Improved catalytic stability of immobilized Candida antarctic lipase B on macroporous resin with organic polymer coating for biodiesel production

Improved catalytic stability of immobilized Candida antarctic lipase B on macroporous resin with organic polymer coating for biodiesel production | 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 Improved catalytic stability of immobilized Candida antarctic lipase B on macroporous resin with organic polymer coating for biodiesel production Jiale Liu, Shufan Zhao, Wan Wei, Shupeng Yu, Zhao Wang, Jianyong Zheng This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4922648/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Lipase is one of the most widely studied and applied biocatalysts. Due to the high enzyme leakage rate of the immobilization method of physical adsorption, we propose a new lipase immobilization method, which based on the combination of macroporous resin adsorption and organic polymer coating. The immobilized Candida antarctic lipase B (CALB@resin-CAB) was prepared by combining the macroporous resin adsorption with cellulose acetate butyrate coating, and its structure was characterized by various analytic methods. Immobilized lipase was applied for biodiesel production using acidified palm oil as the starting material, the conversion rate achieved as high as 98.5% in two steps. Furthermore, immobilized lipase displayed satisfactory stability and reusability in biodiesel production. When the aforementioned reaction was carried out in a continuous flow packed-bed system, the yield of biodiesel was 94.8% and space-time yield was 2.88 g/(mL∙h). CALB@resin-CAB showed high catalytic activity and stability, which has good potential for industrial application in the field of oil processing. Immobilization Lipase Organic polymer Coating Biodiesel Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction The overuse of fossil fuels has led to environmental issues such as global warming, and with the increasing energy consumption, it greatly encouraged the exploration of new energy sources [ 1 , 2 ]. Biodiesel can be used as an alternative to fossil fuels due to its substantial advantages like biodegradable, renewable, non-toxic, and usable by existing engines [ 3 , 4 ]. Biodiesel is mainly produced through chemical ester exchange reactions, which generally require high energy consumption, environmentally harmful catalysts, and a series of complex wastewater treatment measures [ 5 ]. Nevertheless, enzymatic esterification with lipases is a more environmentally friendly method that can avoid some of the problems existed in chemical conversion [ 6 ]. Lipases (EC 3.1.1.3) are enzymes that catalyze the hydrolysis of triacylglycerol ester [ 7 ]. Lipase is one of the most widely used enzymes at both academic and industrial aspects because of their ability to recognize a wide variety of substrates and catalyze different types of reactions while maintaining high stability and activity [ 8 ]. Candida antarctic lipase B (CALB) is an excellent biocatalyst widely used in oil ester processing and preparation of fine chemicals. Enzymatic esterification with CALB has emerged as an effective choice for biodiesel [ 9 , 10 ]. Tan et al. modified CALB employing protein engineering to obtain mutants to improve methanol tolerance and used it as a catalyst for biodiesel production from soybean oil, with a reaction yield of 85% at 24 h [ 11 ]. However, free CALB still has several drawbacks, including poor thermal stability, low yield, and unrecyclability, which limit its application in industrial production [ 12 ]. Fortunately, enzyme immobilization could be the focal point for improving enzyme properties [ 13 ]. Enzymatic immobilization enables the application of lipases in industrial processes [ 14 ]. Appropriate immobilization technologies can achieve the recovery of biocatalysts and minimize or even eliminate product contamination, thereby reducing the costs associated with enzyme use [ 15 , 16 ]. They also bring improved o more convenient operation and better storage stability, as well as better adaptability to pH and temperature changes [ 17 ]. However, although it exhibits many advantages, it also has potential disadvantages. It has been shown that immobilized lipase molecules can passively migrate to the carrier surface which may lead to enzyme leakage under harsh conditions [ 18 , 19 ]. Cross-linking and covalent methods are often used in combination with adsorption methods to enhance the force between the enzyme and the carrier [ 20 , 21 ]. Although immobilized enzymes prepared through cross-linking and covalent adsorption have good stability, the cross-linking or covalent binding process may have the problem of occupying the catalytic active site of the enzyme and may lead to structural changes in the enzyme protein, thereby reducing enzyme activity [ 22 ]. In this work, we present a new core-shell structure concept for lipase immobilization (Fig. 1 ). The novel immobilization method was developed based on the enzyme immobilization strategy of loading followed by encapsulation, a hydrophobic macroporous resin was applied as a carrier for the physical adsorption of lipase, and the organic polymer was used to coat the surface of the macroporous resin by phase change to construct an immobilized lipase with enzyme-organic polymer core-shell structure [ 23 ]. This immobilization method satisfactorily solved the shortcoming of enzyme leakage existing in the adsorption method, while maintaining high catalytic activity [ 24 ]. The organic polymer coating was optimized subsequently to obtain immobilized CALB (CALB@resin-CAB). It was applied to the enzyme-catalyzed biodiesel production by using palm acidified oil as the row material and explored its operational and leaching stability. To simulate the industrial production, CALB@resin-CAB was used for the continuous synthesis of biodiesel. In this study, the lipase immobilization strategy provides new insights to solve the problems of immobilized enzymes in activity, stability, and industrial applicability. 2. Materials and methods 2.1 Materials Lipase B from Candida antarctica (CALB) was supplied from Vland Biotech (Shandong, China, 500 LU/g hydrolytic activity). Macroporous resin (XRZ04B) was purchased from Shanghai Suner Chemical Technology Co., Ltd. (Shanghai, China). Macroporous resins (NKA, D3520, AB-8, and D301) were purchased from Tianjin Nankai Hecheng Sci. &Tech. Co., Ltd. (Tianjin, China). Macroporous resins (D101, NKA-9, and H-20) were purchased from Huayi Technology New Materials Co., Ltd. (Henan, China). Macroporous resins (LXTE-1000, LXTE-705, LXTE-707, LXTE-700s, and HA109M) were purchased from Xi’an Lanxiao Technology New Materials Co., Ltd. (Shanxi, China). Organic polymer Cellulose acetate(CA), cellulose acetate butyrate (CAB), polymethyl methacrylate (PMMA), etc. were purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China). Palm acidified oil (acid value 196) was kindly supplied by Zangyu Technology Group Co., Ltd. (Zhejiang, China). All other reagents and solvents in this study were of analytical grade. 2.2 Preparation procedures of the immobilized lipase CALB The 4 g of macroporous resin XRZ04B was mixed with 16 mL of CALB enzyme solution (16 mg/mL enzyme in deionized water) in a water bath shaker at 25 ℃ for 5 h. After immobilization, the supernatant was removed by filtration to obtain the enzyme-loaded resin. To prevent the enzyme leaching from the carrier during the reaction, the enzyme-loaded resin was coated with an organic polymer [ 25 ]. CA, CAB, PMMA, PSF, PS, and PVDF were selected as coating materials and dissolved in DMF, DMSO, THF, and AC with vigorous stirring to obtain an organic polymer solution (2%-10%); Then, immerse the enzyme loaded resin in a polymer solution for 10 seconds to coat the surface of the enzyme loaded resin with a uniform organic polymer solution The enzyme-loaded resin coated with polymer solution was immersed in deionized water for 20 min. CALB@resin- CAB was obtained by suction filtration, then dried in a vacuum oven at 45°C for 3 h and stored at 4°C. 2.3 Esterification activity assay of the immobilized lipase CALB@resin-CAB 0.03 g immobilized lipase CALB@resin-CAB was added to 10.73 g substrate mixture solution, which included lauric acid (40 mmol), n-propanol (40 mmol), deionized water (3% w/w). The reaction was performed at 60°C and 200 rpm in a water bath shaker for 20 min. Subsequently, 5 µL of the reaction solution was added to 995 µL of ethyl acetate for gas chromatography (GC) analysis. The production of propyl laurate in the reaction system was detected. Gas chromatography detection was performed using the Agilent 6890 GC platform equipped with a flame ionization detector(FID) and a fused silica capillary column (DB-23, 30 m×0.32 mm i.d., 0.1 µm film thickness; Agilent Technologies, Inc.). Nitrogen was used as the carrier gas at a flow rate of 1.0 ml/min. The chromatographic column temperature raised from 100°C to 280°C (2 min), at a heating rate of 10°C/min [ 26 ]. Definition of immobilized lipase methyl esterification activity: Under standard reaction conditions, the amount of enzyme required to produce 1 µmol propyl laurate catalyzed by immobilized enzymes per minute was defined as an enzyme activity unit, expressed as PLU. Protein concentration was determined by the Bradford method using bovine serum albumin as standard. 2.4 Leaching stability assay of the immobilized lipase Leaching stability of the immobilized lipase CALB is characterized by the leakage rate of the protein in high-salt solutions. Add 5 mL of 2 M NaCl aqueous solution to 2 g of immobilized lipase (dry weight), and stir at 30 ℃ for 2 h, then take the supernatant for protein concentration assay. The protein leakage rate is given by Eq. 1 . Y= \(\:\left({A}_{3}*{V}_{2}\right)/\left[\left({A}_{1}-{A}_{2}\right)*{V}_{1}\right]\) ( Eq. 1 ) Where A 1 is enzyme solution protein concentration before immobilization (mg/mL); A 2 is supernatant protein concentration after immobilization (mg/mL); V 1 is the volume of immobilized enzyme solution (mL); A 3 is eluate protein concentration (mg/mL); V 2 is the volume of eluate solution (mL). 2.5 Structure and performance characterizations of CALB @resin-CAB 2.5.1 SEM analysis The morphology of resin XRZ04B, enzyme-loaded resin XRZ04B, and CALB @resin-CAB was characterized with a scanning electron microscope (Hitachi SU-70, Japan) in a field-emission FEI Inspect F operated at 5 kV. Samples were dried, and fixed onto silicon wafers. The samples were then immediately sputter-coated with platinum before observation. 2.5.2 TG analysis The Thermogravimetric analysis was measured in a TGA-DSC simultaneous thermal analyzer (STA449F5, Shanghai, China) under nitrogen with a heating range of 30–900°C and a heating rate of 10°C/min. 2.5.3 Fluorescent labeling assay Free CALB was fluorescently labeled using the fluorescent dye fluorescein isothiocyanate, after that, labeled lipase was adsorbed using a macroporous resin, and prepared enzyme-carrying resin was observed under a fluorescence microscope (EVOS M5000, USA). 2.5.4 Pore size distribution and BET specific surface area assay Sample pore size distribution and BET specific surface area were measured by specific surface area and pore size analyzer (TriStar II Plus 3.03, Shanghai, China). 