共查询到20条相似文献,搜索用时 31 毫秒
1.
Jaya Ram Simkhada Seung Sik Cho Hong Seok Choi Si Wouk Kim Hei Chan Lee Jae Kyung Sohng Jin Cheol Yoo 《Biotechnology and Bioprocess Engineering》2010,15(4):595-602
A phospholipase D (PLD628), constitutively secreted by Streptomyces sp. CS628, was purified by ion exchange with CM Trisacryl and gel filtration with Sepharose CL-6B. The enzyme production
was highest with peptone and starch as nitrogen and carbon sources, and at 30°C with an initial medium pH of 7.5. Molecular
weight, optimum pH, optimum temperature, pH stability, and thermostability of the enzyme were 50 kDa, pH 9.6, 30°C, pH 5.7
∼ 10.6 and ≤30°C, respectively. Detergents and metal ions had varied effects on the enzyme activity. Importantly, PLD628 could not catalyze transphosphatidylation of glycerol, L-serine, myo-inositol or ethanolamine, which are extensively used to assess the activity, suggesting that PLD628 lacks the transphosphatidylation activity. PLD628 could be a novel PLD based on its biochemical characteristics, which are significantly different from previously reported
PLDs, such as thermolability, highest activity at alkaline pH, and lack of transphosphatidylation activity. 相似文献
2.
Jaya Ram Simkhada Hyo Jeong Lee So Young Jang Seung Sik Cho Eun Jung Park Jae Kyung Sohng Jin Cheol Yoo 《Biotechnology letters》2009,31(3):429-435
A 60 kDa phospholipase D (PLD) was obtained from Streptomyces olivochromogenes by one-step chromatography on Sepharose CL-6B. Maximal activity was at pH 8 and 75°C and the enzyme was stable from pH 7
to 13 and from 55 to 75°C. Thermal and pH stability with temperature optimum of the enzyme were highest among Streptomyces PLDs reported so far. The activity was Ca2+-dependent and enhanced by detergents. The Km and Vmax values for phosphatidylcholine were 0.6 mM and 650 μmol min−1 mg−1, respectively. In addition, the enzyme also revealed transphosphatidylation activity, which was optimum at pH 8 and 50°C.
The first 15 amino acid residues of the N terminal sequence were ADYTPGAPGIGDPYY, which are significantly different from the
other known PLDs. The enzyme may therefore be a novel PLD with potential application in the lipid industry. 相似文献
3.
The gene for phospholipase D (PLD) of Streptomyces sp. YU100 was cloned from λ phage library and hetero-logously expressed in Escherichia coli. Using an amplified gene fragment based on the consensus sequences of streptomycetes PLDs, λ phage library of Streptomyces sp. YU100 chromosomal DNA was screened. The sequencing result of BamHI-digested 3.8 kb fragment in a positive phage clone revealed the presence of an open reading frame of a full sequence of
PLD gene encoding a 540-amino acid protein including 33-amino acid signal peptide. The deduced amino acid sequence showed
a high homology with other Streptomyces PLDs, having the highly conserved ‘HKD’ motifs. The PLD gene excluding signal peptide sequence was amplified and subcloned
into a pET-32b(+) expression vector in E. coli BL21(DE3). The recombinant PLD was purified by nickel affinity chromatography and compared the enzyme activity with wild-type
PLD. The results imply that the recombinant PLD produced by E. coli had the nearly same enzyme activity as PLD from Streptomyces sp. YU100. 相似文献
4.
Yozo Nakazawa Rei Suzuki Masataka Uchino Yoshimasa Sagane Takuji Kudo Takeshi Nagai Hiroaki Sato Katsumi Takano 《Current microbiology》2010,60(5):365-372
Previously we isolated six actinomycetes strains, 9-4, 10-1, 10-2, 10-3, 10-6, and 21-4, that produce phospholipase D (PLD)
with high transphosphatidylation activity. In this study, we identified these strains, and the PLD activities were compared
with those of reference strains. 16S rDNA sequences and DNA–DNA hybridization tests indicated taxonomic affiliations of strain
9-6 with Streptomyces senoensis, strains 10-1 and 10-6 with S. vinaceus, and strains 10-2 and 10-3 with S. racemochromogenes. Strain 21-4, though identified as a Streptomyces sp., could not be identified with any known species. Meanwhile, most of the culture supernatants of reference strains demonstrated
no or very weak PLD activity, while those of our strains exhibited significantly higher activity. All of the strains in this
study were identified as Streptomyces species. The PLD activity of our strains exceeded most of the reference Streptomyces strains. The findings in this study imply that the Streptomyces strains, although they are members of the same species, can produce different quantities of PLD enzyme. 相似文献
5.
