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Yu-guo Zheng - One of the best experts on this subject based on the ideXlab platform.
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efficient resolution of cis dimethyl 1 acetylpiperidine 2 3 dicarBoxylate By covalently immoBilized mutant candida antarctica <B>LipaseB> B in Batch and semicontinuous modes
Organic Process Research & Development, 2019Co-Authors: Jiang-wei Shen, Xiao-jian Zhang, Zhi-qiang Liu, Yu-guo ZhengAbstract:Candida antarctica <B>LipaseB> B (CALB) is a roBust Biocatalyst for production of chemicals. In this study, an engineered <B>LipaseB> B (CALB-I189K) was covalently immoBilized onto a glutaraldehyde activated amino resin (ESR-3) and used for the preparation of chiral moxifloxacin precursor though enzymatic resolution of cis-(±)-dimethyl 1-acetylpiperidine-2,3-dicarBoxylate (cis-(±)-1). The immoBilization conditions were optimized, and the immoBilized CALB-I189K exhiBited improved thermal staBility, with a half-life of 247.5 h at 30 °C. It enaBles the resolution of 100 g L–1 cis-(±)-1 for 50 cycles in a stirred tank reactor, with an average productivity of 49.6 g L–1 h–1. The resolution reaction was also performed in a recirculating packed Bed reactor (RPBR) in a semicontinuous mode, and the average productivity reached 59.4 g L–1 h–1 under the optimal operating condition. Moreover, the RPBR Bioconversion system has high operational staBility and conserved over 85.7% of the initial conversion capacity after 50 cycles...
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Efficient Resolution of cis-(±)-Dimethyl 1‑Acetylpiperidine-2,3-dicarBoxylate By Covalently ImmoBilized Mutant Candida antarctica <B>LipaseB> B in Batch and Semicontinuous Modes
2019Co-Authors: Jiang-wei Shen, Xiao-jian Zhang, Zhi-qiang Liu, Yu-guo ZhengAbstract:Candida antarctica <B>LipaseB> B (CALB) is a roBust Biocatalyst for production of chemicals. In this study, an engineered <B>LipaseB> B (CALB-I189K) was covalently immoBilized onto a glutaraldehyde activated amino resin (ESR-3) and used for the preparation of chiral moxifloxacin precursor though enzymatic resolution of cis-(±)-dimethyl 1-acetylpiperidine-2,3-dicarBoxylate (cis-(±)-1). The immoBilization conditions were optimized, and the immoBilized CALB-I189K exhiBited improved thermal staBility, with a half-life of 247.5 h at 30 °C. It enaBles the resolution of 100 g L–1 cis-(±)-1 for 50 cycles in a stirred tank reactor, with an average productivity of 49.6 g L–1 h–1. The resolution reaction was also performed in a recirculating packed Bed reactor (RPBR) in a semicontinuous mode, and the average productivity reached 59.4 g L–1 h–1 under the optimal operating condition. Moreover, the RPBR Bioconversion system has high operational staBility and conserved over 85.7% of the initial conversion capacity after 50 cycles. This efficient Bioconversion process demonstrated the potential of employing immoBilized CALB-I189K for industrial production of chiral moxifloxacin precursor
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enantioselective hydrolysis of diethyl 3 hydroxyglutarate to ethyl s 3 hydroxyglutarate By immoBilized candida antarctica <B>LipaseB> B
Journal of Molecular Catalysis B-enzymatic, 2010Co-Authors: Huaping Dong, Yajun Wang, Yu-guo ZhengAbstract:ABstract Optically pure ethyl (S)-3-hydroxyglutarate [(S)-3-EHG] is used as a key precursor for synthesis of a variety of pharmaceutically important compounds. In this work, we estaBlished an efficient procedure for enantioselectively hydrolyzing diethyl 3-hydroxyglutarate (3-DHG) to optically active (S)-3-EHG employing immoBilized Candida antarctica <B>LipaseB> B (Novozym 435). Under the optimized conditions: pH 7.0, agitation speed 200 rpm, temperature 40 °C, 3-DHG concentration 0.15 mol L−1, and enzyme loading 7 g L−1, (S)-3-EHG was prepared in aBove 95% ee value and 98.5% yield, and the reaction was free from external mass transfer and intra-particle diffusion limitations and kinetically controlled. The inhiBitions of suBstrate (3-DHG) and product (3-EHG) were excluded Because Both displayed no decline in activity at elevated concentrations within the given ranges. In addition, ethanol, a Byproduct of the reaction, inhiBited <B>LipaseB> B following an uncompetitive inhiBition pattern. The kinetic constants were oBtained through non-linear regression, with values of Vmax 1.29 mmol min−1 g−1, Km 0.06 mol L−1, and Ki 0.37 mol L−1, respectively.
