Bioresources and Bioprocessing
·
2025
·Vol. 12
·期 1
·1-16
·
IF 5.1
Li Yuhan,Wu Guowei,Liu Zeqing,Dai Lingmei,Liu Dehua,Du Wei
Mesoporous MIL-88A (Meso-MIL-88A) shows significant potential as an effective carrier for immobilizing large molecules such as lipases. This study investigates Meso-MIL-88A immobilized lipase for the catalysis of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) incorporation into soybean phosphatidylcholine. Phosphatidyl-EPA and phosphatidyl-DHA are known for their superior health benefits and have garnered significant global attention. Firstly, we developed a novel and green water-washing strategy to synthesize Meso-MIL-88A, demonstrating its significant potential as an effective carrier for immobilizing large molecules such as lipases. Nitrogen adsorption/desorption and XPS analyses revealed that water as the eluent yielded larger average pore diameters and lower sulfur residue content compared to ethanol. This optimized Meso-MIL-88A carrier was used to immobilize Candida antarctica lipase B (CalB@Meso-MIL-88A), which was applied to the production of phosphatidyl-EPA and phosphatidyl-DHA. The immobilized CalB@Meso-MIL-88A exhibited exceptional catalytic efficiency, achieving an unprecedented sn -1 positional incorporation rate of 86.8% (44.2% EPA, 42.7% DHA) with 90% EPA/DHA-ethyl ester (EE) donors, while 97% DHA-EE resulted in a record-high 90.1% DHA incorporation at the sn -1 position. Kinetic studies and molecular docking simulations indicated a higher substrate affinity for DHA-EE, attributed to enhanced transfer efficiency of EPA/DHA-EE in organic solvent system. This study presents the first demonstration of the potential of Meso-MIL-88A for industrial lipase immobilization via this optimized route, offering an eco-friendly and highly efficient catalytic application for nutraceutical synthesis. Graphical abstract
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FOOD CHEMISTRY
·
2026
·
IF 10.4
Jiawei Zheng,Xuan Hu,Jiufang Chen,Fengxia Lan,Li Xu,Zeping Lu,Binbin Nian,Nanjing Zhong
CALB@GA-NH 2 -UiO-66 exhibited 97.31% oleic acid conversion and 77.60% DAG content. • CALB@GA-NH 2 -UiO-66 retained full initial activity after 11 reuse cycles. • The immobilized CALB exhibited a single conformational basin. • Covalent enzyme–carrier conjugates were formed via Schiff base linkages. • The immobilization moderately restrained the flexibility of α-helix 5 of CALB. This study aimed to develop a robust biocatalyst for diacylglycerol (DAG) synthesis. Candida antarctica lipase B (CALB) was immobilized on NH₂-UiO-66 via glutaraldehyde (GA) cross-linking. The cross-linked CALB exhibited significantly better performance than the adsorbed counterpart. Oleic acid conversion reached 97.31% with DAG content up to 77.60%. Moreover, the immobilized CALB retained its full initial activity after 11 reuse cycles. The temperature dependence of oleic acid conversion followed the equation ln V₀ = 8.86–4.67 × 10 3 / T , with an activation energy of 38.83 kJ·Mol −1 . Molecular dynamics simulations revealed that the flexibility of α-helix 5 in CALB@GA-NH₂-UiO-66 is moderately restrained, and the immobilized CALB exhibits a single conformational basin. Additionally, immobilization induces slight contraction of the CALB active site pocket, and reduced length and increased bottleneck radius of the tunnel. This study provides molecular insights into the structure–stability relationship of immobilized enzymes, guiding the rational design of robust biocomposites We develop a robust biocatalyst via glutaraldehyde cross-linking of CALB onto NH₂-UiO-66 for DAG production by esterification. Download: Download high-res image (210KB) Download: Download full-size image
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ENZYME AND MICROBIAL TECHNOLOGY
·
2023
·
IF 3.4
Liwen Shu,Xin Zheng,Shuhua Qi,Shuizhi Lin,Yinghua Lu,Chuanyi Yao,Xueping Ling
Docosahexaenoic acid (DHA) enriched with phospholipids (PLs) (DHA-PLs) is a type of structured PL with good physicochemical and nutritional properties. Compared to PLs and DHA, DHA-PLs has higher bioavailability and structural stability and many nutritional benefits. To improve the enzymatic synthesis of DHA-PLs, this study investigated the preparation of phosphatidylcholine (PC) enriched with DHA (DHA-PC) via enzymatic transesterification of algal oil, which is rich in DHA-triglycerides, using immobilized Candida antarctica lipase B (CALB). The optimized reaction system incorporated 31.2% DHA into the acyl chain of PC and converted 43.6% PC to DHA-PC within 72 h at 50 °C, 1:8 PC: algal oil mass ratio, 25% enzyme load (based on total substrate mass), and 0.02 g/mL molecular sieve concentration. Consequently, the side reactions of PC hydrolysis were effectively suppressed and products with high PC content (74.8%) were produced. Molecular structure analysis showed that exogenous DHA was specifically incorporated into the sn-1 site of the PC by immobilized CALB. Furthermore, the evaluation of reusability with eight cycles showed that the immobilized CALB had good operational stability in the present reaction system. Collectively, this study demonstrated the applicability of immobilized CALB as a biocatalyst for synthesizing DHA-PC and provided an improved enzyme-catalyzed method for future DHA-PL synthesis.
