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1.
Biochemical characterization of two haloalkane dehalogenases: DccA from Caulobacter crescentus and DsaA from Saccharomonospora azurea
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Vladimir N. Malashkevich Steven C. Almo Emily C. Mundorff 《Protein science : a publication of the Protein Society》2016,25(4):877-886
Two putative haloalkane dehalogenases (HLDs) of the HLD‐I subfamily, DccA from Caulobacter crescentus and DsaA from Saccharomonospora azurea, have been identified based on sequence comparisons with functionally characterized HLD enzymes. The two genes were synthesized, functionally expressed in E. coli and shown to have activity toward a panel of haloalkane substrates. DsaA has a moderate activity level and a preference for long (greater than 3 carbons) brominated substrates, but little activity toward chlorinated alkanes. DccA shows high activity with both long brominated and chlorinated alkanes. The structure of DccA was determined by X‐ray crystallography and was refined to 1.5 Å resolution. The enzyme has a large and open binding pocket with two well‐defined access tunnels. A structural alignment of HLD‐I subfamily members suggests a possible basis for substrate specificity is due to access tunnel size. 相似文献
2.
Luckarift HR Dalton H Sharma ND Boyd DR Holt RA 《Applied microbiology and biotechnology》2004,65(6):678-685
The kinetic resolution of racemic sulfoxides by dimethyl sulfoxide (DMSO) reductases was investigated with a range of microorganisms. Three bacterial isolates (provisionally identified as Citrobacter braakii, Klebsiella sp. and Serratia sp.) expressing DMSO reductase activity were isolated from environmental samples by anaerobic enrichment with DMSO as terminal electron acceptor. The organisms reduced a diverse range of racemic sulfoxides to yield either residual enantiomer depending upon the strain used. C. braakii DMSO-11 exhibited wide substrate specificity that included dialkyl, diaryl and alkylaryl sulfoxides, and was unique in its ability to reduce the thiosulfinate 1,4-dihydrobenzo-2, 3-dithian-2-oxide. DMSO reductase was purified from the periplasmic fraction of C. braakii DMSO-11 and was used to demonstrate unequivocally that the DMSO reductase was responsible for enantiospecific reductive resolution of racemic sulfoxides. 相似文献
3.
Chaubey A Parshad R Koul S Taneja SC Qazi GN 《Applied microbiology and biotechnology》2006,73(3):598-606
Arthrobacter sp. lipase (ABL, MTCC no. 5125) is being recognized as an efficient enzyme for the resolution of drugs and their intermediates. The immobilization of ABL on various matrices for its enantioselectivity, stability, and reusability has been studied. Immobilization by covalent bonding on sepharose and silica afforded a maximum of 380 and 40 IU/g activity, respectively, whereas sol–gel entrapment provided a maximum of 150 IU/g activity in dry powder. The immobilized enzyme displayed excellent stability in the pH range of 4–10 and even at higher temperature, i.e., 50–60°C, compared to free enzyme, which is unstable under extreme conditions. The resolution of racemic auxiliaries like 1-phenyl ethanol and an intermediate of antidepressant drug fluoxetine, i.e., ethyl 3-hydroxy-3-phenylpropanoate alkyl acylates, provided exclusively R-(+) products (∼99% ee, E=646 and 473), compared to cell free extract/whole cells which gave a product with ∼96% ee (E=106 and 150). The repeated use (ten times) of covalently immobilized and entrapped ABL resulted in no loss in activity, thus demonstrating its prospects for commercial applications. 相似文献
4.
《Biocatalysis and Biotransformation》2013,31(5-6):340-347
Pseudomonas sp. lipase (PSL) was successfully immobilized on a novel hydrophobic polymer support through physical adsorption and the immobilized PSL was used for resolution of (R,S)-2-octanol with vinyl acetate as acyl donor. Enhanced activity and enantioselectivity were observed from the immobilized PSL compared with free PSL. The effects of reaction conditions such as temperature, water activity, substrate molar ratio and the amount of immobilized lipase were investigated. Under optimum conditions, the residual (S)-2-octanol was recovered with 99.5% enantiomeric excess at 52.9% conversion. The results also indicated that the immobilized PSL could maintain 94% of its initial activity even after reusing it five times. 相似文献
5.
MrpA is the multimer resolution protein of the Streptomyces coelicolor A3(2) plasmid SCP2*. Previously, MrpA was found to significantly increase the stability of SCP2*-derived plasmids in Streptomyces lividans. The present report gives a functional characterization of MrpA. A sequence alignment revealed that MrpA shares highly conserved
residues with members of the tyrosine recombinase family. After overexpression and Strep-tag purification, a DNase I footprint
analysis and a gel mobility shift assay allowed for the identification of the 36-bp MrpA binding site mrpS. The mrpS site shows the configuration typical for tyrosine recombinases and contains two MrpA binding sites. The activity of MrpA
was explored in vivo in E. coli cells and in vitro using purified MrpA. Depending on the position and orientation of the mrpS sites, three activities were detected: integration, resolution, and inversion. No accessory sites or proteins were required.
