共查询到20条相似文献,搜索用时 31 毫秒
1.
J.-C. Wang M. Sakakibara J.-Q. Liu T. Dairi N. Itoh 《Applied microbiology and biotechnology》1999,52(3):386-392
The gene encoding phenylacetaldehyde reductase (PAR), a useful biocatalyst for producing chiral alcohols, was cloned from
the genomic DNA of the styrene-assimilating Corynebacterium sp. strain ST-10. The gene contained an opening reading frame consisting of 1,158 nucleotides corresponding to 385 amino
acid residues. The subunit molecular weight was calculated to be 40,299, which was in agreement with that determined by polyacrylamide
gel electrophoresis. The enzyme was sufficiently expressed in recombinant Escherichia coli cells for practical use and purified to homogeneity by three-column chromatography steps. The predicted amino acid sequence
displayed only 20–29% identity with zinc-containing, NAD+-dependent, long-chain alcohol dehydrogenases. Nevertheless, the probable NAD+- and zinc-binding sites are conserved although one of the three catalytic zinc-binding residues of the zinc-containing, long-chain
alcohol dehydrogenases was substituted by Asp in PAR. The protein contains 7.6 mol zinc/mol tetramer. Therefore, the enzyme
was considered as a new member of zinc-containing, long-chain alcohol dehydrogenases with a particular and broad substrate
specificity.
Received: 5 March 1999 / Received last revision: 10 May 1999 / Accepted: 16 May 1999 相似文献
2.
Kazuoka T Oikawa T Muraoka I Kuroda S Soda K 《Extremophiles : life under extreme conditions》2007,11(2):257-267
An NAD+-dependent alcohol dehydrogenase of a psychrotorelant from Antarctic seawater, Flavobacterium frigidimaris KUC-1 was purified to homogeneity with an overall yield of about 20% and characterized enzymologically. The enzyme has an
apparent molecular weight of 160k and consists of four identical subunits with a molecular weight of 40k. The pI value of the enzyme and its optimum pH for the oxidation reaction were determined to be 6.7 and 7.0, respectively. The enzyme
contains 2 gram-atoms Zn per subunit. The enzyme exclusively requires NAD+ as a coenzyme and shows the pro-R stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of NAD+. F. frigidimaris KUC-1 alcohol dehydrogenase shows as high thermal stability as the enzymes from thermophilic microorganisms. The enzyme is
active at 0 to over 85°C and the most active at 70°C. The half-life time and k
cat value at 60°C were calculated to be 50 min and 27,400 min−1, respectively. The enzyme also shows high catalytic efficiency at low temperatures (0–20°C) (k
cat/K
m at 10°C; 12,600 mM−1 min−1) similar to other cold-active enzymes from psychrophiles. The alcohol dehydrogenase gene is composed of 1,035 bp and codes
344 amino acid residues with an estimated molecular weight of 36,823. The sequence identities were found with the amino acid
sequences of alcohol dehydrogenases from Moraxella sp. TAE123 (67%), Pseudomonas aeruginosa (65%) and Geobacillus stearothermophilus LLD-R (56%). This is the first example of a cold-active and thermostable alcohol dehydrogenase. 相似文献
3.
Charles M. H. Hensgens Michael Jansen Manny E. Nienhuis-Kuiper Egbert J. Boekema Jan F. L. Van Breemen Theo A. Hansen 《Archives of microbiology》1995,164(4):265-270
The sulfate-reducing bacterimDesulfovibrio strain HDv (DSM 6830) grew faster on (S)- and on (R, S)-1,2-propanediol (μmax 0.053 h−) than on (R)-propanediol (0.017 h−1) and ethanol (0.027 h−1). From (R, S)-1,2-propanediol-grown cells, an alcohol dehydrogenase was purified. The enzyme was oxygen-labile, NAD-dependent, and decameric;
the subunit mol. mass was 48 kDa. The N-terminal amino acid sequence indicated similarity to alcohol dehydrogenases belonging
to family III of NAD-dependent alcohol dehydrogenases, the first 21 N-terminal amino acids being identical to those of theDesulfovibrio gigas alcohol dehydrogenase. Best substrates were ethanol and propanol (K
m of 0.48 and 0.33 mM, respectively). (R, S)-1,2-Propanediol was a relatively poor substrate for the enzyme, but activities in cell extracts were high enough to account
for the growth rate. The enzyme showed a preference for (S)-1,2-propanediol over (R)-1,2-propanediol. Antibodies raised against the alcohol dehydrogenase ofD. gigas showed cross-reactivity with the alcohol dehydrogenase ofDesulfovibrio strain HDv and with cell extracts of six other ethanol-grown sulfate-reducing bacteria. 相似文献
4.
