Cloning and Characterization of a Carboxylesterase from Bacillus coagulans 81-11

A genomic library of Bacillus coagulans strain 81-11 was screened in Escherichia coli JM83 for lipolytic activity by using tributyrin agar plates. A 2.4 kb DNA fragment was subcloned from a lipolytic-positive clone and completely sequenced. Nucleotide sequence analysis predicted a 723 bp open reading frame (ORF), designated estC1, encoding a protein of 240 amino acids with an estimated molecular mass of 27 528 Da and a pI of 9.15. The deduced amino acid sequence of the estC1 gene exhibited significant amino acid sequence identity with carboxylesterases from thermophilic Geobacillus spp. and sequence analysis showed that the protein contains the signature G-X-S-X-G included in most esterases and lipases. Enzyme assays using p-nitrophenyl (p-NP) esters with different acyl chain lengths as the substrate confirmed the esterase activity. EstC1 exhibited a marked preference for esters of short-chain fatty acids, yielding the highest activity with p-NP butyrate. Maximum activity was found at pH 8 and 50°C, although the enzyme displayed stability at temperatures up to 60°C.     

Identification and Functional Analysis of dTDP-Glucose-4,6-Dehydratase Gene and Its Linked Gene Cluster in an Aminoglycoside Antibiotics Producer of <Emphasis Type="Italic">Streptomyces tenebrarius</Emphasis> H6

Streptomyces tenebrarius H6 produces a variety of aminoglycoside antibiotics, such as apramycin, tobramycin, and kanamycin B. Primers were designed according to the highly conserved sequences of the dTDP-glucose-4,6-dehydratase genes, and a 0.6-kb PCR product was obtained from S. tenebrarius H6 genomic DNA. With the 0.6-kb PCR product as a probe, a BamHI 7.0-kb fragment was isolated. DNA sequence analysis of the 7.0-kb fragment revealed four ORFs and an incomplete ORF. In search of databases, the deduced product of one ORF (orfE) showed 62% identity to the dTDP-glucose-4,6-dehydratase, StrE of S. griseus. Three other ORFs (orfG1, orfG2, and orfGM) showed 55%, 62%, and 42% similarities, respectively, to glycosyltransferase from Clostridium acetobutylicum and mannosyltransferase from Xanthomonas axonopodis pv. citri str. 306 and glycosyltransferase from Pseudomonas putida KT2440. Upstream of the orfE was an incomplete ORF, and the deduced product showed 56% similarity to dTDP-4-dehydrorhamnose, StrL from S. griseus. The function of the orfE gene was studied by targeted gene disruption. The resulting mutant failed to produce tobramycin and kanamycin B, but still produced apramycin, suggesting that the orfE gene and linked gene cluster are essential for the biosynthesis of tobramycin and kanamycin B in S. tenebrarius H6.     

Gene Cloning,Expression, and Substrate Specificity of an Imidase from the Strain <Emphasis Type="Italic">Pseudomonas putida</Emphasis> YZ-26

A gene-encoding imidase was isolated from Pseudomonas putdia YZ-26 genomic DNA using a combination of polymerase chain reaction and activity screening the recombinant. Analysis of the nucleotide sequence revealed that an open reading frame (ORF) of 879 bp encoded a protein of 293 amino acids with a calculated molecular weight of 33712.6 kDa. The deduced amino-acid sequence showed 78% identity with the imidase from Alcaligenes eutrophus 112R4 and 80% identity with N-terminal 20 amino-acid imidase from Blastobacter sp. A17p-4. Next, the ORF was subcloned into vector pET32a to form recombinant plasmid pEI. The enzyme was overexpressed in Escherichia coli and purified to homogeneity by Ni²⁺–NTA column, with 75% activity recovery. The subunit molecular mass of the recombinant imidase as estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis was approximately 36 kDa, whereas its functional unit was approximately 141 kDa with four identical subunits determined by size-exclusion chromatography. The purified enzyme showed the highest activity and affinity toward succinimide, and some other substrates, such as dihydrouracil, hydantoin, succinimide, and maleimde, were investigated.     

