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1.
Clostridium paraputrificum chitinase A (ChiA) was purified from a recombinant Escherichia coli. ChiA was active toward chitin from crab shells, colloidal chitin, glycol chitin, and 4-methylumbelliferyl beta-D-N,N'-diacetylchitobioside [4-MU-(GlcNAc)2]. ChiA showed maximum activity at pH 6.0 and its optimum temperature was 45 degrees C. ChiA was stable between pH 6.0 and 9.0 and at temperatures up to 40 degrees C. The K(m) and V(max) values of ChiA for 4-MU-(GlcNAc)2 were estimated to be 6.9 microM and 43 micromol/min/mg, respectively. Thin-layer chromatography indicated that ChiA hydrolyzes chitooligosaccharides to mainly chitobiose. ChiA was found to adsorb not only chitinous polymers but also cellulosic polymers.  相似文献   

2.
The arabinofuranosidase gene was cloned from the cDNA of Aspergillus sojae. It was found to contain an open reading frame composed of 984 base pairs (bp) and to encode 328 amino acid residues (aa). The cDNA sequence suggested that the mature enzyme is preceded by a 26-aa signal sequence and the molecular mass was predicted to be 32,749 Da. The A. sojae arabinofuranosidase consists of a single catalytic domain; it does not have a specific substrate-binding domain such as the xylan-binding domain reported in an arabinofuranosidase from Streptomyces lividans (Vincent, P. et al.: Biochem. J., 322, 845-852, 1997). The deduced amino acid sequence of the catalytic domain of the mature enzyme exhibits extensive identity with the catalytic domains of Streptomyces coelicolor (74%), Aspergillus niger (75%), S. lividans (74%), and Aspergillus tubingensis (75%), which are enzymes that belong to family 62 of the glycosyl hydrolases. The cloned AFdase gene was expressed in Escherichia coli BL21 (DE3) pLysS as a cellulose-binding domain tag fusion protein. The specific activity of the purified recombinant enzyme was 18.6 units/mg protein, which is one-fourth that of the enzyme purified from a solid-state culture of A. sojae.  相似文献   

3.
A new exo-type cellulase, named exo-cellulase II (Ex-2), was purified from the crude enzyme preparation of Irpex lacteus. Ex-2 was very similar to the previously characterized exo-cellulase I (Ex-1) with respect to enzymatic features such as optimal pH, temperature, heat stability, and catalytic activity. However, Ex-2 exhibited greater pH stability than Ex-1. The molecular mass and carbohydrate content of Ex-2 (56,000, 4.0%) were different from those of Ex-1 (53,000, 2.0%). A cellulase gene (named cel2) encoding both Ex-2 and Ex-1 was isolated from an I. lacteus genomic library. The cel2 gene was found to consist of 1569 bp with an open reading frame encoding 523 amino acids, interrupted by two introns. The deduced amino acid sequences revealed that cel2 ORF has a modular structure consisting of a catalytic domain and a fungal-type cellulose-binding domain (CBD) separated by a serine-rich linker region. The catalytic domain was homologous to those of fungal cellobiohydrolases belonging to family 7 of the glycosyl hydrolases. Northern blot analysis showed that expression of the cel2 gene was induced by various cellulosic substrates and repressed by glucose, fructose, and lactose.  相似文献   

4.
VChti-1 chitinase encoded by the Chlorella virus CVK2 contained two catalytic domains belonging to family 18 glycosyl hydrolases. The first catalytic domain on a C-terminal-truncated derivative of vChti-1 generated exclusively chitobiose from chitotetraose, chitohexaose, and colloidal high-molecular mass chitin in the enzyme reaction, a typical characteristic of an exochitinase. In contrast, N-acetylglucosamine was produced from chitobiose as well as from chitooligosaccharides by the second catalytic domain on an N-terminal-truncated derivative of vChti-1. Therefore, the second domain possessed N-acetylglucosaminidase activity as well as endochitinase activity. The presence of two catalytic domains with different enzymatic properties in the viral enzyme seems to be necessary for hydrolyzing natural substrates in a cooperative fashion.  相似文献   