V. N. Stepanenko R. S. Esipov A. I. Miroshnikov V. L. Andronova G. A. Galegov M. V. Yasko A. A. Gus’kova A. Yu. Skoblov Yu. S. Skoblov 《Russian Journal of Bioorganic Chemistry》2011,37(4):436-440
Thymidine kinase UL23 gene (EC 2.7.1.145) from the L2 acyclovir-sensitive strain of herpes simplex virus type 1 was cloned and expressed in E. coli. The enzyme was purified by chromatography to the purity of 90% according to PAG electrophoresis data. The Michaelis constants
for the reactions with thymidine and acyclovir were determined. The enzyme was found to phosphorylate modified nucleosides,
particularly 3′-deoxythymidine, 3′-deoxy-2′,3′-didehydrothymidine, 2′,3′-dideoxycytidine, 9-[(hydroxyethyl)methyl]guanine,
E-5-(2-bromovinyl-2′-deoxyuridine, 9-(1,3-dihydroxy-2-propoxymethyl)guanine, 2′,3′-dideoxydehydrothymidine, β-L-2′,3′-dideoxy-3′-thiacytidine, and 3′-fluoro-3′-deoxythymidine. Some properties of the purified enzyme were compared with
those of thymidine kinases of other herpes simplex virus strains. It was shown that acyclovir H-phosphonate inhibited the
enzyme. 相似文献
5.
S. Colrat C. Deswarte A. Latché A. Klaébé M. Bouzayen J. Fallot J. P. Roustan 《Planta》1999,207(4):544-550
Eutypine, 4-hydroxy-3-(3-methyl-3-butene-1-ynyl) benzaldehyde, is a toxin produced by Eutypa lata (Pers.: Fr.) Tul., the causal agent of dying arm disease of Vitis vinifera L. (grapevine). Previously, we have shown that eutypine is involved in the development of disease symptoms. In the present
study, the effects of V. vinifera cell-suspension cultures on the biological activity of the toxin were investigated. Eutypine was converted by grapevine tissues
into a single compound, identified by mass spectrometry and nuclear magnetic resonance as 4-hydroxy-3-(3-methyl-3-butene-1-ynyl)
benzyl alcohol, designated eutypinol. This compound was found to be non-toxic for grapevine tissues. Unlike eutypine, eutypinol
failed to affect the oxidation rate or membrane potential of isolated mitochondria. In grapevine cells, reduction of eutypine
into the corresponding alcohol is an NADPH-dependent enzymatic reaction. An enzyme which reduced eutypine was partially purified,
over 1000-fold, using a five-step purification procedure. By gel filtration and sodium dodecyl sulfate-polyacrylamide gel
electrophoresis, the protein was found to have a molecular mass of 54–56 kDa. The enzyme exhibited an apparent K
m for eutypine of 44 μM, and was active between pH 6.8 and 7.5 with a maximum at pH 7.0. The eutypine reductase activity was
improved by Mn2+ and Mg2+ and inhibited by disulfiram and p-hydroxymercuribenzoate. The possible role of the eutypine-detoxification mechanism in the defense reactions of V. vinifera cells is discussed.
Received: 20 April 1998 / Accepted: 22 September 1998 相似文献
6.
