The Experts below are selected from a list of 384 Experts worldwide ranked by ideXlab platform
Jose M Guisan - One of the best experts on this subject based on the ideXlab platform.
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evaluation of different glutaryl acylase mutants to improve the hydolysis of cephalosporin c in the absence of hydrogen peroxide
Advanced Synthesis & Catalysis, 2008Co-Authors: Fernando Lopezgallego, Jose M Guisan, Lorena Betancor, Charles Frederik Sio, Carlos R Reis, Pol Nadal Jimenez, Wim J Quax, Roberto FernandezlafuenteAbstract:2-Oxoadipoyl-7-ACA is an intermediate in the conversion of cephalosporin C (CPC) to 7-Aminocephalosporanic Acid (7-ACA) when using a new route involving D-amino Acid oxidase, catalase and glutaryl acylase. A key point in the reaction design is to avoid the accumulation of hydrogen peroxide in the reaction medium as the yields of 7-ACA decrease in the presence of this compound due to its low stability. Looking for an enzyme with improved activity towards 2-oxoadipoyl-7-ACA, different mutants of glutaryl acylase from Pseudomonas SY-77 with an improved activity towards adipoyl-7-ACA were evaluated. The best results on 2-oxoadipoyl-7-ACA hydrolysis were found with the double mutant Y178F+F375H, which showed a K(cat) increase of 6.5-fold and a K(m) decrease of 3-fold compared to the wild-type (wt) enzyme. When this enzyme was tested in the tri-enzymatic system to convert CPC into 7-ACA, this mutant permitted us to reach more than an 80% yield of 7-ACA using a 3-fold mass excess compared to DAAO; while the wt enzyme gave only a 40% yield. Therefore, the application of this new mutant to the one-pot conversion of CPC to 7-ACA gives very good result in terms of efficiency, yield and rate of the process.
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one pot conversion of cephalosporin c to 7 Aminocephalosporanic Acid in the absence of hydrogen peroxide
Advanced Synthesis & Catalysis, 2005Co-Authors: Fernando Lopezgallego, Lorena Batencor, Aurelio Hidalgo, Cesar Mateo, Roberto Fernandezlafuente, Jose M GuisanAbstract:The main drawback in the production of 7-Aminocephalosporanic Acid (7-ACA) at the industrial level is the inactivation of the enzymes implicated in the process due to the presence of hydrogen peroxide during the reaction. As an alternative, we have developed the conversion of cephalosporin C to 7-ACA in a single reactor without the presence of hydrogen peroxide during the reaction, achieving more than 80% yield. In order to develop this process, D-amino Acid oxidase (DAAO) was co-immobilized with catalase (CAT), which is able to fully eliminate in situ the hydrogen peroxide formed by the neighbouring DAAO molecules. Thus, the product of the reaction is only α-ketoadipyl-7-ACA. This system prevents the inactivation of the oxidase by hydrogen peroxide, solving the main problem of the enzymatic process. Moreover, we have found that α-ketoadipyl-7-ACA is recognized as a substrate by glutaryl acylase (GAC) and hydrolyzed as long as glutaric Acid is absent from the reaction medium (because it is able to inhibit the hydrolysis). The low stability of α-ketoadipyl-7-ACA justifies the use of a single reactor, in which glutaryl acylase is already present when this substrate is generated. Thus, the whole process may (and must) be performed in a single step, and in the absence of hydrogen peroxide that could affect the stabilities of the involved enzymes.