5.
6.
目的将兽疫链球菌透明质酸酶hyl基因在大肠杆菌原核表达系统中高效分泌表达。方法通过PCR方法扩增得兽疫链球菌hyl基因,Xho I和Nco I双酶切后,连入表达载体pET26b(+),并转化入大肠杆菌,经低温异丙基-β-D-硫代半乳糖苷或乳糖诱导表达,DNS法检测胞外透明质酸酶活性。结果成功构建含兽疫链球菌透明质酸酶基因hyl的大肠杆菌基因工程菌株,经IPTG诱导并添加甘氨酸后胞外透明质酸酶活性高达5.3×104 U/mL。结论实现了透明质酸酶在原核系统中的表达,为工业化生产奠定了基础,具有很好的市场前景。  相似文献   

7.
A gene from Vibrio alginolyticus H-8, encoding chitinase, designated as chitinase B, was cloned by the shot-gun method using pUC118 and sequenced. The open reading frame consisted of 846 amino acids including a signal peptide. The molecular mass of the enzyme estimated based on the amino acid sequence data was 90 kDa. The N-terminal amino acid sequence of the enzyme was different from that of chitinase C1 which we had previously reported. This cloned chitinase B was considered one out of four chitinases (A, B, D, and E) which had been newly isolated from the culture broth and cell extract of V. alginolyticus H-8. The gene contained a chitin-binding domain and typical conserved regions of chitinases reported previously. The deduced amino acid sequence of the cloned chitinase B showed high sequence homology with the chitinase from V. parahaemolyticus (84% identity) and the chitinase from V. anguillarum (76.6%), but low sequence homology with the chitinase from V. harveyi (24.4%), and the chitodextrinase from V. furnissii (23.9%). Chitinase E found in cell extract is considered an intracellular chitinase which is different from chitodextrinases.  相似文献   

8.
为研究皱纹盘鲍(Haliotis discus hannai)中脯氨酰内肽酶(Prolyl endopeptidase,Hdh-PEP)的酶学特性与结构特性,利用基因工程技术重组并在大肠杆菌中高效表达了皱纹盘鲍PEP。原核表达的Hdh-PEP分子量为85 kDa,在pH2~6、温度20~60 ℃条件下,Hdh-PEP的表面疏水性明显升高。氨基酸序列同源性分析结果表明,Hdh-PEP催化结构域中有三个高度保守的氨基酸序列:Seq 1:K-D-G-T-K/R-I-P、Seq 2:Y-G-Y-G-G-F和Seq 3:I-R-G-G-E-Y/F。酶动力学研究表明,Hdh-PEP的米氏常数Km为5.32 μmol/L,催化常数kcat值为15.7 s?1。PEP的特异性抑制剂SUAM-14746和ZPP对Hdh-PEP酶活力具有强抑制作用,丝氨酸蛋白酶抑制(PMSF)对Hdh-PEP酶活力也有较大程度的抑制作用。本实验制备了高特异性抗Hdh-PEP多克隆抗体,可检测鲍鱼肌肉中天然PEP的存在情况。Hdh-PEP的体外高效表达和特异性多克隆抗体制备为后续深入研究Hdh-PEP的性质提供了重要参考。  相似文献   

