-14T09:58:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bioresources-and-bioprocessing","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biob","sideBox":"Learn more about [Bioresources and Bioprocessing](http://bioresourcesbioprocessing.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/biob/default.aspx","title":"Bioresources and Bioprocessing","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3cc90d59-e277-4a66-9424-3c9cb39785c4","owner":[],"postedDate":"October 23rd, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-01-08T15:03:22+00:00","versionOfRecord":{"articleIdentity":"rs-3445915","link":"https://doi.org/10.1186/s40643-023-00721-9","journal":{"identity":"bioresources-and-bioprocessing","isVorOnly":false,"title":"Bioresources and Bioprocessing"},"publishedOn":"2024-01-04 15:01:05","publishedOnDateReadable":"January 4th, 2024"},"versionCreatedAt":"2023-10-23 20:31:07","video":"","vorDoi":"10.1186/s40643-023-00721-9","vorDoiUrl":"https://doi.org/10.1186/s40643-023-00721-9","workflowStages":[]},"version":"v1","identity":"rs-3445915","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3445915","identity":"rs-3445915","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}