The extracellular phospholipase D (PLD) gene fromStreptomyces antibioticus was cloned, sequenced, and expressed inEscherichia coli. Analysis of DNA sequence data revealed a putative ribosome-binding site and an open reading frame encoding a 556-amino-acid protein that included amino acid sequences obtained from the purified enzyme. The protein was expressed in an insoluble form inE. coli, but reacted with antibody against PLD. After solubilization of the protein with guanidine-HCI and 2-mercaptoethanol, subsequent dialysis restored the PLD activity. Comparison of the nucleotide sequence data with the N-terminal protein sequence indicates that this secreted protein is synthesized as a larger precursor with a 47-amino-acid N-terminal extension to the mature enzyme of 509 amino acids. The amino acid sequence of the S.antibioticus PLD was extensively compared with other PLDs and phospholipase C (PLC). The deduced amino acid sequence of the cloned PLD was highly homologous to PLDs from S. acidomyceticus andStreptomyces sp., and contained a conserved region with S.chromofuscus PLD. From comparisons of the structural similarity and properties of the various PLDs, a classification of PLDs into two subgroups has been proposed and the highly conserved region designated tentatively region XPLD, which may be important in the catalytic function, has been identified. The homology comparison between our PLD and phosphatidylinositol-specific phospholipase C (PI-PLC) is also discussed. 相似文献
6.
Ya-Wei Shi Li-Xi Niu Xia Feng Jing-Ming Yuan 《World journal of microbiology & biotechnology》2006,22(7):675-680
Summary A d-hydantoinase was expressed in the soluble form by a recombinant E. coli strain, pE-HDT/E. coli BL21 in LB medium. The enzymatic activity of cultured cells reached 5.2–6.5 IU/ml culture at a cell turbidity of 10 at 600 nm.
The expressed enzyme was efficiently purified by three steps, ammonium sulfate fractionation, Phenyl-Sepharose hydrophobic
interaction chromatography and Sephacryl S-200 size-exclusion chromatography. With the above purification process, the enzyme
was purified to more than 95% purity as estimated by SDS-PAGE. The overall recovery of enzymatic activity was 54.4% and the
specific activity for substrate dl-hydantoin achieved 48 U/mg. The purified enzyme appeared as a dimer with a molecular mass of 103 kDa, as measured by size-exclusion
chromatography. The enzyme was stable from pH 6 to 12 with an optimum pH at 9.5 The optimum temperature of the enzyme was
45 °C and it activity was rapidly lost over 55 °C. Divalent metal ions, including Co2+, Mn2+ and Ni 2+ ions obviously enhanced the enzymatic activity, while Zn2+ ion had a slight inhibitory effect. In addition, the dissociation of purified enzyme into its subunits occurred in the presence
of 1 mM Zn2+ ion. The effect of different metal ions on the d-hydantoinase activation/attenuation was discussed. 相似文献
7.
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
8.
S Smaoui F Mathieu L Elleuch Y Coppel G Merlina I Karray-Rebai L Mellouli 《World journal of microbiology & biotechnology》2012,28(3):793-804
A new actinomycete strain designated TN256, producing antimicrobial activity against pathogenic bacteria and fungi, was isolated
from a Tunisian Saharan soil. Morphological and chemical studies indicated that strain TN256 belonged to the genus Streptomyces. Analysis of the 16S rDNA sequence of strain TN256 showed a similarity level ranging between 99.79 and 97.8% within Streptomyces microflavus DSM 40331T and Streptomyces griseorubiginosus DSM 40469T respectively. The comparison of its physiological characteristics showed significant differences with the nearest species.
Combined analysis of the 16 S rRNA gene sequences (FN687758), fatty acids profile, and results of physiological and biochemical
tests indicated that there were genotypic and phenotypic differentiations of that isolate from other Streptomyces species neighbours. These date strongly suggest that strain TN256 represents a novel species with the type strain Streptomyces TN256 (=CTM50228T). Experimental validation by DNA–DNA hybridization would be required for conclusive confirmation. Four active products (1–4)
were isolated from the culture broth of Streptomyces TN256 using various separation and purification steps and procedures. 1: N-[2-(1H-indol-3-yl)-2 oxo-ethyl] acetamide ‘alkaloid’ derivative; 2: di-(2-ethylhexyl) phthalate, a phthalate derivative; 3: 1-Nonadecene and 4: Cyclo (l-Pro-l-Tyr) a diketopiperazine ‘DKP’ derivative. The chemical structure of these four active compounds was established on the basis
of spectroscopic studies NMR and by comparing with data from the literature. According to our biological studies, we showed
in this work that the pure compounds (1–4) possess antibacterial and antifungal activities. 相似文献
9.