Nucleotide sequence and biochemical analysis of d-β-hydroxybutyrate dehydrogenase (EC 1.1.1.30), isolated from Rhodobacter sp., indicate functional oligomers composed of subunits of 257 amino acids with a calculated M
r of 26,800 and a pI of 5.90. Compared to mammalian short-chain alcohol dehydrogenases, the bacterial enzyme lacks a C-terminal
lipid anchor domain and was found to be highly active upon expression in Escherichia coli even without lipid supplement. The recombinant enzyme could be highly enriched using a single chromatography step and was
shown to be stable over a broad range of pH and temperature.
Received: 1 April 1999 / Received last revision: 11 June 1999 / Accepted: 11 June 1999 相似文献
7.
Maurizio Petruccioli Marco Frasconi Daniele Quaratino Stefano Covino Gabriele Favero Franco Mazzei Federico Federici Alessandro D’Annibale 《Journal of biological inorganic chemistry》2009,14(8):1153-1163
A manganese peroxidase (MnP) isoenzyme from Panus tigrinus CBS 577.79 was produced in a benchtop stirred-tank reactor and purified to apparent homogeneity. The purification scheme
involving ultrafiltration, affinity chromatography on concanavalin–A Sepharose, and gel filtration led to a purified MnP,
termed “MnP II,” with a specific activity of 288 IU mg−1 protein and a final yield of 22%. The enzyme turned out to be a monomeric protein with molecular mass of 50.5 kDa, pI of
4.07, and an extent of N-glycosylation of about 5.3% of the high-mannose type. The temperature and pH optima for the formation
of malonate manganic chelates were 45 °C and 5.5, respectively. MnP II proved to be poorly thermostable at 50 and 60 °C, with
half-lives of 11 min and 105 s, respectively. K
m values for H2O2 and Mn2+ were 16 and 124 μM, respectively. Although MnP II was able to oxidize veratryl alcohol and to catalyze the Mn2+-independent oxidation of several phenols, it cannot be assigned to the versatile peroxidase family. As opposed to versatile
peroxidase oxidation, veratryl alcohol oxidation required the simultaneous presence of H2O2 and Mn2+; in addition, low turnover numbers and K
m values higher than 300 μM characterized the Mn2+-independent oxidation of substituted phenols. Kinetic properties and the substrate specificity of the enzyme markedly differed
from those reported for MnP isoenzymes produced by the reference strain P. tigrinus 8/18. To our knowledge, this study reports for the first time a thorough electrochemical characterization of a MnP from this
fungus. 相似文献
8.
Finore Ilaria Kasavi Ceyda Poli Annarita Romano Ida Oner Ebru Toksoy Kirdar Betul Dipasquale Laura Nicolaus Barbara Lama Licia 《World journal of microbiology & biotechnology》2011,27(10):2425-2433
This study reports the purification and biochemical characterization of a raw starch-digesting α-amylase from Geobacillus thermoleovorans subsp. stromboliensis subsp. nov. (strain PizzoT). The molecular weight was estimated to be 58 kDa by SDS–PAGE. The enzyme was highly active over a wide range of pH from
4.0–10.0. The optimum temperature of the enzyme was 70°C. It showed extreme thermostability in the presence of Ca2+, retaining 50% of its initial activity after 90 h at 70°C. The enzyme efficiently hydrolyzed 20% (w/v) of raw starches, concentration
normally used in starch industries. The α-amylase showed an high stability in presence of many organic solvents. In particular
the residual activity was of 73% in presence of 15% (v/v) ethyl alcohol, which corresponds to ethanol yield in yeast fermentation
process. By analyzing its complete amyA gene sequence (1,542 bp), the enzyme was proposed to be a new α-amylase. 相似文献
9.