2.6 Thermal stability assay of the immobilized CALB The thermal stability of CALB@resin-CAB was measured by determining the residual esterification activities after incubation in tert-butanol for a period of time at 50°C, 60°C, and 70°C. The residual activities of the immobilized lipases were determined as described in Section 2.3 . The relative activity of CALB@resin-CAB before incubation was defined as 100%. 2.7 Biosynthesis of biodiesel with palm acidified oil as the starting material 0.01 g CALB @resin-CAB was added to 10 g substrate (9 g palm acidified oil and 1 g methanol). The reaction was performed at 50°C and 200 rpm in a water bath shaker for 8 h. After the reaction, excess methanol and water were removed by spin evaporation, and then the samples were subjected to acid value determination. All assays were conducted in triplicate. The conversion rate of palm acidified oil is given by Eq. 2 . Conversion rate = ( \(\:({E}_{1}-{E}_{2})⁄{E}_{1})*100\%\) ( Eq. 2 ) Where E 1 is the acid value before the reaction, and E 2 is the acid value after the reaction. 2.8 Lipase-catalyzed biodiesel production in the packed bed reactor The packed bed reactor was prepared as followed: 10 g of immobilized lipase CALB@resin-CAB was poured into the column reactor and left a volume of 30 mL in the reactor (height-to-diameter ratio of 5:1) when filling since the dried immobilized enzyme will have some volume expansion during the reaction. Conditions of lipase-catalyzed biodiesel production in packed bed reactor: the substrate was first mixed in a magnetic stirrer and then continuously pumped through the reactor using a high-pressure pump (JJRZ-10004F, Hangzhou) at a certain flow rate (1–20 Bv/h). After pre-running for 30 min, the product was collected, the product was concentrated by rotary evaporation and its acid value was determined [ 27 ]. 3. Results and Discussion 3.1 Conditions optimization of lipase CALB immobilization The properties of the immobilized enzyme carrier, such as: the specific surface area of the carrier, the polarity and the pore size of the carrier, have a great influence on the catalytic activity and stability of the immobilized enzyme [ 28 ]. Therefore, the effect of different types of macroporous resins on the catalytic activity of immobilized lipase was investigated in the previous experiment. As shown in Fig. 2 A, the immobilized lipase CALB prepared with macroporous resin XRZ04B showed the highest esterification activity. According to Fig. 2 B, the specific activity of immobilized lipase was significantly influenced by the ratio of carrier to lipase addition. The enzyme activity of immobilized lipase increased with increasing lipase loading rate. The most efficient immobilization was achieved at a resin addition of 0.25 g/mL, after which the specific activity decreased with increasing lipase loading rate, indicating that the lipase loading rate saturated at a specific concentration, which is consistent with the results of other studies. Adsorption time of the resin plays an important role during the immobilization process (Fig. 2 C). The highest specific enzyme activity of immobilized lipase was achieved at 6 h, which was 13180.6 PLU/g. After that, there was no significant change in specific enzyme activity with increasing immobilization time, which may be attributed to the fact that the immobilized carriers reached its maximum loading at 6 hours. Therefore, 6 hours was chosen as the immobilization time. 3.2 Optimization of organic polymer coating procedure The thickness of the coating and the pore size of the coating surface would affect the enzyme activity and protein leakage rate of the immobilized enzyme, therefore, the organic polymer coating was optimized, including the type of organic polymer, organic polymer concentrations, and organic solvents. 3.2.1 Selection of organic polymers The present study involved the selection of organic polymers (PSF, PS, PVDF, CA, CAB, and PMMA). These polymers were chosen due to their possession of desirable properties such as thermal stability, chemical resistance, and biocompatibility. Hence, this experiment aimed at investigating the impact of six distinct organic polymers on both the catalytic efficiency of immobilized CALB and the rate of protein leakage. The results indicate that the immobilized enzyme exhibited the greatest catalytic efficiency when cellulose acetate butyrate was employed as a coating, yielding a value of 12685.5 PLU/g (Fig. 3 A). The observed phenomenon can be attributed to the more homogeneous dispersion of the coating over the resin when cellulose acetate butyrate was employed, as well as the moderate pore size of the coating, which results in less resistance to mass transfer. The polymer covering composed of cellulose acetate butyrate demonstrated the lowest protein leakage rate of 2.1%, as depicted in Fig. 3 B. According to the figure, it can be observed that there is a correlation between the protein leakage rate and the catalytic activity of the immobilized enzyme. Specifically, when the protein leakage rate increases, the catalytic activity of the immobilized enzyme decreases. This finding suggests that the protein leakage rate is a significant component influencing the catalytic activity of the immobilized enzyme. This is because the leaked protein is mainly a lipase with catalytic activity, and the leakage of the enzyme leads to a decrease in the overall catalytic activity of the immobilized enzyme system. Cellulose acetate butyrate (CAB) is a derivative of cellulose, a highly abundant natural substance. CAB offers several advantages, including its environmentally friendly nature, complete degradability, ample reserves, and renewability. Consequently, CAB is a cost-effective and sustainable polymer. After careful consideration of several factors, CAB was selected as the preferred coating material. 3.2.2 Optimization of organic polymer concentration The concentration of the organic polymer has a direct impact on both the thickness of the coating and the pore size of the coated surface. This change affects the mass transfer resistance and ultimately affects the catalytic activity of immobilized enzymes Thus, the selection of an optimal concentration of organic polymer can effectively regulate the flow of both the substrate and product within the coating, while also intercepting protein molecules released from the resin. This interception mechanism contributes to a reduction in the rate of protein leakage. Therefore, an experimental investigation was conducted to examine the impact of organic polymer concentration on both the catalytic efficiency of immobilized CALB and the rate of protein leakage. (Fig. 4 A and 4 B). The immobilized enzyme activity exhibited two peaks (at concentrations of 40 g/L and 80 g/L) in response to variations in organic polymer concentration. This may be due to the insufficient protection provided by the low organic polymer concentration during the coating process prior to reaching the 40 g/L threshold. Consequently, the enzyme protein in the immobilized enzyme experienced heightened vulnerability to damage from the organic solvent, potentially leading to elution. So at low organic polymer concentration, the activity of the immobilized lipase was even about 15% lower than that of the uncoated immobilized lipase (13180.6 PLU/g). As the concentration of organic polymer increased, the outer layer of immobilized lipase is protected by polymer coatings, and the damage of organic solvent on the immobilized lipase gradually decreased, reaching the first peak of enzyme activity (12,643.4 PLU/g) at an organic polymer concentration of 40 g/L. The main reason for the subsequent effect on the enzymatic activity of the immobilized enzyme may be that the organic polymer concentration changed the homogeneity, thickness, and pore size of the coating, reaching a second peak of enzyme activity (12685.5 PLU/g) at an organic polymer concentration of 80 g/L. As the concentration of organic polymer increased, the protein leakage rate of the immobilized enzyme decreased. At the organic polymer concentration of 100 g/L, the protein leakage rate was reduced to 2%. However, the activity of the immobilized lipase was found to be low due to the increased concentration leading to the formation of a thicker coating, smaller pore size, and greater mass transfer resistance. At the organic polymer concentrations of 40 g/L and 80 g/L, it was seen that the immobilized lipase activity remained comparable. However, it is noteworthy that the protein leakage rate was further reduced at the higher concentration of 80 g/L, specifically measuring at 2.1%. This suggests that the immobilized enzyme exhibits an extended operational lifespan when the concentration of organic polymer is at 80 g/L. On balance, a concentration of 80 g/L was chosen for CAB. 3.2.3 Selection of organic solvents The impact of organic solvents on immobilized CALB primarily stems from the varying solubility of organic polymers in various organic solvents, as well as the distinct distribution patterns of organic polymers on the resin surface after the phase transition of the coating. Furthermore, there exists variation in the toxicity levels of diverse organic reagents towards the protein of the immobilized enzyme. Therefore, the effects of organic solvent types on the catalytic efficiency of immobilized CALB and the protein leakage rate were investigated, and the results are shown in Table 1 . The catalytic activity of the immobilized enzyme was influenced by different organic reagents, among which the highest catalytic activity of 12685.5 PLU/g was obtained when acetone was used as the organic solvent, which was 1.26 times higher than that of Novozym 435. In terms of protein leakage rate, different organic reagents had little effect on it. Hence, acetone was chosen as the organic solvent for cellulose acetate butyrate. Table 1 Effect of organic solvents on the enzymatic activity and protein leakage rate of the immobilized lipase CALB. Organic solvent Specific enzyme activity (PLU/g) Protein leakage rate (%) DMF 8529.3 ± 2.1 2.3 ± 0.1 DMSO 9224.4 ± 3.2 2.2 ± 0.2 Tetrahydrofuran 9783.4 ± 2.3 2.1 ± 0.1 Acetone 12685.5 ± 2.6 2.1 ± 0.3 Fernandez et al. [ 29 ] used polyethyleneimine and dextran sulfate coating of CALB immobilized on octylated agarose gels, which resulted in a significant increase in stability, this could be attributed to the fact that the coating caused physical intermolecular cross-linking of the CALB molecules with the polymer, reducing the desorption of the enzyme from the carrier. This further demonstrated that polymer coatings do improve the stability of immobilized enzyme. In addition, the esterification activity and protein leakage rate of the immobilized CALB (enzyme-loaded resin), CALB@resin-CAB, Novozym 435, and coated Novozym 435 were characterized ( Table S1 ). The specific enzyme activity of immobilized CALB was higher than Novozym 435, about 1.26 times the esterification activity of Novozym 435. After organic polymer coating, the protein leakage rate of the enzyme was reduced to one-quarter of the pre-coating rate. This demonstrates that the use of organic polymer coatings may be a successful strategy for mitigating protein leakage in immobilized enzymes. 