Richard A. Gross - One of the best experts on this subject based on the ideXlab platform.
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candida antarctica <B>LipaseB> B chemically immoBilized on epoxy activated micro and nanoBeads catalysts for polyester synthesis
Biomacromolecules, 2008Co-Authors: Bo Chen, Elizabeth M Miller, Wenchun Xie, Minmin Cai, Richard A. GrossAbstract:Candida antarctica <B>LipaseB> B (CALB) was covalently immoBilized onto epoxy-activated macroporous poly(methyl methacrylate) AmBerzyme Beads (235 µm particle size, 220 A pore size) and nanoparticles (nanoPSG, diameter 68 nm) with a poly(glycidyl methacrylate) outer region. AmBerzyme Beads allowed CALB loading up to 0.16 g of enzyme per gram of support. IR microspectroscopy generated images of AmBerzyme−CALB Beads showed CALB is localized within a 50 µm thick loading front. IR microspectroscopy images, recorded prior to and after treatment of AmBerzyme−CALB with DMSO/aqueous Triton X-100, are similar, confirming that CALB is largely chemically linked to AmBerzyme. The activity of CALB immoBilized on AmBerzyme, Lewatit (i.e., Novozym 435 catalyst), and nanoPSG was assessed for lactone ring-opening and step-condensation polymerizations. For example, the percent conversion of ϵ-caprolactone using the same amount of enzyme catalyzed By AmBerzym−CALB, Novozym 435, and nanoPSG−CALB for 20 min was 7.0, 16, and 65%, r...
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effects of porous polystyrene resin parameters on candida antarctica <B>LipaseB> B adsorption distriBution and polyester synthesis activity
Langmuir, 2007Co-Authors: Bo Chen, And Elizabeth M Miller, Richard A. GrossAbstract:Polystyrene resins with varied particle sizes (35 to 350−600 μm) and pore diameters (300−1000 A) were employed to study the effects of immoBilization resin particle size and pore diameter on Candida antarctica <B>LipaseB> B (CALB) loading, distriBution within resins, fraction of active sites, and catalytic properties for polyester synthesis. CALB adsorBed rapidly (saturation time ≤ 4 min) for particle sizes ≤ 120 μm (pore size = 300 A). Infrared microspectroscopy showed that CALB forms protein loading fronts regardless of resin particle size at similar enzyme loadings (∼8%). From the IR images, the fractions of total surface area availaBle to the enzyme are 21, 33, 35, 37, and 88% for particle sizes 350−600, 120, 75, 35 μm (pore size 300 A), and 35 μm (pore size 1000 A), respectively. Titration with methyl p-nitrophenyl n-hexylphosphate (MNPHP) showed that the fraction of active CALB molecules adsorBed onto resins was ∼60%. The fraction of active CALB molecules was invariaBle as a function of resin particle an...
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effects of macroporous resin size on candida antarctica <B>LipaseB> B adsorption fraction of active molecules and catalytic activity for polyester synthesis
Langmuir, 2007Co-Authors: Bo Chen, Elizabeth M Miller, Lisa Karen Miller, John J Maikner, Richard A. GrossAbstract:Methyl methacrylate resins with identical average pore diameter (250 A) and surface area (500 m2/g) But with varied particle size (35 to 560−710 μm) were employed to study how immoBilization resin particle size influences Candida antarctica <B>LipaseB> B (CALB) loading, fraction of active sites, and catalytic properties for polyester synthesis. CALB adsorBed more rapidly on smaller Beads. Saturation occurred in less than 30 s and 48 h for Beads with diameters 35 and 560−710 μm, respectively. Linearization of adsorption isotherm data By the Scatchard analysis showed for the 35 μm resin that: (i) CALB loading at saturation was well Below that required to form a monolayer and fully cover the support surface and (ii) CALB has a high affinity for this resin surface. Infrared microspectroscopy showed that CALB forms protein loading fronts for resins with particle sizes 560−710 and 120 μm. In contrast, CALB appears evenly distriButed throughout 35 μm resins. By titration with p-nitrophenyl n-hexyl phosphate (MNPHP),...
