Cloning and expression of a conjugated bile acid hydrolase gene from Lactobacillus plantarum by using a direct plate assay.

The conjugated bile acid hydrolase gene from the silage isolate Lactobacillus plantarum 80 was cloned and expressed in Escherichia coli MC1061. For the screening of this hydrolase gene within the gene bank, a direct plate assay developed by Dashkevicz and Feighner (M. P. Dashkevicz and S. D. Feighner, Appl. Environ. Microbiol. 53:331-336, 1989) was adapted to the growth requirements of E. coli. Because of hydrolysis and medium acidification, hydrolase-active colonies were surrounded with big halos of precipitated, free bile acids. This phenomenon was also obtained when the gene was cloned into a multicopy shuttle vector and subsequently reintroduced into the parental Lactobacillus strain. The cbh gene and surrounding regions were characterized by nucleotide sequence analysis. The deduced amino acid sequence was shown to have 52% similarity with a penicillin V amidase from Bacillus sphaericus. Preliminary characterization of the gene product showed that it is a cholylglycine hydrolase (EC 3.5.1.24) with only slight activity against taurine conjugates. The optimum pH was between 4.7 and 5.5. Optimum temperature ranged from 30 to 45 degrees C. Southern blot analysis indicated that the cloned gene has similarity with genomic DNA of bile acid hydrolase-active Lactobacillus spp. of intestinal origin.     

Molecular cloning and characterization of a bile salt hydrolase from Lactobacillus acidophilus PF01

Phenotypic screening for bile salt hydrolase (BSH) activity was performed on Lactobacillus acidophilus PF01 isolated from piglet feces. A gene encoding BSH was identified and cloned from the genomic library of L. acidophilus PF01. The bsh gene and surrounding regions were characterized by nucleotide sequence analysis and were found to contain a single open reading frame (ORF) of 951 nucleotides encoding a 316 amino acid protein. The potential bsh promoter region was located upstream of the start codon. The protein deduced from the complete ORF had high similarity with other BSHs, and four amino acid motifs located around the active site, FGRNXD, AGLNF, VLTNXP, and GXGXGXXGXPGD, were highly conserved. The bsh gene was cloned into the pET21b expression vector and expressed in Escherichia coli BLR(DE3) by induction with 0.1mM of isopropylthiogalactopyranoside. The BSH enzyme was purified with apparent homogeneity using a Ni2+-NTA agarose column and characterized. The overexpressed recombinant BSH enzyme of L. acidophilus PF01 exhibited hydrolase activity against tauroconjugated bile salts, but not glycoconjugated bile salts. It showed the highest activity against taurocholic acid. The maximum BSH activity occurred at approximately 40oC. The enzyme maintained approximately 70% of its maximum activity even at 60 degrees , whereas its activity rapidly decreased at below 37 degrees . The optimum pH was 6, and BSH activity was rapidly inactivated below pH 5 and above pH 7.     

Cloning and characterization of a conjugated bile acid hydrolase gene from Clostridium perfringens.

The gene encoding a conjugated bile acid hydrolase (CBAH) from Clostridium perfringens 13 has been cloned and expressed in Escherichia coli, and its nucleotide sequence has been determined. Nucleotide and predicted amino acid sequence analyses indicated that the gene product is related to two previously characterized amidases, a CBAH from Lactobacillus plantarum (40% identity) and a penicillin V amidase from Bacillus sphaericus (34% identity). The product is apparently unrelated to a CBAH from C. perfringens for which N-terminal sequence information was determined. The gene product was purified from recombinant E. coli and used to raise antibody in rabbits. The presence of the protein in C. perfringens was then confirmed by immunoblot analysis. The protein was shown to have a native molecular weight of 147,000 and a subunit molecular weight of 36,100, indicating its probable existence as a tetramer. Disruption of the chromosomal C. perfringens CBAH gene with a chloramphenicol resistance cartridge resulted in a mutant strain which retained partial CBAH activity. Polyacrylamide gel electrophoresis followed by enzymatic activity staining and immunoblotting indicated that the mutant strain no longer expressed the cloned CBAH (CBAH-1) but did express at least one additional CBAH (CBAH-2). CBAH-2 was immunologically distinct from CBAH-1, and its mobility on native polyacrylamide gels was different from that of CBAH-1. Furthermore, comparisons of pH optima and substrate specificities of CBAH activities from recombinant E. coli and wild-type and mutant C. perfringens provided further evidence for the presence of multiple CBAH activities in C. perfringens.     