3.3 Characterization of the immobilized lipase CALB@resin-CAB 3.3.1 SEM analysis Characterization of the surfaces of resin XRZ04B, enzyme-loaded resin XRZ04B, and CALB@resin-CAB by scanning electron microscopy to derive their microscopic surface variations. Figure 5 A and Fig. 5 B show the microscopic surface changes of the macroporous resin XRZ04B before and after the adsorption of CALB with a magnification of 150, 000. It can be seen that the former surface is quite rough and has larger pores. While the latter surface is smoother and the pores have been partially filled by protein molecules. It indicates that the lipase was successfully adsorbed onto the macroporous resin XRZ04B. Figure 5 C and Fig. 5 D show the microscopic surface changes before and after the coating of the enzyme-carrying resin XRZ04B with a magnification of 25, 000. It can be seen that an organic polymer coating was successfully formed on the surface of the carrier, and the thickness of the coating was about 0.5% of the radius of the resin XRZ04B. This coating structure can effectively reduce the leakage rate of protein on the carrier while ensuring the catalytic activity of the immobilized enzyme. 3.3.2 EDS analysis The surface of resin XRZ04B, enzyme-loaded resin XRZ04B, and CALB@resin-CAB were characterized by EDS elemental analysis to derive the variation of nitrogen on their microscopic surfaces leading to the distribution of enzyme proteins on the resin ( Fig. S1 A-C ). Comparing the changes in nitrogen density on the resin surface before and after immobilization showed that CALB was successfully immobilized on the carrier. 3.3.3 Thermogravimetric analysis TG analysis was performed on resin XRZ04B, enzyme-loaded resin XRZ04B, and CALB@resin-CAB, respectively. TG analysis was performed under dry nitrogen to reduce the oxidation products that may affect the experimental results. Degradation or depolymerization of the resin and protein starts at 250 ℃, the main degradation step is at 350 ℃, and the degradation ends at 450 ℃ ( Fig. S2 ). The thermal distribution of degradation was not significantly different for the three samples, and the relative masses of the three samples were slightly different from 30 to 200 ℃. This is because the change in weight in this temperature interval is mainly due to the moisture in the samples, including the bound water of the molecules. The main temperature range for sample weight loss was from 200–600 ℃, which was attributed to the removal of organic components (C, H, O, and N) in this temperature range. The heat loss curves of the enzyme-loaded resin XRZ04B and the CALB@resin-CAB were smoother due to the adsorption of lipase by XRZ04B and the coating of an organic polymer on the resin surface. All three samples were finally charred at 800 ℃. About 1.1% of the residue of the carrier XRZ04B was in the form of char, about 3.9% of the residue of the carrier resin XRZ04B, and about 5.5% of the residue of the CALB@resin-CAB. From the relative weights of the final residues of the three samples, it is known that the enzyme protein was successfully immobilized on the resin as well as the organic polymer coating was successfully applied to the resin surface. 3.3.4 Fluorescent labeling analysis First, the free CALB was fluorescently labeled using the fluorescent dye fluorescein isothiocyanate (FITC); then, the labeled lipase was adsorbed using a macroporous resin; finally, the prepared enzyme-loaded resin was observed under a fluorescence microscope. The fluorescence of the immobilized CALB surface ( Fig. S3A-B ) shows that the CALB is uniformly distributed on the resin surface. The distribution of CALB inside the resin can be seen from the fluorescence of the immobilized CALB cross section. CALB was mostly immobilized on the outer surface of the resin, with less distribution inside the resin. Therefore, the organic polymer coating of the enzyme-loaded resin XRZ04B can effectively prevent the leakage of CALB on the outer surface of the resin. 3.3.5 Pore size distribution and BET specific surface area analysis The changes in pore size and specific surface area of the resin and immobilized CALB before and after immobilization were analyzed. The N2 adsorption-desorption isotherm ( Fig. S4A-C ) of the carrier XRZ04B belongs to the type II isotherm feature, which indicates that XRZ04B is a macroporous material. Due to the adsorption of CALB and the pore structure of the organic polymer coating, there was a significant decrease in the BET specific surface area of the enzyme-loaded resin XRZ04B and an increase in the BET specific surface area after the organic polymer coating. As shown in Table S2 , the BET specific surface areas of resin XRZ04B, enzyme carrier XRZ04B, and CALB@resin-CAB were 161.17, 3.24 and 34.00 m 2 ·g − 2 , respectively; the pore capacities were 0.44, 0.01 and 0.09 cm 3 ·g − 1 , respectively. As shown in Fig. S5 , the pore size of the carrier XRZ04B was 20–50 nm and the most probable pore size was 22.49 nm; after adsorption of CALB, the pore size became 10–20 nm and the most probable pore size was 16.04 nm; after organic polymer coating, the pore size increased to 15–30 nm and the most probable pore size was 21.13 nm. The most probable pore size is the one with the highest probability of occurrence in the sample. Based on the BET analysis chart, we infer that the decrease in specific surface area in the first stage is due to the enzyme occupying the pores on the surface of the macroporous resin after adsorption, completing the immobilization process of the enzyme in the first step; The increase in specific surface area in the second stage is due to the use of ethyl acetate cellulose coating, which endows the surface of the macroporous resin after adsorbing enzymes with a porous polymer protective layer. This protective layer not only prevents enzyme leakage, but also reduces substrate mass transfer limitations, allowing the immobilized enzyme system to achieve higher catalytic activity. 3.4 Thermal stability of immobilized lipase CALB@resin-CAB In this study, the thermal stability of CALB@resin-CAB was investigated by incubating the immobilized lipase in tert-butanol at different temperatures (50°C, 60°C and 70°C) for different times. CALB@resin- CAB maintained a high residual activity with increasing temperature (Fig. 6 ). The decrease of the residual enzyme activity of CALB@resin-CAB accelerated with the increase of storage temperature. The thermal stability of CALB@resin-CAB was satisfactory at 50°C and 60°C, and the relative enzyme activity remained at about 100% after 10 h of storage; the residual enzyme activity was still about 88% after 10 h of storage at 70°C, which indicated CALB@resin-CAB showed excellent thermal stability. 3.5 Immobilized lipase CALB catalyzed production of biodiesel from palm acidified oil 3.5.1 Conditions optimization of lipase-catalyzed biodiesel production Immobilized lipase CALB@resin-CAB was applied to the biocatalyzed production of biodiesel by using palm acidified oil as the starting material, and the key affecting factors of lipase-catalyzed process were optimized. The optimal conditions for the reaction were determined as follows ( Fig. S6A-C ): reaction temperature of 50°C, a molar ratio of oil to alcohol of 1:3 ( Table S3 ), organic solvent tert-butanol with concentration of 20% (v/v). As illustrated in Fig. 7 , after the first enzyme-catalyzed reaction (12 h), the conversion of substrate reached a level of 91.2% with an acid value of 17.5. This phenomenon occurred due to the generation of a substantial quantity of water and a little quantity of glycerol, which would inhibit the continuation of the reaction [ 30 ]. In order to enhance the conversion of the substrate, a further enzymatic reaction was conducted on the resulting product subsequent to the first reaction. Firstly, the reaction was left to stratify to remove large amounts of water and glycerol; subsequently, the remaining reaction mixture was subjected to spin evaporation to eliminate any leftover water content. Lastly, methanol and CALB@resin-CAB were reintroduced to sustain the progression of the reaction. As can be seen from Fig. 7 , the substrate conversion was enhanced to 98.5% in the second reaction catalyzed by the enzyme, resulting in an acid value of 3.1. Therefore, the CALB@resin-CAB exhibits an outstanding level of catalytic activity in the enzymatic process involved in the synthesis of biodiesel from fats and oils with high acid values. 3.5.2 Batch stability of the immobilized lipase CALB@resin-CAB Excellent batch stability is essential for the application of immobilized enzymes in industrial production [ 31 ]. Therefore, the batch stability of CALB@resin-CAB for the synthesis of biodiesel from palm acidified oil was investigated ( Fig. S7 ). The results showed that CALB@resin-CAB exhibited good batch stability during the enzymatic production of biodiesel using palm acidified oil. The enzyme activity of CALB@resin-CAB remained close to the initial enzyme activity value after 10 batches of enzyme-catalyzed reactions. 3.6 Immobilized CALB catalyzed biodiesel production in the packed bed reactor A continuous flow catalytic reaction was carried out in the packed bed reactor filled with CALB@resin-CAB under the optimized conditions. This packed bed reactor uses a continuous flow reaction mode with continuous feed and continuous discharge to simulate the form of immobilized lipase application in industrial production [ 32 , 33 ]. The effect of flow rate on the conversion of palm acidified oil was investigated by flowing a substrate flow rate of 1–20 Bv/h through a packed bed reactor filled with immobilized lipase CALB@resin-CAB, respectively ( Fig. S8 ). At a flow rate less than 4 Bv/h, the conversion of palm acidified oil reached a steady state and conversion of the substrate did not increase as the flow rate decreased. When the flow rate was larger than 4 Bv/h, the conversion of palm acidified oil decreased with increasing flow rate, and at a flow rate of 20 Bv/h, the conversion of palm acidified oil decreased to about 80%. Therefore, a substrate flow rate of 4 Bv/h was selected, at which time the conversion of palm acidified oil was 94.8%, and the space-time yield of biodiesel was 2.88 g/(mL∙h). 