scholarly journals Cloning and Characterization of a Novel Esterase from Rhodococcus sp. for Highly Enantioselective Synthesis of a Chiral Cilastatin Precursor

2014 ◽  
Vol 80 (23) ◽  
pp. 7348-7355 ◽  
Author(s):  
Yan Zhang ◽  
Jiang Pan ◽  
Zheng-Jiao Luan ◽  
Guo-Chao Xu ◽  
Sunghoon Park ◽  
...  
Keyword(s):  
Enantiomeric Excess ◽  
Green Manufacturing ◽  
Enantioselective Hydrolysis ◽  
Content Type ◽  
Chiral Building Block ◽  
Chain Acyl ◽  
Novel Esterase ◽  
Optically Pure ◽  
Hydrolysis Of

ABSTRACTA novel nonheme chloroperoxidase (RhEst1), with promiscuous esterase activity for enantioselective hydrolysis of ethyl (S)-2,2-dimethylcyclopropanecarboxylate, was identified from a shotgun library ofRhodococcussp. strain ECU1013.RhEst1 was overexpressed inEscherichia coliBL21(DE3), purified to homogeneity, and functionally characterized. Fingerprinting analysis revealed thatRhEst1 preferspara-nitrophenyl (pNP) esters of short-chain acyl groups.pNP esters with a cyclic acyl moiety, especially that with a cyclobutanyl group, were also substrates forRhEst1. TheKmvalues for methyl 2,2-dimethylcyclopropanecarboxylate (DmCpCm) and ethyl 2,2-dimethylcyclopropane carboxylate (DmCpCe) were 0.25 and 0.43 mM, respectively.RhEst1 could serve as an efficient hydrolase for the bioproduction of optically pure (S)-2,2-dimethyl cyclopropane carboxylic acid (DmCpCa), which is an important chiral building block for cilastatin. As much as 0.5 M DmCpCe was enantioselectively hydrolyzed into (S)-DmCpCa, with a molar yield of 47.8% and an enantiomeric excess (ee) of 97.5%, indicating an extremely high enantioselectivity (E= 240) of this novel and unique biocatalyst for green manufacturing of highly valuable chiral chemicals.

scholarly journals Characterization of Glycoside Hydrolase Family 5 Proteins in Schizosaccharomyces pombe

2010 ◽  
Vol 9 (11) ◽  
pp. 1650-1660 ◽  
Author(s):  
Encarnación Dueñas-Santero ◽  
Ana Belén Martín-Cuadrado ◽  
Thierry Fontaine ◽  
Jean-Paul Latgé ◽  
Francisco del Rey ◽  
...  
Keyword(s):  
Cell Wall ◽  
Schizosaccharomyces Pombe ◽  
Protein Characterization ◽  
Wall Material ◽  
Glycoside Hydrolase Family ◽  
Content Type ◽  
Hydrolysis Of ◽  
Controlled Hydrolysis ◽  
Hydrolase Family

ABSTRACT In yeast, enzymes with β-glucanase activity are thought to be necessary in morphogenetic events that require controlled hydrolysis of the cell wall. Comparison of the sequence of the Saccharomyces cerevisiae exo-β(1,3)-glucanase Exg1 with the Schizosaccharomyces pombe genome allowed the identification of three genes that were named exg1 + (locus SPBC1105.05), exg2 + (SPAC12B10.11), and exg3 + (SPBC2D10.05). The three proteins have different localizations: Exg1 is secreted to the periplasmic space, Exg2 is a membrane protein, and Exg3 is a cytoplasmic protein. Characterization of the biochemical activity of the proteins indicated that Exg1 and Exg3 are active only against β(1,6)-glucans while no activity was detected for Exg2. Interestingly, Exg1 cleaves the glucans with an endohydrolytic mode of action. exg1 + showed periodic expression during the cell cycle, with a maximum coinciding with the septation process, and its expression was dependent on the transcription factor Sep1. The Exg1 protein localizes to the septum region in a pattern that was different from that of the endo-β(1,3)-glucanase Eng1. Overexpression of Exg2 resulted in an increase in cell wall material at the poles and in the septum, but the putative catalytic activity of the protein was not required for this effect.

