RSC Advances
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2018
·Vol. 8
·期 72
·41247-41254
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IF 2.936
Jiong-feng Zhao,Zhe Wang,Fei-long Gao,Jian-ping Lin,Li-rong Yang,Mian-bin Wu
Rhizopus oryzae lipase (ROL) is important because of its extreme sn-1,3-regioselectivity, but it shows poor thermostability, which severely restricts its application. In this work, the thermostability of ROL was greatly enhanced by rational design. First, several sites that may affect the thermostability of ROL were identified by multiple-sequence alignment. The half-lives of mutants V209L and D262G at 55 °C were about 4.38- and 4.2-times those of the wild-type, respectively. Then, a disulfide bond was introduced between positions 190 and 238 based on the prediction of Disulfide by Design 2, which greatly improved the thermostability of the protein. The activity of variant E190C/E238C retained about 58.2% after incubation at 55 °C for 720 min, whereas the half-life of wild type ROL was only about 11.7 min. On the basis of the results obtained by the two methods, we carried out a combined mutation. Quadruple mutant V209L/D262G/E190C/E238C was constructed and the thermostability was improved even further. The half-lives at 55 °C and 65 °C were 102.5- and 20-times those of the wild-type ROL. This improvement in thermostability will give ROL wider industrial applicability, especially in the preparation of structured lipids.
研究领域:
生物化学
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酶工程
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PubMed
高影响因子
BIOTECHNOLOGY LETTERS
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2019
·Vol. 42
·期 2
·269-276
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IF 2.154
Cai Xinyong,Zhang Mengjie,Wei Wei,Zhang Yinjun,Wang Zhao,Zheng Jianyong
A novel enzyme immobilization method employing metal–organic framework (MOF) encapsulation and macroporous resin adsorption was developed in this study. Candida antarctica lipase B (CALB) was firstly encapsulated onto metal–organic frame structures (Zeolitic imidazole framework-8, ZIF-8) and further bonded to macroporous resin by physical adsorption. Under optimized immobilization conditions, the activity of the prepared immobilized lipase (CALB-ZIF-8@D101) determined via the methyl esterification of oleic acid was 38.4 U/mg. Compared with free lipase, the immobilized lipase exhibited improved thermal and operational stability and organic solvent tolerance. These results demonstrate that the immobilization method of ZIF-8 encapsulation and macroporous resin adsorption enhanced enzyme properties at a superior level.
研究领域:
生物化学
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酶工程
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材料科学
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PubMed
高影响因子
Frontiers in Bioengineering and Biotechnology
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2022
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IF 6.064
Xue Yu,Zhang Xiao-Guang,Lu Ze-Ping,Xu Chao,Xu Hua-Jin,Hu Yi
Various betaine ionic liquids composed of different chain lengths and different anions were designed and synthesized to modify Candida antarctica lipase B (CALB). The results showed that the catalytic activity of all modified lipases improved under different temperature and pH conditions, while also exhibiting enhanced thermostability and tolerance to organic solvents. With an increase in ionic liquid chain length, the modification effect was greater. Overall, CALB modified by [BetaineC16][H2PO4] performed best, with the modified CALB enzyme activity increased 3-fold, thermal stability increased 1.5-fold when stored at 70°C for 30 min, with tolerance increased 2.9-fold in 50% DMSO and 2.3-fold in 30% mercaptoethanol. Fluorescence and circular dichroism (CD) spectroscopic analysis showed that the introduction of an ionic liquid caused changes in the microenvironment surrounding some fluorescent groups and the secondary structure of the CALB enzyme protein. In order to establish the enzyme activity and stability change mechanisms of the modified CALB, the structures of CALB modified with [BetaineC4][Cl] and [BetaineC16][Cl] were constructed, while the reaction mechanisms were studied by molecular dynamics simulations. Results showed that the root mean square deviation (RMSD) and total energy of modified CALB were less than those of native CALB, indicating that modified CALB has a more stable structure. Root mean square fluctuation (RMSF) calculations showed that the rigidity of modified CALB was enhanced. Solvent accessibility area (SASA) calculations exhibited that both the hydrophilicity and hydrophobicity of the modified enzyme-proteins were improved. The increase in radial distribution function (RDF) of water molecules confirmed that the number of water molecules around the active sites also increased. Therefore, modified CALB has enhanced structural stability and higher hydrolytic activity.