4. Conclusion In this study, a novel CALB immobilization method based on macroporous resin adsorption and organic polymer coating was developed. The immobilized lipase CALB@resin-CAB was applied to the biocatalyzed production of biodiesel with acidified palm oil as the starting material and the conversion rate reached 98.5%. Moreover, the immobilized lipase showed excellent batch stability in biodiesel production. When the aforementioned biocatalytic reaction was carried out in a continuous flow packed-bed system, the yield of biodiesel was 94.8% and space-time yield was 2.88 g/(mL∙h). In conclusion, the immobilized lipase CALB@resin-CAB has high catalytic activity and stability, which has good prospects for industrial applications in enzyme-catalyzed biodiesel production. Declarations Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author Contribution Jiale Liu: Resources, Conceptualization, Methodology, Investigation, Validation, Data curation, Formal analysis, Writing - original draft. Shufan Zhao: Investigation, Validation, Data curation, Formal analysis, Writing - original draft. Wan Wei: Formal analysis, Validation. Shupeng Yu: Formal analysis, Validation. Zhao Wang: Conceptualization, Software, Supervision, Writing - review & editing. Jianyong Zheng: Data curation, Formal analysis, Writing - review & editing, Project administration. Acknowledgement This research was financially supported by the technology innovation and application development special key project of Chongqing (cstc2021jscx-jbgsX0002). Data Availability The data are available from the corresponding author on reasonable request. Appendix A. Supplementary data E-supplementary data of this work can be found in online version of the paper. References Ravichandran P, Rajendran N, Al-Ghanim KA, Govindarajan M, Gurunathan M (2023) Investigations on evaluation of marine macroalgae Dictyota bartayresiana oil for industrial scale production of biodiesel through technoeconomic analysis. Bioresour Technol 374:128769 Vimali E, Gunaseelan S, Devi VC, Mothil S, Arumugam M, Ashokkumar B, Moorthy IMG, Pugazhendhi A, Varalakshmi P (2022) Comparative study of different catalysts mediated FAME conversion from macroalga Padina tetrastromatica biomass and hydrothermal liquefaction facilitated bio-oil production. Chemosphere 292:133485 Cavalcante FTT, Neto FS, De Aguiar Falcão IR, Da Silva Souza JEE, De Moura Junior LS, Da Silva Sousa P, Rocha TG, De Sousa IG (2021) Opportunities for improving biodiesel production via lipase catalysis. Fuel 288:119577 Sangeetha B, Priya SM, Pravin R, Tamilarasan K, Baskar G (2023) Process optimization and technoeconomic assessment of biodiesel production by one-pot transesterification of Ricinus communis seed oil. Bioresour Technol 376:128880 Liu J, Chen G, Yan B, Yi W, Yao J (2022) Biodiesel production in a magnetically fluidized bed reactor using whole-cell biocatalysts immobilized within ferroferric oxide-polyvinyl alcohol composite beads. Bioresour Technol 355:127253 López-Fernández J, Benaiges MD, Valero F (2021) Second-and third-generation biodiesel production with immobilised recombinant Rhizopus oryzae lipase: Influence of the support, substrate acidity and bioprocess scale-up. Bioresour Technol 334:125233 Salihu A, Alam MZ (2015) Solvent tolerant lipases: a review. Process Biochem 50: 86-96 Casas-Godoy L, Gasteazoro F, Duquesne S, Bordes F, Marty A, Sandoval G (2018) Lipases: an overview. Methods Mol Biol 1835:3-38, Adachi D, Hama S, Nakashima K, Bogaki T, Ogino C, Kondo A (2013) Production of biodiesel from plant oil hydrolysates using an Aspergillus oryzae whole-cell biocatalyst highly expressing Candida antarctica lipase B. Bioresour Technol 135:410-416 Sarmah N, Revathi D, Sheelu G, Yamuna Rani K, Sridhar S, Mehtab V, Sumana C (2018) Recent advances on sources and industrial applications of lipases. Biotechnol Prog 34:5-28 Tan Z, Li X, Shi H, Yin X, Zhu X, Bilal M, Onchari MM (2022) Enhancing the methanol tolerance of Candida antarctica lipase B by saturation mutagenesis for biodiesel preparation. 3 Biotech 12:22 Filho DG, Silva AG, Guidini CZ (2015) Lipases: sources, immobilization methods, and industrial applications. Appl Microbiol Biotechnol 103:7399-7423 Zhang Y, Ge J, Liu Z (2015) Enhanced activity of immobilized or chemically modified enzymes. ACS Catal 5:4503-4513 Abdulmalek SA, Yan Y (2022) Recent developments of lipase immobilization technology and application of immobilized lipase mixtures for biodiesel production. Biofuel Bioprod Biorefin 16:1062-1094 Rodrigues RC, Ortiz C, Berenguer-Murcia Á, Torres R, Fernández-Lafuente R (2013) Modifying enzyme activity and selectivity by immobilization. Chem Soc Rev 42:6290-6307 Souza PM, Carballares D, Gonçalves LR, Fernandez-Lafuente R, Rodrigues S (2022) Immobilization of Lipase B from Candida antarctica in Octyl-Vinyl Sulfone Agarose: Effect of the Enzyme-Support Interactions on Enzyme Activity, Specificity, Structure and Inactivation Pathway. Int J Mol Sci 23:14268 Sheldon RA, Basso A, Brady D (2021) New frontiers in enzyme immobilisation: robust biocatalysts for a circular bio-based economy. Chem Soc Rev 50: 5850-5862 Rueda N, Dos Santos JC, Torres R, Ortiz C, Barbosa O (2015) Improved performance of lipases immobilized on heterofunctional octyl-glyoxyl agarose beads. RSC Adv 5:11212-11222 Virgen-Ortíz JJ, Tacias-Pascacio VG, Hirata DB, Torrestiana-Sanchez B, Rosales-Quintero A, Fernandez-Lafuente R (2017) Relevance of substrates and products on the desorption of lipases physically adsorbed on hydrophobic supports. Enzyme Microb Technol 96:30-35 Qian J, Huang A, Zhu H, Ding J, Zhang W, Chen Y (2023) Immobilization of lipase on silica nanoparticles by adsorption followed by glutaraldehyde cross-linking. Bioprocess Biosyst Eng 46:25-38 Tan T, Lu J, Nie K, Deng L, Wang F (2010) Biodiesel production with immobilized lipase: a review. Biotechnol Adv 28:628-634 Silva JMF, Dos Santos KP, Dos Santos ES, Rios NS, Gonçalves LRB (2023) Immobilization of Thermomyces lanuginosus lipase on a new hydrophobic support (Streamline phenyl™): Strategies to improve stability and reusability. Enzyme Microb Technol 163:110166 Qin L, Li C, Li X, Zhang X, Shen C, Meng Q, Shen L, Lu Y, Zhang G (2020) Confined encapsulation of living cells in self-assembled fiber macrospheres with micro/nanoporous polymer shells for the transformation of contaminants to green energy. J Mater Chem A 8:1929-1938 Zhong L, Feng Y, Hu H, Xu J, Wang Z, Du Y, Cui J (2021) Enhanced enzymatic performance of immobilized lipase on metal organic frameworks with superhydrophobic coating for biodiesel production. J Colloid Interface Sci 602: 426-436 Wiemann LO, Nieguth RE, Eckstein M, Naumann M, Thum O, Ansorge-Schumacher MB, Composite particles of novozyme 435 and silicone: advancing technical applicability of macroporous enzyme carriers. ChemCatChem 1:455-462 Cai X, Zhang M, Wei W, Zhan Y, Wang Z, Zheng J (2020) The Immobilization of Candida antarctica lipase B by ZIF-8 encapsulation and macroporous resin adsorption: preparation and characterizations. Biotechnol Lett 42:269-276 Cao L, Asad Abbas S, Hyeon Jeong S, Seo D, Min Nam K, Kyoon Park J (2023) Chemoselective nitro reduction using nitrogen-doped carbon-encapsulated Ni catalyst and Y-type packed bed column for continuous flow reaction. Adv Synth Catal 365:2230-2239 Rodrigues J, Canet A, Rivera I, Osório N, Sandoval G, Valero F, Ferreira-Dias S (2016) Biodiesel production from crude Jatropha oil catalyzed by non-commercial immobilized heterologous Rhizopus oryzae and Carica papaya lipases. Bioresour Technol 213:88-95 Fernandez-Lopez L, Virgen-OrtÍz JJ, Pedrero SG, Lopez-Carrobles N, Gorines BC, Otero C, Fernandez-Lafuente R (2017) Optimization of the coating of octyl-CALB with ionic polymers to improve stability and decrease enzyme leakage. Biocatal Biotransfor 36:47-56 Sankaran R, Show PL, Chang JS (2016) Biodiesel production using immobilized lipase: feasibility and challenges. Biofuel Bioprod Biorefin 10:896-916 Chandra P, Enespa, Singh R, Arora PK (2020) Microbial lipases and their industrial applications: a comprehensive review. Microb Cell Fact 19:1-42 Britton J, Majumda Sr S, Weiss GA (2018) Continuous flow biocatalysis. Chem Soc Rev 47: 5891-5918 Kuo CH, Tsai ML, Wang HMD, Liu YC, Hsieh C, Tsai YH, Dong CD, Huang CY, Shieh CJ (2022) Continuous production of DHA and EPA ethyl esters via lipase-catalyzed transesterification in an ultrasonic packed-bed bioreactor. Catalysts 12:404 Additional Declarations Competing interest reported. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 23 Sep, 2024 Reviews received at journal 22 Sep, 2024 Reviewers agreed at journal 05 Sep, 2024 Reviewers invited by journal 05 Sep, 2024 Editor assigned by journal 19 Aug, 2024 Submission checks completed at journal 19 Aug, 2024 First submitted to journal 16 Aug, 2024 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-4922648","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":352716304,"identity":"8baed8e9-c034-4edd-a5ef-187906eb8570","order_by":0,"name":"Jiale Liu","email":"","orcid":"","institution":"Zhejiang University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Jiale","middleName":"","lastName":"Liu","suffix":""},{"id":352716305,"identity":"7e2d3114-080f-47c5-94d6-0ca558c4e805","order_by":1,"name":"Shufan Zhao","email":"","orcid":"","institution":"Zhejiang University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Shufan","middleName":"","lastName":"Zhao","suffix":""},{"id":352716306,"identity":"9f77a486-7691-47ab-a7f0-545def327a88","order_by":2,"name":"Wan Wei","email":"","orcid":"","institution":"Zhejiang University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Wan","middleName":"","lastName":"Wei","suffix":""},{"id":352716307,"identity":"063cd981-f9b9-43ee-8ff9-c3c088678f3a","order_by":3,"name":"Shupeng Yu","email":"","orcid":"","institution":"Zhejiang University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Shupeng","middleName":"","lastName":"Yu","suffix":""},{"id":352716308,"identity":"01e5864e-8716-4b86-b927-7849ac03b77c","order_by":4,"name":"Zhao Wang","email":"","orcid":"","institution":"Zhejiang University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Zhao","middleName":"","lastName":"Wang","suffix":""},{"id":352716309,"identity":"f2dc3495-9e5c-4254-8c8c-c34c5dee96f2","order_by":5,"name":"Jianyong Zheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/UlEQVRIie3QMWrDMBSA4WcE7vISrzIu8RWeMHQy5CqvFDwHcoC6ZMjSAzgkh2hvYBOoO/gA7qZSaJcOgSwdAo1FabfIHgvRPwnxPh4SgMv1Lys9vaMUg4sc6OeinwhVzLLL8L7sCA8i4Ee426bUmvEhJL5rEkISCC8fej76gsm4ZW8/sxAvb5SW5KO3Zk6QIQlbFlFhIQK6LUSIIuLSkOuHlv1uqeUh0FxJJol+WOWG3PYSNKQ0a6QAQ5j6iISnucqJUWIGapNJtWpeF5GNxMX28e1w+J5O6/qdPtM0Htc31d5GQD7T37vIbDW/aAMAwVL/HoU+OeVyuVxn3RFCBUjl/UsNRgAAAABJRU5ErkJggg==","orcid":"","institution":"Zhejiang University of Technology","correspondingAuthor":true,"prefix":"","firstName":"Jianyong","middleName":"","lastName":"Zheng","suffix":""}],"badges":[],"createdAt":"2024-08-16 05:44:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4922648/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4922648/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":64594812,"identity":"9bdcff07-38ea-4e37-a34d-64a06c4e306e","added_by":"auto","created_at":"2024-09-16 10:30:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":122771,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustration of the preparation strategy of CALB@resin-CAB\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4922648/v1/6f30a3381fd2ae47cf6476d4.png"},{"id":64594427,"identity":"95267edd-9d06-4672-8bcc-847138d4ffb9","added_by":"auto","created_at":"2024-09-16 10:22:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":72670,"visible":true,"origin":"","legend":"\u003cp\u003eOptimization of CALB immobilization conditions. (A) various macroporous resin (B) resin addition (specific enzyme activity■, total enzyme activity▲) (C) adsorption time\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4922648/v1/f4610c0880d8dc4868e5937c.png"},{"id":64594818,"identity":"365f9d26-81e4-4608-89ca-e50fd38aac66","added_by":"auto","created_at":"2024-09-16 10:30:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":112538,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of the organic polymer types on the activity of the immobilized CALB (A) and the protein leakage rate of the immobilized CALB (B).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4922648/v1/6d775d11e43ff414bd3d5e39.png"},{"id":64594429,"identity":"7b4a5137-e917-45e6-a155-a074f8eb5ef0","added_by":"auto","created_at":"2024-09-16 10:22:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":44055,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of the organic polymer concentration on the activity of the immobilized CALB (A) and the protein leakage rate of the immobilized CALB (B).