Isolation of an esterase-producing Trichosporon brassicae and its catalytic performance in kinetic resolution of ketoprofen

2001 ◽  
Vol 47 (12) ◽  
pp. 1101-1106 ◽  
Author(s):  
Duan Shen ◽  
Jian-He Xu ◽  
Peng-Fei Gong ◽  
Hui-Yuan Wu ◽  
You-Yan Liu
Keyword(s):  
Ethyl Ester ◽  
Sole Carbon Source ◽  
Kinetic Resolution ◽  
Enantiomeric Excess ◽  
Catalytic Performance ◽  
Soil Samples ◽  
Enantioselective Hydrolysis ◽  
Resting Cells ◽  
Enantiomeric Ratio ◽  
Hydrolysis Of

A yeast strain CGMCC 0574, identified as Trichosporon brassicae, was selected from 92 strains for its high (S) selectivity in the hydrolysis of ketoprofen ethyl ester. The effective strains of the microorganisms were isolated from soil samples with the ester as the sole carbon source. The ethyl ester proved to be the best substrate for resolution of ketoprofen among several ketoprofen esters examined. The resting cells of CGMCC 0574 could catalyze the hydrolysis of ketoprofen ethyl ester with an enantiomeric ratio of 44.9, giving (S)-ketoprofen an enantiomeric excess of 91.5% at 42% conversion.Key words: ketoprofen, biocatalytic resolution, enantioselective hydrolysis, microbial esterase, Trichosporon brassicae.

scholarly journals GlcNAc De-N-Acetylase from the Hyperthermophilic ArchaeonSulfolobus solfataricus

2018 ◽  
Vol 85 (2) ◽  
Author(s):  
Roberta Iacono ◽  
Andrea Strazzulli ◽  
Luisa Maurelli ◽  
Nicola Curci ◽  
Angela Casillo ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Recombinant Enzyme ◽  
Mass Spectrometry Analysis ◽  
Genomic Context ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Additional Information ◽  
The One ◽  
Hydrolysis Of

ABSTRACTSulfolobus solfataricusis an aerobic crenarchaeal hyperthermophile with optimum growth at temperatures greater than 80°C and pH 2 to 4. Within the crenarchaeal group ofSulfolobales,N-acetylglucosamine (GlcNAc) has been shown to be a component of exopolysaccharides, forming their biofilms, and of theN-glycan decorating some proteins. The metabolism of GlcNAc is still poorly understood inArchaea, and one approach to gaining additional information is through the identification and functional characterization of carbohydrate active enzymes (CAZymes) involved in the modification of GlcNAc. The screening ofS. solfataricusextracts allowed the detection of a novel α-N-acetylglucosaminidase (α-GlcNAcase) activity, which has never been identified inArchaea. Mass spectrometry analysis of the purified activity showed a protein encoded by thesso2901gene. Interestingly, the purified recombinant enzyme, which was characterized in detail, revealed a novel de-N-acetylase activity specific for GlcNAc and derivatives. Thus, assays to identify an α-GlcNAcase found a GlcNAc de-N-acetylase instead. The α-GlcNAcase activity observed inS. solfataricusextracts did occur when SSO2901 was used in combination with an α-glucosidase. Furthermore, the inspection of the genomic context and the preliminary characterization of a putative glycosyltransferase immediately upstream ofsso2901(sso2900) suggest the involvement of these enzymes in the GlcNAc metabolism inS. solfataricus.IMPORTANCEIn this study, a preliminary screening of cellular extracts ofS. solfataricusallowed the identification of an α-N-acetylglucosaminidase activity. However, the characterization of the corresponding recombinant enzyme revealed a novel GlcNAc de-N-acetylase, which, in cooperation with the α-glucosidase, catalyzed the hydrolysis of O-α-GlcNAc glycosides. In addition, we show that the product of a gene flanking the one encoding the de-N-acetylase is a putative glycosyltransferase, suggesting the involvement of the two enzymes in the metabolism of GlcNAc. The discovery and functional analysis of novel enzymatic activities involved in the modification of this essential sugar represent a powerful strategy to shed light on the physiology and metabolism ofArchaea.

scholarly journals Gene Cloning and Characterization of Two NADH-Dependent 3-Quinuclidinone Reductases from Microbacterium luteolum JCM 9174

2012 ◽  
Vol 79 (4) ◽  
pp. 1378-1384 ◽  
Author(s):  
Kentaro Isotani ◽  
Junji Kurokawa ◽  
Fumiko Suzuki ◽  
Syunsuke Nomoto ◽  
Takashi Negishi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Residues ◽  
Recombinant Enzymes ◽  
Content Type ◽  
Genes Encoding ◽  
Short Chain Alcohol ◽  
Optically Pure ◽  
High Stereoselectivity ◽  
Suitable Organism