植物乳杆菌JPP2胆盐水解酶相关基因克隆和表达

通过PCR方法从植物乳杆菌JPP2中扩增出胆盐水解酶（BSH）相关基因bsh3,利用中间克隆载体pMD19-T将其构建于表达载体pET-28b上,并转化入表达宿主菌E．coli BL21（DE3）,成功构建重组BSH的工程菌。核苷酸及推导的氨基酸序列分析表明,正确克隆出目的基因。诱导表达后,SDS-PAGE电泳结果显示出特异性蛋白质条带,其分子量约为38kDa。此单克隆体系的构建为进一步研究BSH的功能奠定基础。     

一种新的乙酰高丝氨酸内酯水解酶基因的克隆和表达

目的 获得新的降解革兰阴性细菌数量阈值感应信号分子乙酰高丝氨酸内酯类化合物(AHL)的水解酶基因。方法 选择性富集和培养土壤中耐热细菌,抽取细菌总DNA作为模板,特异性聚合酶链反应扩增乙酰高丝氨酸内酯水解酶基因,进行克隆和DNA序列分析及原核表达。结果 得到1个新的AHL水解酶基因,该基因与已知基因的核苷酸序列和对应的氨基酸序列同源性最高分别为87％和94％。该基因在原核表达系统中表达,得到了与预期相对分子质鲢(Mr)一致的蛋白质。结论 证实乙酰高丝氨酸内酯水解酶广泛存在于环境微生物中。为进一步研究提供条件。     

Identification and cloning of gusA, encoding a new beta-glucuronidase from Lactobacillus gasseri ADH

The gusA gene, encoding a new beta-glucuronidase enzyme, has been cloned from Lactobacillus gasseri ADH. This is the first report of a beta-glucuronidase gene cloned from a bacterial source other than Escherichia coli. A plasmid library of L. gasseri chromosomal DNA was screened for complementation of an E. coli gus mutant. Two overlapping clones that restored beta-glucuronidase activity in the mutant strain were sequenced and revealed three complete and two partial open reading frames. The largest open reading frame, spanning 1,797 bp, encodes a 597-amino-acid protein that shows 39% identity to beta-glucuronidase (GusA) of E. coli K-12 (EC 3.2.1.31). The other two complete open reading frames, which are arranged to be separately transcribed, encode a putative bile salt hydrolase and a putative protein of unknown function with similarities to MerR-type regulatory proteins. Overexpression of GusA was achieved in a beta-glucuronidase-negative L. gasseri strain by expressing the gusA gene, subcloned onto a low-copy-number shuttle vector, from the strong Lactobacillus P6 promoter. GusA was also expressed in E. coli from a pET expression system. Preliminary characterization of the GusA protein from crude cell extracts revealed that the enzyme was active across an acidic pH range and a broad temperature range. An analysis of other lactobacilli identified beta-glucuronidase activity and gusA homologs in other L. gasseri isolates but not in other Lactobacillus species tested.     

The bile acid-inducible baiB gene from Eubacterium sp. strain VPI 12708 encodes a bile acid-coenzyme A ligase.

The baiB gene from Eubacterium sp. strain VPI 12708 was previously cloned, sequenced, and shown to be part of a large bile acid-inducible operon encoding polypeptides believed to be involved in bile acid 7 alpha-dehydroxylation. In the present study, the baiB gene was subcloned and expressed in Escherichia coli and shown to encode a bile acid-coenzyme A (CoA) ligase. This ligase required a C-24 bile acid with a free carboxyl group, ATP, Mg2+, and CoA for synthesis of the final bile acid-CoA conjugate. Product analysis by reverse-phase high-performance liquid chromatography revealed final reaction products that comigrated with cholyl-CoA and AMP. A putative bile acid-AMP intermediate was detected when CoA was omitted from the reaction mixture. The bile acid-CoA ligase has amino acid sequence similarity to several other polypeptides involved in the ATP-dependent linking of AMP or CoA to cyclic carboxylated compounds. The bile acid-CoA ligation is believed to be the initial step in the bile acid 7 alpha-dehydroxylation pathway in Eubacterium sp. strain VPI 12708.     