9.
10.
Bifidobacterium bifidum was found to produce a specific 1,2-alpha-L-fucosidase. Its gene (afc A) has been cloned and the DNA sequence was determined. The Afc A protein consisting of 1959 amino acid residues with a predicted molecular mass of 205 kDa can be divided into three domains; the N-terminal function-unknown domain (576 aa), the catalytic domain (898 aa), and the C-terminal bacterial Ig-like domain (485 aa). The recombinant catalytic domain specifically hydrolyzed the terminal alpha-(1-->2)-fucosidic linkages of various oligosaccharides and sugar chains of glycoproteins. The primary structure of the catalytic domain exhibited no similarity to those of any glycoside hydrolases but showed similarity to those of several hypothetical proteins in a database, which resulted in establishment of a novel glycoside hydrolase family (GH family 95). Several bifidobacteria were found to produce a specific endo-alpha-N-acetylgalactosaminidase, which is the endoglycosidase liberating the O-glycosidically linked galactosyl beta1-->3 N-acetylgalactosamine disaccharide from mucin glycoprotein. The molecular cloning of endo-alpha-N-acetylgalactosaminidase was carried out on Bifidobacterium longum based on the information in the database. The gene was found to comprise 1966 amino acid residues with a predicted molecular mass of 210 kDa. The recombinant protein released galactosyl beta1-->3 N-acetylgalactosamine disaccharide from natural glycoproteins. This enzyme of B. longum is believed to be involved in the catabolism of oligosaccharide of intestinal mucin glycoproteins. Both 1,2-alpha-L-fucosidase and endo-alpha-N-acetylgalactosaminidase are novel and specific enzymes acting on oligosaccharides that exist mainly in mucin glycoproteins. Thus, it is reasonable to conclude that bifidobacteria produce these enzymes to preferentially utilize the oligosaccharides present in the intestinal ecosystem.  相似文献   

11.
D-塔格糖3-差向异构酶是生物法生产新型功能性因子D-阿洛酮糖最为有效的酶。一种新型的能够编码D-塔格糖3-差向异构酶的基因CLOBOL00069被克隆,它来源于Clostridium bolteae ATCC BAA-613。以pUC57为克隆载体,以pET-22b(+)为载体质粒,E.coli BL21(DE3)为宿主细胞,构建了基因重组工程菌。IPTG诱导剂诱导目的蛋白的表达;通过镍柱亲和层析,杂蛋白与目的蛋白得到了很好的分离。对纯化的重组蛋白样品进行SDS-PAGE分析,在约32ku处出现明显的特征条带。通过活性研究表明,Clostridium bolteae ATCC BAA-613 DTEase属于DTEase家族,并具有较高的生物转化率,反应10h后转化率达到20%。  相似文献   

12.
In this paper, we present the expression and characterization of two novel enzymes from the alpha-amylase family exhibiting cyclomaltodextrinase specificity. The nucleotide sequences encoding the enzymes were isolated from the genomic DNA of two thermophilic bacterial strains originating from Icelandic hot springs and belonging to the genera Anoxybacillus (AfCda13) and Laceyella (LsCda13). The genes were amplified using a consensus primer strategy utilizing two of the four conserved regions present in glycoside hydrolase family 13. No identifiable signal peptides were present in open reading frames encoding the enzymes, indicating an intracellular location of both enzymes, and their physiological function to be intracellular cyclodextrin degradation. The domain structures of both enzymes were also similar, including an N-terminal domain, the catalytic module composed of the A- and B-domains, and a C-terminal domain. Despite the similarity in domain composition, the two enzymes displayed differences in the oligomeric state with AfCda13 being a dimeric protein, whereas LsCda13 was monomeric. The two enzymes also displayed significantly different activity profiles, despite being active on the same range of substrates. It was shown that the enzyme displaying the highest activity on cyclodextrin was dimeric (AfCda13). Moreover, a fraction of the dimeric enzyme could be converted to a monomeric state in the presence of KCl and this fraction retained only 23% of its activity on alpha-cyclodextrin while its activity on starch was not significantly affected, indicating that the oligomeric state is an important factor for a high activity on cyclodextrin substrates.  相似文献   