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solvent free adipic acid 1 8 octanediol condensation polymerizations catalyzed By candida antartica <B>LipaseB> B
Macromolecules, 2004Co-Authors: Anil Mahapatro, Bhanu Kalra, Ajay Kumar, Richard A. GrossAbstract:Bulk condensation polymerizations of adipic acid and octanediol, catalyzed By Candida antartica <B>LipaseB> B (CALB), were investigated. The polymers formed By 8 and 24 h polymerizations using CALB immoBilized on Accurel and Lewatit had similar molecular weights (e.g., Mn at 24 h ≈15 000). CALB “free” of the immoBilization resin was also active for the polymerization But, relative to its immoBilized forms, gave slower chain growth (Mn ≈ 10 000 By 48 h). For all three catalyst systems at degree of polymerization (DP) ≥ 20, dispersity (Mw/Mn) was ≤1.5. Since random processes of step-growth condensation polymerizations give dispersity values ≥ 2, the dispersity of products oBtained using CALB as the catalyst is Believed to result from the unique chain length or mass selectivity of the <B>LipaseB>. Gel permeation chromatograms showed that Between 15 min and 4 h chain growth occurred rapidly so that the fraction of product with Mp values > 2910 increased from 28 to 78%. At 70 °C the catalyst activity at 4 h remained unc...
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kinetics and mechanism of candida antarctica <B>LipaseB> B catalyzed solution polymerization of e caprolactone
Macromolecules, 2003Co-Authors: Ying Mei, And Ajay Kumar, Richard A. GrossAbstract:Studies of the kinetics and mechanism of Candida antarctica <B>LipaseB> B (CALB) catalyzed e-caprolactone (e-CL) polymerizations in toluene were performed. The kinetic plot of ln ([M]0/[M]t) vs time was carried out to 96% e-CL conversion and Mn 11 970. The plot is linear (r2 = 0.998), indicating that termination did not occur and the propagation rate is first order with respect to monomer concentration. Changes in the water (e.g., initiator) concentration did not change the polymerization rate But did change the numBer of chains [R−OH]. Thus, the polymerization is zero order with respect to [R−OH] and initiator concentration. A plot of ln kapp vs ln [enzyme] gave 0.7 as the reaction order of the enzyme concentration. The apparent activation energy for Novozyme-435 catalyzed e-CL polymerization in toluene is 2.88 kcal mol-1. This is well Below 10.3 kcal mol-1, the activation energy for aluminum alkoxide catalyzed e-CL polymerization in toluene. Upward deviation from linearity for Mn vs fractional e-CL conversio...
Jiang-wei Shen - One of the best experts on this subject based on the ideXlab platform.
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efficient resolution of cis dimethyl 1 acetylpiperidine 2 3 dicarBoxylate By covalently immoBilized mutant candida antarctica <B>LipaseB> B in Batch and semicontinuous modes
Organic Process Research & Development, 2019Co-Authors: Jiang-wei Shen, Xiao-jian Zhang, Zhi-qiang Liu, Yu-guo ZhengAbstract:Candida antarctica <B>LipaseB> B (CALB) is a roBust Biocatalyst for production of chemicals. In this study, an engineered <B>LipaseB> B (CALB-I189K) was covalently immoBilized onto a glutaraldehyde activated amino resin (ESR-3) and used for the preparation of chiral moxifloxacin precursor though enzymatic resolution of cis-(±)-dimethyl 1-acetylpiperidine-2,3-dicarBoxylate (cis-(±)-1). The immoBilization conditions were optimized, and the immoBilized CALB-I189K exhiBited improved thermal staBility, with a half-life of 247.5 h at 30 °C. It enaBles the resolution of 100 g L–1 cis-(±)-1 for 50 cycles in a stirred tank reactor, with an average productivity of 49.6 g L–1 h–1. The resolution reaction was also performed in a recirculating packed Bed reactor (RPBR) in a semicontinuous mode, and the average productivity reached 59.4 g L–1 h–1 under the optimal operating condition. Moreover, the RPBR Bioconversion system has high operational staBility and conserved over 85.7% of the initial conversion capacity after 50 cycles...