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4922648/v1/57739cbf684105d8c8a902fc.png"},{"id":64594433,"identity":"b467570f-f3a4-4558-b6ed-393910720dce","added_by":"auto","created_at":"2024-09-16 10:22:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":399642,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of macroporous resin XRZ04B (A); enzyme-loaded resin XRZ04B (B); enzyme-loaded resin XRZ04B(C); CALB@resin-CAB (D). The magnification of Fig. 5A and Fig. 5B is 150, 000, Fig. 5C and Fig. 5D is 25, 000.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4922648/v1/d5514c383c4cd60e77f0ee28.png"},{"id":64594430,"identity":"e073e5c0-925f-41be-ac5a-5ce4e35b6769","added_by":"auto","created_at":"2024-09-16 10:22:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":10885,"visible":true,"origin":"","legend":"\u003cp\u003eThermal stability analysis of immobilized lipaseCALB@resin-CAB at 50 °C, 60 °C and 70 °C.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4922648/v1/71464ef259b9cb65c8706ab4.png"},{"id":64595233,"identity":"4cb8eca1-5ef8-4de8-9fed-4e523f89762d","added_by":"auto","created_at":"2024-09-16 10:38:28","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":70231,"visible":true,"origin":"","legend":"\u003cp\u003eTime course of lipase-catalyzed biodiesel production with a two-step reaction.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4922648/v1/df79b3285e33e2c2851a6f3a.png"},{"id":64595719,"identity":"c6b4528c-fd19-4bd4-916c-8fe910c04b58","added_by":"auto","created_at":"2024-09-16 10:46:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1654910,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4922648/v1/4a218446-31ae-4b3d-bf2b-0781b1a8c102.pdf"},{"id":64594434,"identity":"fc30a753-578c-420b-b529-a92abab29d43","added_by":"auto","created_at":"2024-09-16 10:22:28","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9531445,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4922648/v1/6e0eb5d426158a8d2d2d966d.docx"}],"financialInterests":"Competing interest reported. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.","formattedTitle":"Improved catalytic stability of immobilized Candida antarctic lipase B on macroporous resin with organic polymer coating for biodiesel production","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe overuse of fossil fuels has led to environmental issues such as global warming, and with the increasing energy consumption, it greatly encouraged the exploration of new energy sources [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Biodiesel can be used as an alternative to fossil fuels due to its substantial advantages like biodegradable, renewable, non-toxic, and usable by existing engines [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Biodiesel is mainly produced through chemical ester exchange reactions, which generally require high energy consumption, environmentally harmful catalysts, and a series of complex wastewater treatment measures [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Nevertheless, enzymatic esterification with lipases is a more environmentally friendly method that can avoid some of the problems existed in chemical conversion [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLipases (EC 3.1.1.3) are enzymes that catalyze the hydrolysis of triacylglycerol ester [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Lipase is one of the most widely used enzymes at both academic and industrial aspects because of their ability to recognize a wide variety of substrates and catalyze different types of reactions while maintaining high stability and activity [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. \u003cem\u003eCandida antarctic\u003c/em\u003e lipase B (CALB) is an excellent biocatalyst widely used in oil ester processing and preparation of fine chemicals. Enzymatic esterification with CALB has emerged as an effective choice for biodiesel [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Tan et al. modified CALB employing protein engineering to obtain mutants to improve methanol tolerance and used it as a catalyst for biodiesel production from soybean oil, with a reaction yield of 85% at 24 h [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, free CALB still has several drawbacks, including poor thermal stability, low yield, and unrecyclability, which limit its application in industrial production [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Fortunately, enzyme immobilization could be the focal point for improving enzyme properties [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEnzymatic immobilization enables the application of lipases in industrial processes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Appropriate immobilization technologies can achieve the recovery of biocatalysts and minimize or even eliminate product contamination, thereby reducing the costs associated with enzyme use [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. They also bring improved o more convenient operation and better storage stability, as well as better adaptability to pH and temperature changes [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, although it exhibits many advantages, it also has potential disadvantages. It has been shown that immobilized lipase molecules can passively migrate to the carrier surface which may lead to enzyme leakage under harsh conditions [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Cross-linking and covalent methods are often used in combination with adsorption methods to enhance the force between the enzyme and the carrier [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Although immobilized enzymes prepared through cross-linking and covalent adsorption have good stability, the cross-linking or covalent binding process may have the problem of occupying the catalytic active site of the enzyme and may lead to structural changes in the enzyme protein, thereby reducing enzyme activity [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this work, we present a new core-shell structure concept for lipase immobilization (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The novel immobilization method was developed based on the enzyme immobilization strategy of loading followed by encapsulation, a hydrophobic macroporous resin was applied as a carrier for the physical adsorption of lipase, and the organic polymer was used to coat the surface of the macroporous resin by phase change to construct an immobilized lipase with enzyme-organic polymer core-shell structure [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. This immobilization method satisfactorily solved the shortcoming of enzyme leakage existing in the adsorption method, while maintaining high catalytic activity [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The organic polymer coating was optimized subsequently to obtain immobilized CALB (CALB@resin-CAB). It was applied to the enzyme-catalyzed biodiesel production by using palm acidified oil as the row material and explored its operational and leaching stability. To simulate the industrial production, CALB@resin-CAB was used for the continuous synthesis of biodiesel. In this study, the lipase immobilization strategy provides new insights to solve the problems of immobilized enzymes in activity, stability, and industrial applicability.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eLipase B from \u003cem\u003eCandida antarctica\u003c/em\u003e (CALB) was supplied from Vland Biotech (Shandong, China, 500 LU/g hydrolytic activity). Macroporous resin (XRZ04B) was purchased from Shanghai Suner Chemical Technology Co., Ltd. (Shanghai, China). Macroporous resins (NKA, D3520, AB-8, and D301) were purchased from Tianjin Nankai Hecheng Sci. \u0026amp;Tech. Co., Ltd. (Tianjin, China). Macroporous resins (D101, NKA-9, and H-20) were purchased from Huayi Technology New Materials Co., Ltd. (Henan, China). Macroporous resins (LXTE-1000, LXTE-705, LXTE-707, LXTE-700s, and HA109M) were purchased from Xi\u0026rsquo;an Lanxiao Technology New Materials Co., Ltd. (Shanxi, China). Organic polymer Cellulose acetate(CA), cellulose acetate butyrate (CAB), polymethyl methacrylate (PMMA), etc. were purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China). Palm acidified oil (acid value 196) was kindly supplied by Zangyu Technology Group Co., Ltd. (Zhejiang, China). All other reagents and solvents in this study were of analytical grade.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Preparation procedures of the immobilized lipase CALB\u003c/h2\u003e \u003cp\u003eThe 4 g of macroporous resin XRZ04B was mixed with 16 mL of CALB enzyme solution (16 mg/mL enzyme in deionized water) in a water bath shaker at 25 ℃ for 5 h. After immobilization, the supernatant was removed by filtration to obtain the enzyme-loaded resin. To prevent the enzyme leaching from the carrier during the reaction, the enzyme-loaded resin was coated with an organic polymer [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. CA, CAB, PMMA, PSF, PS, and PVDF were selected as coating materials and dissolved in DMF, DMSO, THF, and AC with vigorous stirring to obtain an organic polymer solution (2%-10%); Then, immerse the enzyme loaded resin in a polymer solution for 10 seconds to coat the surface of the enzyme loaded resin with a uniform organic polymer solution The enzyme-loaded resin coated with polymer solution was immersed in deionized water for 20 min. CALB@resin- CAB was obtained by suction filtration, then dried in a vacuum oven at 45\u0026deg;C for 3 h and stored at 4\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Esterification activity assay of the immobilized lipase CALB@resin-CAB\u003c/h2\u003e \u003cp\u003e0.03 g immobilized lipase CALB@resin-CAB was added to 10.73 g substrate mixture solution, which included lauric acid (40 mmol), n-propanol (40 mmol), deionized water (3% w/w). The reaction was performed at 60\u0026deg;C and 200 rpm in a water bath shaker for 20 min. Subsequently, 5 \u0026micro;L of the reaction solution was added to 995 \u0026micro;L of ethyl acetate for gas chromatography (GC) analysis. The production of propyl laurate in the reaction system was detected. Gas chromatography detection was performed using the Agilent 6890 GC platform equipped with a flame ionization detector(FID) and a fused silica capillary column (DB-23, 30 m\u0026times;0.32 mm i.d., 0.1 \u0026micro;m film thickness; Agilent Technologies, Inc.). Nitrogen was used as the carrier gas at a flow rate of 1.0 ml/min. The chromatographic column temperature raised from 100\u0026deg;C to 280\u0026deg;C (2 min), at a heating rate of 10\u0026deg;C/min [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDefinition of immobilized lipase methyl esterification activity: Under standard reaction conditions, the amount of enzyme required to produce 1 \u0026micro;mol propyl laurate catalyzed by immobilized enzymes per minute was defined as an enzyme activity unit, expressed as PLU.\u003c/p\u003e \u003cp\u003eProtein concentration was determined by the Bradford method using bovine serum albumin as standard.