13.
D(-)-3-hydroxybutyrate dehydrogenase (BDH; EC 1.1.1.30) from a poly(D(-)-3-hydroxybutyrate) (PHB) degrading bacterium, Acidovorax sp. SA1, was purified using Toyopearl DEAE-650M, red-Sepharose CL-4B, and Q Sepharose FF. The molecular mass of the enzyme was estimated as 27 kDa by SDS-PAGE and 110 kDa by gel filtration. The gene encoding BDH was cloned and sequenced, and expressed in Escherichia coli. The gene product was purified in two steps with a high yield. The N-terminal amino acid sequence of the enzyme purified from E. coli agreed with that of the purified enzyme from strain SA1. The BDH of strain SA1 had high amino acid sequence homology to that of Ralstonia eutropha H16. The Km values for D(-)-3-hydroxybutyrate and NAD+ in the oxidation reaction were 4.5 x 10(-4) M and 8.9 x 10(-5) M, respectively. The Km values for acetoacetate and NADH in the reduction reaction were 2.4 x 10(-4) M and 2.9 x 10(-5) M, respectively.  相似文献   

14.
A gene encoding deacetylase DA1 that is specific for N, N'-diacetylchitobiose was cloned using the shot-gun method with pUC118 and sequenced. The open reading frame encoded a protein of 427 amino acids including the signal peptide. The molecular mass of the mature enzyme estimated from the amino acid sequence data was 44.7 kDa, which is approximately similar to that, estimated by SDS-PAGE (48.0 kDa), of the purified enzyme reported previously. The N-terminal amino acid sequence deduced from the cloned deacetylase gene showed partial sequence homology with the Nod B protein from Rhizobium sp. (37% identity) and chitin deacetylase from Mucor rouxii (28%). It contained a domain, which showed homology with a chitin-binding domain of chitinase A from Bacillus circulans (39%).  相似文献   

15.
A cDNA encoding the alpha-galactosidase of Absidia corymbifera IFO 8084 was cloned and sequenced. The cloned DNA has a single open-reading frame consisting of 2190 base pairs, and the deduced amino acid sequence revealed that the mature enzyme consisted of 730 amino acid residues with a molecular mass of 82,712 Da. The native structure of the alpha-galactosidase of A. corymbifera IFO 8084 was determined to be a tetramer. Comparison with amino acid sequences of other alpha-galactosidase showed high homology with sequences of members of family 36. An expression vector, pET32Trx/galalpha, was constructed by introducing the cDNA coding region into a thioredoxin fusion system, pET32-Ek/LIC. The resulting transformant, pET32Trx/galalpha, overproduced the active enzyme as a thioredoxin fused form in the host Escherichia coli. By using His-binding metal affinity chromatography, recombinant alpha-galactosidase was purified to homogeneity in a single step. The purified recombinant fusion alpha-galactosidase showed properties very similar to the native alpha-galactosidase from A. corymbifera IFO 8084.  相似文献   

16.
α 阿拉伯糖苷酶 /木糖苷酶对来源于被子植物的木聚糖类半纤维素的生物降解和转化是必不可少的。文中首次报道了国内对该酶的研究。α 阿拉伯糖苷酶 /木糖苷酶的基因工程菌在发酵罐中以LB为基质进行生长 ,以乳糖为诱导剂 ,所产生的α 阿拉伯糖苷酶 /木糖苷酶在 70℃热处理 3 0min后、经DEAE Sephacel阴离子柱层析、金属Ni2 + 的亲和层析等提纯步骤 ,达到了电泳纯 ,提纯倍数为 49 3倍 ,收率为 2 0 4%。SDS PAGE法测定α 阿拉伯糖苷酶/木糖苷酶的分子质量为 85ku ,与理论推算值相吻合  相似文献   

17.
Multiple ethyl 4-chloro-3-oxobutanoate (COBE)-reducing enzymes were isolated from a cell-free extract of Candida magnoliae. A NADPH-dependent COBE-reducing enzyme, distinct from the carbonyl reductase and aldehyde reductase previously reported, was purified to homogeneity using five steps, including polyethylene glycol treatment. The relative molecular mass of the enzyme was estimated to be 86,000 on high performance gel-permeation chromatography and 29,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme catalyzed the reduction of COBE to the corresponding (S)-alcohol with a 51% enantiomeric excess. The substrate specificity of the enzyme was different from those of the other COBE-reducing enzymes of the same strain. The partial amino acid sequences of the enzyme showed that it belongs to the short chain alcohol dehydrogenase/reductase (SDR) family. This is the first report of multiple COBE-reducing enzymes with various stereoselectivities occurring in the same strain but belonging to different (super)families.  相似文献   