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Efficient Resolution of cis-(±)-Dimethyl 1‑Acetylpiperidine-2,3-dicarBoxylate By Covalently ImmoBilized Mutant Candida antarctica <B>LipaseB> B in Batch and Semicontinuous Modes
2019Co-Authors: Jiang-wei Shen, Xiao-jian Zhang, Zhi-qiang Liu, Yu-guo ZhengAbstract:Candida antarctica <B>LipaseB> B (CALB) is a roBust Biocatalyst for production of chemicals. In this study, an engineered <B>LipaseB> B (CALB-I189K) was covalently immoBilized onto a glutaraldehyde activated amino resin (ESR-3) and used for the preparation of chiral moxifloxacin precursor though enzymatic resolution of cis-(±)-dimethyl 1-acetylpiperidine-2,3-dicarBoxylate (cis-(±)-1). The immoBilization conditions were optimized, and the immoBilized CALB-I189K exhiBited improved thermal staBility, with a half-life of 247.5 h at 30 °C. It enaBles the resolution of 100 g L–1 cis-(±)-1 for 50 cycles in a stirred tank reactor, with an average productivity of 49.6 g L–1 h–1. The resolution reaction was also performed in a recirculating packed Bed reactor (RPBR) in a semicontinuous mode, and the average productivity reached 59.4 g L–1 h–1 under the optimal operating condition. Moreover, the RPBR Bioconversion system has high operational staBility and conserved over 85.7% of the initial conversion capacity after 50 cycles. This efficient Bioconversion process demonstrated the potential of employing immoBilized CALB-I189K for industrial production of chiral moxifloxacin precursor
Jinho Seo - One of the best experts on this subject based on the ideXlab platform.
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simple amino acid tags improve Both expression and secretion of candida antarctica <B>LipaseB> B in recomBinant escherichia coli
Biotechnology and Bioengineering, 2015Co-Authors: Sunki Kim, Wonki Min, Yongcheol Park, Hyung Ho Lee, Seung Taeg Jeon, Jinho SeoAbstract:Escherichia coli is the Best-estaBlished microBial host strain for production of proteins and chemicals, But has a weakness for not secreting high amounts of active heterologous proteins to the extracellular culture medium, of which origins Belong to whether prokaryotes or eukaryotes. In this study, Candida antarctica <B>LipaseB> B (CalB), a popular eukaryotic enzyme which catalyzes a numBer of Biochemical reactions and Barely secreted extracellularly, was expressed functionally at a gram scale in culture medium By using a simple amino acid-tag system of E. coli. New fusion tag systems consisting of a pelB signal sequence and various anion amino acid tags facilitated Both intracellular expression and extracellular secretion of CalB. Among them, the N-terminal five aspartate tag changed the quaternary structure of the dimeric CalB and allowed production of 1.9 g/L active CalB with 65 U/mL activity in culture medium, which exhiBited the same enzymatic properties as the commercial CalB. This PelB-anion amino acid tag-Based expression system for CalB can Be extended to production of other industrial proteins hardly expressed and exported from E. coli, thereBy increasing target protein concentrations and minimizing purification steps.
