共查询到20条相似文献,搜索用时 78 毫秒
1.
Jin Zhou Ju Chu Yong-Hong Wang Si-Liang Zhang Ying-Ping Zhuang Zhong-Yi Yuan 《World journal of microbiology & biotechnology》2008,24(6):789-796
An intracellular S-adenosylmethionine synthetase (SAM-s) was purified from the fermentation broth of Pichia pastoris GS115 by a sequence chromatography column. It was purified to apparent homogeneity by (NH4)2SO4 fractionation (30–60%), anion exchange, hydrophobic interaction, anion exchange and gel filtration chromatography. HPLC showed
the purity of purified SAM-s was 91.2%. The enzyme was purified up to 49.5-fold with a final yield of 20.3%. The molecular
weight of the homogeneous enzyme was 43.6 KDa, as determined by electro-spray ionization mass spectrometry (ESI-MS). Its isoelectric
point was approximately 4.7, indicating an acidic character. The optimum pH and temperature for the enzyme reaction were 8.5
and 35 °C, respectively. The enzyme was stable at pH 7.0–9.0 and was easy to inactivate in acid solution (pH ≤ 5.0). The temperature
stability was up to 45 °C. Metal ions, such as, Mn2+ and K+ at the concentration of 5 mM had a slight activation effect on the enzyme activity and the Mg2+ activated the enzyme significantly. The enzyme activity was strongly inhibited by heavy metal ions (Cu2+ and Ag2+) and EDTA. The purified enzyme from the transformed Pichia pastoris synthesized S-adenosylmethionine (SAM) from ATP and l-methionine in vitro with a K
m of 120 and 330 μM and V
max of 8.1 and 23.2 μmol/mg/min for l-methionine and ATP, respectively. 相似文献
2.
Kemel Jellouli Ali Bougatef Laila Manni Rym Agrebi Rayda Siala Islem Younes Moncef Nasri 《Journal of industrial microbiology & biotechnology》2009,36(7):939-948
A protease-producing bacterium was isolated from an alkaline wastewater of the soap industry and identified as Vibrio metschnikovii J1 on the basis of the 16S rRNA gene sequencing and biochemical properties. The strain was found to over-produce proteases
when it was grown at 30°C in media containing casein as carbon source (14,000 U ml−1). J1 enzyme, the major protease produced by V. metschnikovii J1, was purified by a three-step procedure, with a 2.1-fold increase in specific activity and 33.3% recovery. The molecular
weight of the purified protease was estimated to be 30 kDa by SDS-PAGE and gel filtration. The N-terminal amino acid sequence
of the first 20 amino acids of the purified J1 protease was AQQTPYGIRMVQADQLSDVY. The enzyme was highly active over a wide
range of pH from 9.0 to 12.0, with an optimum at pH 11.0. The optimum temperature for the purified enzyme was 60°C. The activity
of the enzyme was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine protease. The kinetic
constants K
m and K
cat of the purified enzyme using N-succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide were 0.158 mM and 1.14 × 105 min−1, respectively. The catalytic efficiency (K
cat
/K
m) was 7.23 × 108 min−1 M−1. The enzyme showed extreme stability toward non-ionic surfactants and oxidizing agents. In addition, it showed high stability
and compatibility with some commercial liquid and solid detergents. The aprJ1 gene, which encodes the alkaline protease from V. metschnikovii J1, was isolated, and its DNA sequence was determined. The deduced amino acid sequence of the preproenzyme differs from that
of V. metschnikovii RH530 detergent-stable protease by 12 amino acids, 7 located in the propeptide and 5 in the mature enzyme. 相似文献
3.
An N-acetylglucosaminidase produced by Streptomyces cerradoensis was partially purified giving, by SDS-PAGE analysis, two main protein bands with Mr of 58.9 and 56.4 kDa. The Km and Vmax values for the enzyme using p-nitrophenyl-β-N-acetylglucosaminide as substrate were of 0.13 mM and 1.95 U mg−1 protein, respectively. The enzyme was optimally activity at pH 5.5 and at 50 °C when assayed over 10 min. Enzyme activity
was strongly inhibited by Cu2+ and Hg2+ at 10 mM, and was specific to substrates containing acetamide groups such as p-nitrophenyl-β-N-acetylglucosaminide and p-nitrophenyl-β-D-N,N′-diacetylchitobiose. 相似文献
4.