南极嗜冷假单胞菌7197 S- 腺苷同型半胱氨酸水解酶 ( SAHH) 基因的克隆与序列分析

从南极普利兹湾深海沉积物中筛选到一株耐冷菌株7197,其16S rDNA序列分析表明该菌株属于假单胞菌属（Pseudomonas）。作者通过设计引物,从该菌的全基因组DNA中克隆到编码S-腺苷-L-高半胱氨酸（SAHH）的完整ORF,全长为1424bp。使用DNAMAN（5,1）软件对全长ORF为1424bp的SAHH基因进行分析,SAHH基因编码一个由474AA残基组成、分子量预计为52523Da的SAHH蛋白质,与Psychrobacter sp．273—4的SAHH有96．84％的相似性;与Acinetobacter sp．ADP1的SAHH有79％的相似性;与Pseudomonas fluorescens Pf-5的SAHH有75％的相似性。     

Expression and nucleotide sequence of the Lactobacillus bulgaricus beta-galactosidase gene cloned in Escherichia coli.

The Lactobacillus bulgaricus beta-galactosidase gene was cloned on a ca. 7-kilobase-pair HindIII fragment in the vector pKK223-3 and expressed in Escherichia coli by using its own promoter. The nucleotide sequence of the gene and approximately 400 bases of 3'- and 5'-flanking sequences was determined. The amino acid sequence of the beta-galactosidase, deduced from the nucleotide sequence of the gene, yielded a monomeric molecular mass of ca. 114 kilodaltons, slightly smaller than the E. coli lacZ and Klebsiella pneumoniae lacZ enzymes but larger than the E. coli evolved (ebgA) beta-galactosidase. The cloned beta-galactosidase was found to be indistinguishable from the native enzyme by several criteria. From amino acid sequence alignments, the L. bulgaricus beta-galactosidase has a 30 to 34% similarity to the E. coli lacZ, E. coli ebgA, and K. pneumoniae lacZ enzymes. There are seven regions of high similarity common to all four of these beta-galactosidases. Also, the putative active-site residues (Glu-461 and Tyr-503 in the E. coli lacZ beta-galactosidase) are conserved in the L. bulgaricus enzyme as well as in the other two beta-galactosidases mentioned above. The conservation of active-site amino acids and the large regions of similarity suggest that all four of these beta-galactosidases evolved from a common ancestral gene. However, these enzymes are quite different from the thermophilic beta-galactosidase encoded by the Bacillus stearothermophilus bgaB gene.     

Molecular cloning and sequence analysis of a gene encoding an extracellular proteinase from Lactobacillus helveticus CP790

A 2.6-kilobase HaeIII DNA fragment corresponding to an extracellular proteinase gene (prtY) was cloned from chromosomal DNA of Lactobacillus helveticus CP790 in Escherichia coli using a pKK223-3 vector. The transformant expressed a 48-kDa protein that reacts with monoclonal antibodies specific to the proteinase and seemed to be a pre-proproteinase, but had no proteolytic activity. About 1.6 kilobases of the 2.6-kilobase DNA fragment, which contained the complete gene for the proteinase was sequenced. Sequence analysis found an open reading frame with a capacity to encode a protein of 449 amino acids. The coding region contained a Gram-positive-type signal peptide of 30 amino acids. The N-terminal sequences of the proproteinase and the mature proteinase have been observed in the polypeptide at position + 31 and + 38. The putative amino acid sequence showed a significant similarity to a surface layer protein of L. helveticus and Lactobacillus acidophilus in the amino terminal signal sequence and carboxyl terminus.     