ABSTRACTWe used the resting-cell reaction to screen approximately 200 microorganisms for biocatalysts which reduce 3-quinuclidinone to optically pure (R)-(−)-3-quinuclidinol.Microbacterium luteolumJCM 9174 was selected as the most suitable organism. The genes encoding the protein products that reduced 3-quinuclidinone were isolated fromM. luteolumJCM 9174. ThebacCgene, which consists of 768 nucleotides corresponding to 255 amino acid residues and is a constituent of the bacilysin synthetic gene cluster, was amplified by PCR based on homology to known genes. Theqnrgene consisted of 759 nucleotides corresponding to 252 amino acid residues. Both enzymes belong to the short-chain alcohol dehydrogenase/reductase (SDR) family. The genes were expressed inEscherichia colias proteins which were His tagged at the N terminus, and the recombinant enzymes were purified and characterized. Both enzymes showed narrow substrate specificity and high stereoselectivity for the reduction of 3-quinuclidinone to (R)-(−)-3-quinuclidinol.

scholarly journals Protein Engineering of a Nitrilase from Burkholderia cenocepacia J2315 for Efficient and Enantioselective Production of (R)-o-Chloromandelic Acid

2015 ◽  
Vol 81 (24) ◽  
pp. 8469-8477 ◽  
Author(s):  
Hualei Wang ◽  
Wenyuan Gao ◽  
Huihui Sun ◽  
Lifeng Chen ◽  
Lujia Zhang ◽  
...  
Keyword(s):  
Protein Engineering ◽  
Hot Spots ◽  
Enantiomeric Excess ◽  
Random Mutagenesis ◽  
Burkholderia Cenocepacia ◽  
Saturation Mutagenesis ◽  
Wild Type ◽  
High Concentration ◽  
Content Type ◽  
Optically Pure

ABSTRACTThe nitrilase-mediated pathway has significant advantages in the production of optically pure aromatic α-hydroxy carboxylic acids. However, low enantioselectivity and activity are observed on hydrolyzingo-chloromandelonitrile to produce optically pure (R)-o-chloromandelic acid. In the present study, a protein engineering approach was successfully used to enhance the performance of nitrilase obtained fromBurkholderia cenocepaciastrain J2315 (BCJ2315) in hydrolyzingo-chloromandelonitrile. Four hot spots (T49, I113, Y199, and T310) responsible for the enantioselectivity and activity of BCJ2315 were identified by random mutagenesis. An effective double mutant (I113M/Y199G [encoding the replacement of I with M at position 113 and Y with G at position 199]), which demonstrated remarkably enhanced enantioselectivity (99.1% enantiomeric excess [ee] compared to 89.2%eefor the wild type) and relative activity (360% of the wild type), was created by two rounds of site saturation mutagenesis, first at each of the four hot spots and subsequently at position 199 for combination with the selected beneficial mutation I113M. Notably, this mutant also demonstrated dramatically enhanced enantioselectivity and activity toward other mandelonitrile derivatives and, thus, broadened the substrate scope of this nitrilase. Using an ethyl acetate-water (1:9) biphasic system,o-chloromandelonitrile (500 mM) was completely hydrolyzed in 3 h by this mutant with a small amount of biocatalyst (10 g/liter wet cells), resulting in a high concentration of (R)-o-chloromandelic acid with 98.7%ee, to our knowledge the highest ever reported. This result highlights a promising method for industrial production of optically pure (R)-o-chloromandelic acid. Insight into the source of enantioselectivity and activity was gained by homology modeling and molecular docking experiments.

scholarly journals Identification and Characterization of a New 7-Aminocephalosporanic Acid Deacetylase from Thermophilic Bacterium Alicyclobacillus tengchongensis

2015 ◽  
Vol 198 (2) ◽  
pp. 311-320 ◽  
Author(s):  
Jun-Mei Ding ◽  
Ting-Ting Yu ◽  
Nan-Yu Han ◽  
Jia-Lin Yu ◽  
Jun-Jun Li ◽  
...  
Keyword(s):  
Building Blocks ◽  
Hypothetical Protein ◽  
Catalytic Triad ◽  
Thermophilic Bacterium ◽  
Starting Material ◽  
Main Method ◽  
Content Type ◽  
Enzymatic Transformation ◽  
Chain Acyl