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Leaching stability assay of the immobilized lipase\u003c/h2\u003e \u003cp\u003eLeaching stability of the immobilized lipase CALB is characterized by the leakage rate of the protein in high-salt solutions. Add 5 mL of 2 M NaCl aqueous solution to 2 g of immobilized lipase (dry weight), and stir at 30 ℃ for 2 h, then take the supernatant for protein concentration assay. The protein leakage rate is given by \u003cb\u003eEq.\u0026nbsp;1\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eY= \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\left({A}_{3}*{V}_{2}\\right)/\\left[\\left({A}_{1}-{A}_{2}\\right)*{V}_{1}\\right]\\)\u003c/span\u003e\u003c/span\u003e (\u003cb\u003eEq.\u0026nbsp;1\u003c/b\u003e)\u003c/p\u003e \u003cp\u003eWhere A\u003csub\u003e1\u003c/sub\u003e is enzyme solution protein concentration before immobilization (mg/mL); A\u003csub\u003e2\u003c/sub\u003e is supernatant protein concentration after immobilization (mg/mL); V\u003csub\u003e1\u003c/sub\u003e is the volume of immobilized enzyme solution (mL); A\u003csub\u003e3\u003c/sub\u003e is eluate protein concentration (mg/mL); V\u003csub\u003e2\u003c/sub\u003e is the volume of eluate solution (mL).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Structure and performance characterizations of CALB @resin-CAB\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1 SEM analysis\u003c/h2\u003e \u003cp\u003eThe morphology of resin XRZ04B, enzyme-loaded resin XRZ04B, and CALB @resin-CAB was characterized with a scanning electron microscope (Hitachi SU-70, Japan) in a field-emission FEI Inspect F operated at 5 kV. Samples were dried, and fixed onto silicon wafers. The samples were then immediately sputter-coated with platinum before observation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2 TG analysis\u003c/h2\u003e \u003cp\u003eThe Thermogravimetric analysis was measured in a TGA-DSC simultaneous thermal analyzer (STA449F5, Shanghai, China) under nitrogen with a heating range of 30\u0026ndash;900\u0026deg;C and a heating rate of 10\u0026deg;C/min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.5.3 Fluorescent labeling assay\u003c/h2\u003e \u003cp\u003eFree CALB was fluorescently labeled using the fluorescent dye fluorescein isothiocyanate, after that, labeled lipase was adsorbed using a macroporous resin, and prepared enzyme-carrying resin was observed under a fluorescence microscope (EVOS M5000, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.5.4 Pore size distribution and BET specific surface area assay\u003c/h2\u003e \u003cp\u003eSample pore size distribution and BET specific surface area were measured by specific surface area and pore size analyzer (TriStar II Plus 3.03, Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Thermal stability assay of the immobilized CALB\u003c/h2\u003e \u003cp\u003eThe thermal stability of CALB@resin-CAB was measured by determining the residual esterification activities after incubation in tert-butanol for a period of time at 50\u0026deg;C, 60\u0026deg;C, and 70\u0026deg;C. The residual activities of the immobilized lipases were determined as described in Section \u003cspan refid=\"Sec5\" class=\"InternalRef\"\u003e2.3\u003c/span\u003e. The relative activity of CALB@resin-CAB before incubation was defined as 100%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Biosynthesis of biodiesel with palm acidified oil as the starting material\u003c/h2\u003e \u003cp\u003e0.01 g CALB @resin-CAB was added to 10 g substrate (9 g palm acidified oil and 1 g methanol). The reaction was performed at 50\u0026deg;C and 200 rpm in a water bath shaker for 8 h. After the reaction, excess methanol and water were removed by spin evaporation, and then the samples were subjected to acid value determination. All assays were conducted in triplicate. The conversion rate of palm acidified oil is given by \u003cb\u003eEq.\u0026nbsp;2\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eConversion rate = (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:({E}_{1}-{E}_{2})\u0026frasl;{E}_{1})*100\\%\\)\u003c/span\u003e\u003c/span\u003e (\u003cb\u003eEq.\u0026nbsp;2\u003c/b\u003e)\u003c/p\u003e \u003cp\u003eWhere E\u003csub\u003e1\u003c/sub\u003e is the acid value before the reaction, and E\u003csub\u003e2\u003c/sub\u003e is the acid value after the reaction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Lipase-catalyzed biodiesel production in the packed bed reactor\u003c/h2\u003e \u003cp\u003eThe packed bed reactor was prepared as followed: 10 g of immobilized lipase CALB@resin-CAB was poured into the column reactor and left a volume of 30 mL in the reactor (height-to-diameter ratio of 5:1) when filling since the dried immobilized enzyme will have some volume expansion during the reaction.\u003c/p\u003e \u003cp\u003eConditions of lipase-catalyzed biodiesel production in packed bed reactor: the substrate was first mixed in a magnetic stirrer and then continuously pumped through the reactor using a high-pressure pump (JJRZ-10004F, Hangzhou) at a certain flow rate (1\u0026ndash;20 Bv/h). After pre-running for 30 min, the product was collected, the product was concentrated by rotary evaporation and its acid value was determined [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Conditions optimization of lipase CALB immobilization\u003c/h2\u003e \u003cp\u003eThe properties of the immobilized enzyme carrier, such as: the specific surface area of the carrier, the polarity and the pore size of the carrier, have a great influence on the catalytic activity and stability of the immobilized enzyme [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Therefore, the effect of different types of macroporous resins on the catalytic activity of immobilized lipase was investigated in the previous experiment. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, the immobilized lipase CALB prepared with macroporous resin XRZ04B showed the highest esterification activity. According to Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, the specific activity of immobilized lipase was significantly influenced by the ratio of carrier to lipase addition. The enzyme activity of immobilized lipase increased with increasing lipase loading rate. The most efficient immobilization was achieved at a resin addition of 0.25 g/mL, after which the specific activity decreased with increasing lipase loading rate, indicating that the lipase loading rate saturated at a specific concentration, which is consistent with the results of other studies. Adsorption time of the resin plays an important role during the immobilization process (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The highest specific enzyme activity of immobilized lipase was achieved at 6 h, which was 13180.6 PLU/g. After that, there was no significant change in specific enzyme activity with increasing immobilization time, which may be attributed to the fact that the immobilized carriers reached its maximum loading at 6 hours. Therefore, 6 hours was chosen as the immobilization time.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Optimization of organic polymer coating procedure\u003c/h2\u003e \u003cp\u003eThe thickness of the coating and the pore size of the coating surface would affect the enzyme activity and protein leakage rate of the immobilized enzyme, therefore, the organic polymer coating was optimized, including the type of organic polymer, organic polymer concentrations, and organic solvents.\u003c/p\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1 Selection of organic polymers\u003c/h2\u003e \u003cp\u003eThe present study involved the selection of organic polymers (PSF, PS, PVDF, CA, CAB, and PMMA). These polymers were chosen due to their possession of desirable properties such as thermal stability, chemical resistance, and biocompatibility. Hence, this experiment aimed at investigating the impact of six distinct organic polymers on both the catalytic efficiency of immobilized CALB and the rate of protein leakage. The results indicate that the immobilized enzyme exhibited the greatest catalytic efficiency when cellulose acetate butyrate was employed as a coating, yielding a value of 12685.5 PLU/g (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The observed phenomenon can be attributed to the more homogeneous dispersion of the coating over the resin when cellulose acetate butyrate was employed, as well as the moderate pore size of the coating, which results in less resistance to mass transfer. The polymer covering composed of cellulose acetate butyrate demonstrated the lowest protein leakage rate of 2.1%, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eB. According to the figure, it can be observed that there is a correlation between the protein leakage rate and the catalytic activity of the immobilized enzyme. Specifically, when the protein leakage rate increases, the catalytic activity of the immobilized enzyme decreases. This finding suggests that the protein leakage rate is a significant component influencing the catalytic activity of the immobilized enzyme. This is because the leaked protein is mainly a lipase with catalytic activity, and the leakage of the enzyme leads to a decrease in the overall catalytic activity of the immobilized enzyme system. Cellulose acetate butyrate (CAB) is a derivative of cellulose, a highly abundant natural substance. CAB offers several advantages, including its environmentally friendly nature, complete degradability, ample reserves, and renewability. Consequently, CAB is a cost-effective and sustainable polymer. After careful consideration of several factors, CAB was selected as the preferred coating material.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2 Optimization of organic polymer concentration\u003c/h2\u003e \u003cp\u003eThe concentration of the organic polymer has a direct impact on both the thickness of the coating and the pore size of the coated surface. This change affects the mass transfer resistance and ultimately affects the catalytic activity of immobilized enzymes Thus, the selection of an optimal concentration of organic polymer can effectively regulate the flow of both the substrate and product within the coating, while also intercepting protein molecules released from the resin. This interception mechanism contributes to a reduction in the rate of protein leakage. Therefore, an experimental investigation was conducted to examine the impact of organic polymer concentration on both the catalytic efficiency of immobilized CALB and the rate of protein leakage. (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The immobilized enzyme activity exhibited two peaks (at concentrations of 40 g/L and 80 g/L) in response to variations in organic polymer concentration. This may be due to the insufficient protection provided by the low organic polymer concentration during the coating process prior to reaching the 40 g/L threshold. Consequently, the enzyme protein in the immobilized enzyme experienced heightened vulnerability to damage from the organic solvent, potentially leading to elution. So at low organic polymer concentration, the activity of the immobilized lipase was even about 15% lower than that of the uncoated immobilized lipase (13180.6 PLU/g). As the concentration of organic polymer increased, the outer layer of immobilized lipase is protected by polymer coatings, and the damage of organic solvent on the immobilized lipase gradually decreased, reaching the first peak of enzyme activity (12,643.4 PLU/g) at an organic polymer concentration of 40 g/L. The main reason for the subsequent effect on the enzymatic activity of the immobilized enzyme may be that the organic polymer concentration changed the homogeneity, thickness, and pore size of the coating, reaching a second peak of enzyme activity (12685.5 PLU/g) at an organic polymer concentration of 80 g/L. As the concentration of organic polymer increased, the protein leakage rate of the immobilized enzyme decreased. At the organic polymer concentration of 100 g/L, the protein leakage rate was reduced to 2%. However, the activity of the immobilized lipase was found to be low due to the increased concentration leading to the formation of a thicker coating, smaller pore size, and greater mass transfer resistance. At the organic polymer concentrations of 40 g/L and 80 g/L, it was seen that the immobilized lipase activity remained comparable. However, it is noteworthy that the protein leakage rate was further reduced at the higher concentration of 80 g/L, specifically measuring at 2.1%. This suggests that the immobilized enzyme exhibits an extended operational lifespan when the concentration of organic polymer is at 80 g/L. On balance, a concentration of 80 g/L was chosen for CAB.