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optimization of an industrial biocatalyst of glutaryl acylase stabilization of the enzyme by multipoint covalent attachment onto new amino epoxy sepabeads
Journal of Biotechnology, 2004Co-Authors: Fernando Lopezgallego, Aurelio Hidalgo, Cesar Mateo, Jose M Guisan, Lorena Betancor, Roberto FernandezlafuenteAbstract:Abstract Glutaryl-7-Aminocephalosporanic Acid acylase (GA), an industrially relevant enzyme, has been immobilized onto very different supports, including glyoxyl agarose, heterofunctional epoxy Sepabeads, glutaraldehyde and cyanogen bromide (CNBr) activated supports. Immobilization onto amino-epoxy Sepabeads rendered the most thermo stable preparation of GA, with a half-life time eight times higher than the soluble enzyme, keeping 80% of the enzyme activity. Several parameters that affect the enzyme-support interaction (pH and incubation time) were studied. It was found that after immobilization onto amino-epoxy Sepabeads, incubation at alkaline pH and low temperature exerted dramatic stabilizing effects, increasing the half-life time of the derivative 130 times with respect to the soluble enzyme, while keeping unaltered its intrinsic activity. The loading capacity of the amino-epoxy Sepabeads proved to be very good with a maximum load of 62 mg of protein per g of support with 85 IU/g at 25 °C and 200 IU/g at 37 °C which makes it a biocatalyst of possible industrial application.
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modulation of penicillin acylase properties via immobilization techniques one pot chemoenzymatic synthesis of cephamandole from cephalosporin c
Bioorganic & Medicinal Chemistry Letters, 2001Co-Authors: Marco Terreni, Cesar Mateo, Roberto Fernandezlafuente, Giuseppe Pagani, Daniela Ubiali, Jose M GuisanAbstract:Abstract The modulation of penicillin G acylase (PGA) properties via immobilization techniques has been performed studying the acylation of 7-Aminocephalosporanic Acid with R -mandelic Acid methyl ester. PGA from Escherichia coli , immobilized onto agarose activated with glycidol (glyoxyl–agarose), has been used for the design of a novel one-pot synthesis of Cephamandole in aqueous medium and without isolation of intermediates, through three consecutive biotransformations catalyzed by d -amino Acid oxidase, glutaryl acylase and PGA.
Roberto Fernandezlafuente - One of the best experts on this subject based on the ideXlab platform.
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evaluation of different glutaryl acylase mutants to improve the hydolysis of cephalosporin c in the absence of hydrogen peroxide
Advanced Synthesis & Catalysis, 2008Co-Authors: Fernando Lopezgallego, Jose M Guisan, Lorena Betancor, Charles Frederik Sio, Carlos R Reis, Pol Nadal Jimenez, Wim J Quax, Roberto FernandezlafuenteAbstract:2-Oxoadipoyl-7-ACA is an intermediate in the conversion of cephalosporin C (CPC) to 7-Aminocephalosporanic Acid (7-ACA) when using a new route involving D-amino Acid oxidase, catalase and glutaryl acylase. A key point in the reaction design is to avoid the accumulation of hydrogen peroxide in the reaction medium as the yields of 7-ACA decrease in the presence of this compound due to its low stability. Looking for an enzyme with improved activity towards 2-oxoadipoyl-7-ACA, different mutants of glutaryl acylase from Pseudomonas SY-77 with an improved activity towards adipoyl-7-ACA were evaluated. The best results on 2-oxoadipoyl-7-ACA hydrolysis were found with the double mutant Y178F+F375H, which showed a K(cat) increase of 6.5-fold and a K(m) decrease of 3-fold compared to the wild-type (wt) enzyme. When this enzyme was tested in the tri-enzymatic system to convert CPC into 7-ACA, this mutant permitted us to reach more than an 80% yield of 7-ACA using a 3-fold mass excess compared to DAAO; while the wt enzyme gave only a 40% yield. Therefore, the application of this new mutant to the one-pot conversion of CPC to 7-ACA gives very good result in terms of efficiency, yield and rate of the process.
