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Improved Catalytic Performance of Carrier-Free Immobilized Lipase by Advanced Cross-Linked Enzyme Aggregates Technology

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Improved Catalytic Performance of Carrier-Free Immobilized Lipase by Advanced Cross-Linked Enzyme Aggregates Technology Improved Catalytic Performance of Carrier-Free Immobilized Lipase by Advanced Cross-Linked Enzyme Aggregates Technology Xia Jiaojiao Jiangsu University of Science and Technology: Jiangsu University Yan Yan Jiangsu University Bin Zou ( [email protected] ) Jiangsu University https://orcid.org/0000-0003-3816-8776 Adesanya Idowu Onyinye Jiangsu University Research Article Keywords: lipase, carrier-free, immobilization, ionic liquid, stability Posted Date: May 24th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-505612/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/20 Abstract The cross-linked enzyme aggregates (CLEAs) are one of the technologies that quickly immobilize the enzyme without a carrier. This carrier-free immobilization method has the advantages of simple operation, high reusability and low cost. In this study, ionic liquid with amino group (1-aminopropyl-3- methylimidazole bromideIL) was used as the novel functional surface molecule to modify industrialized lipase (Candida rugosa lipase, CRL). The enzymatic properties of the prepared CRL-FIL-CLEAs were investigated. The activity of CRL-FIL-CLEAs (5.51 U/mg protein) was 1.9 times higher than that of CRL- CLEAs without surface modication (2.86 U/mg protein). After incubation at 60℃ for 50 min, CRL-FIL- CLEAs still maintained 61% of its initial activity, while the value for CRL-CLEAs was only 22%. After repeated use for ve times, compared with the 22% residual activity of CRL-CLEAs, the value of CRL-FIL- CLEAs was 51%. Further kinetic analysis indicated that the Km values for CRL-FIL-CLEAs and CRL-CLEAs were 4.80 mM and 8.06 mM, respectively, which was inferred that the anity to substrate was increased after modication. Based on the above results, it was indicated that this method provided a new idea for the effective synthesis of immobilized enzyme. Highlights Novel immobilized lipase was prepared with carrier-free immobilization method. The aggregates of ionic liquid and lipase proved to be an ecient catalyst. The activity and stability of carrier-free immobilized lipase were improved. The activity of the novel aggregates reached 1.9 times of conventional lipase. 1. Introduction Lipase (EC 3.1.1.3) is a very unique biocatalyst with interface activation mechanism [1]. Its active center at the hydrophobic end is a catalytic triad composed of serine, histidine and aspartic acid. Usually, the active center is covered by a polypeptide chain lid. When the lipase is at hydrophobic interface, the lid opens and the active center exhibits catalytic activities [2]. Among them, Candida rugosa lipase (CRL) is widely applied in bioengineering industries, such as the production of fatty acids and the synthesis of various esters. However, the low stability limited the application of free CRL in catalytic reactions. Immobilization technique is an effective way to solve these problems. After immobilization, the enzymatic properties regarding to the storage stability, reusability and resistance to organic solvents are signicantly improved [3]. Unfortunately, common immobilization methods usually require the assistance of external carriers. Inert carriers often hinder the contact between the lipase and the substrate, thereby affecting the reaction rate and reducing production capacity. In addition, additional immobilization carrier also increased the cost of immobilized lipase. In recent years, carrier-free immobilization technology has been rapidly developed. Lipase can be self- immobilized through aggregation and cross-linking to form water-insoluble aggregates under mild Page 2/20 conditions [4]. Carrier-free immobilized lipase do not require expensive carriers, which greatly reduces the cost. The lipase molecules are connected by cross-linking agent, the reusability could be improved. At the same time, the particles of carrier-free immobilized lipase are generally smaller, and the dispersion in the solvent is more uniform. Carrier-free immobilization technologies mainly include cross-linked enzyme crystals (CLECs) technology [5] and cross-linked enzyme aggregates (CLEAs) technology [6]. Although CLECs have excellent operational stability, the high purity requirements of crystallase lead to enhanced costs [7]. CLEAs do not require high-purity lipases, the preliminary purication could be integrated with immobilization process simultaneously. Therefore, CLEAs have more broad application prospects. Many different enzymes such as esterase [8], lipase [9], cellulase [10] and xylanase [11] have been immobilized by CLEAs technology and used in various biocatalytic applications. However, when there are fewer amino groups on the surface of the lipase protein, the cross-linking