Nucleotide and Deduced Amino Acid Sequences of a New Subtilisin from an Alkaliphilic <Emphasis Type="Italic">Bacillus</Emphasis> Isolate

The gene for a new subtilisin from the alkaliphilic Bacillus sp. KSM-LD1 was cloned and sequenced. The open reading frame of the gene encoded a 97 amino-acid prepro-peptide plus a 307 amino-acid mature enzyme that contained a possible catalytic triad of residues, Asp³², His⁶⁶, and Ser²²⁴. The deduced amino acid sequence of the mature enzyme (LD1) showed approximately 65% identity to those of subtilisins SprC and SprD from alkaliphilic Bacillus sp. LG12. The amino acid sequence identities of LD1 to those of previously reported true subtilisins and high-alkaline proteases were below 60%. LD1 was characteristically stable during incubation with surfactants and chemical oxidants. Interestingly, an oxidizable Met residue is located next to the catalytic Ser²²⁴ of the enzyme as in the cases of the oxidation-susceptible subtilisins reported to date.Received: 19 November 2002 / Accepted: 19 December 2002     

Cloning of the thermostable phytase gene (phy) from Bacillus sp. DS11 and its overexpression in Escherichia coli

Phytase hydrolyzes phytate to release inorganic phosphate, which would decrease the addition of phosphorus to feedstuffs for monogastric animals and thus reduce environmental pollution. The gene encoding phytase from Bacillus sp. DS11 was cloned in Escherichia coli and its sequence determined. A 560-bp DNA fragment was used as a probe to screen the genomic library. It was obtained through PCR of Bacillus sp. DS11 chromosomal DNA and two oligonucleotide primers based on N-terminal amino acid sequences of the purified protein and the cyanogen bromide-cleaved 21-kDa fragment. The phy cloned was encoded by a 2.2-kb fragment. This gene comprises 1152 nucleotides and encodes a polypeptide of 383 amino acids with a deduced molecular mass of 41 808 Da. Phytase was produced to 20% content of total soluble proteins in E. coli BL21 (DE3) using the pET22b(+) vector with the inducible T7 promoter. This is the first nucleic sequence report on phytase from a bacterial strain.     

Gene Cloning,Sequencing, and Expression of an Esterase from <Emphasis Type="Italic">Acinetobacter lwoffii</Emphasis> I6C-1

The esterase-encoding gene, estA, was cloned from Acinetobacter lwoffii I6C-1 genomic DNA into Escherichia coli BL21(DE3) with plasmid vector pET-22b (pEM1). pEM1 has a 4.4-kb EcoRI insert that contained the complete estA gene. A 2.4-kb AvaI-SphI DNA fragment was subcloned (pEM3) and sequenced. estA gene encodes a protein of 366 amino acids (40,687 Da) with a pI of 9.17. The EstA signal peptide was 31 amino acids long, and the mature esterase sequence is 335 amino acids long (37.5 kDa). The conserved catalytic serine residue of EstA is in position 210. The EstA sequence was similar to that of the carboxylesterase from Acinetobacter calcoaceticus (75% identity, 85% similarity), Archaeoglobus fulgidus (37% identity, 59% similarity), and Mycobacterium tuberculosis (35% identity, 51% similarity). These enzymes contained the conserved motif G-X₁-S-X₂-G carrying the active-site serine of hydrolytic enzyme. The EstA activity in A. lwoffii I6C-1 remains constant throughout the stationary phase, and the activity in E. coil BL21 (DE3) with pEM1 was similar to A. lwoffii I6C-1. Received: 4 June 2002 / Accepted: 5 July 2002     

Cloning,purification and characterization of an alkali-stable endoxylanase from thermophilic <Emphasis Type="Italic">Geobacillus</Emphasis> sp. 71