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one pot conversion of cephalosporin c to 7 Aminocephalosporanic Acid in the absence of hydrogen peroxide
Advanced Synthesis & Catalysis, 2005Co-Authors: Fernando Lopezgallego, Lorena Batencor, Aurelio Hidalgo, Cesar Mateo, Roberto Fernandezlafuente, Jose M GuisanAbstract:The main drawback in the production of 7-Aminocephalosporanic Acid (7-ACA) at the industrial level is the inactivation of the enzymes implicated in the process due to the presence of hydrogen peroxide during the reaction. As an alternative, we have developed the conversion of cephalosporin C to 7-ACA in a single reactor without the presence of hydrogen peroxide during the reaction, achieving more than 80% yield. In order to develop this process, D-amino Acid oxidase (DAAO) was co-immobilized with catalase (CAT), which is able to fully eliminate in situ the hydrogen peroxide formed by the neighbouring DAAO molecules. Thus, the product of the reaction is only α-ketoadipyl-7-ACA. This system prevents the inactivation of the oxidase by hydrogen peroxide, solving the main problem of the enzymatic process. Moreover, we have found that α-ketoadipyl-7-ACA is recognized as a substrate by glutaryl acylase (GAC) and hydrolyzed as long as glutaric Acid is absent from the reaction medium (because it is able to inhibit the hydrolysis). The low stability of α-ketoadipyl-7-ACA justifies the use of a single reactor, in which glutaryl acylase is already present when this substrate is generated. Thus, the whole process may (and must) be performed in a single step, and in the absence of hydrogen peroxide that could affect the stabilities of the involved enzymes.
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optimization of an industrial biocatalyst of glutaryl acylase stabilization of the enzyme by multipoint covalent attachment onto new amino epoxy sepabeads
Journal of Biotechnology, 2004Co-Authors: Fernando Lopezgallego, Aurelio Hidalgo, Cesar Mateo, Jose M Guisan, Lorena Betancor, Roberto FernandezlafuenteAbstract:Abstract Glutaryl-7-Aminocephalosporanic Acid acylase (GA), an industrially relevant enzyme, has been immobilized onto very different supports, including glyoxyl agarose, heterofunctional epoxy Sepabeads, glutaraldehyde and cyanogen bromide (CNBr) activated supports. Immobilization onto amino-epoxy Sepabeads rendered the most thermo stable preparation of GA, with a half-life time eight times higher than the soluble enzyme, keeping 80% of the enzyme activity. Several parameters that affect the enzyme-support interaction (pH and incubation time) were studied. It was found that after immobilization onto amino-epoxy Sepabeads, incubation at alkaline pH and low temperature exerted dramatic stabilizing effects, increasing the half-life time of the derivative 130 times with respect to the soluble enzyme, while keeping unaltered its intrinsic activity. The loading capacity of the amino-epoxy Sepabeads proved to be very good with a maximum load of 62 mg of protein per g of support with 85 IU/g at 25 °C and 200 IU/g at 37 °C which makes it a biocatalyst of possible industrial application.
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modulation of penicillin acylase properties via immobilization techniques one pot chemoenzymatic synthesis of cephamandole from cephalosporin c
Bioorganic & Medicinal Chemistry Letters, 2001Co-Authors: Marco Terreni, Cesar Mateo, Roberto Fernandezlafuente, Giuseppe Pagani, Daniela Ubiali, Jose M GuisanAbstract:Abstract The modulation of penicillin G acylase (PGA) properties via immobilization techniques has been performed studying the acylation of 7-Aminocephalosporanic Acid with R -mandelic Acid methyl ester. PGA from Escherichia coli , immobilized onto agarose activated with glycidol (glyoxyl–agarose), has been used for the design of a novel one-pot synthesis of Cephamandole in aqueous medium and without isolation of intermediates, through three consecutive biotransformations catalyzed by d -amino Acid oxidase, glutaryl acylase and PGA.
Fernando Lopezgallego - One of the best experts on this subject based on the ideXlab platform.