Alankar Vaidya Gayatri Gera Sonti Ramakrishna 《World journal of microbiology & biotechnology》2008,24(12):2987-2995
Commercial available lipases viz. Lipozyme™, Novozyme-735 and Candida antartica lipase-B (CAL-B) were immobilized on seven different supports by simple adsorption process. The importance of suitable enzyme–support
combination in esterification of lauric acid and iso-propanol was validated experimentally. Effect of long chain fatty acids
(C4–C18) and small chain monohydric alcohols (C1–C6) on specific activities of different immobilized lipases were evaluated.
Lauric acid (C12) was found to be the most preferred fatty acid and t-amyl alcohol (C5) being the best alcohol. CAL-B adsorbed on Lewatit was the most efficient immobilized enzyme for esterification
reaction. Selectivity constant for lauric acid (3.4) was the highest among all fatty acids tested, whereas there was not much
difference in selectivity between different alcohols. Furthermore, increase in fatty acid unsaturation leads to decrease catalytic
efficiency of immobilized CAL-B. The optimum conditions for t-amyllaurate synthesis were as follows: lauric acid—0.5 M, t-amyl
alcohol—0.3 M and amount of immobilized enzyme—150 mg. Finally, CAL-B adsorbed on Lewatit was reused for three consecutive
cycles. 相似文献
10.
We have cloned a glucansucrase from the type strain of Leuconostoc mesenteroides (NRRL B-1118; ATCC 8293) and successfully expressed the enzyme in Escherichia coli. The recombinant processed enzyme has a putative sequence identical to the predicted secreted native enzyme (1,473 amino
acids; 161,468 Da). This enzyme catalyzed the synthesis of a water-insoluble α-D-glucan from sucrose (K
M 12 mM) with a broad pH optimum between 5.0 and 5.7 in the presence of calcium. Removal of calcium with dialysis resulted
in lower activity in the acidic pH range, effectively shifting the pH optimum to 6.0–6.2. The enzyme was quickly inactivated
at temperatures above approximately 45°C. The presence of dextran offered some protection from thermal inactivation between
room temperature and 40°C but had little effect above 45°C. NMR and methylation analysis of the water-insoluble α-d-glucan revealed that it had approximately equal amounts of α(1 → 3)-linked and α(1 → 6)-linked d-glucopyranosyl units and a low degree of branching. 相似文献
11.
Xue Z Chao Y Wang D Wang M Qian S 《Journal of industrial microbiology & biotechnology》2011,38(12):1931-1938
Rhodococcus erythropolis AJ270 metabolizes a wide range of nitriles via the two-step nitrile hydratase/amidase pathway. In this study, an amidase
gene from R. erythropolis AJ270 was cloned and expressed in Escherichia coli BL21 (DE3). The activity reached the highest level of 22.04 U/ml in a complex auto-inducing medium using a simplified process
of fermentation operation. The recombinant amidase was purified to more than 95% from the crude lysate using Ni-NTA affinity
chromatography and Superose S10-300 gel filtration. The V
max and K
m values of the purified enzyme with acetamide (50 mM) were 6.89 μmol/min/mg protein and 4.12 mM, respectively, which are similar
to those of the enzyme from the wild-type cell. The enzyme converted racemic α-substituted amides, O-benzylated β-hydroxy amides, and N-benzylated β-amino amides to the corresponding (S)-acids with remarkably high enantioselectivity. The ionic liquid [BMIm][PF6] (1-butyl-3-methylimidazolium hexafluorophosphate) enhanced the activity by 1.5-fold compared with water. The adequate expression
of the enzyme and excellent enantioselectivity of the recombinant amidase to a broad spectrum of amides suggest that the enzyme
has prospective industrial-scale practical applications in pharmaceutical chemistry. 相似文献
12.