The gene encoding a xylanase from Geobacillus sp. 71 was isolated, cloned, and sequenced. Purification of the Geobacillus sp 7.1 xylanase, XyzGeo71, following overexpression in E. coli produced an enzyme of 47 kDa with an optimum temperature of 75°C. The optimum pH of the enzyme is 8.0, but it is active over a broad pH range. This protein showed the highest sequence identity (93%) with the xylanase from Geobacillus thermodenitrificans NG80-2. XyzGeo71 contains a catalytic domain that belongs to the glycoside hydrolase family 10 (GH10). XyzGeo71 exhibited good pH stability, remaining stable after treatment with buffers ranging from pH 7.0 to 11.0 for 6 h. Its activity was partially inhibited by Al³⁺ and Cu²⁺ but strongly inhibited by Hg²⁺. The enzyme follows Michaelis–Menten kinetics, with K_m and V_max values of 0.425 mg xylan/ml and 500 μmol/min.mg, respectively. The enzyme was free from cellulase activity and degraded xylan in an endo fashion. The action of the enzyme on oat spelt xylan produced xylobiose and xylotetrose.     

Cloning,characterization and insertional inactivation of theLactobacillus helveticus D(−) lactate dehydrogenase gene

A plasmid, designated pSUW100, encoding the D(-)lactate dehydrogenase [D(-)-LDH; NAD⁺ oxidoreductase, EC 1.1.1.28] fromLactobacillus helveticus CNRZ32 was identified from a genomic library by complementation ofEscherichia coli FMJ39. The D(-)LDH gene was localized by Tn5 mutagenesis and subcloning to a 1.4-kb region of pSUW100. A 2-kbDraI fragment of pSUW100 encoding D(-)LDH activity was subcloned and its nucleotide sequence determined. Analysis of this sequence identified a putative 1,014-bp D(-) LDH open reading frame that encodes a polypeptide of 337 amino acid residues with a deduced molecular mass of 38 kDa. The distribution of homology to the CNRZ32 D(-)LDH gene in several lactic acid bacteria was determined by Southern hybridization using an internal fragment of the D(-)LDH gene as a probe. Hybridization was detected in leuconostocs and pediococci but not in lactococci orLactobacillus casei. An integration plasmid was constructed from pSA3 and a 0.60-kb internal fragment of the D(-)LDH gene. This plasmid was used to construct a D(-)LDH-negative derivative ofL. helveticus CNRZ 32 by gene disruption; this derivative was determined as producing only L(+)lactic acid. No significant difference in growth or total lactic acid production was observed between CNRZ32 and its D(-)LDH mutant.     

Cloning of the Escherichia coli endo-1,4-D-glucanase gene and identification of its product

A plasmid (pYP17) containing a genomic DNA insert from Escherichia coli K-12 that confers the ability to hydrolyze carboxymethylcellulose (CMC) was isolated from a genomic library constructed in the cosmid vector pLAFR3 in E. coli DH5α. A small 1.65-kb fragment, designated bcsC (pYP300), was sequenced and found to contain an ORF of 1,104 bp encoding a protein of 368 amino acid residues, with a calculated molecular weight of 41,700 Da. BcsC carries a typical prokaryotic signal peptide of 21 amino acid residues. The predicted amino acid sequence of the BcsC protein is similar to that of CelY of Erwinia chrysanthemi, CMCase of Cellulomonas uda, EngX of Acetobacter xylinum, and CelC of Agrobacterium tumefaciens. Based on these sequence similarities, we propose that the bcsC gene is a member of glycosyl hydrolase family 8. The apparent molecular mass of the protein, when expressed in E. coli, is approximately 40 kDa, and the CMCase activity is found mainly in the extracellular space. The enzyme is optimally active at pH 7 and a temperature of 40° C. Received: 6 February 1998 / Accepted: 6 November 1998     