The xylose reductase (XR) gene (xyl1) from Candida shehatae was cloned and expressed in Escherichia coli, and purified as a His6-tagged fusion protein. The recombinant XR had Km values for NADH than NADPH of 150 μM and 20 μM, respectively. The optimal reaction was at pH 6.5 and 35°C. The enzyme was
specific for d-xylese. 相似文献
5.
K. Mochizuki 《Archives of microbiology》1999,172(3):182-185
An enzyme cleaving l-2-oxothiazolidine-4-carboxylic acid to l-cysteine was purified 75-fold with 8% recovery to near homogeneity from crude extracts of Paecilomyces varioti F-1, which had been isolated as a fungus able to assimilate l-2-oxothiazolidine-4-carboxylic acid. The molecular mass was estimated to be 260 kDa by gel filtration. The purified preparation
migrated as a single band of molecular mass 140 kDa upon SDS-PAGE. The maximum activity was observed at a range of pH 7.0–8.0
and at 50 °C. The enzyme activity was completely inhibited by SH-blocking reagents such as AgNO3, p-chloromercuribenzoic acid, N-ethylmaleimide, and N-bromosuccinimide. The enzyme required ATP, Mg2+, and KCl for the cleavage of l-2-oxothiazolidine-4-carboxylic acid. The enzyme also cleaved 5-oxo-l-proline to l-glutamic acid and is considered to be 5-oxo-l-prolinase.
Received: 23 March 1999 / Accepted: 22 June 1999 相似文献
6.
Nakazawa Y Sagane Y Kikuchi T Uchino M Nagai T Sato H Toeda K Takano K 《The protein journal》2010,29(8):598-608
We previously isolated Streptomyces racemochromogenes strain 10-3, which produces a phospholipase D (PLD) with high transphosphatidylation activity. Here, we purified and cloned
the PLD (PLD103) from the strain. PLD103 exerted the highest hydrolytic activity at a slightly alkaline pH, which is in contrast
to the majority of known Streptomyces PLDs that have a slightly acidic optimum pH. PLD103 shares only 71–76% amino acid sequence identity with other Streptomyces PLDs that have a slightly acidic optimum pH; thus, the diversity in the primary structure might explain the discrepancy observed
in the optimum pH. The purified PLD displayed high transphosphatidylation activity in the presence of glycerol, l-serine, and 2-aminoethanol hydrochloride with a conversion rate of 82–97% in a simple one-phase system, which was comparable
to the rate of other Streptomyces PLDs in a complicated biphasic system. 相似文献
7.
Miake F Satho T Takesue H Yanagida F Kashige N Watanabe K 《Archives of microbiology》2000,173(1):65-70
α-l-Rhamnosidase was extracted and purified from the cells of Pseudomonas paucimobilis FP2001 with a 19.5% yield. The purified enzyme, which was homogeneous as shown by SDS-PAGE and isoelectric focusing, had
a molecular weight of 112,000 and an isoelectric point of 7.1. The enzyme activity was accelerated by Ca2+ and remained stable for several months when stored at –20 °C. The optimum pH was 7.8; the optimum temperature was 45 °C.
The K
m, V
max and k
cat for p-nitrophenyl α-l-rhamnopyranoside were 1.18 mM, 92.4 μM · min–1 and 117,000 · min–1, respectively. Examination of the substrate specificity using various synthetic and natural l-rhamnosyl glycosides showed that this enzyme had a relatively broader substrate specificity than those reported so far.
Received: 24 May 1999 / Accepted: 7 October 1999 相似文献
8.