Purification, immobilization and characterization of linoleic acid isomerase on modified palygorskite 总被引:2,自引:0,他引:2
Linoleic acid isomerase from Lactobacillus delbrueckii subsp. bulgaricus 1.1480 was purified by DEAE ion-exchange chromatography and gel filtration chromatography. An overall 5.1% yield and purification
of 93-fold were obtained. The molecular weight of the purified protein was ~41 kDa which was analyzed by SDS-PAGE. The purified
enzyme was immobilized on palygorskite modified with 3-aminopropyltriethoxysilane. The immobilized enzyme showed an activity
of 82 U/g. The optimal temperature and pH for the activity of the free enzyme were 30 °C and pH 6.5, respectively; whereas
those for the immobilized enzyme were 35 °C and pH 7.0, respectively. The immobilized enzyme was more stable than the free
enzyme at 30–60 °C, and the operational stability result showed that more than 85% of its initial activity was retained after
incubation for 3 h. The K
m and V
max values of the immobilized enzyme were found to be 0.0619 mmol l−1 and 0.147 mmol h−1 mg−1, respectively. The immobilized enzyme had high operational stability and retained high enzymatic activity after seven cycles
of reuse at 37 °C. 相似文献
13.
(S)(E)-2-{3-[3-[2-(7-chloro-2-quinolinyl)ethenyl]-phenyl]-3-hydroxypropyl} benzoic acid methyl ester,␣a key intermediate in the
synthesis of the anti-asthma drug, Montelukast, was prepared from the corresponding ketone (keto ester M) by microbial transformation.
The biotransforming organism, Microbacterium campoquemadoensis (MB5614), was discovered as a result of an extensive screening program and was used for the isolation and purification of
the responsible enzyme. The enzyme is a soluble cytoplasmic protein which was purified as a complex with a low-molecular-mass
molecule that had a visible-light absorption maximum at 460 nm. The purified enzyme has an apparent molecular mass of 60 kDa,
when denatured, and is isolated in the native state as an oligomer. The isolated enzyme requires NADPH for its activity and
reduces the keto ester M to the desired (S)-hydroxy ester with an enantiomeric excess greater than 95% at the optimum temperature of 30 °C and pH 8. The enzyme was
immobilized on oxirane-activated acrylamide beads with some loss of activity, but it was fully active in a two-phase (water/hexane
25:75) solvent system, both as a free solution and in an immobilized form.
Received: 31 October 1997 / Received revision: 8 January 1998 / Accepted: 24 January 1998 相似文献
14.
Wang SS Morton LM Bergen AW Lan EZ Chatterjee N Kvale P Hayes RB Chanock SJ Caporaso NE 《Human genetics》2007,122(1):41-49
Catechol-O-methyltransferase (COMT) is an important modulator in the catabolism of extraneural dopamine, which plays an important role
in drug reward mechanisms. It is hypothesized that genetic variations in the COMT gene, which can result in a three to fourfold difference in COMT enzyme activity, may be associated with several reward-motivated
behaviors. The aim of our study was to examine the relationship between COMT polymorphisms with smoking, obesity and alcohol. Three single nucleotide polymorphisms (SNPs) in COMT were genotyped in 2,371 participants selected randomly from the screening arm of the PLCO Cancer Screening Trial after stratifying
by sex, age, and smoking status. Smoking, obesity, and alcohol consumption were assessed by questionnaire. SNP and haplotype
associations were estimated using odds ratios (ORs) and 95% confidence intervals (CIs) derived from conditional logistic regression
models, adjusted for race/ethnicity. The COMT Ex4-76C > G (Leu136Leu) polymorphism was statistically significantly associated with individuals who had >30% increases in
BMI from ages 20 to 50 years, compared to those with 0–5% increase in BMI (0–5%) over the same age period: (CC is referent;
ORCG = 1.42, ORGG = 1.46, P
trend = 0.06). By sex, the increased risk was further pronounced among females (ORCG = 1.50, ORGG = 2.10, P
trend = 0.03). Consistent with our analyses of single polymorphisms, individuals whose BMI increased >30% from ages 20 to 50 years
were more likely than individuals with 0–5% increases in BMI to possess COMT haplotypes [COMT Ex3-104C > T–COMT Ex4-76 C > G–COMT Ex4-12 A > G] that included the variant allele for COMT Ex4-76 C > G: C-G-G (T-C-A is referent: ORC-G-G
= 1.33, 95% CI 1.01–1.77) and C-G-A (ORC-G-A = 1.79, 95% CI 0.72–4.49). We observed no association between any of the COMT polymorphisms with smoking behavior or alcohol intake. The COMT Ex4-76C > G (Leu136Leu) polymorphism appears to play a role in large increases in BMI. The null association with smoking
and alcohol and the pronounced association with increasing BMI among women further implicates COMT’s role in estrogen metabolism as a potentially culpable pathway. Our results support a need for comprehensive evaluation
of COMT variations and their functional relevance as COMT may be an important molecular target to evaluate for new treatments regarding
obesity. 相似文献
15.