Cloning and sequencing of the gene for the DNA-binding 17 K protein of Escherichia coli

The skp gene encoding the 17 K protein, a basic DNA-binding nucleoid-associated protein of Escherichia coli, was cloned as part of a 2.3-kb genomic fragment. The gene was sequenced and a polypeptide of 161 amino acids (aa) was deduced from the nucleotide sequence. The primary translation product was processed by cutting off the N-terminal 20 aa residues, yielding a mature polypeptide of 141 aa. The M_r of the mature polypeptide was 15674. An E. coli transformant containing the skp gene on the plasmid pGAH317 was shown to overproduce the gene product some 20-fold.     

Cloning and Characterization of a Novel Oligoalginate Lyase from a Newly Isolated Bacterium Sphingomonas sp. MJ-3

A bacterium possessing alginate-degrading activity was isolated from marine brown seaweed soup liquefied by salted and fermented anchovy. The isolated strain was designated as Sphingomonas sp. MJ-3 based on the analyses of 16S ribosomal DNA sequences, 16S-23S internal transcribed spacer region sequences, biochemical characteristics, and cellular fatty acid composition. A novel alginate lyase gene was cloned from genomic DNA library and then expressed in Escherichia coli. When the deduced amino acid sequence was compared with the sequences on the databases, interestingly, the cloned gene product was predicted to consist of AlgL (alginate lyase L)-like and heparinase-like protein domain. The MJ-3 alginate lyase gene shared below 27.0% sequence identity with exolytic alginate lyase of Sphingomonas sp. A1. The optimal pH and temperature for the recombinant MJ-3 alginate lyase were 6.5 and 50°C, respectively. The final degradation products of alginate oligosaccharides were analyzed by electrospray ionization mass spectrometry and proved to be alginate monosaccharides. Based on the results, the recombinant alginate lyase from Sphingomonas sp. MJ-3 is regarded as an oligoalginate lyase that can degrade oligoalginate and alginate into alginate monosaccharides.     

Molecular cloning of the phospholipase D gene from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. YU100 and its expression in <Emphasis Type="Italic">Escherichia coli</Emphasis>

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.     

MEI1, an Arabidopsis gene required for male meiosis: isolation and characterization

 The T-DNA tagged mutant gene of Arabidopsis thaliana, mei1, produces after meiosis an abnormal tetrad, consisting of five to eight microspores of varying sizes and DNA contents. Plant DNA flanking the inserted T-DNA was isolated by inverse PCR. An approximately 16-kb DNA fragment spanning the T-DNA insertion site was isolated by screening a wild-type genomic library, using the plant flanking DNA as a probe. Using RT-PCR and RNA isolated from very young flower buds, a cDNA fragment was obtained. Nucleotide sequence comparison of the cDNA and the genomic sequence in this region indicated a gene which contained two introns. The 5′ and 3′ splice sites of neither intron comply with the :GU...AG: rule. In the mutant, the T-DNA had inserted into one of the introns. The deduced sequence of the MEI1 wild-type gene, which contains 89 amino acids, shows possible similarity with the human acrosin-trypsin inhibitor, HUSI-II, and is about the same size. Two wild-type DNA fragments, both extending over the T-DNA insertion site, were introduced into mutant plants by Agrobacterium-mediated transformation and plants were selected for both hygromycin and kanamycin resistance. Several independent male-fertile transformants were obtained with one of the DNA fragments. The fragment showing complementation of the mutant phenotype indicated that the sequence with similarity to the acrosin-trypsin inhibitor is MEI1. Within the 16-kb genomic fragment two other genes were identified; one showed no overall similarity to any protein sequence in the database and the other had almost complete identity with an Arabidopsis-transcribed sequence tag with similarity to ACC oxidase. Double mutants between mei1 and qrt1 were made, permitting better characterization of the mei1 phenotype because the individual microspores continued to be held together after callose dissolution. Received: 21 April 1998 / Revision accepted: 11 June 1998     