研究领域:
生物化学
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酶工程
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PubMed
高影响因子
JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE
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2025
·Vol. 105
·期 7
·3931-3941
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IF 3.5
Jiawei Zheng,Qiangyue Zhang,Nanjing Zhong
BACKGROUND Immobilized enzyme possessing both high activity and good selectivity is important in practice. In this study, Candida antarctica lipase B (CALB) was immobilized onto the macroporous resin ADS-17 for triacylglycerol (TAG) synthesis through esterification of oleic acid and glycerol. The reaction conditions were optimized by single-factor study and orthogonal test, and the reusability of the immobilized CALB (CALB@ADS-17) was evaluated. In addition, the mechanism of lipase immobilization was studied and the catalytic mechanism of CALB@ADS-17 was investigated. RESULTS Oleic acid conversion up to 99.20% and TAG content at 91.58 wt% could be obtained under optimal conditions. In addition, the CALB@ADS-17 retained 84.28% of its initial activity after 11 cycles of reuse. The mechanism of lipase immobilization was through hydrophobic adsorption. The relationship between temperature and oleic acid conversion was ln V 0 = 6.3316 − 4.3321/ T , and the activation energy ( E a ) was 36.02 kJ mol −1 . CALB@ADS-17 did not exhibit an obvious interfacial activation phenomenon. Its kinetic behavior can be described by the Michaelis–Menten model, whose kinetic parameters of v max , k cat , K m , K i , and k ca t /K m were 0.01265 μmol L −1 s −1 , 9310.72 s −1 , 0.4907 mmol L −1 , 3.997 mmol L −1 , and 1.90 × 10 4 L mmol −1 s −1 , respectively. CONCLUSION CALB@ADS-17 showed good esterification performance and exhibited good selectivity towards TAG generation. In addition, CALB@ADS-17 exhibited good reusability in esterification reactions and has potential in practical applications. © 2025 Society of Chemical Industry.
研究领域:
生物化学
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酶工程
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高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2018
·
IF 3.909
Yue Li,Nanjing Zhong,Ling-Zhi Cheong,Jianrong Huang,Hongxiao Chen,Shaoyan Lin
In this study, SBA-15 was modified by a series of silane coupling reagents and later used to immobilize Candida antartica lipase B (CALB). The enzymatic properties of the immobilized CALB samples were studied. In addition, the catalytic performance in glycerolysis of soybean oil for diacylglycerols (DAG) production was also investigated. The highest enzymatic activity up to 6100.00 ± 246.41 U/g was observed from the propyl methacrylate group modified SBA-15 supported CALB. No loss of activity was observed from the propyl methacrylate group modified SBA-15 supported CALB, but a higher-than-initial activity was notably found from 3-aminopropyl group and n -octyl group modified SBA-15 supported CALB after a 4-h incubation in air at 70 °C. 1-isocyanatopropane group modified SBA-15 supported CALB exhibited selectivity for DAG production. DAG content up to 61.90 ± 2.38 wt% and a DAG/MAG ratio at 3.11 ± 0.08 was obtained after a 24-h reaction at 60 °C in a solvent-free system.
研究领域:
生物化学
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酶工程
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食品科学
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PubMed
高影响因子
CHEMICAL ENGINEERING JOURNAL
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2025
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IF 13.2
Qian Wang,Xinyuan Zheng,Meiling Ge,Rong Chen,Fangpei Ma,Jianming Pan,Fu Yang
Maintaining the long-term stability of flexible polymer-immobilized lipase remains a significant challenge in enzyme catalysis. The deformation of the polymer during the process often damages the lipase, severely limiting its practical application. In this work, we in situ constructed rigid Zeolitic Imidazolate Framework-8 (ZIF-8) in porous hydrophobic polymer PVIm as “rigid and flexible” composite PVIm/ZIF-8 through a self-assemble strategy. The implanted rigid ZIF-8 improves the network stability of the hydrophobic polymer PVIm. Additionally, it acts as a co-factor, leveraging defective Zn sites to immobilize lipase through bonding with its amino acid moieties. This approach represents a typical two-birds-with-one-stone strategy. The optimal biocatalyst CalA@PVIm/ZIF-8(0.3) affords the highest yield of biodiesel of 82 % with a TON of 136.6 g (biodiesel) /g (CalA) from waste cooking oil under 30 °C and 24 h. Compared to CalA@PVIm/ZIF-8(0.3), the TON of free CalA is only 25 % of that value. Compared to the ZIF-8-free CalA@PVIm, CalA@PVIm/ZIF-8(0.3) exhibits near 1.2 folds enhancement of performance, further unveiling the co-factor role of ZIF-8 in the reaction. More importantly, this incorporation of ZIF-8 makes the resulted CalA@PVIm/ZIF-8(0.3) afford at least 44 days operation period and retains 83 % original activity after 16 running recycles, superior to almost all the reported immobilized lipase researches, far outperforming that (24.1 % activity after 16 cycles) of CalA@PVIm. The wide flexibility in various biodiesel synthesis can be further demonstrated, affording the practical application value of developed catalyst.