Shinji Tokuyama Hiroyuki Miya Kazunori Hatano Takeshi Takahashi 《Applied microbiology and biotechnology》1994,40(6):835-840
A novel enzyme, N-acylamino acid racemase, was purified to homogeneity from Streptomyces atratus Y-53 and characterized. This enzyme catalyzes the interconversion of optically active N-acylamino acids. The relative molecular mass (Mr) of the enzyme was estimated to be about 41 000 and 244 000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively, indicating that the enzyme is composed of six subunits with an equal Mr. The enzyme showed a broad substrate specificity toward N-acylamino acids, such as N-acetylmethionine, N-chloroacetylphenylalanine and N-chloroacetylvaline. The apparent Michaelis constant (Km) values for N-acetyl-l-methionine and N-acetyl-d-methionine were calculated to be 15.2 and 5.6 mm, respectively. Enzyme activity was markedly enhanced by divalent metal ions, such as Co2+, Mg2+ and Mn2+, and was inhibited by metal-chelating reagent, indicating that the enzyme is a metalloenzyme. We propose to name the enzyme N-acylamino acid racemase (acylamino acid racemase).
Correspondence to: S. Tokuyama 相似文献
9.
Poonperm W Takata G Okada H Morimoto K Granström TB Izumori K 《Applied microbiology and biotechnology》2007,76(6):1297-1307
The l-rhamnose isomerase gene (L
-rhi) encoding for l-rhamnose isomerase (l-RhI) from Bacillus pallidus Y25, a facultative thermophilic bacterium, was cloned and overexpressed in Escherichia coli with a cooperation of the 6×His sequence at a C-terminal of the protein. The open reading frame of L
-rhi consisted of 1,236 nucleotides encoding 412 amino acid residues with a calculated molecular mass of 47,636 Da, showing a
good agreement with the native enzyme. Mass-produced l-RhI was achieved in a large quantity (470 mg/l broth) as a soluble protein. The recombinant enzyme was purified to homogeneity
by a single step purification using a Ni-NTA affinity column chromatography. The purified recombinant l-RhI exhibited maximum activity at 65°C (pH 7.0) under assay conditions, while 90% of the initial enzyme activity could be
retained after incubation at 60°C for 60 min. The apparent affinity (K
m) and catalytic efficiency (k
cat/K
m) for l-rhamnose (at 65°C) were 4.89 mM and 8.36 × 105 M−1 min−1, respectively. The enzyme demonstrated relatively low levels of amino acid sequence similarity (42 and 12%), higher thermostability,
and different substrate specificity to those of E. coli and Pseudomonas stutzeri, respectively. The enzyme has a good catalyzing activity at 50°C, for d-allose, l-mannose, d-ribulose, and l-talose from d-psicose, l-fructose, d-ribose and l-tagatose with a conversion yield of 35, 25, 16 and 10%, respectively, without a contamination of by-products. These findings
indicated that the recombinant l-RhI from B. pallidus is appropriate for use as a new source of rare sugar producing enzyme on a mass scale production. 相似文献
10.
Mochizuki K 《Archives of microbiology》2003,180(6):490-493
A bacterium, strain 314B, able to assimilate (S)-5-oxo-2-tetrahydrofurancarboxylic acid was isolated from soil and identified as Erwinia cypripedii. A lactonase hydrolyzing (S)-5-oxo-2-tetrahydrofurancarboxylic acid to l--hydroxyglutaric acid was purified 63-fold with 2% recovery from crude extracts of this bacterium to homogeneity as judged by SDS-PAGE. The molecular masses estimated by SDS-PAGE and gel filtration were 41 kDa and 79 kDa, respectively. The maximum activity was observed at pH 6.5–7.5 and 35–45 °C. The enzyme showed lower activity toward dl-2-oxotetrahydrofuran-4,5-dicarboxylic acid, but did not act on (R)-5-oxo-2-tetrahydrofurancarboxylic acid and other natural and synthetic lactones tested. 相似文献
11.