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evaluation of different glutaryl acylase mutants to improve the hydolysis of cephalosporin c in the absence of hydrogen peroxide
Advanced Synthesis & Catalysis, 2008Co-Authors: Fernando Lopezgallego, Jose M Guisan, Lorena Betancor, Charles Frederik Sio, Carlos R Reis, Pol Nadal Jimenez, Wim J Quax, Roberto FernandezlafuenteAbstract:2-Oxoadipoyl-7-ACA is an intermediate in the conversion of cephalosporin C (CPC) to 7-Aminocephalosporanic Acid (7-ACA) when using a new route involving D-amino Acid oxidase, catalase and glutaryl acylase. A key point in the reaction design is to avoid the accumulation of hydrogen peroxide in the reaction medium as the yields of 7-ACA decrease in the presence of this compound due to its low stability. Looking for an enzyme with improved activity towards 2-oxoadipoyl-7-ACA, different mutants of glutaryl acylase from Pseudomonas SY-77 with an improved activity towards adipoyl-7-ACA were evaluated. The best results on 2-oxoadipoyl-7-ACA hydrolysis were found with the double mutant Y178F+F375H, which showed a K(cat) increase of 6.5-fold and a K(m) decrease of 3-fold compared to the wild-type (wt) enzyme. When this enzyme was tested in the tri-enzymatic system to convert CPC into 7-ACA, this mutant permitted us to reach more than an 80% yield of 7-ACA using a 3-fold mass excess compared to DAAO; while the wt enzyme gave only a 40% yield. Therefore, the application of this new mutant to the one-pot conversion of CPC to 7-ACA gives very good result in terms of efficiency, yield and rate of the process.
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one pot conversion of cephalosporin c to 7 Aminocephalosporanic Acid in the absence of hydrogen peroxide
Advanced Synthesis & Catalysis, 2005Co-Authors: Fernando Lopezgallego, Lorena Batencor, Aurelio Hidalgo, Cesar Mateo, Roberto Fernandezlafuente, Jose M GuisanAbstract:The main drawback in the production of 7-Aminocephalosporanic Acid (7-ACA) at the industrial level is the inactivation of the enzymes implicated in the process due to the presence of hydrogen peroxide during the reaction. As an alternative, we have developed the conversion of cephalosporin C to 7-ACA in a single reactor without the presence of hydrogen peroxide during the reaction, achieving more than 80% yield. In order to develop this process, D-amino Acid oxidase (DAAO) was co-immobilized with catalase (CAT), which is able to fully eliminate in situ the hydrogen peroxide formed by the neighbouring DAAO molecules. Thus, the product of the reaction is only α-ketoadipyl-7-ACA. This system prevents the inactivation of the oxidase by hydrogen peroxide, solving the main problem of the enzymatic process. Moreover, we have found that α-ketoadipyl-7-ACA is recognized as a substrate by glutaryl acylase (GAC) and hydrolyzed as long as glutaric Acid is absent from the reaction medium (because it is able to inhibit the hydrolysis). The low stability of α-ketoadipyl-7-ACA justifies the use of a single reactor, in which glutaryl acylase is already present when this substrate is generated. Thus, the whole process may (and must) be performed in a single step, and in the absence of hydrogen peroxide that could affect the stabilities of the involved enzymes.
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optimization of an industrial biocatalyst of glutaryl acylase stabilization of the enzyme by multipoint covalent attachment onto new amino epoxy sepabeads
Journal of Biotechnology, 2004Co-Authors: Fernando Lopezgallego, Aurelio Hidalgo, Cesar Mateo, Jose M Guisan, Lorena Betancor, Roberto FernandezlafuenteAbstract:Abstract Glutaryl-7-Aminocephalosporanic Acid acylase (GA), an industrially relevant enzyme, has been immobilized onto very different supports, including glyoxyl agarose, heterofunctional epoxy Sepabeads, glutaraldehyde and cyanogen bromide (CNBr) activated supports. Immobilization onto amino-epoxy Sepabeads rendered the most thermo stable preparation of GA, with a half-life time eight times higher than the soluble enzyme, keeping 80% of the enzyme activity. Several parameters that affect the enzyme-support interaction (pH and incubation time) were studied. It was found that after immobilization onto amino-epoxy Sepabeads, incubation at alkaline pH and low temperature exerted dramatic stabilizing effects, increasing the half-life time of the derivative 130 times with respect to the soluble enzyme, while keeping unaltered its intrinsic activity. The loading capacity of the amino-epoxy Sepabeads proved to be very good with a maximum load of 62 mg of protein per g of support with 85 IU/g at 25 °C and 200 IU/g at 37 °C which makes it a biocatalyst of possible industrial application.