研究领域:
生物化学
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催化材料
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可再生能源
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高影响因子
BIORESOURCE TECHNOLOGY
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2019
·
IF 6.669
Konglong Feng,Zaocheng Huang,Bo Peng,Weijie Dai,Yunqi Li,Xiaoai Zhu,Yunjiao Chen,Xing Tong,Yaqi Lan,Yong Cao
Deacidification of high-acid soy sauce residue (SSR) oil is crucial to utilization of SSR oil. Aspergillus niger lipase (ANL) has been widely applied for such purpose while its immobilization still has large room for improvement. ANL was immobilized onto six different macroporous acrylic resins, accounting the effect of the different textural properties of resins on stability and their potential for application in enzymatic deacidification. The resin MARE with lower porosity, higher bulk density, and medium hydrophobicity, was chosen as the best carrier for the best thermostability and reusability. ANL-MARE is a promising catalyst than Novozym 40086, which not only exhibited higher deacidification activity and good thermostability, but also was continuously reused for 15 cycles and efficiently catalyzed from high-acid SSR oil into diacylglycerol-enriched oil. Therefore, immobilized ANL was a novel, low-cost and recyclable biocatalyst that could be used as a good alternative to higher-cost commercial lipases in industrial applications.
研究领域:
生物化学
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食品科学
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PubMed
高影响因子
BIOPROCESS AND BIOSYSTEMS ENGINEERING
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2022
·Vol. 45
·期 4
·749-759
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IF 3.434
Zhang Xiao-guang,Xue Yu,Lu Ze-ping,Xu Hua-jin,Hu Yi
In this study, a series of proline ionic liquids with different lengths of hydrophobic alkyl on the side chain were used to modify the Candida Antarctic lipase B (CALB). The catalytic activity, thermal stability and tolerance to methanol and DMSO of the modified enzyme were all improved simultaneously. The optimum temperature changed from 55 to 60 ℃. The hydrophobicity and anion type of the modifier have important influence on the catalytic performance of CALB. CALB modified by [ProC 12 ][H 2 PO 4 ] has a better effect. Under the optimal conditions, its hydrolysis activity was 3.0 times than that of the native enzyme, the catalytic efficiency Kcat/Km improved 2.8 times in aqueous phase, and the tolerance to organic solvent with strong polarity (50% methanol 2 h) was increased by 6.8 times. Fluorescence spectra and circular dichroism (CD) spectroscopy showed that the introduction of ionic liquids changed the microenvironment near the fluorophores of the enzyme protein, the α-helix decreased and β-sheet increased in the secondary structure of the modified enzymes. The root mean square deviation (RMSD), residue root mean square fluctuation (RMSF), radius of gyration (Rg), and solution accessible surface area (SASA) of [ProC 2 ][Br]-CALB, [ProC 12 ][Br]-CALB and native CALB were obtained for comparison by molecular dynamics simulation. The results of dynamics simulation were in good agreement with enzymology experiment. The introduction of ionic liquids can keep CALB in a better active conformation, and proline ionic liquids with long hydrophobic chains can significantly improve the surface hydrophobicity and overall rigidity of CALB. This research offers a new idea for rapid screening of efficient modifiers and provision of enzymes with high stability and activity for industrial application.
研究领域:
生物化学
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酶工程
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工业生物技术
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高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2024
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IF 7.7
Ruirui Li,Wei Zhuang,Yan Gao,Yahan Bai,Keke Zhang,Zhenfu Wang
Herein, UiO-66 was selected as the immobilization carrier of Rhizomucor miehei lipase (RML). After etching and hydrophobic modification, the functionalized UIO-66 (H-UIO-66-OPA) was utilized for RML immobilization and the obtained RML@H-UIO-66-OPA showed about 70 % relative activity after incubation at 60 °C, which was much better than RML (20 %). RML@H-UIO-66-OPA was used in the synthesis of 1,3-dioleic acid glycerol (1,3-DAG) and the effects of reaction conditions (temperature, enzyme addition, substrate molar ratio, and time) on 1,3-DAG yield were investigated. Under the optimal conditions (temperature of 65 °C, enzyme addition of 2 wt%, substrate molar ratio of 2:1, and reaction time of 4 h), 1,3-DAG yield reached 77 %. Finally, Box-Behnken experimental design method was utilized to optimize the process parameters for the preparation of 1,3-DAG, and the final 1,3-DAG content was 79.80 %, which demonstrated that the model could predict the relationship between the factors and be suitable for the process optimization in 1,3-DAG preparation.