Yoshimoto Tadashi Nakanishi Toshihiro Fukumoto Juichiro Tsuru Daisuke 《Bioscience, biotechnology, and biochemistry》2013,77(11):1775-1782
About 100 soil samples were subjected to screening for microorganisms which were capable of producing lytic enzyme toward Staphylococcus aureus. A strain belonging to Streptomyces was isolated and found to produce lytic enzyme(s) noninduciblly, when grown aerobically at 37°C for 25 hr in a medium containing 7.5% soybean cake extract, 2% dextrin, 0.6% K2HPO4, 0.02% each of MgSO4·7H2O and KCl, pH 7.0. The crude enzyme preparation was active at pH values of 8.5 and 5.8 toward S. aureus, B. subtilis, L. bulgaricus and Str. faecalis but was completely inert against M. lysodeikticus, indicating the enzyme(s) to be distinguished from other bacteriolytic enzymes of Streptomyces so far reported. 相似文献
12.
W. Chungool W. Thongkam P. Raweesri A. Thamchaipenet P. Pinphanichakarn 《World journal of microbiology & biotechnology》2008,24(4):549-556
Acetyl esterase was produced by Streptomyces sp. PC22 at comparable levels of about 0.3 U ml−1 using either 1.0% (w/v) birchwood xylan or 1.5% (w/v) corn husks as a carbon source and cultivating at 45 °C, at pH 9 for
3 or 2 days, respectively. The enzyme was purified from culture filtrate to about 54-fold purity by ammonium sulfate precipitation,
followed by consecutive chromatography using a Macro-Prep DEAE, t-butyl hydrophobic interaction and hydroxyapatite, respectively. The approximate molecular weight of the purified enzyme was
155 kDa as analyzed by gel filtration, and it contained four identical 34 kDa subunits, as assessed by SDS-PAGE. It had K
m and V
max values for p-nitrophenyl acetate of 0.43 mM and 70.78 U mg−1 and 7.8 mM and 1,027 U mg−1 for α-naphthyl acetate, respectively. Its optimal pH and temperature were 6.5–7.0 and 50 °C, respectively. It was stable for 30 min
at a broad range of pH values, from 5.0 to 9.0, and at temperatures up to 60 °C. The purified enzyme had no other xylanolytic
activities. It showed cooperative action on birchwood xylan degradation, when used in combination with xylanase from the same
strain and β-xylosidase from Streptomyces sp. CH7. Enhancement was 1.4-fold, compared to the expected amount of individual enzymes alone. This indicates that the enzyme
has potential industrial applications, especially for utilizing renewable hemicelluloses containing acetyl xylan for the production
of biofuels or other fermentation products. 相似文献
13.
The purpose of this study was to identify the seleno-l-methionine (l-SeMet) α,γ-elimination enzyme that catalyzes l-SeMet to generate methylselenol (CH3SeH), a notable intermediate for the metabolism of selenium compounds, in mammalian tissues. The enzyme purified from ICR
mouse liver was separated by one-dimensional gel electrophoresis, and the specific band was subjected to in-gel trypsin digestion
followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometric analysis. In the peptide mass fingerprinting
search, the mass numbers of 14 peptides produced by tryptic digestion of the enzyme were consistent with the theoretical mass
numbers calculated from the amino acid sequence of murine cystathionine γ-lyase (E.C. 4.4.1.1). The peptide sequence tags
search was also performed to obtain the amino acid sequence data of five tryptic peptides. These peptides were significantly
identical to the partial amino acid sequences of cystathionine γ-lyase. This enzyme was clearly shown to catalyze the α, γ-elimination
reaction of l-cystathionine by the enzymological research. The K
m
value for the catalysis of l-cystathionine was 0.81 mM and V
max was. 0.0013 unit/mg protein. These results suggested that cystathionine γ-lyase catalyzes l-SeMet to generate CH3SeH by its α,γ-elimination reaction. 相似文献
14.