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
10.
Yoshiko Uesugi Tadashi Hatanaka 《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2009,1791(9):962-969
Phospholipase D (PLD) plays various roles in important biological processes and physiological functions, including cell signaling. Streptomyces PLDs show significant sequence similarity and belong to the PLD superfamily containing two catalytic HKD motifs. These PLDs have conserved catalytic regions and are among the smallest PLD enzymes. Therefore, Streptomyces PLDs are thought to be suitable models for studying the reaction mechanism among PLDs from other sources. Furthermore, Streptomyces PLDs present advantages related to their broad substrate specificity and ease of enzyme preparation. Moreover, the tertiary structure of PLD has been elucidated only for PLD from Streptomyces sp. PMF. This article presents a review of recently reported studies of the mechanism of the catalytic reaction, substrate recognition, substrate specificity and stability of Streptomyces PLD using various protein engineering methods and surface plasmon resonance analysis. 相似文献
11.
Yi-Guang Chen Jun Chen Qi-Hui Chen Shu-Kun Tang Yu-Qin Zhang Jian-Wu He Wen-Jun Li Yan-Qi Liu 《Antonie van Leeuwenhoek》2010,98(3):395-401
A novel Gram-stain-positive, slightly halophilic, facultatively alkaliphilic, non-motile, non-sporulating, catalase-positive,
oxidase-negative, aerobic bacterium, designated strain JSM 070026T, was isolated from non-saline forest soil in China. Growth occurred with 0–20% (w/v) NaCl (optimum, 2–4%) and at pH 6.0–10.5
(optimum, pH 8.0) and 5–40°C (optimum, 30°C). Good growth also occurred in the presence of 0–28% (w/v) KCl (optimum, 2–5%)
or 0–25% (w/v) MgCl2·6H2O (optimum, 1–4%). The peptidoglycan type was A4α (l-Lys–Gly–l-Glu). Cell-wall sugars contained mannose and xylose. The major cellular fatty acids were anteiso-C15:0 and iso-C15:0. Strain
JSM 070026T contained menaquinone 8 as the major respiratory quinone and diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol
as the major polar lipids. The DNA G + C content of strain JSM 070026T was 56.7 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain JSM 070026T was a member of the suborder Micrococcineae and most closely related to Yaniella flava YIM 70178T (sequence similarity 99.4%) and Yaniella halotolerans YIM 70085T (97.9%). The three strains formed a distinct branch in the phylogenetic tree. The combination of phylogenetic analysis, DNA–DNA
relatedness values, phenotypic characteristics and chemotaxonomic data supports the proposal that strain JSM 070026T represents a novel species of the genus Yaniella, for which the name Yaniella soli sp. nov. is proposed. The type strain is JSM 070026T (=DSM 22211T = KCTC 13527T). 相似文献
12.
Marui J Matsushita-Morita M Tada S Hattori R Suzuki S Amano H Ishida H Yamagata Y Takeuchi M Kusumoto K 《Applied microbiology and biotechnology》2012,93(2):655-669
The gdaA gene encoding S12 family glycine–d-alanine aminopeptidase (GdaA) was found in the industrial fungus Aspergillus oryzae. GdaA shares 43% amino acid sequence identity with the d-aminopeptidase of the Gram-negative bacterium Ochrobactrum anthropi. GdaA purified from an A. oryzae gdaA-overexpressing strain exhibited high d-stereospecificity and efficiently released N-terminal glycine and d-alanine of substrates in a highly specific manner. The optimum pH and temperature were 8 to 9 and 40°C, respectively. This
enzyme was stable under alkaline conditions at pH 8 to 11 and relatively resistant to acidic conditions until pH 5.0. The
chelating reagent EDTA, serine protease inhibitors such as AEBSF, benzamidine, TPCK, and TLCK, and the thiol enzyme inhibitor
PCMB inhibited the enzyme. The aminopeptidase inhibitor bestatin did not affect the activity. GdaA was largely responsible
for intracellular glycine and d-alanine aminopeptidase activities in A. oryzae during stationary-phase growth in liquid media. In addition, the activity increased in response to the depletion of nitrogen
or carbon sources in the growth media, although the GdaA-independent glycine aminopeptidase activity highly increased simultaneously.