The extremely thermophilic archaeon Thermococcus hydrothermalis, isolated from a deep-sea hydrothermal vent in the East Pacific Rise at 21°N, produced an extracellular pullulanase. This
enzyme was purified 97-fold to homogeneity from cell-free culture supernatant. The purified pullulanase was composed of a
single polypeptide chain having an estimated molecular mass of 110 kDa (gel filtration) or 128 kDa (sodium dodecyl sulfate/polyacryl
amide gel electrophoresis). The enzyme showed optimum activity at pH 5.5 and 95 °C. The thermostability and the thermoactivity
were considerably increased in the presence of Ca2+. The enzyme was activated by 2-mercaptoethanol and dithiothreitol, whereas N-bromosuccinimide and α-cyclodextrin were inhibitors. This enzyme was able to hydrolyze, in addition to the α-1,6-glucosidic
linkages in pullulan, α-1,4-glucosidic linkages in amylose and soluble starch, and can therefore be classified as a type II
pullulanase or an amylopullulanase. The purified enzyme displayed Michaelis constant (K
m) values of 0.95 mg/ml for pullulan and 3.55 mg/ml for soluble starch without calcium and, in the presence of Ca2+, 0.25 mg/ml for pullulan and 1.45 mg/ml for soluble starch.
Received: 19 November 1997 / Received revision: 9 March 1998 / Accepted: 14 March 1998 相似文献
16.
Purification and characterization of dye degrading of veratryl alcohol oxidase from Pseudomonas aeruginosa strain BCH 总被引:1,自引:0,他引:1
Phugare Swapnil S. Waghmare Shailesh R. Jadhav Jyoti P. 《World journal of microbiology & biotechnology》2011,27(10):2415-2423
In the present study we have purified the intracellular veratryl alcohol oxidase (VAO) enzyme from Pseudomonas aeruginosa strain BCH to evaluate its dye decolorizing potential. The enzyme was purified by ion exchange chromatography using DEAE
cellulose followed by gel filtration chromatography using Biogel P-100. The molecular weight of the purified enzyme was estimated
by polyacrylamide gel electrophoresis (PAGE) analysis. The VAO was purified up to 12 and 16.3-fold by ion exchange and gel
filtration chromatography respectively. VAO was estimated to be about 85 kDa by SDS–PAGE. The optimum pH and temperature for
purified VAO was 3 and 55°C respectively. The purified enzyme exerted its optimal activity with veratryl alcohol and also
oxidized various other substrates, whereas diminished activity was noted in case of tryptophan and xylidine. The metal ions
Mn++ and Hg++ were found to suppress the oxidase activity. The purified enzyme decolorized different dyes with variable decolorization
rates and efficiencies. Decolorization mechanism of Remazol Black by purified enzyme was studies in detail using various analytical
techniques like HPLC, GC–MS and FTIR. This study is useful for understanding the precise role of Pseudomonas aeruginosa strain BCH in the decolorization of textile dyes containing industrial wastewater. 相似文献
17.