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BIOTECHNOLOGY LETTERS
·
2019
·Vol. 42
·期 2
·269-276
·
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.
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ORGANIC & BIOMOLECULAR CHEMISTRY
·
2013
·Vol. 11
·期 41
·7192-7198
·
IF 3.568
Ru Jia,Yi Hu,Luo Liu,Ling Jiang,He Huang
Various imidazolium-functional ionic liquids (ILs) composed of different cations and anions were grafted onto Candida antarctica lipase B (CALB) through lysine coupling, and 4–6 of the 9 primary amino groups of the lysine residues were modified. The catalytic activity and stability were investigated in a p-nitrophenyl palmitate hydrolysis reaction. After modification, CALB was activated and achieved a high catalytic efficiency in the aqueous phase (e.g., [HOOCMMIm][Cl] modification showed a 1.5-fold increase in the catalytic efficiency). The thermostability and organic solvent tolerance were significantly increased with the ILs modification (e.g., [HOOCBMIm][Cl] owned kosmotropic cation and chaotropic anion modification showed a 7-fold thermostability increase at 70 °C, 1-fold increase in 50% aqueous dimethylformamide and 5-fold increase in 50% aqueous methanol). Conformation changes were confirmed by fluorescence spectroscopy, circular dichroism spectroscopy and attenuated total reflection Fourier transform infrared spectroscopy.
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Catalysts
·
2026
·Vol. 16
·期 5
·377
·
IF 4.5
Sining Wei,Mahwish Aziz,Yilin Zhang,Jian Xiong,Cheng Cheng,Bin Wu
This study addressed the issue of insufficient activity in CALB lipase during the catalytic synthesis of key chiral intermediates for moxifloxacin. A structure-guided protein engineering strategy was employed to systematically modify its functional domains. Through molecular dynamics simulations of CALB-I189K, multiple regions exhibiting high conformational flexibility were preliminarily identified. Subsequently, by integrating 3D structural alignment with active site pocket distance analysis, the functionally most critical region (143–146) was selected. A site-directed saturation mutation library was constructed specifically targeting this region. Building upon the previously reported CALB-I189K, a mutant I189K/L144R/A146K was ultimately obtained through high-throughput screening combined with chiral HPLC validation. This mutant maintains excellent stereoselectivity (E = 206.52) while enhancing catalytic efficiency (kcat/Κm) to 273.73 min−1·mM−1, approximately 4.5-fold that of I189K. At a substrate concentration of 1 M, it achieves 50% conversion within 2.6 h, demonstrating kinetic resolution capabilities approaching industrial standards. Molecular simulation analysis indicates that the L144R and A146K mutations synergistically enhance catalytic performance primarily by optimizing spatial distances between catalytic residues. This study not only provides a high-performance catalyst for the efficient biosynthesis of moxifloxacin chiral intermediates but also offers new insights for enzyme rational design based on dynamic structural information.