Zhongyao Shen - One of the best experts on this subject based on the ideXlab platform.
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optimization of cephalosporin c acylase expression in escherichia coli by high throughput screening a constitutive promoter mutant library
Applied Biochemistry and Biotechnology, 2021Co-Authors: Hongxu Sun, Hui Luo, Yanhong Chang, Tianjiao Liu, Zihao Nie, Zhongyao ShenAbstract:Cephalosporin C acylase (CCA) is capable of catalyzing cephalosporin C (CPC) to produce 7-Aminocephalosporanic Acid (7-ACA), an intermediate of semi-synthetic cephalosporins. Inducible expression is usually used for CCA. To improve the efficiency of CCA expression without gene induction, three recombinant strains regulated by constitutive promoters BBa_J23105, PLtetO1, and tac were constructed, respectively. Among them, BBa_J23105 was the best promoter and its mutant libraries were established using saturation mutagenesis. In order to obtain the mutants with enhanced activity, a high-throughput screening method based on flow cytometric sorting techniques was developed by using green fluorescent protein (GFP) as the reporter gene. A series of mutants were screened at 28 °C, 200 rpm, and 24-h culture condition. The study of mutants showed that the enzyme activity, fluorescence intensity, and promoter transcriptional strength were positively correlated. The enzyme activity of the optimal mutant obtained by screening reached 12772 U/L, 3.47 times that of the original strain.
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high throughput screening of t7 promoter mutants for soluble expression of cephalosporin c acylase in e coli
Applied Biochemistry and Biotechnology, 2020Co-Authors: Zihao Nie, Hui Luo, Yanhong Chang, Hongxu Sun, Tianjiao Liu, Ying Xiao, Ruiqi Jia, Zhongyao ShenAbstract:Cephalosporin C acylase (CCA) is the key enzyme in the production of 7-Aminocephalosporanic Acid (7-ACA) via a one-step enzymatic process. To improve the soluble expression level of CCA in recombinant Escherichia coli at elevated temperatures, a library of T7 promoter mutants was created by site-saturation mutagenesis, and a series of mutated promoters were subsequently screened. Green fluorescent protein (GFP) was fused to the C-terminus of CCA to facilitate library screening, and the expression of the CCA and GFP fusion proteins was investigated under the control of the T7 promoter. Twenty-four mutants were selected by detecting the fluorescence intensity of colonies on agar plates to form a library with different expression levels. The enzyme activities of the mutants were positively correlated with their fluorescence intensities. The highest enzyme activity among these mutant promoters was 1.3-fold higher than the enzyme activity resulting from the wild-type promoter when the cells were cultured at 32 °C for 16 h. In addition, the transcription and expression levels of several typical promoters were discussed, and the effects of GFP fusion on the enzyme activity of CCA were investigated.
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oriented immobilization and characterization of a poly lysine tagged cephalosporin c acylase on glyoxyl agarose support
Applied Biochemistry and Biotechnology, 2015Co-Authors: Hui Luo, Huan Zhao, Yanhong Chang, Qixin Wang, Zhongyao ShenAbstract:Cephalosporin C acylase (CCA), an important industrial enzyme for the production of 7-Aminocephalosporanic Acid, has very limited and scattered surface lysine residues. A mutant of cephalosporin C acylase (mCCA) has been designed to fuse a poly-lysine tag to the C-terminal of the β-subunit, which is far away from the active site. The free mCCA showed a near equal specific activity with the wild-type CCA, while a much higher activity recovery was obtained for the mCCA than its wild-type counterpart after immobilization on glyoxyl agarose support (73.3 versus 53.3 %). The mCCA’s oriented immobilization enables it to obtain a higher substrate affinity and even a higher thermal stability than the wild-type enzyme. The improvement of stability might be attributed to the multipoint covalent attachment by the oriented enzyme immobilization via the adhered poly-lysine tag, which prevents the dissociation of the β-subunit of CCA from the support.