Aminopeptidases of A. oryzae attract attention because the enzymatic release of a variety of amino acids and peptides is important for the enhancement
of the palatability of fermented foods. GdaA activity was found in extracts of a solid-state rice culture of A. oryzae (rice koji), which is widely used as a starter culture for Japanese traditional fermented foods, and was largely responsible
for the glycine and d-alanine aminopeptidase activity detected at a pH range of 6 to 9. 相似文献
13.
An N-acetyl-d-lactosamine (LacNAc) specific lectin from tubers of Alocasia cucullata was purified by affinity chromatography on asialofetuin-linked amino activated silica. The pure lectin showed a single band
in SDS-PAGE at pH 8.8 and was a homotetramer with a subunit molecular mass of 13.5 kDa and native molecular mass of 53 kDa.
It was heat stable up to 55 °C for 15 min and showed optimum hemagglutination activity from pH 2 to 11. The lectin was affected
by denaturing agents such as urea (2 m), thiourea (2 m) and guanidine–HCl (0.5 m) and did not require Ca2+ and Mn2+ for its activity. It was a potent mitogen at 10 μg/ml towards human peripheral blood mononuclear cells with 50% growth inhibitory
potential towards SiHa (human cervix ) cancer cell line at 100 μg/ml. 相似文献
14.
Johanna Mansfeld Renate Ulbrich-Hofmann 《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2009,1791(9):913-926
Most phospholipases D (PLDs) occurring in microorganisms, plants and animals belong to a superfamily which is characterized by several conserved regions of amino acid sequence including the two HKD motifs necessary for catalytic activity. Most eukaryotic PLDs possess additional regulatory structures such as the Phox and Pleckstrin homology domains in mammalian PLDs and the C2 domain in most plant PLDs. Owing to recombinant expression techniques, an increasing number of PLDs from different organisms has been obtained in purified form, allowing the investigation of specific and unspecific interactions of the enzymes with regulatory components in vitro. The present paper gives an overview on different factors which can modulate PLD activity and compares their influence on the enzymes from different sources. While no biological regulator can be recognized for extracellular bacterial PLDs, the most prominent specific activator of eukaryotic PLDs is phosphatidylinositol-4,5-bisphosphate (PIP2). In a sophisticated interplay PIP2 seems to cooperate with several regulatory proteins in mammalian PLDs, whereas in plant PLDs it mainly acts in concert with Ca2+ ions. Moreover, curvature, charges and heterogeneities of membrane surfaces are assessed as unspecific modulators. A possible physiological role of the transphosphatidylation reaction catalyzed by PLDs in competition with phospholipid hydrolysis is discussed. 相似文献
15.
Lerchner A Mansfeld J Schäffner I Schöps R Beer HK Ulbrich-Hofmann R 《Biochimica et biophysica acta》2005,1737(2-3):94-101
The genes of two phospholipase D (PLD) isoenzymes, PLD1 and PLD2, from poppy seedlings (2829 and 2828 bp) were completely sequenced. The two genes have 96.9% identity in the encoding region and can be assigned to the alpha-type of plant PLDs. The corresponding amino acid sequences do not contain any signal sequences. One Asn-glycosylation site, six and two phosphorylation sites for protein kinase C and tyrosine kinase, respectively, and two phosphatidylinositol-4,5-bisphosphate binding motifs could be identified. Like in most plant PLDs, two HKD motifs and one C2 domain are present. PLD1 and PLD2 have ten and nine cysteine residues. The two enzymes were expressed in E. coli and purified to homogeneity by Ca2+ ion-mediated hydrophobic interaction chromatography. The Ca2+ ion concentration needed for carrier binding of the two enzymes in chromatography as well as for optimum activity was found to be considerably higher (>100 mM) than with other alpha-type plant PLDs. Although PLD1 and PLD2 differ in eleven amino acids only, they showed remarkable differences in their transphosphatidylation activity. Two amino acid exchanges within and near the first HKD motif contribute to this difference as shown by the A349E/E352Q-variant of PLD2. 相似文献
16.