Kohei Imai Tohru Kikuta Mikihiko Kobayashi Kazuo Matsuda 《Bioscience, biotechnology, and biochemistry》2013,77(8):1339-1346
An α-l,3-glucanase was detected in the culture supernatant of a micro-organism, which was isolated from soil on agar medium containing α-l,3-glucan as sole carbon source. The isolated strain was characterized as a strain of Streptomyces, tentatively named KI-8. This enzyme required α-l,3-glucosidic linkage as an inducer. The optimum conditions for enzyme production were studied.The enzyme was purified by (NH4)2SO4 precipitation, column chromatography on DEAE-cellulose and P(phospho)-cellulose. To eliminate the concomitant β-l,3-glucanase activity, partially purified enzyme preparation was passed through a column packed with pachyman. Final purification was accomplished by the adsorption chromatography using Sephadex G-150 from which the α-l,3-glucanase was eluted with a solution of α-1,3-linked gluco-oligo-saccharides. The purified enzyme was electrophoretically homogeneous and had a molecular weight of approximately 78,000 by SDS-polyacrylamide gel electrophoresis. 相似文献
15.
d-Xylulose-forming d-arabitol dehydrogenase (aArDH) is a key enzyme in the bio-conversion of d-arabitol to xylitol. In this study, we cloned the NAD-dependent d-xylulose-forming d-arabitol dehydrogenase gene from an acetic acid bacterium, Acetobacter suboxydans sp. The enzyme was purified from A. suboxydans sp. and was heterogeneously expressed in Escherichia coli. The native or recombinant enzyme was preferred NAD(H) to NADP(H) as coenzyme. The active recombinant aArDH expressed in
E. coli is a homodimer, whereas the native aArDH in A. suboxydans is a homotetramer. On SDS–PAGE, the recombinant and native aArDH give one protein band at the position corresponding to 28 kDa.
The optimum pH of polyol oxidation and ketone reduction is found to be pH 8.5 and 5.5 respectively. The highest reaction rate
is observed when d-arabitol is used as the substrate (K
m = 4.5 mM) and the product is determined to be d-xylulose by HPLC analysis. 相似文献
16.
Jiro Arima Masanori Chiba Tsuyoshi Ichiyanagi Yukinori Yabuta Nobuhiro Mori Tadanori Aimi 《Applied microbiology and biotechnology》2010,87(5):1791-1801
An aminopeptidase that has peptide bond formation activity was identified in the cell-free extract of carpophore of Pleurotus eryngii. The enzyme, redesignated as eryngase, was purified for homogeneity and characterized. Eryngase had a molecular mass of approximately
79 kDa. It showed somewhat high stability with respect to temperature and pH; it was inhibited by iodoacetate. Among hydrolytic
activities toward aminoacyl-p-nitroanilides (aminoacyl-pNAs), eryngase mainly hydrolyzed hydrophobic l-aminoacyl-pNAs and exhibited little activity toward d-Ala-pNA and d-Leu-pNA. In terms of peptide bond formation activity, eryngase used various aminoacyl derivatives as acyl donors and acceptors.
The products were all dipeptidyl derivatives. Investigation of time dependence on peptide synthesis revealed that some peptides
that are not recognized as substrates for hydrolytic activity of eryngase could become good targets for synthesis. Furthermore,
eryngase has produced opioid dipeptides––l-kyotorphin (l-Tyr-l-Arg) and d-kyotorphin (l-Tyr-d-Arg)––using l-Tyr-NH2 and d- and l-Arg-methyl ester respectively as an acyl donor and acceptor. Yield evaluation of kyotorphin synthesis indicated that the
conversion ratio of substrate to kyotorphin was moderate: the value was estimated as greater than 20%. 相似文献
17.
Thermotoga hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90°C. It uses carbohydrates and peptides as carbon
and energy sources to produce acetate, CO2, H2, l-alanine and ethanol as end products. Alcohol dehydrogenase activity was found to be present in the soluble fraction of T. hypogea. The alcohol dehydrogenase was purified to homogeneity, which appeared to be a homodimer with a subunit molecular mass of
40 ± 1 kDa revealed by SDS-PAGE analyses. A fully active enzyme contained iron of 1.02 ± 0.06 g-atoms/subunit. It was oxygen
sensitive; however, loss of enzyme activity by exposure to oxygen could be recovered by incubation with dithiothreitol and
Fe2+. The enzyme was thermostable with a half-life of about 10 h at 70°C, and its catalytic activity increased along with the
rise of temperature up to 95°C. Optimal pH values for production and oxidation of alcohol were 8.0 and 11.0, respectively.
