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1.
J Microbiol Biotechnol ; 32(5): 672-679, 2022 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-35354762

RESUMO

Microbial lipases are used widely in the synthesis of various compounds due to their substrate specificity and position specificity. 4-Ethyl malate (4-EM) made from diethyl malate (DEM) is an important starting material used to make argon fluoride (ArF) photoresist. We tested several microbial lipases and found that Photobacterium lipolyticum M37 lipase position-specifically hydrolyzed DEM to produce 4-EM. We purified the reaction product through silica gel chromatography and confirmed that it was 4-EM through nuclear magnetic resonance analysis. To mass-produce 4-EM, DEM hydrolysis reaction was performed using an enzyme reactor system that could automatically control the temperature and pH. Effects of temperature and pH on the reaction process were investigated. As a result, 50°C and pH 4.0 were confirmed as optimal reaction conditions, meaning that M37 was specifically an acid lipase. When the substrate concentration was increased to 6% corresponding to 0.32 M, the reaction yield reached almost 100%. When the substrate concentration was further increased to 12%, the reaction yield was 81%. This enzyme reactor system and position-specific M37 lipase can be used to mass-produce 4-EM, which is required to synthesize ArF photoresist.


Assuntos
Lipase , Malatos , Concentração de Íons de Hidrogênio , Hidrólise , Lipase/metabolismo , Photobacterium/metabolismo , Especificidade por Substrato , Temperatura
2.
J Microbiol Biotechnol ; 20(9): 1300-6, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20890095

RESUMO

Ethyl (R, S)-4-chloro-3-hydroxybutanoate (ECHB) is a useful chiral building block for the synthesis of L-carnitine and hypercholesterolemia drugs. The yeast reductase, YOL151W (GenBank locus tag), exhibits an enantioselective reduction activity, converting ethyl-4-chlorooxobutanoate (ECOB) exclusively into (R)-ECHB. YOL151W was generated in Escherichia coli cells and purified via Ni- NTA and desalting column chromatography. It evidenced an optimum temperature of 45 degrees C and an optimum pH of 6.5-7.5. Bacillus subtilis glucose dehydrogenase (GDH) was also expressed in Escherichia coli, and was used for the recycling of NADPH, required for the reduction reaction. Thereafter, Escherichia coli cells co-expressing YOL151W and GDH were constructed. After permeablization treatment, the Escherichia coli whole cells were utilized for ECHB synthesis. Through the use of this system, the 30 mM ECOB substrate could be converted to (R)-ECHB.


Assuntos
Acetoacetatos/metabolismo , Bacillus subtilis/enzimologia , Biotecnologia/métodos , Butiratos/metabolismo , Escherichia coli/metabolismo , Glucose 1-Desidrogenase/genética , Oxirredutases/genética , Proteínas de Saccharomyces cerevisiae/genética , Expressão Gênica , Engenharia Genética , NADP/metabolismo , Proteínas Recombinantes/genética
3.
Biochim Biophys Acta ; 1804(9): 1841-9, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20601218

RESUMO

Ethyl (S)-4-chloro-3-hydroxy butanoate (ECHB) is a building block for the synthesis of hypercholesterolemia drugs. In this study, various microbial reductases have been cloned and expressed in Escherichia coli. Their reductase activities toward ethyl-4-chloro oxobutanoate (ECOB) have been assayed. Amidst them, Baker's yeast YDL124W, YOR120W, and YOL151W reductases showed high activities. YDL124W produced (S)-ECHB exclusively, whereas YOR120W and YOL151W made (R)-form alcohol. The homology models and docking models with ECOB and NADPH elucidated their substrate specificities and enantioselectivities. A glucose dehydrogenase-coupling reaction was used as NADPH recycling system to perform continuously the reduction reaction. Recombinant E. coli cell co-expressing YDL124W and Bacillus subtilis glucose dehydrogenase produced (S)-ECHB exclusively.


Assuntos
Butiratos/síntese química , Butiratos/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Proteínas de Saccharomyces cerevisiae/síntese química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Bacillus subtilis/metabolismo , Butiratos/química , Escherichia coli/metabolismo , Glucose 1-Desidrogenase/metabolismo , Modelos Químicos , NADP/metabolismo , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Estereoisomerismo , Especificidade por Substrato
4.
J Microbiol Biotechnol ; 20(2): 325-31, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20208436

RESUMO

Rhodococcus erythropolis amidase was expressed in Escherichia coli cells. The crude amidase in the cell-free extract was immobilized using the cross-linked enzyme aggregate (CLEA) method. The crude amidase was mixed with bovine serum albumin and then precipitated with ammonium sulfate. The resultant precipitant was subsequently cross-linked with glutaraldehyde. Scanning electron microscopy revealed that this co-CLEA had a ball-like shape of a diameter of approximately 1 micronm. This co-CLEA evidenced hydrolytic activity toward a variety of amide substrates. The amidase co-CLEA evidenced an optimum temperature of 60 degrees and an optimum pH of 8.0, results that were similar to those of the soluble amidase. The reaction stability of the co-CLEA was increased. That is, it was stable up to 50 degrees and in a pH range of 5.0-12.0. Additionally, the co-CLEA could be recovered by centrifugation, and retained 96% activity after 3 repeated cycles. This amidase co-CLEA may prove useful as a substitute for soluble amidase as a biocatalyst in the pharmaceutical and chemical industries.