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polycationic amino acid tags enhance soluBle expression of candida antarctica <B>LipaseB> B in recomBinant escherichia coli
Bioprocess and Biosystems Engineering, 2011Co-Authors: Hyunjung Jung, Sunki Kim, Wonki Min, Sungsuk Lee, Kyungmoon Park, Yongcheol Park, Jinho SeoAbstract:<B>LipaseB> (EC 3.1.1.3) is a popular enzyme used as an ingredient in detergents and Biocatalyst in many Biochemical reactions. <B>LipaseB> is usually expressed in Escherichia coli as an inactive inclusion Body and at a low level. In this study, Candida antarctica <B>LipaseB> B (CalB) was fused with various polycationic amino acid tags and expressed in E. coli in order to increase a soluBle expression level. By induction with 1.0 mM IPTG, the authentic and fused CalBs were expressed at 27–56% of total protein. The 10-arginine and 10-lysine tags fused at the C-terminal of CalB significantly increased the soluBility of CalB By five- to ninefold, relative to the case of the authentic CalB expressed in a recomBinant E. coli Origami 2TM (DE3) strain. Among a series of the C-terminal poly-arginine tags, the recomBinant CalB comBined with the 10-arginine tag (CalB-R10) possessed the highest <B>LipaseB> specific activity of 9.5 ± 0.03 U/mg protein, corresponding to a fourfold enhancement compared with the authentic CalB.
Roberto Fernandezlafuente - One of the best experts on this subject based on the ideXlab platform.
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staBilization of candida antarctica <B>LipaseB> B calB immoBilized on octyl agarose By treatment with polyethyleneimine pei
Molecules, 2016Co-Authors: Sara Peirce, Jose J Virgenortiz, Veymar G Taciaspascacio, Maria Russo, Antonio Marzocchella, Roberto FernandezlafuenteAbstract:<B>LipaseB> B from Candida antarctica (CALB) was immoBilized on octyl agarose (OC) and physically modified with polyethyleneimine (PEI) in order to confer a strong ion exchange character to the enzyme and thus enaBle the immoBilization of other enzymes on its surface. The enzyme activity was fully maintained during the coating and the thermal staBility was marginally improved. The enzyme release from the support By incuBation in the non-ionic detergent Triton X-100 was more difficult after the PEI-coating, suggesting that some intermolecular physical crosslinking had occurred, making this desorption more difficult. Thermal staBility was marginally improved, But the staBility of the OCCALB-PEI was significantly Better than that of OCCALB during inactivation in mixtures of aqueous Buffer and organic cosolvents. SDS-PAGE analysis of the inactivated Biocatalyst showed the OCCALB released some enzyme to the medium during inactivation, and this was partially prevented By coating with PEI. This effect was oBtained without preventing the possiBility of reuse of the support By incuBation in 2% ionic detergents. That way, this modified CALB not only has a strong anion exchange nature, while maintaining the activity, But it also shows improved staBility under diverse reaction conditions without affecting the reversiBility of the immoBilization.
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tuning <B>LipaseB> B from candida antarctica c c Bond promiscuous activity By immoBilization on poly styrene divinylBenzene Beads
RSC Advances, 2014Co-Authors: Diana F Izquierdo, Roberto Fernandezlafuente, Pedro Lozano, Oveimar Barbosa, Isabel M Burguete, Santiago V Luis, Eduardo GarciaverdugoAbstract:<B>LipaseB> B from Candida antarctica (CALB) is aBle to catalyze C–C Bond formation. After immoBilization onto a hydrophoBic PS-DVB support, the activity increases when compared to that of the soluBle or tan – the commercially availaBle Novozyme 435 (Being up to 6 fold more active). Our results show that although this activity is not related to the catalytic group, the promiscuous activity of CALB may Be tuned via immoBilization. In addition, we have show that the secondary structure of Both immoBilized enzymes is quite different, using FT-ATR-IR spectroscopy.
