The Experts below are selected from a list of 921 Experts worldwide ranked by ideXlab platform
Zhongyi Jiang - One of the best experts on this subject based on the ideXlab platform.
-
monolithic biocatalytic systems with enhanced stabilities constructed through biomimetic silicification induced enzyme immobilization on rgo feooh hydrogel
Biochemical Engineering Journal, 2017Co-Authors: Dong Yang, Jiafu Shi, Xueyan Wang, Shaohua Zhang, Zhongyi Jiang, Jingjing ZhaoAbstract:Abstract In this study, we present a green and facile method of utilizing biomimetic silicification to trigger enzyme immobilization on the surface of the rGO/FeOOH hydrogel for constructing stable monolithic biocatalytic systems. In brief, the rGO/FeOOH hydrogel is firstly prepared through metal ion-induced reduction/assembly of graphene oxide (GO) nanosheets, which is then utilized to adsorb cationic polyethyleneimine (PEI). This cationic PEI, as the mineralization-inducing agent, catalyzes the condensation of silicate to form silica (biomimetic silicification) on the rGO surface, where enzyme is simultaneously entrapped. The resultant rGO/FeOOH/silica hydrogel shows an extraordinary three-dimensional (3D) porous structure. The silica content on the rGO surface can be facilely tailored through changing the silica precursor concentration. Combined with monolithic macroscale of the rGO/FeOOH/silica hydrogel, the acquired monolithic biocatalytic systems display easy recyclability and elevated pH/thermal/recycling/storage stabilities during the catalytic production of 6-Aminopenicillanic Acid (6-APA) in comparison to enzyme in free form and enzyme adsorbed on rGO/FeOOH hydrogel. Notably, the activity can be retained up to 93.3% of its initial activity after 11 reaction cycles for our biocatalytic systems.
-
Enhancing 6‑APA Productivity and Operational Stability of Penicillin G Acylase via Rapid Surface Capping on Commercial Resins
2016Co-Authors: Dong Yang, Hua Liu, Jiafu Shi, Xueyan Wang, Shaohua Zhang, Hongjian Zou, Zhongyi JiangAbstract:In this study, immobilized penicillin G acylase (PGA) was prepared via a facile and rapid approach of generating the TA-TiIV layer on PGA-adsorbed commercial resins (PGA@Resins). In brief, the TA-TiIV layer was constructed through coordination-enabled self-assembly of tannic Acid (TA) and titanium(IV) bis(ammonium lactate) dihydroxide (Ti-BALDH). In comparison to PGA@Resins, TA-TiIV-capped PGA@Resins exhibited higher 6-Aminopenicillanic Acid (6-APA) productivity and enhanced operational stability along with comparable activity recovery during the catalytic hydrolysis of penicillin G potassium (PGK). Particularly, TA-TiIV-capped PGA@Resins exhibited relative activities of 103.7% and 81.51%, respectively, after 68-day storage and 20 cycles, indicating significantly enhanced storage and recycling stabilities compared to PGA@Resins (68.98% and 62.88%). Both immobilized PGA were further packed into a glass column for hydrolyzing PGK in a continuous flow reactor, where TA-TiIV-capped PGA@Resins displayed a much higher 6-APA yield (initial yield: 49.22% vs 28.99%; yield after 10 days: 17.39% vs 6.11%) than PGA@Resins
Jiafu Shi - One of the best experts on this subject based on the ideXlab platform.
-
monolithic biocatalytic systems with enhanced stabilities constructed through biomimetic silicification induced enzyme immobilization on rgo feooh hydrogel
Biochemical Engineering Journal, 2017Co-Authors: Dong Yang, Jiafu Shi, Xueyan Wang, Shaohua Zhang, Zhongyi Jiang, Jingjing ZhaoAbstract:Abstract In this study, we present a green and facile method of utilizing biomimetic silicification to trigger enzyme immobilization on the surface of the rGO/FeOOH hydrogel for constructing stable monolithic biocatalytic systems. In brief, the rGO/FeOOH hydrogel is firstly prepared through metal ion-induced reduction/assembly of graphene oxide (GO) nanosheets, which is then utilized to adsorb cationic polyethyleneimine (PEI). This cationic PEI, as the mineralization-inducing agent, catalyzes the condensation of silicate to form silica (biomimetic silicification) on the rGO surface, where enzyme is simultaneously entrapped. The resultant rGO/FeOOH/silica hydrogel shows an extraordinary three-dimensional (3D) porous structure. The silica content on the rGO surface can be facilely tailored through changing the silica precursor concentration. Combined with monolithic macroscale of the rGO/FeOOH/silica hydrogel, the acquired monolithic biocatalytic systems display easy recyclability and elevated pH/thermal/recycling/storage stabilities during the catalytic production of 6-Aminopenicillanic Acid (6-APA) in comparison to enzyme in free form and enzyme adsorbed on rGO/FeOOH hydrogel. Notably, the activity can be retained up to 93.3% of its initial activity after 11 reaction cycles for our biocatalytic systems.