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.2.3 Selection of organic solvents\u003c/h2\u003e \u003cp\u003eThe impact of organic solvents on immobilized CALB primarily stems from the varying solubility of organic polymers in various organic solvents, as well as the distinct distribution patterns of organic polymers on the resin surface after the phase transition of the coating. Furthermore, there exists variation in the toxicity levels of diverse organic reagents towards the protein of the immobilized enzyme. Therefore, the effects of organic solvent types on the catalytic efficiency of immobilized CALB and the protein leakage rate were investigated, and the results are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The catalytic activity of the immobilized enzyme was influenced by different organic reagents, among which the highest catalytic activity of 12685.5 PLU/g was obtained when acetone was used as the organic solvent, which was 1.26 times higher than that of Novozym 435. In terms of protein leakage rate, different organic reagents had little effect on it. Hence, acetone was chosen as the organic solvent for cellulose acetate butyrate.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of organic solvents on the enzymatic activity and protein leakage rate of the immobilized lipase CALB.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOrganic solvent\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpecific enzyme activity (PLU/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eProtein leakage rate (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e8529.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMSO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e9224.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTetrahydrofuran\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e9783.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\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=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e12685.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\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\u003eFernandez et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] used polyethyleneimine and dextran sulfate coating of CALB immobilized on octylated agarose gels, which resulted in a significant increase in stability, this could be attributed to the fact that the coating caused physical intermolecular cross-linking of the CALB molecules with the polymer, reducing the desorption of the enzyme from the carrier. This further demonstrated that polymer coatings do improve the stability of immobilized enzyme. In addition, the esterification activity and protein leakage rate of the immobilized CALB (enzyme-loaded resin), CALB@resin-CAB, Novozym 435, and coated Novozym 435 were characterized (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). The specific enzyme activity of immobilized CALB was higher than Novozym 435, about 1.26 times the esterification activity of Novozym 435. After organic polymer coating, the protein leakage rate of the enzyme was reduced to one-quarter of the pre-coating rate. This demonstrates that the use of organic polymer coatings may be a successful strategy for mitigating protein leakage in immobilized enzymes.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Characterization of the immobilized lipase CALB@resin-CAB\u003c/h2\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1 SEM analysis\u003c/h2\u003e \u003cp\u003eCharacterization of the surfaces of resin XRZ04B, enzyme-loaded resin XRZ04B, and CALB@resin-CAB by scanning electron microscopy to derive their microscopic surface variations. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eB show the microscopic surface changes of the macroporous resin XRZ04B before and after the adsorption of CALB with a magnification of 150, 000. It can be seen that the former surface is quite rough and has larger pores. While the latter surface is smoother and the pores have been partially filled by protein molecules. It indicates that the lipase was successfully adsorbed onto the macroporous resin XRZ04B. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eD show the microscopic surface changes before and after the coating of the enzyme-carrying resin XRZ04B with a magnification of 25, 000. It can be seen that an organic polymer coating was successfully formed on the surface of the carrier, and the thickness of the coating was about 0.5% of the radius of the resin XRZ04B. This coating structure can effectively reduce the leakage rate of protein on the carrier while ensuring the catalytic activity of the immobilized enzyme.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2 EDS analysis\u003c/h2\u003e \u003cp\u003eThe surface of resin XRZ04B, enzyme-loaded resin XRZ04B, and CALB@resin-CAB were characterized by EDS elemental analysis to derive the variation of nitrogen on their microscopic surfaces leading to the distribution of enzyme proteins on the resin (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-C\u003c/b\u003e). Comparing the changes in nitrogen density on the resin surface before and after immobilization showed that CALB was successfully immobilized on the carrier.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e3.3.3 Thermogravimetric analysis\u003c/h2\u003e \u003cp\u003eTG analysis was performed on resin XRZ04B, enzyme-loaded resin XRZ04B, and CALB@resin-CAB, respectively. TG analysis was performed under dry nitrogen to reduce the oxidation products that may affect the experimental results. Degradation or depolymerization of the resin and protein starts at 250 ℃, the main degradation step is at 350 ℃, and the degradation ends at 450 ℃ (\u003cb\u003eFig. S2\u003c/b\u003e). The thermal distribution of degradation was not significantly different for the three samples, and the relative masses of the three samples were slightly different from 30 to 200 ℃. This is because the change in weight in this temperature interval is mainly due to the moisture in the samples, including the bound water of the molecules. The main temperature range for sample weight loss was from 200\u0026ndash;600 ℃, which was attributed to the removal of organic components (C, H, O, and N) in this temperature range. The heat loss curves of the enzyme-loaded resin XRZ04B and the CALB@resin-CAB were smoother due to the adsorption of lipase by XRZ04B and the coating of an organic polymer on the resin surface. All three samples were finally charred at 800 ℃. About 1.1% of the residue of the carrier XRZ04B was in the form of char, about 3.9% of the residue of the carrier resin XRZ04B, and about 5.5% of the residue of the CALB@resin-CAB. From the relative weights of the final residues of the three samples, it is known that the enzyme protein was successfully immobilized on the resin as well as the organic polymer coating was successfully applied to the resin surface.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003e3.3.4 Fluorescent labeling analysis\u003c/h2\u003e \u003cp\u003eFirst, the free CALB was fluorescently labeled using the fluorescent dye fluorescein isothiocyanate (FITC); then, the labeled lipase was adsorbed using a macroporous resin; finally, the prepared enzyme-loaded resin was observed under a fluorescence microscope. The fluorescence of the immobilized CALB surface (\u003cb\u003eFig. S3A-B\u003c/b\u003e) shows that the CALB is uniformly distributed on the resin surface. The distribution of CALB inside the resin can be seen from the fluorescence of the immobilized CALB cross section. CALB was mostly immobilized on the outer surface of the resin, with less distribution inside the resin. Therefore, the organic polymer coating of the enzyme-loaded resin XRZ04B can effectively prevent the leakage of CALB on the outer surface of the resin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003e3.3.5 Pore size distribution and BET specific surface area analysis\u003c/h2\u003e \u003cp\u003eThe changes in pore size and specific surface area of the resin and immobilized CALB before and after immobilization were analyzed. The N2 adsorption-desorption isotherm (\u003cb\u003eFig. S4A-C\u003c/b\u003e) of the carrier XRZ04B belongs to the type II isotherm feature, which indicates that XRZ04B is a macroporous material. Due to the adsorption of CALB and the pore structure of the organic polymer coating, there was a significant decrease in the BET specific surface area of the enzyme-loaded resin XRZ04B and an increase in the BET specific surface area after the organic polymer coating. As shown in \u003cb\u003eTable S2\u003c/b\u003e, the BET specific surface areas of resin XRZ04B, enzyme carrier XRZ04B, and CALB@resin-CAB were 161.17, 3.24 and 34.00 m\u003csup\u003e2\u003c/sup\u003e\u0026middot;g\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, respectively; the pore capacities were 0.44, 0.01 and 0.09 cm\u003csup\u003e3\u003c/sup\u003e\u0026middot;g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. As shown in \u003cb\u003eFig. S5\u003c/b\u003e, the pore size of the carrier XRZ04B was 20\u0026ndash;50 nm and the most probable pore size was 22.49 nm; after adsorption of CALB, the pore size became 10\u0026ndash;20 nm and the most probable pore size was 16.04 nm; after organic polymer coating, the pore size increased to 15\u0026ndash;30 nm and the most probable pore size was 21.13 nm. The most probable pore size is the one with the highest probability of occurrence in the sample. Based on the BET analysis chart, we infer that the decrease in specific surface area in the first stage is due to the enzyme occupying the pores on the surface of the macroporous resin after adsorption, completing the immobilization process of the enzyme in the first step; The increase in specific surface area in the second stage is due to the use of ethyl acetate cellulose coating, which endows the surface of the macroporous resin after adsorbing enzymes with a porous polymer protective layer. This protective layer not only prevents enzyme leakage, but also reduces substrate mass transfer limitations, allowing the immobilized enzyme system to achieve higher catalytic activity.