Substitution of the conserved tyrosine (Y354F) by site-directed mutagenesis resulted in a complete loss of recombination activity
but it still allowed the binding of MrpA to mrpS. The results define MrpA as a new site-specific tyrosine recombinase that acts with mrpS. In addition, we suggest that Y354 provides the nucleophile for DNA cleavage during recombination. 相似文献
6.
Several new microorganisms have been isolated from soil samples with high epoxide hydrolase activity toward ethyl 3,4-epoxybutyrate.
Screening was performed by enrichment culture on alkenes as sole carbon source, followed by chiral gas chromatography. Eight
strains were discovered with enantioselectivity from moderate to high level and identified as bacterial and yeast species.
Cells were cultivated under aerobic condition at 30°C using glucose as carbon source and resting cells were used as biocatalysts
for kinetic resolution of ethyl 3,4-epoxybutyrate. Among isolated microorganisms, Acinetobacter baumannii showed highest enantioselectivity for (S)-enantiomer, resulting in (R)-ethyl-3,4-epoxybutyrates (>99%ee, 46% yield). It is the first report on the fact that epoxide hydrolases originating from
bacterial species of A. baumannii was applied to kinetic resolution of ethyl 3,4-epoxybutyrate in order to obtain enantiopure high-value-added (R)-ethyl-3,4-epoxybutyrate. 相似文献
7.
Masahiro Goto Sadafumi Noda Noriho Kamiya Fumiyuki Nakashio 《Biotechnology letters》1996,18(7):839-844
Summary Surfactant-coated lipases have been utilized as a biocatalyst for the resolution of racemic ibuprofen. S-(+)-ibuprofen was selectively transferred to the ester form by Mucor javanicus or Candida rugosa lipase. The enzymatic activity of upases in organic media was remarkably enhanced by coating with a nonionic surfactant. The reaction rates of the coated lipases were increased around 100-fold that of the powder lipases. 相似文献
8.
《Biocatalysis and Biotransformation》2013,31(4):315-329
Three novel lipase-producing microorganisms have been isolated from 526 actinomycete strains by employing screening techniques on solid media. Time-course and scale-up of enzyme production were analyzed. The lipases, produced by microorganisms belonging to the Streptomyces genus, were tested in several reactions in organic medium using unnatural substrates. The lyophilized crude lipases are stable at least for 1 month at 4°C (100% recovered activity). The lipase activity per milliliter of cell culture broth was higher than described in the literature for other lipases from actinomycetes. The three selected lipases displayed better activity than commercial lipase from Candida rugosa in the resolution of chiral secondary alcohols. The lipase from S. halstedii also displayed very good activity in the synthesis of carbamates. 相似文献
9.
A thermostable esterase from the hyperthemophilic archaeonSulfolobus solfataricus was partially purified 590-fold with 16.2% recovery. The partially purified esterase had a specific activity of 29.5μmol
min−1 mg−1 when the enzyme activity was determined usingp-nitrophenyl butyrate as a substrate. The apparent molecular weight was about 100 kDa, while the optimum temperature and pH
for esterase were 75°C and 8.0, respectively. The enzyme showed high thermal stability and solvent tolerance in comparison
to its mesophilic counterpart. The enzyme also showed chiral resolution activity for (S)-ibuprofen, indicating thatS. solfataricus esterase can be used for the production of commercially important chiral drugs. 相似文献
10.
Wessel Van Leeuwen Marc J. M. Hagendoorn Tom Ruttink Remco Van Poecke Linus H. W. Van Der Plas Alexander R. Van Der Krol 《Plant Molecular Biology Reporter》2000,18(2):143-144
The properties of the firefly luciferase (LUC) make it a very good nondestructive reporter to quantify and image transgene
promoter activity in plants. The short half-life of the LUC mRNA and protein, and the very limited regeneration of the LUC
protein after reacting with luciferin, enables monitoring of changes in gene activity with a high time resolution. However,
the ease at which luciferase activity is measuredin planta, using a light sensitive camera system (2D-luminometer), contrasts sharply with the complications that arise from interpreting
the results. A variegated pattern of luciferase activity, that is often observed inin planta measurements, might either be caused by differences in influx, availability of the substrates (luciferin, oxygen, ATP) or
by local differences in reporter gene activity. Here we tested the possible contribution of differences in the availability
of each substrate to the variegatedin planta luciferase activity, and we show whenin planta luciferase activity is measured under substrate equilibrium conditions and can be related to the promoter activity of the
reporter gene. Furthermore, we demonstrate the effects of protein stability, apparent half-life of luciferase activity, regeneration
of luciferase and pH on thein vivo andin vitro luciferase measurements. The combined results give the prerequisites for the correct utilisation of the luciferase reporter
system, especially forin vivo gene expression studies in plant research. 相似文献
11.