研究领域:
生物化学
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酶工程
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PubMed
高影响因子
FOOD CHEMISTRY
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2023
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IF 8.8
Lili Yin,Kunpeng Gao,Xiangzhao Mao,Yang Hu
Lipase B from Candida antarctica (CALB) plays a prominent role as a biocatalyst in several industries, especially for biphasic conversion of functional lipids. Herein, an amphiphilic Janus halloysite nanosheet (JHNS) was fabricated and employed simultaneously as a solid surfactant for stabilizing Pickering emulsion and as a carrier for immobilizing CALB, with the aim to realize highly efficient biphasic bioconversion. The obtained JHNS could stabilize Pickering emulsion for at least 7 days. Immobilization of CALB on JHNS improved the substrate affinity, catalytic efficiency, thermal stability, and alkaline tolerance of the enzyme. Moreover, JHNS-based immobilized CALB was exploited as a biocatalytic platform for the conversion of retinyl acetate, with almost twice increase in conversion efficiency. Taken together, the JHNS-based immobilized CALB paves the way for the design of efficient biphasic conversion system for the production of added-value lipids.
研究领域:
生物技术
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化学工程
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材料科学
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PubMed
高影响因子
INDUSTRIAL & ENGINEERING CHEMISTRY RESEARCH
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2024
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IF 3.8
Jun Liang,Huiting Xu,Zhixi Zhang,Yaqian Bian,Qiang Zhang,Weiyi Su,Yuqi Hu,Chunli Li,Xiong Yu,Honghai Wang
Enzymatic processes coupled with continuous microfluidic techniques have significant application prospects in pharmaceuticals and fine chemicals. It is important to stably load enzymes in microreactors. This study reported a sol–gel method for preparing wall-coated immobilized enzyme microreactors (W-IMERs) by entrapment of Candida antarctica lipase B (CALB). The W-IMERs were simply constructed in one step by a flow coating process in a capillary. Compared with the free enzyme, the tolerance of immobilized enzyme to ethanol and temperature is significantly improved, and the yield of immobilized enzyme activity is 76.12% in the batch reactor. In W-IMERs, the yield of immobilized enzyme activity increased to 82.95%. The W-IMERs maintained stable activity in 10 cycles for up to 30 days. A CFD model was also established to explore the relationship between the internal reaction and the mass transfer in W-IMERs. The relationship between the internal and external diffusion limits of W-IMERs and the overall reaction rate was analyzed by dimensionless numbers. The results indicated that sol–gel flow coating is a simple and effective method for the preparation of enzymatic microreactors. An in-depth understanding of the relationship between diffusion limitation and reaction rate will have a significant impact on the overall performance of enzyme-catalyzed microreactors.
研究领域:
生物化学
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生物工程
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高影响因子
JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE
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2021
·Vol. 102
·期 5
·1812-1822
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IF 3.639
Wenyi Chen,Li Xu,Nanjing Zhong
BACKGROUND Enzymatic esterification is attracting for particular high-acid oil deacidification. In this study, Candida antarctica lipase B (CALB) was encapsulated into a series of nucleotide-hybrid metal coordination polymers (CPs), which were constructed by guanosine 5′-monophosphate (GMP) and various metals. RESULTS We here found that, most of the present CPs encapsulated CALB (CALB@CPs) samples were highly selective for esterification while poor in glycerolysis reaction. They exhibited quite poor performance in glycerolysis, with triacylglycerols (TAGs) conversion lower than 5%, despite this considerable enzymatic hydrolysis activities were observed. However, they (most of them) showed good performance in esterification of fatty acids and glycerol for TAG synthesis. In addition, the GMP/Tb (CPs constructed by GMP and Tb 3+ ) encapsulated CALB (CALB@GMP/Tb) transformed over 98% of oleic acid into glycerides in the high-acid oil deacidification process, and TAG content from 87 to 89% was obtained. Moreover, the CALB@GMP/Tb showed good reusability in the esterification system. CONCLUSION The present CALB@CPs samples are selective for esterification and suitable for high-acid oils deacidification. This work provides a new system for enzymatic selectivity improvement study. © 2021 Society of Chemical Industry.
研究领域:
生物化学
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酶工程
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催化化学
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高影响因子
危险品化学品经营许可证（带存储）