ABSTRACTDeacetylation of 7-aminocephalosporanic acid (7-ACA) at position C-3 provides valuable starting material for producing semisynthetic β-lactam antibiotics. However, few enzymes have been characterized in this process before now. Comparative analysis of the genome of the thermophilic bacteriumAlicyclobacillus tengchongensisrevealed a hypothetical protein (EstD1) with typical esterase features. The EstD1 protein was functionally cloned, expressed, and purified fromEscherichia coliBL21(DE3). It indeed displayed esterase activity, with optimal activity at around 65°C and pH 8.5, with a preference for esters with short-chain acyl esters (C2to C4). Sequence alignment revealed that EstD1 is an SGNH hydrolase with the putative catalytic triad Ser15, Asp191, and His194, which belongs to carbohydrate esterase family 12. EstD1 can hydrolyze acetate at the C-3 position of 7-aminocephalosporanic acid (7-ACA) to form deacetyl-7-ACA, which is an important starting material for producing semisynthetic β-lactam antibiotics. EstD1 retained more than 50% of its initial activity when incubated at pH values ranging from 4 to 11 at 65°C for 1 h. To the best of our knowledge, this enzyme is a new SGNH hydrolase identified from thermophiles that is able to hydrolyze 7-ACA.IMPORTANCEDeacetyl cephalosporins are highly valuable building blocks for the industrial production of various kinds of semisynthetic β-lactam antibiotics. These compounds are derived mainly from 7-ACA, which is obtained by chemical or enzymatic processes from cephalosporin C. Enzymatic transformation of 7-ACA is the main method because of the adverse effects chemical deacylation brought to the environment. SGNH hydrolases are widely distributed in plants. However, the tools for identifying and characterizing SGNH hydrolases from bacteria, especially from thermophiles, are rather limited. Here, our work demonstrates that EstD1 belongs to the SGNH family and can hydrolyze acetate at the C-3 position of 7-ACA. Moreover, this study can enrich our understanding of the functions of these enzymes from this family.

An easy access to optically pure (R)-malic acid via enantioselective hydrolysis of diethyl malate byRhizopus lipase

1992 ◽  
Vol 14 (9) ◽  
pp. 795-800 ◽  
Author(s):  
Kazutoshi Ushio ◽  
Kouzou Nakagawa ◽  
Katsuhiko Nakagawa ◽  
Kunio Watanabe
Keyword(s):  
Malic Acid ◽  
Easy Access ◽  
Enantioselective Hydrolysis ◽  
Optically Pure ◽  
Hydrolysis Of

scholarly journals Genetic and Kinetic Characterization of the Novel AmpC β-Lactamases DHA-6 and DHA-7

2014 ◽  
Vol 58 (11) ◽  
pp. 6544-6549 ◽  
Author(s):  
Francisco José Pérez-Llarena ◽  
Laura Zamorano ◽  
Frédéric Kerff ◽  
Alejandro Beceiro ◽  
Patricia García ◽  
...  
Keyword(s):  
Enterobacter Cloacae ◽  
Slight Reduction ◽  
The Novel ◽  
Incompatibility Group ◽  
Content Type ◽  
E Coli ◽  
Natural Variants ◽  
Tertiary Protein Structure ◽  
Hydrolysis Of

ABSTRACTDuring a Spanish surveillance study, two natural variants of DHA β-lactamases, DHA-6 and DHA-7, were found, with the replacements Ala226Thr and Phe322Ser, respectively, with respect to DHA-1. The DHA-6 and DHA-7 enzymes were isolated fromEscherichia coliandEnterobacter cloacaeclinical isolates, respectively. The aim of this study was to genetically, microbiologically, and biochemically characterize the DHA-6 and DHA-7 β-lactamases. TheblaDHA-6andblaDHA-7genes were located in the I1 and HI2 incompatibility group plasmids of 87.3 and 310.4 kb, respectively. The genetic contexts ofblaDHA-6andblaDHA-7were similar to that already described for theblaDHA-1gene and included theqnrB4andaadAgenes. The MICs for cephalothin, aztreonam, cefotaxime, and ceftazidime were 8- to 32-fold lower for DHA-6 than for DHA-1 or DHA-7 expressed in the same isogenicE. coliTG1 strain. Interestingly, the MIC for cefoxitin was higher in the DHA-6-expressing transformant than in DHA-1 or DHA-7. Biochemical studies with pure β-lactamases revealed slightly lower catalytic efficiencies of DHA-6 against cephalothin, ceftazidime, and cefotaxime than those of DHA-1 and DHA-7. To understand this behavior, stability experiments were carried out and showed that the DHA-6 protein displayed significantly higher stability than the DHA-1 and DHA-7 enzymes. The proximity of Thr226 to the N terminus in the tertiary protein structure in DHA-6 may promote this stabilization and, consequently, may induce a slight reduction in the dynamic of this enzyme that primarily affects the hydrolysis of some of the bulkiest antibiotics.

Sign in / Sign up

Export Citation Format

Share Document