Kaieda M Nagayoshi M Hama S Kondo A Fukuda H 《Applied microbiology and biotechnology》2004,65(3):301-305
In the present study, we used gene manipulation to construct a recombinant Aspergillus oryzae strain overexpressing lipase and investigated its application to the optical resolution of chiral compounds. A. oryzae niaD300, which was derived from the wild-type strain RIB40, was used as the host strain. The tglA gene, which encodes a triacylglycerol lipase, was cloned from the A. oryzae niaD300 chromosomal genome, then reintroduced, with and without a secretion-signal sequence, into the genome and expressed under the control of the improved glaA promoter of plasmid pNGA142. The resulting recombinant strain overexpressing A. oryzae lipase was immobilized within biomass-support particles and used as a whole-cell biocatalyst. The immobilized lipase-overexpressing strain with secretion-signal sequence showed high activity and was used to selectively synthesize (R)-1-phenylethyl acetate from (RS)-1-phenylethanol and vinyl acetate. After 48 h reaction at 30°C with molecular sieve 4A, the yield and enantiomeric excess (%ee) of (R)-1-phenylethyl acetate reached approximately 90 and 95%ee, respectively. The whole-cell biocatalyst for optical resolution of chiral compounds produced in this study maintained its activity over 25 batch-reaction cycles. 相似文献
12.
Kinetic resolution of N‐acetyl‐DL‐alanine methyl ester using immobilized Escherichia coli cells bearing recombinant esterase from Bacillus cereus
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D‐alanine is widely used in medicine, food, additives, cosmetics, and other consumer items. Esterase derived from Bacillus cereus WZZ001 exhibits high hydrolytic activity and stereoselectivity. In this study, we expressed the esterase gene in Escherichia coli BL21 (DE3). We analyzed the biocatalytic resolution of N‐acetyl‐DL‐alanine methyl ester by immobilized whole E. coli BL21 (DE3) cells, which were prepared through embedding and cross‐linking. We analyzed biocatalytic resolution under the optimal conditions of pH of 7.0, temperature of 40°C and substrate concentration of at 700 mM with an enantiomeric excess of 99.99% and e.e.p of 99.50%. The immobilized recombinant B. cereus esterase E. coli BL21 (DE3) cells exhibited excellent reusability and retained 86.04% of their initial activity after 15 cycles of repeated reactions. The immobilized cells are efficient and stable biocatalysts for the preparation of N‐acetyl‐D‐alanine methyl esters. 相似文献
13.
Gen-Hai Zhao Hui Li Wei Liu Wei-Guo Zhang Fei Zhang Qian Liu Qing-Cai Jiao 《Amino acids》2011,40(1):215-220
In this research, an improved method for preparation of optically pure β-hydroxy-α-amino acids, catalyzed by serine hydroxymethyl
transferase with threonine aldolase activity, is reported. Using recombinant serine hydroxymethyl transferase (SHMT), an enzymatic
resolution process was established. A series of new substrates, β-phenylserine, β-(nitrophenyl) serine and β-(methylsulfonylphenyl)
serine were used in the resolution process catalyzed by immobilized Escherichia coli cells with SHMT activity. It was observed that the K
m for l-threonine was 28-fold higher than that for l-allo-threonine, suggesting that this enzyme can be classified as a low-specificity l-allo-threonine aldolase. The results also shows that SHMT activity with β-phenylserine as substrate was about 1.48-fold and 1.25-fold
higher than that with β-(methylsulfonylphenyl) serine and β-(nitrophenyl) serine as substrate, respectively. Reaction conditions
were optimized by using 200 mmol/l β-hydroxy-α-amino acid, and 0.1 g/ml of immobilized SHMT cells at pH 7.5 and 45°C. Under
these conditions, the immobilized cells were continuously used 10 times, yielding an average conversion rate of 60.4%. Bead
activity did not change significantly the first five times they were used, and the average conversion rate during the first
five instances was 84.1%. The immobilized cells exhibited favourable operational stability. 相似文献
14.