Assuntos
Ácidos/metabolismo , Amidoidrolases/metabolismo , Aminas/metabolismo , Proteínas de Bactérias/metabolismo , Enzimas Imobilizadas/metabolismo , Rhodococcus/enzimologia , Amidoidrolases/química , Amidoidrolases/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biotransformação , Reagentes de Ligações Cruzadas/química , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/genética , Glutaral/química , Rhodococcus/genética
5.
Appl Microbiol Biotechnol ; 87(1): 185-93, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20111861

RESUMO

3-Chloro-1-phenyl-1-propanol is used as a chiral intermediate in the synthesis of antidepressant drugs. Various microbial reductases were expressed in Escherichia coli, and their activities toward 3-chloro-1-phenyl-1-propanone were evaluated. The yeast reductase YOL151W (GenBank locus tag) exhibited the highest level of activity and exclusively generated the (S)-alcohol. Recombinant YOL151W was purified by Ni-nitrilotriacetic acid (Ni-NTA) and desalting column chromatography. It displayed an optimal temperature and pH of 40 degrees C and 7.5-8.0, respectively. The glucose dehydrogenase coupling reaction was introduced as an NADPH regeneration system. NaOH solution was occasionally added to maintain the reaction solution pH within the range of 7.0-7.5. By using this reaction system, the substrate (30 mM) could be completely converted to the (S)-alcohol product with an enantiomeric excess value of 100%. A homology model of YOL151W was constructed based on the structure of Sporobolomyces salmonicolor carbonyl reductase (Protein Data Bank ID: 1Y1P). A docking model of YOL151W with NADPH and 3-chloro-1-phenyl-1-propanone was then constructed, which showed that the cofactor and substrate bound tightly to the active site of the enzyme in the lowest free energy state and explained how the (S)-alcohol was produced exclusively in the reduction process.


Assuntos
Hidrocarbonetos Clorados/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Propanóis/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sítios de Ligação , Estabilidade Enzimática , Hidrocarbonetos Clorados/química , Conformação Molecular , NADP/química , NADP/metabolismo , Oxirredutases/genética , Oxirredutases/isolamento & purificação , Propanóis/química , Ligação Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Estereoisomerismo , Especificidade por Substrato
6.
J Microbiol Biotechnol ; 18(3): 552-9, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18388476

RESUMO

Ethyl (S)-4-chloro-3-hydroxybutyrate is an intermediate for the synthesis of Atorvastatin, a chiral drug used for hypercholesterolemia. A Rhodococcus erythropolis strain (No. 7) able to convert 4-chloro-3-hydroxybutyronitrile into 4-chloro-3-hydroxybutyric acid has recently been isolated from soil. This activity has been regarded as having been caused by the successive actions of the nitrile hydratase and amidase. In this instance, the corresponding amidase gene was cloned from the R. erythropolis strain and expressed in Escherichia coli cells. A soluble active form of amidase enzyme was obtained at 18 degrees . The Ni column-purified recombinant amidase was found to have a specific activity of 3.89 U/mg toward the substrate isobutyramide. The amidase was found to exhibit a higher degree of activity when used with midchain substrates than with short-chain ones. Put differently, amongst the various amides tested, isobutyramide and butyramide were found to be hydrolyzed the most rapidly. In addition to amidase activity, the enzyme was found to exhibit acyltransferase activity when hydroxyl amine was present. This dual activity has also been observed in other enzymes belonging to the same amidase group (E.C. 3.5.1.4). Moreover, the purified enzyme was proven to be able to enantioselectively hydrolyze 4-chloro-3-hydroxybutyramide into the corresponding acid. The e.e. value was measured to be 52% when the conversion yield was 57%. Although this e.e. value is low for direct commercial use, molecular evolution could eventually result in this amidase being used as a biocatalyst for the production of ethyl (S)-4-chloro-3-hydroxybutyrate.


Assuntos
Amidoidrolases/metabolismo , Expressão Gênica , Hidroxibutiratos/metabolismo , Rhodococcus/enzimologia , Amidas/metabolismo , Amidoidrolases/química , Amidoidrolases/genética , Amidoidrolases/isolamento & purificação , Sequência de Aminoácidos , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Hidroliases/metabolismo , Hidrólise , Cinética , Dados de Sequência Molecular , Filogenia , Rhodococcus/classificação , Rhodococcus/genética , Rhodococcus/isolamento & purificação , Alinhamento de Sequência , Microbiologia do Solo , Especificidade por Substrato
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