-
Enhancing 6‑APA Productivity and Operational Stability of Penicillin G Acylase via Rapid Surface Capping on Commercial Resins
2016Co-Authors: Dong Yang, Hua Liu, Jiafu Shi, Xueyan Wang, Shaohua Zhang, Hongjian Zou, Zhongyi JiangAbstract:In this study, immobilized penicillin G acylase (PGA) was prepared via a facile and rapid approach of generating the TA-TiIV layer on PGA-adsorbed commercial resins (PGA@Resins). In brief, the TA-TiIV layer was constructed through coordination-enabled self-assembly of tannic Acid (TA) and titanium(IV) bis(ammonium lactate) dihydroxide (Ti-BALDH). In comparison to PGA@Resins, TA-TiIV-capped PGA@Resins exhibited higher 6-Aminopenicillanic Acid (6-APA) productivity and enhanced operational stability along with comparable activity recovery during the catalytic hydrolysis of penicillin G potassium (PGK). Particularly, TA-TiIV-capped PGA@Resins exhibited relative activities of 103.7% and 81.51%, respectively, after 68-day storage and 20 cycles, indicating significantly enhanced storage and recycling stabilities compared to PGA@Resins (68.98% and 62.88%). Both immobilized PGA were further packed into a glass column for hydrolyzing PGK in a continuous flow reactor, where TA-TiIV-capped PGA@Resins displayed a much higher 6-APA yield (initial yield: 49.22% vs 28.99%; yield after 10 days: 17.39% vs 6.11%) than PGA@Resins
Janssen, Dick B. - One of the best experts on this subject based on the ideXlab platform.
-
The Penicillium chrysogenum aclA gene encodes a broad-substrate-specificity acyl-coenzyme A ligase involved in activation of adipic Acid, a side-chain precursor for cephem antibiotics
2010Co-Authors: Koetsier, Martijn J., Gombert, Andreas K., Fekken Susan, Bovenberg, Roel A.l., Berg, Marco A. Van Den, Kiel, Jan A.k.w., Jekel, Peter A., Janssen, Dick B., Pronk, Jack T., Klei, Ida J. Van DerAbstract:Activation of the cephalosporin side-chain precursor to the corresponding CoA-thioester is an essential step for its incorporation into the β-lactam backbone. To identify an acyl-CoA ligase involved in activation of adipate, we searched in the genome database of Penicillium chrysogenum for putative structural genes encoding acyl-CoA ligases. Chemostat-based transcriptome analysis was used to identify the one presenting the highest expression level when cells were grown in the presence of adipate. Deletion of the gene renamed aclA, led to a 32% decreased specific rate of adipate consumption and a threefold reduction of adipoyl-6-Aminopenicillanic Acid levels, but did not affect penicillin V production. After overexpression in Escherichia coli, the purified protein was shown to have a broad substrate range including adipate. Finally, protein-fusion with cyan-fluorescent protein showed co-localization with microbody-borne acyl-transferase. Identification and functional characterization of aclA may aid in developing future metabolic engineering strategies for improving the production of different cephalosporins.