S. S. Sudge K. B. Bastawde D. V. Gokhale U. R. Kalkote T. Ravindranathan 《Applied microbiology and biotechnology》1998,49(5):594-599
About 1000 bacterial colonies isolated from sea water were screened for their ability to convert dl-5-phenylhydantoin to d(−)N-carbamoylphenylglycine as a criterion for the determination of hydantoinase activity. The strain M-1, out of 11 hydantoinase-producing
strains, exhibited the maximum ability to convert dl-5-phenylhydantoin to d(−)N-carbamoylphenylglycine. The strain M-1 appeared to be a halophilic Pseudomonas sp. according to morphological and physiological characteristics. Optimization of the growth parameters revealed that nutrient
broth with 2% NaCl was the preferred medium for both biomass and enzyme production. d-Hydantoinase of strain M-1 was not found to be inducible by the addition of uracil, dihydrouracil, β-alanine etc. The optimum
temperature for enzyme production was about 25 °C and the organism showed a broad pH optimum (pH 6.5–9.0) for both biomass
and hydantoinase production. The organism seems to have a strict requirement of NaCl for both growth and enzyme production.
The optimum pH and temperature of enzyme activity were 9–9.5 and 30 °C respectively. The biotransformation under the alkaline
conditions allowed the conversion of 80 g l−1
dl-5-phenylhydantoin to 82 g l−1
d(−)N-carbamoylphenylglycine within 24 h with a molar yield of 93%.
Received: 15 September 1997 / Received revision: 5 January 1998 / Accepted: 6 January 1998 相似文献
17.
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
18.
We purified phospholipase D (PLD) enzyme from peanut seeds, and the PLD enzyme eluted as two distinct peak fractions on Mono-Q chromatography, the first of which was characterized. N-terminal sequencing indicated that the N-terminus was blocked. The molecular mass of the purified enzyme was estimated to be 92 kDa by SDS-PAGE. The pH optimum of the enzyme was 5.0, and the K
m value against its substrate phosphatidylcholine (PC), in the presence of 10 mM CaCl2 and 4 mM deoxycholate, was estimated to be 0.072 mM. The enzyme catalyzed two reactions, i.e., hydrolysis of PC generating phosphatidic acid (PA) and choline, and transphosphatidylation of the PA-moiety in the PC molecule to the acceptor glycerol, generating phosphatidylglycerol. Furthermore, we cloned two types of full-length cDNA, Ahpld1 and Ahpld2, each encoding distinct PLD molecules having 794 and 807 residues, respectively. The partial amino acid sequence of the purified PLD was consistent with the deduced sequence of AhPLD2. 相似文献
19.
Lu L Zhao M Zhang BB Yu SY Bian XJ Wang W Wang Y 《Applied microbiology and biotechnology》2007,74(6):1232-1239
The white rot fungus Pycnoporus sanguineus produced high amount of laccase in the basal liquid medium without induction. Laccase was purified using ultrafiltration,
anion-exchange chromatography, and gel filtration. The molecular weight of the purified laccase was estimated as 61.4 kDa
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme oxidized typical substrates of laccases including
2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate), 2,6-dimethoxyphenol, and syringaldazine. The optimum pH and temperature for the purified
laccase were 3.0 and 65°C, respectively. The enzyme was stable up to 40°C, and high laccase activity was maintained at pH 2.0–5.0.
Sodium azide, l-cysteine, and dithiothreitol strongly inhibited the laccase activity. The purified enzyme efficiently decolorized Remazol
Brilliant Blue R in the absence of added redox mediators. The high production of P. sanguineus laccase as well as its decolorization ability demonstrated its potential applications in dye decolorization. 相似文献
20.
l-2-Amino-Δ2-thiazoline-4-carboxylic acid hydrolase (ATC hydrolase) was purified and characterized from the crude extract of Escherichia coli, in which the gene for ATC hydrolase of Pseudomonas sp. strain ON-4a was expressed. The results of SDS–polyacrylamide gel electrophoresis and gel filtration on Sephacryl S-200 suggested that the ATC hydrolase was a tetrameric enzyme consisted of identical 25-kDa subunits. The optimum pH and temperature of the enzyme activity were pH 7.0 and 30–35°C, respectively. The enzyme did not require divalent cations for the expression of the activity, and Cu2+ and Mn2+ ions strongly inhibited the enzyme activity. An inhibition experiment by diethylpyrocarbonic acid, 2-hydroxy-5-nitrobenzyl bromide, and N-bromosuccinimide suggested that tryptophan, cysteine, or/and histidine residues may be involved in the catalytic site of this enzyme. The enzyme was strictly specific for the l-form of d,l-ATC and exhibited high activity for the hydrolysis of l-ATC with the values of K
m (0.35 mM) and V
max (69.0 U/mg protein). This enzyme could not cleave the ring structure of derivatives of thiazole, thiazoline, and thiazolidine tested, except for d,l- and l-ATC. These results show that the ATC hydrolase is a novel enzyme cleaving the carbon–sulfur bond in a ring structure of l-ATC to produce N-carbamoyl-l-cysteine. 相似文献