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Thermal stability of immobilized lipase CALB@resin-CAB\u003c/h2\u003e \u003cp\u003eIn this study, the thermal stability of CALB@resin-CAB was investigated by incubating the immobilized lipase in tert-butanol at different temperatures (50\u0026deg;C, 60\u0026deg;C and 70\u0026deg;C) for different times. CALB@resin- CAB maintained a high residual activity with increasing temperature (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The decrease of the residual enzyme activity of CALB@resin-CAB accelerated with the increase of storage temperature. The thermal stability of CALB@resin-CAB was satisfactory at 50\u0026deg;C and 60\u0026deg;C, and the relative enzyme activity remained at about 100% after 10 h of storage; the residual enzyme activity was still about 88% after 10 h of storage at 70\u0026deg;C, which indicated CALB@resin-CAB showed excellent thermal stability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Immobilized lipase CALB catalyzed production of biodiesel from palm acidified oil\u003c/h2\u003e \u003cdiv id=\"Sec29\" class=\"Section3\"\u003e \u003ch2\u003e3.5.1 Conditions optimization of lipase-catalyzed biodiesel production\u003c/h2\u003e \u003cp\u003eImmobilized lipase CALB@resin-CAB was applied to the biocatalyzed production of biodiesel by using palm acidified oil as the starting material, and the key affecting factors of lipase-catalyzed process were optimized. The optimal conditions for the reaction were determined as follows (\u003cb\u003eFig. S6A-C\u003c/b\u003e): reaction temperature of 50\u0026deg;C, a molar ratio of oil to alcohol of 1:3 (\u003cb\u003eTable S3\u003c/b\u003e), organic solvent tert-butanol with concentration of 20% (v/v).\u003c/p\u003e \u003cp\u003eAs illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e7\u003c/span\u003e, after the first enzyme-catalyzed reaction (12 h), the conversion of substrate reached a level of 91.2% with an acid value of 17.5. This phenomenon occurred due to the generation of a substantial quantity of water and a little quantity of glycerol, which would inhibit the continuation of the reaction [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In order to enhance the conversion of the substrate, a further enzymatic reaction was conducted on the resulting product subsequent to the first reaction. Firstly, the reaction was left to stratify to remove large amounts of water and glycerol; subsequently, the remaining reaction mixture was subjected to spin evaporation to eliminate any leftover water content. Lastly, methanol and CALB@resin-CAB were reintroduced to sustain the progression of the reaction. As can be seen from Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e7\u003c/span\u003e, the substrate conversion was enhanced to 98.5% in the second reaction catalyzed by the enzyme, resulting in an acid value of 3.1. Therefore, the CALB@resin-CAB exhibits an outstanding level of catalytic activity in the enzymatic process involved in the synthesis of biodiesel from fats and oils with high acid values.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section3\"\u003e \u003ch2\u003e3.5.2 Batch stability of the immobilized lipase CALB@resin-CAB\u003c/h2\u003e \u003cp\u003eExcellent batch stability is essential for the application of immobilized enzymes in industrial production [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Therefore, the batch stability of CALB@resin-CAB for the synthesis of biodiesel from palm acidified oil was investigated (\u003cb\u003eFig. S7\u003c/b\u003e). The results showed that CALB@resin-CAB exhibited good batch stability during the enzymatic production of biodiesel using palm acidified oil. The enzyme activity of CALB@resin-CAB remained close to the initial enzyme activity value after 10 batches of enzyme-catalyzed reactions.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Immobilized CALB catalyzed biodiesel production in the packed bed reactor\u003c/h2\u003e \u003cp\u003eA continuous flow catalytic reaction was carried out in the packed bed reactor filled with CALB@resin-CAB under the optimized conditions. This packed bed reactor uses a continuous flow reaction mode with continuous feed and continuous discharge to simulate the form of immobilized lipase application in industrial production [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The effect of flow rate on the conversion of palm acidified oil was investigated by flowing a substrate flow rate of 1\u0026ndash;20 Bv/h through a packed bed reactor filled with immobilized lipase CALB@resin-CAB, respectively (\u003cb\u003eFig. S8\u003c/b\u003e). At a flow rate less than 4 Bv/h, the conversion of palm acidified oil reached a steady state and conversion of the substrate did not increase as the flow rate decreased. When the flow rate was larger than 4 Bv/h, the conversion of palm acidified oil decreased with increasing flow rate, and at a flow rate of 20 Bv/h, the conversion of palm acidified oil decreased to about 80%. Therefore, a substrate flow rate of 4 Bv/h was selected, at which time the conversion of palm acidified oil was 94.8%, and the space-time yield of biodiesel was 2.88 g/(mL∙h).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn this study, a novel CALB immobilization method based on macroporous resin adsorption and organic polymer coating was developed. The immobilized lipase CALB@resin-CAB was applied to the biocatalyzed production of biodiesel with acidified palm oil as the starting material and the conversion rate reached 98.5%. Moreover, the immobilized lipase showed excellent batch stability in biodiesel production. When the aforementioned biocatalytic reaction was carried out in a continuous flow packed-bed system, the yield of biodiesel was 94.8% and space-time yield was 2.88 g/(mL∙h). In conclusion, the immobilized lipase CALB@resin-CAB has high catalytic activity and stability, which has good prospects for industrial applications in enzyme-catalyzed biodiesel production.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJiale Liu: Resources, Conceptualization, Methodology, Investigation, Validation, Data curation, Formal analysis, Writing - original draft. Shufan Zhao: Investigation, Validation, Data curation, Formal analysis, Writing - original draft. Wan Wei: Formal analysis, Validation. Shupeng Yu: Formal analysis, Validation. Zhao Wang: Conceptualization, Software, Supervision, Writing - review \u0026amp; editing. Jianyong Zheng: Data curation, Formal analysis, Writing - review \u0026amp; editing, Project administration.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis research was financially supported by the technology innovation and application development special key project of Chongqing (cstc2021jscx-jbgsX0002).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data are available from the corresponding author on reasonable request.\u003c/p\u003e\u003cp\u003eAppendix A. Supplementary data\u003c/p\u003e\n\u003cp\u003eE-supplementary data of this work can be found in online version of the paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRavichandran P, Rajendran N, Al-Ghanim KA, Govindarajan M, Gurunathan M (2023) Investigations on evaluation of marine macroalgae \u003cem\u003eDictyota bartayresiana\u003c/em\u003e oil for industrial scale production of biodiesel through technoeconomic analysis. Bioresour Technol 374:128769\u003c/li\u003e\n\u003cli\u003eVimali E, Gunaseelan S, Devi VC, Mothil S, Arumugam M, Ashokkumar B, Moorthy IMG, Pugazhendhi A, Varalakshmi P (2022) Comparative study of different catalysts mediated FAME conversion from macroalga \u003cem\u003ePadina tetrastromatica\u003c/em\u003e biomass and hydrothermal liquefaction facilitated bio-oil production. 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Catalysts 12:404\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":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bioprocess-and-biosystems-engineering","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Bioprocess and Biosystems Engineering](https://www.springer.com/journal/449)","snPcode":"449","submissionUrl":"https://submission.nature.com/new-submission/449/3","title":"Bioprocess and Biosystems Engineering","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Immobilization, Lipase, Organic polymer, Coating, Biodiesel","lastPublishedDoi":"10.21203/rs.3.rs-4922648/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4922648/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLipase is one of the most widely studied and applied biocatalysts. Due to the high enzyme leakage rate of the immobilization method of physical adsorption, we propose a new lipase immobilization method, which based on the combination of macroporous resin adsorption and organic polymer coating. The immobilized \u003cem\u003eCandida antarctic\u003c/em\u003e lipase B (CALB@resin-CAB) was prepared by combining the macroporous resin adsorption with cellulose acetate butyrate coating, and its structure was characterized by various analytic methods. Immobilized lipase was applied for biodiesel production using acidified palm oil as the starting material, the conversion rate achieved as high as 98.5% in two steps. Furthermore, immobilized lipase displayed satisfactory stability and reusability in biodiesel production. When the aforementioned reaction was carried out in a continuous flow packed-bed system, the yield of biodiesel was 94.8% and space-time yield was 2.88 g/(mL∙h). CALB@resin-CAB showed high catalytic activity and stability, which has good potential for industrial application in the field of oil processing.\u003c/p\u003e","manuscriptTitle":"Improved catalytic stability of immobilized Candida antarctic lipase B on macroporous resin with organic polymer coating for biodiesel production","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-16 10:22:23","doi":"10.21203/rs.3.rs-4922648/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-23T13:29:20+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-22T10:06:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"200137433971602942565940458580788276574","date":"2024-09-06T00:05:25+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-05T23:59:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-19T08:06:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-19T06:25:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Bioprocess and Biosystems Engineering","date":"2024-08-16T05:42:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bioprocess-and-biosystems-engineering","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Bioprocess and Biosystems Engineering](https://www.springer.com/journal/449)","snPcode":"449","submissionUrl":"https://submission.nature.com/new-submission/449/3","title":"Bioprocess and Biosystems Engineering","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"dd377ec8-224f-41c8-87c4-4c59a7b994d5","owner":[],"postedDate":"September 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-10-11T00:53:13+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-16 10:22:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4922648","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4922648","identity":"rs-4922648","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}