The enzyme had a broad specificity to use primary alcohols and aldehydes as substrates. Apparent K
m values for ethanol and 1-butanol were much higher than that of acetaldehyde and butyraldehyde. It was concluded that the
physiological role of this enzyme is likely to catalyze the reduction of aldehydes to alcohols. 相似文献
18.
Eun-Seon Seo Yu-Ri Lim Yeong-Su Kim Chang-Su Park Deok-Kun Oh 《Biotechnology and Bioprocess Engineering》2010,15(4):590-594
The bacterium Bacillus licheniformis, which exhibits high hydrolytic activity toward arabinan, was isolated from soil, and its gene encoding endo-1,5-α-l-arabinanase was cloned and sequenced. The gene has an open reading frame that encodes 328 amino acids, including a signal
peptide of 37 amino acids. Endo-1,5-α-l-arabinanase, a member of glycosyl hydrolase family 43, was expressed in Escherichia coli and purified as a 34-kD monomer with a specific activity of 27 U/mg. Optimal activity toward debranched arabinan (linear
1,5-α-l-arabinan) occurred at pH 6.0 and 35°C, with a k
cat of 160/sec and a K
m of 19 mg/mL. 相似文献
19.
Jei Oh Yon Ji Seon Lee Bo Geum Kim Sang Dal Kim Doo Hyun Nam 《Biotechnology and Bioprocess Engineering》2008,13(1):102-107
The immobilization of phospholipase D produced by Streptomyces sp. YU100 was evaluated to see it would be practical for industrial applications. To accomplish this, the purified enzyme,
which contained 53 unit/mg of protein, was subjected to immobilization on various matrices. When immobilization supports including
calcium alginate gel, polyacrylamide gel, and macroporous resin were evaluated, the highest enzyme retention ratio (> 42%)
was observed on a Dowex MSA-2 macro-porous resin. This may have occurred as a result of the ability of the hydrophobic domain
of phospholipase D to interact with the polystyrene backbone of the resin, as well as the ability of the dimethylethanolamine
group of the MSA-2 resin to retain the enzyme by forming hydrogen bonds with the acidic residues of the enzyme. Upon the operation
of a reactor packed with enzyme that had been immobilized on a Dowex MSA-2 resin, greater than 80% of the initial enzyme activity
was retained for 16 days. During the reaction, phosphatidylcholine became bound to the immobilized resin and interfered with
the enzyme reaction, therefore, the resin was washed with ethyl ether every 2 h. A process for recovering excessive l-serine from phospholipids using the Dowex MR-3 resin was designed, and the separated l
-serine was employed again after replacing the amount that was used. 相似文献
20.
Prabhu P Tiwari MK Jeya M Gunasekaran P Kim IW Lee JK 《Applied microbiology and biotechnology》2008,81(2):283-290
Based on analysis of the genome sequence of Bacillus licheniformis ATCC 14580, an isomerase-encoding gene (araA) was proposed as an l-arabinose isomerase (L-AI). The identified araA gene was cloned from B. licheniformis and overexpressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,422 bp, capable of encoding a polypeptide of 474 amino acid residues
with a calculated isoelectric point of pH 4.8 and a molecular mass of 53,500 Da. The gene was overexpressed in E. coli, and the protein was purified as an active soluble form using Ni–NTA chromatography. The molecular mass of the purified enzyme
was estimated to be ~53 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and 113 kDa by gel filtration chromatography,
suggesting that the enzyme is a homodimer. The enzyme required a divalent metal ion, either Mn2+or Co2+, for enzymatic activity. The enzyme had an optimal pH and temperature of 7.5 and 50°C, respectively, with a k
cat of 12,455 min−1 and a k
cat/K
m of 34 min−1 mM−1 for l-arabinose, respectively. Although L-AIs have been characterized from several other sources, B. licheniformis L-AI is distinguished from other L-AIs by its wide pH range, high substrate specificity, and catalytic efficiency for l-arabinose, making B. licheniformis L-AI the ideal choice for industrial applications, including enzymatic synthesis of l-ribulose. This work describes one of the most catalytically efficient L-AIs characterized thus far. 相似文献