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overexpression of synthesized cephalosporin c acylase containing mutations in the substrate transport tunnel
Journal of Bioscience and Bioengineering, 2012Co-Authors: Ying Wang, Hui Luo, Wensi Song, Jing Zhang, Zhongyao ShenAbstract:Abstract Cephalosporin C (CPC) acylase converts CPC into 7-Aminocephalosporanic Acid (7-ACA) by single-step enzymatic catalysis. An optimized CPC acylase gene with substituted codons and a reduced GC content was artificially designed, synthesized and overexpressed in recombinant Escherichia coli. The synthetic CPC acylase (sCPCAcy) exhibited 2.3 times more CPC specific deacylation activity with substrate CPC than with substrate glutaryl-7-ACA (GL-7-ACA). Site-directed mutagenesis of the residues around the active center showed that not only the residues that were adjacent to the CPC D -α-aminoadipyl moiety, but also the residues that were in the substrate transport tunnel (Leu666, Ala675, Leu677), played crucial roles in catalysis as the ones locating in active center. Mutant sCPCAcyLeu666Phe and sCPCAcyLeu677Ala exhibited significantly reduced specific enzymatic activity, while mutant sCPCAcyAla675Gly demonstrated enhanced activity. The specific activity of purified sCPCAcy and sCPCAcyAla675Gly was 10.0 U/mg and 11.3 U/mg, respectively. The optimal CPC acylase productivity of mutant sCPCAcyAla675Gly reached 5349 U/l after 24 h in culture, which was a 35% increase over the activity of sCPCAcy.
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characteristic of immobilized cephalosporin c acylase and its application in one step enzymatic conversion of cephalosporin c to 7 Aminocephalosporanic Acid
World Journal of Microbiology & Biotechnology, 2011Co-Authors: Xiangwei Zhu, Hui Luo, Yanhong Chang, Zhongyao ShenAbstract:Cephalosporin C (CPC) acylase is an enzyme which hydrolyzes CPC to 7-Aminocephalosporanic Acid (7-ACA) directly, and therefore has great potential in industrial application. In this study, the CPC acylase from a recombinant Escherichia coli was purified to high purity by immobilized metal affinity chromatography, and the CPC acylase was covalently attached to three kinds of epoxy supports, BB-2, ES-V-1 and LX-1000EP. The immobilized CPC acylase with LX-1000EP as the support shows the highest activity (81 U g−1) suggesting its potential in industrial 7-ACA production. The activity of immobilized enzyme was found to be optimal at pH between 8.5 and 9.5 and to increase with temperature elevation until 55 °C. Immobilized CPC acylase showed good stability at pH between 8.0 and 9.5 and at temperature up to 40 °C. To avoid product degradation, the production of 7-ACA utilizing immobilized enzyme was carried out at 25 °C, pH 8.5 in a designed reactor. Under optimal reaction conditions, a very high 7-ACA yield of 96.7% was obtained within 60 min. In the results of repeated batch production of 7-ACA, 50% activity of the initial cycle was maintained after being recycled 24 times and the average conversion rate of CPC reached 98%.
Qiang Tan - One of the best experts on this subject based on the ideXlab platform.