-
The Penicillium chrysogenum aclA gene encodes a broad-substrate-specificity acyl-coenzyme A ligase involved in activation of adipic Acid, a side-chain precursor for cephem antibiotics
2010Co-Authors: Koetsier, Martijn J., Gombert, Andreas K., Fekken Susan, Jekel, Peter A., Janssen, Dick B., Pronk, Jack T., Bovenberg, Roel A. L., Van Den Berg, Marco A., Kiel, Jan A. K. W., Van Der Klei, Ida J.Abstract:Activation of the cephalosporin side-chain precursor to the corresponding CoA-thioester is an essential step for its incorporation into the P-lactam backbone. To identify an acyl-CoA ligase involved in activation of adipate, we searched in the genome database of Penicillium chrysogenum for putative structural genes encoding acyl-CoA ligases. Chemostat-based transcriptome analysis was used to identify the one presenting the highest expression level when cells were grown in the presence of adipate. Deletion of the gene renamed aclA, led to a 32% decreased specific rate of adipate consumption and a threefold reduction of adipoyl-6-Aminopenicillanic Acid levels, but did not affect penicillin V production. After overexpression in Escherichia coli, the purified protein was shown to have a broad substrate range including adipate. Finally, protein-fusion with cyan-fluorescent protein showed co-localization with microbody-borne acyl-transferase. Identification and functional characterization of aclA may aid in developing future metabolic engineering strategies for improving the production of different cephalosporins. (C) 2009 Elsevier Inc. All rights reserved
-
The Penicillium chrysogenum aclA gene encodes a broad-substrate-specificity acyl-coenzyme A ligase involved in activation of adipic Acid, a side-chain precursor for cephem antibiotics
ACADEMIC PRESS INC ELSEVIER SCIENCE, 2010Co-Authors: Koetsier, Martijn J., Gombert, Andreas K., Fekken Susan, Berg, Marco A. Van Den, Jekel, Peter A., Janssen, Dick B., Pronk, Jack T., Bovenberg, Roel A. L., Kiel, Jan A. K. W., Klei, Ida J. Van DerAbstract:Activation of the cephalosporin side-chain precursor to the corresponding CoA-thioester is an essential step for its incorporation into the P-lactam backbone. To identify an acyl-CoA ligase involved in activation of adipate, we searched in the genome database of Penicillium chrysogenum for putative structural genes encoding acyl-CoA ligases. Chemostat-based transcriptome analysis was used to identify the one presenting the highest expression level when cells were grown in the presence of adipate. Deletion of the gene renamed aclA, led to a 32% decreased specific rate of adipate consumption and a threefold reduction of adipoyl-6-Aminopenicillanic Acid levels, but did not affect penicillin V production. After overexpression in Escherichia coli, the purified protein was shown to have a broad substrate range including adipate. Finally, protein-fusion with cyan-fluorescent protein showed co-localization with microbody-borne acyl-transferase. Identification and functional characterization of aclA may aid in developing future metabolic engineering strategies for improving the production of different cephalosporins. (C) 2009 Elsevier Inc. All rights reserved.Dutch ministry of economic affairsNetherlands organization for Scientific Research (NWO
-
Increasing the synthetic performance of penicillin acylase PAS2 by structure-inspired semi-random mutagenesis
2004Co-Authors: Gabor, Esther M., Janssen, Dick B.Abstract:A semi-random mutagenesis approach was followed to increase the performance of penicillin acylase PAS2 in the kinetically controlled synthesis of ampicillin from 6-Aminopenicillanic Acid (6-APA) and activated D-phenylglycine derivatives. We directed changes in amino Acid residues to positions close to the active site that are expected to affect the catalytic performance of penicillin acylase: αR160, αF161 and βF24. From the resulting triple mutant gene bank, six improved PAS2 mutants were recovered by screening only 700 active mutants with an HPLC-based screening method. A detailed kinetic analysis of the three most promising mutants, T23, TM33 and TM38, is presented. These mutants allowed the accumulation of ampicillin at 4–5 times higher concentrations than the wild-type enzyme, using D-phenylglycine methyl ester as the acyl donor. At the same time, the loss of activated acyl donor due to the competitive hydrolytic side reactions could be reduced to 80% when using wild-type PAS2. Although catalytic activity dropped by a factor of 5–10, the enhanced synthetic performance of the recovered penicillin acylase variants makes them interesting biocatalysts for the production of β-lactam antibiotics.
-
Characterization of the β-lactam binding site of penicillin acylase of Escherichia coli by structural and site-directed mutagenesis studies
2000Co-Authors: Alkema, Wynand B.l., Hensgens, Charles M.h., Kroezinga, Els H., Erik De ,vries, Floris René, Laan, Jan-metske Van Der, Dijkstra, Bauke W., Janssen, Dick B.Abstract:The binding of penicillin to penicillin acylase was studied by X-ray crystallography. The structure of the enzyme–substrate complex was determined after soaking crystals of an inactive βN241A penicillin acylase mutant with penicillin G. Binding of the substrate induces a conformational change, in which the side chains of αF146 and αR145 move away from the active site, which allows the enzyme to accommodate penicillin G. In the resulting structure, the β-lactam binding site is formed by the side chains of αF146 and βF71, which have van der Waals interactions with the thiazolidine ring of penicillin G and the side chain of αR145 that is connected to the carboxylate group of the ligand by means of hydrogen bonding via two water molecules. The backbone oxygen of βQ23 forms a hydrogen bond with the carbonyl oxygen of the phenylacetic Acid moiety through a bridging water molecule. Kinetic studies revealed that the site-directed mutants αF146Y, αF146A and αF146L all show significant changes in their interaction with the β-lactam substrates as compared with the wild type. The αF146Y mutant had the same affinity for 6-Aminopenicillanic Acid as the wild-type enzyme, but was not able to synthesize penicillin G from phenylacetamide and 6-Aminopenicillanic Acid. The αF146L and αF146A enzymes had a 3–5-fold decreased affinity for 6-Aminopenicillanic Acid, but synthesized penicillin G more efficiently than the wild type. The combined results of the structural and kinetic studies show the importance of αF146 in the β-lactam binding site and provide leads for engineering mutants with improved synthetic properties.