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Catalysis Science & Technology
·
2018
·Vol. 8
·期 18
·4718-4725
·
IF 5.365
Jiang-Wei Shen,Jia-Mei Qi,Xiao-Jian Zhang,Zhi-Qiang Liu,Yu-Guo Zheng
Candida antarctica lipase B (CALB) is a versatile and robust lipase with high activity and enantioselectivity in the resolution of alcohols and amines. Poor specific activity toward carboxylic esters that are branched or substituted in close proximity to the carboxyl group limits the application of CALB. In this study, a semi-rational engineering approach was successfully applied to tailor CALB for efficient synthesis of the moxifloxacin chiral intermediate through enzymatic resolution of cis-(±)-dimethyl 1-acetylpiperidine-2,3-dicarboxylate. The combinatorial active-site saturation test was performed on CALB and two key residue sites, 189 and 190, were identified in the determination of hydrolytic activity. The specific activity and catalytic efficiency (kcat/Km) of the best variant mut-I189K were increased by 286- and 193-fold, respectively, without impacting its enantioselectivity. With 0.1 g L−1 of purified mut-I189K loading, 1 M substrate was asymmetrically hydrolyzed with 49.9% conversion within 5 h, resulting in a space–time yield of 583.8 g L−1 d−1. These results demonstrated that mut-I189K is a competitive biocatalyst for large-scale preparation of the moxifloxacin chiral intermediate.
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BIOPROCESS AND BIOSYSTEMS ENGINEERING
·
2024
·
IF 3.5
Liu Jiale,Zhao Shufan,Wei Wan,Yu Shupeng,Wang Zhao,Zheng Jianyong
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, based on the combination of macroporous resin adsorption and organic polymer coating. The immobilized Candida antarctica 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, the 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). The immobilized lipase CALB@resin-CAB showed high catalytic activity and stability, which has good potential for industrial application in the field of oil processing.
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LANGMUIR
·
2026
·
IF 4.4
Yudan Chai,Bin Wang,Jun Xu,Jian Zhang
Microbial lipases are versatile biocatalysts that are essential for hydrolyzing ester bonds in triglycerides. Among these enzymes, Pseudomonas cepacia lipase (PCL) possesses a canonical lid domain, whereas Candida antarctica lipase B (CALB) features a rigid lid-like structure. Understanding how these different structures catalyze mechanisms differently at the interface is crucial. Experimental assays demonstrated high activity for both enzymes in aqueous systems, but CALB exhibited lower activity than PCL in biphasic water–organic solvent systems. Molecular dynamics simulations were employed to investigate the conformational dynamics and interfacial interactions. The results reveal that the PCL undergoes polarity-induced conformational changes leading to channel shortening and bottleneck widening, thereby facilitating substrate access. In contrast, CALB retains a rigid architecture with fixed distances between the lid-like region and the catalytic triad, thereby limiting interfacial adaptability. PCL further employs a lid-mediated adsorption mechanism that enhances interfacial binding and exposes hydrophobic residues. Conversely, CALB relies on an active-site-oriented approach with a weaker interfacial affinity. Additionally, the aqueous environment promotes lid closure in lipase PCL by stabilizing hydrogen bond networks and inducing conformational shifts in key flexible regions. This study elucidates polarity-driven interfacial activation mechanisms distinguishing lid-containing and lid-like lipases, offering atomistic insights for rational lipase engineering in nonaqueous environments.
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INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
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.
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ENZYME AND MICROBIAL TECHNOLOGY
·
2014
·
IF 2.966
Chuan-Yang Shang,Wei-Xun Li,Rui-Feng Zhang
ZnO nanowires were successfully introduced into a macroporous SiO 2 by in situ hydrothermal growth in 3D pores. The obtained composites were characterized by SEM and XRD, and used as supports to immobilize Candida antarctica lipase B (CALB) through adsorption. The high specific surface area (233 m 2 /g) and strong electrostatic interaction resulted that the average loading amount of the composite supports (196.8 mg/g) was 3–4 times of that of macroporous SiO 2 and approximate to that of a silica-based mesoporous material. Both adsorption capacity and the activity of the CALB immobilized on the composite supports almost kept unchanged as the samples were soaked in buffer solution for 48 h. The chiral resolution of 2-octanol was catalyzed by immobilized CALB. A maximum molar conversion of 49.1% was achieved with 99% enantiomeric excess of ( R )-2-octanol acetate under the optimal condition: a reaction using 1.0 mol/L ( R , S )-2-octanol, 2.0 mol/L vinyl acetate and 4.0 wt.% water content at 60 °C for 8 h. After fifteen recycles the immobilized lipase could retain 96.9% of relative activity and 93.8% of relative enantioselectivity .
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JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE
·
2025
·Vol. 105
·期 7
·3931-3941
·
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.
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