eciency may decrease. At the same time, prolonged exposure to cross-linking agent will damage the lipase activity. By adding more functional molecules with amino groups (for example: bovine serum albumin) in lipase cross-linking process, the loss of lipase activity can be reduced. Simultaneously, using functional molecules with amino groups to modify the surface of the lipase could affect the opening degree of the "lid" and speed up the substrate to enter the active center. Razib et al. studied the effect of adding bovine serum albumin (BSA) in CLEAs process [12].The results showed that the recovery activity of CLEA-elastase-SB after adding BSA was 80%, while the recovery activity of CLEA-elastase was only 60%. The CLEA immobilization of elastase enhanced the stability of the enzyme at high temperature and at a broader pH. In this work, the industrialized lipase (Candida rugosa lipase, CRL) was selected as the research object. Based on excellent biocompatibility of ionic liquid solvent to lipase[13, 14], a novel ionic liquid with amino groups (1-aminoethyl-3 -methylimidazole bromide) was chosen as a modier. Surface chemical modication of the lipase was carried out simultaneously with CLEAs preparation process (Fig. 1). Compared to CRL-CLEAs without modify agent, the effects of reported modier BSA and new ionic liquids on the performance of CRL-CLEAs were studied. The differences in thermal stability, pH stability, organic solvent stability, and storage stability of CRL-CLEAs, CRL-BSA-CLEAs and CRL-FIL-CLEAs are discussed. FT-IR and SEM were used to characterize above lipase to explore the mechanism of improved lipase performance. This study is expected to enhance the activity and stability of CRL low-costly. 2. Experiment 2.1 materials P-nitrophenyl palmitate (PNPP) was purchased from Aladdin (Shanghai, China). Candida rugosa lipase (CRL, type VII, L-1754) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (USA) and stored at 4 ℃.1-aminoethyl-3-methylimidazole bromide were purchased from LanZhou Greenchem ILs, Ammonium sulfate, Anhydrous dimethyl sulfoxide (DMSO), isopropanol (HPLC grade), ammonium Page 3/20 sulfate, sodium dihydrogen phosphate, and disodium hydrogen phosphate were purchased from SCRC (China). All other reagents used in the experiment were of analytical grade. 2.2 Preparation of immobilized CRL 2.2.1 Preparation of CRL-CLEAs Ammonium sulfate (0.56 g) and 1 mL lipase solution (100 mg/mL) were mixed in a 2 mL centrifuge tube and allowed to stand for 30 mins until the lipase protein precipitated. 100 µl of glutaraldehyde (5%, v/v) was added to the centrifuge tube, and cross-linked in a shaker for 1 h at room temperature. The centrifuge tube was centrifuged at 10,000 rpm for 5 mins. The supernatant was removed, and washed with phosphate buffer solution (PBS, pH 7.5). The precipitate was retained for activity determination of lipase. 2.2.2 Preparation of CRL-BSA-CLEAs 1 mL of lipase solution (100 mg/mL) was thoroughly mixed with 0 mg, 5 mg, 20 mg and 50 mg BSA respectively in a 2 mL centrifuge tube. Ammonium sulfate (0.56g) was added to the centrifuge tube and mixed solution, allowed to stand for 30 mins until the lipase protein precipitated. The other steps are the same as Sect. 2.2.1. 2.2.3 Preparation of CRL-FIL-CLEAs 1 mL of enzyme solution (100 mg/mL) was thoroughly mixed with 100 mg, 200 mg, 300 mg, 400 mg, 500 mg 1-aminoethyl-3-methylimidazole bromide respectively in a 2 mL centrifuge tube. The other steps are the same as Sect. 2.2.2. 2.3 Activities assay of immobilized lipase In a water bath shaker at 40°C, 0.03g PNPP was dissolved in 5 ml isopropanol solution. The obtained solution was stored at 4°C. PNPP isopropanol solution (16.5 mmol/L) and PBS (pH 7.5) were mixed into a milky white substrate solution at a ratio of 1: 9 (v/v). 0.02 g sample and 0.5 mL p-NPP substrate solution were added to a 2 mL centrifuge tube. The mixture was reacted at 35℃ and 150 rpm for 10 mins, and then placed in a centrifuge at 12,000 rpm for 5 mins. 250 µL of the diluted supernatant was pipetted onto the microplate. The OD value was measured at 405 nm by a microplate reader (type: Tecan Innite 200 Pro).The enzyme activity was dened as the protein amount of lipase required to catalyze the hydrolysis of PNPP to 1 µmol of PNP per minute at 35°C and pH 7.5. Enzymatic activity = (A1-A0 + C0)/K (µmol/ml) × V1 (mL) × n / [t (min) × M (g)] Where A1 and A0 represented the OD value of the lipase sample and control solution, respectively. K and C0 were the slope and intercept of the PNP standard curve, respectively. V1 was the volume of the reaction solution (mL). n was the dilution factor. t was the reaction time (min). M was the mass of immobilized lipase in the reaction medium (g). Page 4/20 2.4 Effect of modier amount and reaction temperature on lipase activity At 35℃, the enzyme activity of free CRL, CRL-CLEAs, and CRL-BSA-CLEAs and CRL-FIL-CLEAs with different modiers content (BSA and FIL) was determined.
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