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progress in one pot bioconversion of cephalosporin c to 7 Aminocephalosporanic Acid
Current Pharmaceutical Biotechnology, 2018Co-Authors: Qiang Tan, Jie Qiu, Xiang Luo, Yewang Zhang, Yuting Liu, Yuquan Chen, Jing Yuan, Wei LiaoAbstract:Background Cephalosporins are the most widely used semisynthetic antibiotics, which acted on bacterial cell wall (peptidoglycan) synthesis. The key intermediate for fabricating about twothirds of cephalosporins in clinical use is 7-Aminocephalosporanic Acid (7-ACA), which is derived from chemical or enzymatic deacylation of the natural antibiotic cephalosporin C (CPC). The chemical deacylation process has been replaced by the enzymatic deacylation process because the chemical process required harsh conditions and released toxic waste. Methods A two-step enzymatic process that utilized D-amino Acid oxidase (DAAO) and 7-β-(4carboxybutanamido)-cephalosporanic Acid acylase (GLA) for two successive reactions has been applied for the conversion of CPC to 7-ACA in an industrial scale. Results To simplify the process and lower costs, the one-pot enzymatic processes were developed by the application of the mono-enzymatic process (application of cephalosporin C acylase or the variants of GLA), di-enzymatic process (simultaneous action of DAAO and GLA) or the tri-enzymatic process (simultaneous action of DAAO, GLA and catalase) for direct conversion of CPC to 7-ACA. Conclusion Here, we mainly focused on the description of these one-pot enzymatic processes and emphasized on the preparation of the involved biocatalysts.
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single pot conversion of cephalosporin c to 7 Aminocephalosporanic Acid in the absence of hydrogen peroxide
World Journal of Microbiology & Biotechnology, 2010Co-Authors: Qiang Tan, Yewang Zhang, Qingxun Song, Dongzhi WeiAbstract:In this study, d-amino Acid oxidase (DAAO) and catalase (CAT) in the permeabilized recombinant Pichia pastori cells were well investigated. It appeared that their thermal stability was negatively correlated with the apparent enzymatic activities. The frozen-melted cells presented the best stability and the lowest apparent activities of DAAO and CAT, whereas the cetyltrimethylammonium bromide (CTAB) permeabilized cells displayed the weakest stability and the highest apparent activities of the two enzymes. Simultaneous action of DAAO and CAT in the CTAB-permeabilized cells and glutaryl-7-Aminocephalosporanic Acid acylase (GA) immobilized on carrier contributed to the conversion of cephalosporin C (CPC) to 7-Aminocephalosporanic Acid (7-ACA) with a yield of 76.2%. During such a reaction cycle, no visible activity loss occurred at the immobilized GA, whereas the loss rates of DAAO and CAT activities were about 0.029 and 1.13 U min−1, respectively. Nevertheless, this problem could be easily solved by continuous feeding of the new permeabilized cell suspension at the rate of 6 ml h−1 to the reactor. Following such a fed-batch strategy, these permeabilized cells and the immobilized GA could be efficiently reused for 6 and 15 reaction cycles, respectively, yielding around 76% 7-ACA at each reaction cycle.
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single pot conversion of cephalosporin c to 7 Aminocephalosporanic Acid using cell bound and support bound enzymes
Enzyme and Microbial Technology, 2006Co-Authors: Qiang Tan, Qingxun Song, Dongzhi WeiAbstract:Abstract The two enzymes in two disparate forms, d -amino Acid oxidase (DAAO) in the permeabilized Pichia pastoris cells and immobilized glutaryl-7-Aminocephalosporanic Acid acylase (GA) on support, were employed to convert cephalosporin C (CPC) to 7-Aminocephalosporanic Acid (7-ACA) in a single reactor. As a catalyst used in the reaction, DAAO in the permeabilized cells was relatively stable and its half-life was up to 14.5 days at 30 °C. In this study, CPC could be converted to 90.9% 7-ACA, 5% α-ketoadipyl-7-ACA (AKA-7-ACA) and 4.1% unidentified by-product within 2.5 h. During the reaction process, the loss of DAAO activity in the reactor was at an average rate of 0.07 U min −1 , but it could be compensated by continuous addition of the new permeabilized cell suspension. At the end of reaction, the stable immobilized GA was intercepted in a specially designed reactor, and reused for the next conversions. The permeabilized cells were separated outside the reactor by centrifugation and reused. Thus, the consecutive production of 7-ACA from CPC in a single reactor is achieved by a fed-batch strategy. In the reaction system, the used permeabilized cells and immobilized GA were renewed every four cycles and every seven cycles, respectively. The yield of 7-ACA reached around 90% at each reaction cycle.