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chemical amination of <B>LipaseB> B from candida antarctica is an efficient solution for the preparation of crosslinked enzyme aggregates
Process Biochemistry, 2012Co-Authors: Magaly Galvis, Oveimar Barbosa, Monica Ruiz, Jennifer Cruz, Claudia Ortiz, Rodrigo Torres, Roberto FernandezlafuenteAbstract:<B>LipaseB> B from Candida antarctica (CALB) is not very adequate to prepare crosslinked enzyme aggregates (CLEAs). Although the precipitation step is easy using different precipitants, the crosslinking step Becomes a proBlem due to the low amount of Lys residues in this enzyme. In this paper, we have enriched the enzyme in amino groups By chemical amination of the enzyme using ethylenediamine and carBodiimide. The modification was performed using a solid phase strategy modifying the enzyme adsorBed on octyl-Sepharose. After desorption from the support, the enzyme was more active at pH 7.0 than the unmodified enzyme. This modified enzyme showed to Be suitaBle to produce CLEAs. Using this modified enzyme, precipitation is also effective But the crosslinking step did not fail in giving an intense intermolecular crosslinking. This way, the CLEA did not release enzyme molecules even if Boiled in SDS. StaBility of this CLEA was higher in Both thermal and cosolvent inactivation experiments than that of the coCLEA produced By coagregation of BSA and CALB; another alternative to produce a CLEA of this interesting enzyme. The strategy may Be of high interest for many other enzymes as a way to Both permit the production of CLEAs and to improve enzyme staBility during CLEA production.
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immoBilization of <B>LipaseB> B from candida antarctica on porous styrene divinylBenzene Beads improves Butyl acetate synthesis
Biotechnology Progress, 2012Co-Authors: Natalia G Graebin, Roberto Fernandezlafuente, Cristina Garciagalan, Andrea B Martins, Andre Soibelmann Glock Lorenzoni, Marco Antonio Zachia Ayub, Rafael C. RodriguesAbstract:A new Biocatalyst of <B>LipaseB> B from Candida antarctica (MCI-CALB) immoBilized on styrene–divinylBenzene Beads (MCI GEL CHP20P) was compared with the commercial Novozym 435 (immoBilized <B>LipaseB>) in terms of their performances as Biocatalysts for the esterification of acetic acid and n-Butanol. The effects of experimental conditions on reaction rates differed for each Biocatalyst, showing different optimal values for water content, temperature, and suBstrate molar ratio. MCI-CALB could Be used at higher acid concentrations, up to 0.5 M, while Novozym 435 Became inactivated at these acid concentrations. Although Novozym 435 exhiBited 30% higher initial activity than MCI-CALB for the Butyl acetate synthesis, the reaction course was much more linear using the new preparation, meaning that the MCI-CALB allows for higher productivities per cycle. Both preparations produced around 90% of yield conversions after only 2 h of reaction, using 10% (mass fraction) of enzyme. However, the main advantage of the new Biocatalyst was the superior performance during reuse. While Novozym 435 was fully inactivated after only two Batches, MCI-CALB could Be reused for six consecutive cycles without any washings and keeping around 70% of its initial activity. It is proposed that this effect is due to the higher hydrophoBicity of the new support, which does not retain water or acid in the enzyme environment. MCI-CALB has shown to Be a very promising Biocatalyst for the esterification of small-molecule acids and alcohols. © 2012 American Institute of Chemical Engineers Biotechnol. Prog.,, 2012
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simple and efficient immoBilization of <B>LipaseB> B from candida antarctica on porous styrene divinylBenzene Beads
Enzyme and Microbial Technology, 2011Co-Authors: Karel Hernandez, Cristina Garciagalan, Roberto FernandezlafuenteAbstract:ABstract Two commercial porous styrene–divinylBenzene Beads (Diaion HP20LX and MCI GEL CHP20P) have Been evaluated as supports to immoBilize <B>LipaseB> B from Candida antarctica (CALB). MCI GEL CHP20P rapidly immoBilized the enzyme, permitting a very high loading capacity: around 110 mg CALB/wet g of support compared to the 50 mg oBtained using decaoctyl SepaBeads. Although enzyme specificity of the enzyme immoBilized on different supports was quite altered By the support used in the immoBilization, specific activity of the enzyme immoBilized on MCI GEL CHP20P was always higher than those found using decaoctyl SepaBeads for all assayed suBstrates. Thus, a CALB Biocatalyst having 3–8 folds (depending on the suBstrate) higher activity/wet gram of support than the commercial Novozym 435 was oBtained. Half-live of CAL-Diaion HP20LX at 60 °C was 2–3 higher than the one of Novozym 435, it was 30–40 higher in the presence of 50% acetonitrile and it was around 100 folds greater in the presence of 10 M hydrogen peroxide. Results indicate that styrene–divinylBenzene supports may Be promising alternatives as supports to immoBilize CALB.