Identification and characterization of a novel intracellular alkaline alpha-amylase from the hyperthermophilic bacterium Thermotoga maritima MSB8

The gene for a novel alpha-amylase, designated AmyC, from the hyperthermophilic bacterium Thermotoga maritima was cloned and heterologously overexpressed in Escherichia coli. The putative intracellular enzyme had no amino acid sequence similarity to glycoside hydrolase family (GHF) 13 alpha-amylases, yet the range of substrate hydrolysis and the product profile clearly define the protein as an alpha-amylase. Based on sequence similarity AmyC belongs to a subgroup within GHF 57. On the basis of amino acid sequence similarity, Glu185 and Asp349 could be identified as the catalytic residues of AmyC. Using a 60-min assay, the maximum hydrolytic activity of the purified enzyme, which was dithiothreitol dependent, was found to be at 90 degrees C. AmyC displayed a remarkably high pH optimum of pH 8.5 and an unusual sensitivity towards both ATP and EDTA.     

Cloning,sequencing, and overexpression in Escherichia coli of a phenylserine dehydratase gene from Ralstonia pickettii PS22

The structural gene coding for phenylserine dehydratase from Ralstonia pickettii PS22 was cloned into Escherichia coli cells, and the nucleotide sequence was identified. The predicted amino acid sequence had high sequence similarity to biodegradative and biosynthetic threonine dehydratases from E. coli and serine dehydratase from human liver. Transformed E. coli cells overproduced phenylserine dehydratase, and the recombinant enzyme was purified to homogeneity with a high yield and characterized.     

Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding an alpha-amino acid ester hydrolase from Acetobacter turbidans.

The alpha-amino acid ester hydrolase from Acetobacter turbidans ATCC 9325 is capable of hydrolyzing and synthesizing beta-lactam antibiotics, such as cephalexin and ampicillin. N-terminal amino acid sequencing of the purified alpha-amino acid ester hydrolase allowed cloning and genetic characterization of the corresponding gene from an A. turbidans genomic library. The gene, designated aehA, encodes a polypeptide with a molecular weight of 72,000. Comparison of the determined N-terminal sequence and the deduced amino acid sequence indicated the presence of an N-terminal leader sequence of 40 amino acids. The aehA gene was subcloned in the pET9 expression plasmid and expressed in Escherichia coli. The recombinant protein was purified and found to be dimeric with subunits of 70 kDa. A sequence similarity search revealed 26% identity with a glutaryl 7-ACA acylase precursor from Bacillus laterosporus, but no homology was found with other known penicillin or cephalosporin acylases. There was some similarity to serine proteases, including the conservation of the active site motif, GXSYXG. Together with database searches, this suggested that the alpha-amino acid ester hydrolase is a beta-lactam antibiotic acylase that belongs to a class of hydrolases that is different from the Ntn hydrolase superfamily to which the well-characterized penicillin acylase from E. coli belongs. The alpha-amino acid ester hydrolase of A. turbidans represents a subclass of this new class of beta-lactam antibiotic acylases.     

Cloning, sequencing and expression of the L-2-hydroxyisocaproate dehydrogenase-encoding gene of Lactobacillus confusus in Escherichia coli

The gene (L-HicDH) encoding L-2-hydroxyisocaproate dehydrogenase (L-HicDH) from Lactobacillus confusus was cloned in Escherichia coli. A 69-mer oligodeoxyribonucleotide probe, derived to be complementary to the N-terminal amino acid (aa) coding sequence, was used for screening. The complete nucleotide (nt) sequence of the L-HicDH gene was determined. The 5'-end of the mRNA was mapped by primer extension and the promoter identified. Downstream from the L-HicDH gene is a typical Rho-independent terminator. The aa sequence of L-HicDH, deduced from the nt sequence, has an overall similarity of 30% to the aa sequence of L-lactate dehydrogenase (L-LDH) from Lactobacillus casei. The aa residues involved in binding of coenzyme and substrate are highly conserved in L-HicDH with respect to prokaryotic and eukaryotic L-LDHs. The L-HicDH gene could be expressed under control of phage lambda 'Leftward' and 'rightward' promoters in E. coli up to 35% of total cell protein. The enzyme produced under these conditions exhibits full specific activity and is found exclusively in soluble form.     