Alexandra Cristina Blaga - One of the best experts on this subject based on the ideXlab platform.
-
engineering aspects of penicillin g transfer and conversion to 6 aminopenicillanic Acid in a bioreactor with a mobile bed of immobilized penicillin amidase
Chemical Engineering Communications, 2014Co-Authors: Anca-irina Galaction, Marius Turnea, Alexandra Cristina Blaga, Ramona Mihaela Matran, Dan CaşcavalAbstract:This article presents studies on the external and internal mass transfers of penicillin G for 6-Aminopenicillanic Acid enzymatic production using a bioreactor with a stirred bed of immobilized penicillin amidase. By means of the substrate mass balance for a single particle of biocatalyst and considering the kinetic model adapted for competitive and noncompetitive inhibitions, specific mathematical models were developed for describing the profiles of penicillin G concentration in the outer and inner regions of biocatalyst and for estimating its mass flows in the liquid boundary layer surrounding the particle and inside the particle. The values of the mass flows are significantly influenced by the internal diffusion velocity and rate of the enzymatic conversion of substrate. These cumulated influences led to the appearance of an enzymatic inactive region near the particle center, its magnitude varying from 0 to 9.2% of the overall volume of particles.
-
6 aminopenicillanic Acid production in stationary basket bioreactor with packed bed of immobilized penicillin amidase penicillin g mass transfer and consumption rate under internal diffusion limitation
Biochemical Engineering Journal, 2012Co-Authors: Dan Caşcaval, Marius Turnea, Anca-irina Galaction, Alexandra Cristina BlagaAbstract:Abstract The external and internal mass transfers of Penicillin G in the process of its enzymatic hydrolysis to 6-Aminopenicillanic Acid under competitive and non-competitive inhibitions using a bioreactor with stationary basket bed of immobilized penicillin amidase have been analyzed. By means of the Penicillin G mass balance for a single particle of biocatalysts, considering the specific kinetic model proposed by Warburton et al., mathematical expressions have been developed for describing the profiles of Penicillin G concentrations and mass flows in the outer and inner regions of biocatalyst particles, as well as for estimating the influence of internal diffusion on its hydrolysis rate. The results indicated that very low values of internal mass flow could be reached in the particles centre. The corresponding region was considered an “enzymatic inactive region”, its extent varying from 0 to 51% from the overall volume of each biocatalyst. By enzyme immobilization and using the basket bed, the rate of enzymatic reaction is reduced over 160 times compared to the process with free enzyme
D E Kerr - One of the best experts on this subject based on the ideXlab platform.
-
a colorimetric assay for penicillin v amidase
Analytical Biochemistry, 1993Co-Authors: D E KerrAbstract:Abstract The hydrolysis of penicillin-V to phenoxyacetic Acid and 6-Aminopenicillanic Acid by the fungal enzyme penicillin-V amidase is of industrial importance since the 6-Aminopenicillanic Acid produced is an intermediate for semisynthetic penicillins. A rapid colorimetric assay of penicillin-V amidase was developed which uses 2-nitro-5-(phenoxyacetamido)-benzoic Acid as a substrate. The released chromophore, 2-amino-5-nitro-benzoic Acid, was detected at 405 nm. Using penicillin-V amidase from the fungus Fusarium oxysporum, the KM and Vmax for this substrate were 0.89 mM and 2.6 μmol/min/mg enzyme, respectively. Hydrolysis could be competitively inhibited by penicillin-V with a Ki of 4 mM. The change in the initial velocity of hydrolysis of 2-nitro-5-(phenoxyacetamido)-benzoic Acid at 500 μM was linear over the range of 0.5 to 10 μg/ml enzyme. These results show that this new compound is useful in determining the presence and levels of penicillin-V amidase.