18.
A thermophilic and cyanide ion-tolerant bacterium, Bacillus stearothermophilus CN3 isolated from a hot spring in Japan, was found to produce thermostable beta-cyano-L-alanine synthase. The enzyme catalyzes the synthesis of beta-cyano-L-alanine from O-acetyl-L-serine and cyanide ions. The purified enzyme has a molecular mass of approximately 70 kDa and consists of two identical subunits. It was stable in the pH range of 6.0 to 10.0 and up to 70 degrees C. The enzyme also catalyzes the synthesis of various beta-substituted-L-alanine derivatives from O-acetyl-L-serine and nucleophilic reagents. The gene encoding the beta-cyano-L-alanine synthase was isolated from B. stearothermophilus CN3. Sequence homology analysis revealed that the beta-cyano-L-alanine synthase of the bacterium is O-acetyl-L-serine sulfhydrylase. A recombinant plasmid, constructed by ligation of the cloned gene and an expression vector, pKK223-3, was introduced into E. coli JM109. The transformed E. coli cells overexpressed beta-cyano-L-alanine synthase. Heat stable beta-cyano-L-alanine synthase can be applied to the synthesis of [4-11C]l-2,4-diaminobutyric acid as a tracer for positron emission tomography.  相似文献   

19.
A NAD+-dependent medium-chain alcohol dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernix K1 was expressed in Escherichia coli and purified. The recombinant enzyme was a homotetramer of molecular mass 1.6 x 10(2) kDa. The optimum pH for the oxidative reaction was around 10.5 and that for the reductive reaction was around 8.0. The enzyme had a broad substrate specificity including aliphatic and aromatic alcohols, aliphatic and aromatic ketones, and benzylaldehyde. This enzyme produced (S)-alcohols from the corresponding ketones. The enzyme was thermophilic and the catalytic activity increased up to 95 degrees C. It maintained 24% of the original catalytic activity after incubation for 30 min at 98 degrees C, indicating that this enzyme is highly thermostable.  相似文献   

20.
A 1.4-kbp DNA fragment, including the NADH-linked acetylacetoin reductase/2,3-butanediol dehydrogenase (AACRII/BDH) gene from the chromosomal DNA of Bacillus cereus YUF-4, was cloned in Escherichia coli DH5alpha after its insertion into pUC119, and the resulting plasmid was named pAACRII119. The AACRII/BDH gene had an open reading frame consisting of 1047 bp encoding 349 amino acids. The enzyme exhibited not only AACR activity, but also BDH activity. However, the gene was not located in a 2,3-butanediol (BD) operon, as is the case in the BDH gene of Klebsiella pneumoniae and that of K. terrigena. In addition, there was no BD-cycle-related enzyme gene in the region surrounding the AACRII/BDH gene. The AACR and BDH activities in E. coli DH5alpha/pAACRII119 were 200-fold higher than those in the original B. cereus YUF-4. The characteristics of the AACRII/BDH from E. coli DH 5alpha/pAACRII119 are similar to those of the AACRII/BDH from B. cereus YUF-4. The AACRII/BDH was considered to belong to the NAD(P)- and zinc-dependent long-chain alcohol dehydrogenase (group I ADH) family on the basis of the following distinctive characteristics: it possessed 14 strictly conserved residues of microbial group I ADH and consisted of about 350 amino acids. The enzymatic and genetic characteristics of AACRII/BDH were completely different from those of BDHs belonging to the short-chain dehydrogenase/reductase family. These findings indicated that the AACRII/BDH could be considered a new type of BDH.  相似文献   

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