Characterization of the Bacillus subtilis ywtD gene,whose product is involved in gamma-polyglutamic acid degradation

The ywtD gene, which codes for an enzyme that degrades gamma-polyglutamic acid (PGA), was cloned from Bacillus subtilis IFO16449. The gene is located immediately downstream of ywsC and ywtABC, a PGA operon involved in PGA biosynthesis, and it showed partial similarity to genes coding for DL-endopeptidase, a peptidoglycan-degrading enzyme. The ywtD gene, from which signal sequence is excised, was inserted into pET15b, and the recombinant plasmid was then transformed into Escherichia coli. Histidine-tagged YwtD was purified from sonicated cells of the transformant. The purified YwtD degraded PGA to yield two hydrolyzed products, a high-molecular-mass product (490 kDa with nearly 100% L-glutamic acid) and an 11-kDa product (with D-glutamic acid and L-glutamic acid in an 80:20 ratio). This finding and results of enzymatic analysis of the two products with carboxypeptidase G suggest that YwtD is a novel enzyme cleaving the gamma-glutamyl bond only between D- and L-glutamic acids of PGA, and it may be designated gamma-DL-glutamyl hydrolase.     

Cloning, sequencing and genetic mapping of a Bacillus subtilis cell wall hydrolase gene

We have cloned DNA fragments from Bacillus subtilis 168S into Escherichia coli, which produced a lytic zone on an agar medium containing B. subtilis cell wall. Sequencing of the fragments showed the presence of an open reading frame (ORF) which encodes a polypeptide of 272 amino acids with a molecular mass of 29919 Da. The deduced amino acid sequence showed considerable homology with that of the cell wall hydrolase gene of Bacillus sp. (Potvin, C., Leclerc, D., Tremblay, G., Asselin, A. & Bellemare, G. (1988). Molecular and General Genetics 214, 241-248). Accordingly, the gene was designated cwlA, for cell wall lysis. The N-terminal amino acid sequence of cwlA gene product prepared from a E. coli clone was AIKVVKNLVSKSKYGLKCPN, which is consistent with that of the deduced sequence starting from Ala at second position from the initiation codon of the cwlA gene. A presumed sigma A promoter and a rho-independent terminator were found upstream and downstream of the ORF, respectively. A chloramphenicol-resistance determinant integrated into the ORF was mapped by PBS1 transduction, which indicated the gene sequence dnaE-aroD-cwlA.     

Characterization of a Paenibacillus woosongensis beta-Xylosidase/alpha-Arabinofuranosidase produced by recombinant Escherichia coli

A gene encoding the beta-xylosidase/alpha-arabinofuranosidase (XylC) of Paenibacillus woosongensis was cloned into Escherichia coli. This xylC gene consisted of 1,425 nucleotides, encoding a polypeptide of 474 amino acid residues. The deduced amino acid sequence exhibited an 80% similarity with those of both Clostridium stercorarium beta-xylosidase/alpha-N-arabinosidase and Bacillus cellulosilyticus alpha-arabinofuranosidase, belonging to the glycosyl hydrolase family 43. The structural gene was subcloned with a Cterminal His-tag into a pET23a(+) expression vector. The His-tagged XylC, purified from a cell-free extract of a recombinant E. coli BL21(DE3) Codon Plus carrying a xylC gene by affinity chromatography, was active on paranitrophenyl- alpha-arabinofuranoside (pNPA) as well as paranitrophenyl- beta-xylopyranoside (pNPX). However, the enzymatic activities for the substrates were somewhat incongruously influenced by reaction pHs and temperatures. The enzyme was also affected by various chemicals at different levels. SDS (5 mM) inhibited the enzymatic activity for pNPX, while enhancing the enzymatic activity for pNPA. Enzyme activity was also found to be inhibited by addition of pentose or hexose. The Michaelis constant and maximum velocity of the purified enzyme were determined for hydrolysis of pNPX and pNPA, respectively.