Abdul Rahman Mohd Basyaruddin Jarmi Nor Izuan Chaibakhsh Naz Basri Mahiran 《Journal of industrial microbiology & biotechnology》2011,38(1):229-234
Esterification of succinic acid with oleyl alcohol catalyzed by immobilized Candida antarctica lipase B (Novozym 435) was investigated in this study. Response surface methodology (RSM) based on a five-level, four-variable
central composite design (CCD) was used to model and analyze the reaction. A total of 21 experiments representing different
combinations of the four parameters including temperature (35–65°C), time (30–450 min), enzyme amount (20-400 mg), and alcohol:acid
molar ratio (1:1-8:1) were generated. A partial cubic equation could accurately model the response surface with a R2 of 0.9853. The effect and interactions of the variables on the ester synthesis were also studied. Temperature was found to
be the most significant parameter that influenced the succinate ester synthesis. At the optimal conditions of 41.1°C, 272.8 min,
20 mg enzyme amount and 7.8:1 alcohol:acid molar ratio, the esterification percentage was 85.0%. The model can present a rapid
means for estimating the conversion yield of succinate ester within the selected ranges. 相似文献
18.
P. Wahl P. Walser-Volken K. Laumen M. Kittelmann O. Ghisalba 《Applied microbiology and biotechnology》1999,53(1):12-18
Rhodococcus globerulus K1/1 was found to express an inducible (S)-specific N-acetyl-2-amino-1-phenyl-4-pentene amidohydrolase. Optimal bacterial growth and amidohydrolase expression were both observed
at about pH 6.5. Purification of the enzyme to a single band in a Coomassie blue-stained SDS-PAGE gel was achieved by nucleic
acid and ammonium sulfate precipitation of Rhodococcus globerulus K1/1 crude extract and column chromatography on TSK Butyl-650(S) Fractogel and Superose 12HR. The amidohydrolase was purified to a homogeneity leading to a tenfold increase of the specific
activity with a recovery rate of 65%. At pH 7.0 and 23 °C the enzyme showed no loss of activity after 30 days incubation.
The amidohydrolase was stable up to 55 °C. The enzyme was inhibited strongly only by 10 mM Zn2+ among the tested metal cations and was inhibited 100% by 0.01 mM phenylmethanesulfonyl fluoride. The molecular weight of
the native enzyme was estimated to be 92 kDa by gel filtration and 55 kDa by SDS-PAGE, suggesting a homodimeric structure.
Received: 8 February 1999 / Received revision: 3 May 1999 / Accepted: 7 May 1999 相似文献
19.
An anti-Prelog alcohol dehydrogenase from Oenococcus oeni that reduces 2-octanone to (R)-2-octanol was purified by 26-fold to homogeneity. The enzyme had a homodimeric structure consisting
of 49 kDa subunits, required NADPH, but not NADH, as a cofactor and was a Zn-independent short-chain dehydrogenase. Aliphatic
methyl ketones (chain length ≥6 carbon atoms) and aromatic methyl ketones were the preferred substrates for the enzyme, the
best being 2-octanone. Maximum enzyme activity with 2-octanone was at 45°C and at pH 8.0. 相似文献
20.
The enzyme oxaloacetate hydrolase (EC 3.7.1.1), which is involved in oxalate formation, was purified from Aspergillus niger. The native enzyme has a molecular mass of 360–440 kDa, and the denatured enzyme has a molecular mass of 39 kDa, as determined
by gel electrophoresis. Enzyme activity is maximal at pH 7.0 and 45 °C. The fraction containing the enzyme activity contained
at least five proteins. The N-terminal amino acid sequences of four of these proteins were determined. The amino acid sequences
were aligned with EST sequences from A. niger, and an EST sequence that showed 100% identity to all four sequences was identified. Using this EST sequence the gene encoding
oxaloacetate hydrolase (oah) was cloned by inverse PCR. It consists of an ORF of 1227 bp with two introns of 92 and 112 bp, respectively. The gene encodes
a protein of 341 amino acids with a molecular mass of 37 kDa. Under the growth conditions tested, the highest oah expression was found for growth on acetate as carbon source. The gene was expressed only at pH values higher than 4.0.
Received: 9 May 1999 / Accepted: 30 November 1999 相似文献