1-aminocyclopropane-1-carboxylate synthase (ACS) is a key enzyme in the biosynthesis of the plant hormone ethylene. Recently, a new biological role for ACS has been found in Cucumis melo where a single point mutation (A57V) of one isoform of the enzyme, causing reduced activity, results in andromonoecious plants. We present here a straightforward structural basis for the reduced activity of the A57V mutant, based on our work on Malus domestica ACS, including a new structure of the unliganded apple enzyme at 1.35 Å resolution. 相似文献
15.
The phage N15 protelomerase enzyme (TelN) is essential for the replication of its genome by resolution of its telRL domain, located within a telomerase occupancy site (tos), into hairpin telomeres. Isolation of TelN for in vitro processing of tos, however, is a highly complex process, requiring multiple purification steps. In this study a simplified protocol for crude total protein extraction is described that retains the tos-cleaving activity of TelN for at least 4 weeks, greatly simplifying in vitro testing of its activity. This protocol may be extended for functional analysis of other phage and bacterial proteins, particularly DNA-processing enzymes. 相似文献
16.
Adrian P. Ward 《The Journal of eukaryotic microbiology》1985,32(2):357-359
Electrophoretic separation of the water-soluble proteins from seven Acanthamoeba strains, followed by lactic dehydrogenase (LDH) visualization by the zymogram method, has indicated a single LDH activity in each extract. To account for this observation, three alternatives are considered: inadequate electrophoretic resolution, two gene-encodement of LDH with constraints relating to subunit assembly or oligomer stability, and one gene-LDH encodement. 相似文献
17.
Seungha Hwang Hyejin Hyun Byoungju Lee Youngseub Park Eun Yeol Lee Chayong Choi 《Biotechnology and Bioprocess Engineering》2006,11(4):282-287
ACaulobacter crescentus epoxide hydrolase (CCEH) from a recombinantEscherichia coli was purified to homogeneity using a three-step procedure. The CCEH protein was purified 7.3-fold with a 22.9% yield in overall
activity. The optimal reaction temperature and pH were determined to be 37°C and pH 8.0, respectively. The addition of 10%
(v/v) dimethylsulfoxide as a cosolvent improved the enantioselectivity of CCEH for a batch kinetic resolution of racemic indene
oxide. 相似文献
18.
Marcio Voloch Michael R. Ladisch Victor W. Rodwell George T. Tsao 《Biotechnology and bioengineering》1981,23(6):1289-1296
Fermentation of xylose by Klebsiella pneumoniae (ATCC 8724) producers meso and nonmeso 2,3-butaneodiol. The enzyme Kinetic of 2,3-butanediol stereoisomer formation from acetone is currently under study in our laboratory. Modeling of these kinetics requires resolution of meso and racemic 2,3-butanediol and positive identification of these resolved components. We report their resolution by aqueous liquid chromatography on both an analytical and a preparative scale. The resolved stereoisomer were identified by a combination of gas chromatography, gas chromatography/mass spectroscopy, 13C-NMR spectroscopy, optical activity, and, melting points of the m-dinitrobenzoyl eaters of meso and racemic 2,3-butanediol. An aqueous liquid chromatographic technique for resolving and qualifying major components of a butanediol fermentation mixture in 40 min is presented. 相似文献
19.
《Biocatalysis and Biotransformation》2013,31(2-3):47-53
AbstractPseudomonas sp. lipase (PSL) immobilization was performed using three different protocols. Lipase immobilized on Diaion HP20 (HP20-PSL) exhibited the highest catalytic activity and stability in the kinetic resolution of racemic 2-octanol. The reaction rate of HP20-PSL was approximately 20 times higher than that of free PSL and the residual activities of HP20-PSL and free PSL were respectively 84% and 19% after incubation in the reaction medium for 72 h. A study of the effect of different reaction parameters on HP20-PSL-catalyzed resolution of (R,S)-2octanol showed that the optimal water content of the immobilized matrix and the optimal molar ratio of vinyl acetate to 2-octanol were 60 ± 5% and 2.5:1, respectively. Under the optimized reaction conditions, (S)-2-octanol of high optically purity (enantiomeric excess > 99%) could be recovered at 53% conversion rate, and HP20-PSL could be reused for ten cycles without significant decrease in its activity and enantioselectivity. 相似文献
20.
Wheat germ lipase catalyzed kinetic resolution of secondary alcohols in non-aqueous media 总被引:1,自引:0,他引:1
The lipase from wheat germ was used for the kinetic resolution of secondary alcohols. It has the opposite enantioselectivity
against the Kazlauskas rule and acts as an anti-Kazlauskas catalyst. The effect of initial water activity, organic solvent,
acyl donor and temperature were investigated. Wheat germ lipase had a high activity and enantioselectivity only in n-hexane with a high initial water activity (αw = 0.97), especially with 1-phenylethanol (C 32%, E > 200). Its performance changed little with the chain length of acyl donor and temperature.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献