The gene for indole-3-acetyl-L-aspartic acid hydrolase from Enterobacter agglomerans : molecular cloning, nucleotide sequence, and expression in Escherichia coli

A 5.5-kb DNA fragment containing the indole-3-acetyl-aspartic acid (IAA-asp) hydrolase gene (iaaspH) was isolated from Enterobacter agglomerans strain GK12 using a hybridization probe based on the N-terminal amino acid sequence of the protein. The DNA sequence of a 2.4-kb region of this fragment was determined and revealed a 1311-nucleotide ORF large enough to encode the 45-kDa IAA-asp hydrolase. A 1.5-kb DNA fragment containing iaaspH was subcloned into the Escherichia coli expression plasmid pTTQ8 to yield plasmid pJCC2. Extracts of IPTG-induced E. coli cultures containing the pJCC2 recombinant plasmid showed IAA-asp hydrolase levels 5 to 10-fold higher than those in E. agglomerans extracts. Homology searches revealed that the IAA-asp hydrolase was similar to a variety of amidohydrolases. In addition, IAA-asp hydrolase showed 70% sequence identity to a putative thermostable carboxypeptidase of E. coli. Received: 12 March 1998 / Accepted: 30 March 1998     

Purification,Characterization and Gene Analysis of N-Acetylglucosaminidase from Enterobacter sp. G-1

Enterobacter sp. G-1 is a bacterium isolated previously as a chitinase-producing bacterium. We found this bacterium also produced N-acetylglucosaminidase and characterized that in this study. Extracellular N-acetylglucosaminidase of 92.0 kDa was purified near homogeneity by 8.57-fold from Enterobacter sp. G-1. The optimum temperature and the optimum pH of the purified N-acetylglucosaminidase was 45°C and 6.0, respectively. The N-terminal amino acid sequence of 23 residues of N-acetylglucosaminidase was identified. Based on the N-terminal sequence, we amplified pieces of the DNA fragments by PCR. Using these PCR products as probes, we screened the genomic library and successfully isolated the entire N-acetyl-glucosaminidase gene (designated nag1) from Enterobacter sp. G-1. The nucleotide sequence of the nag1 gene was found to consist of 2,655 bp encoding a protein of 885 amino acid residues. Comparison of the deduced amino acid sequence from the nag1 gene found 97.3% identity with chitobiase from Serratia marcescens, 54.4% identity with N,N′-diacetylchitobiase from Vibrio harveyi, and 42.7% identity with N-acetylglucosaminidase (ExoI) from Vibrio furnissii. Enzymatic activity assay of N-acetylglucosaminidase indicated stronger activity toward PNP-GlcNAc than PNP-(GlcNAc)₂ or PNP-(GlcNAc)₃.     

Molecular Cloning of the leuB Gene from Bacteroides fragilis by Functional Complementation in Escherichia coli

Clones containing the Bacteroides fragilis leuB-complementing gene were isolated by screening of a B. fragilis genomic library constructed in Escherichia coli. One recombinant clone, designated pOT865, with the smallest DNA insert (4.5 kb) could complement three independent leuB mutations in E. coli and the leuB-complementing determinant in pOT865 was localized to a region of 1.5-kb DNA. The results of Southern blot analysis suggested that a single copy of the cloned gene was present in the B. fragilis genome. The cloned fragment appeared to contain a sequence that could function as a promoter in E. coli and direct the synthesis of a 42-kDa protein. These results suggest that the cloned segment contains the structural gene for β-isopropylmalate dehydrogenase (leuB).     

Buchnera aphidicola (Aphid-Endosymbiont) glyceraldehyde-3-phosphate dehydrogenase: Molecular cloning and sequence analysis

Buchnera aphidicola is an endosymbiont of the aphid Schizaphis graminum. A 3.9-kb B. aphidicola DNA fragment was sequenced and found to contain two open reading frames (ORFs). The deduced amino acid sequence of one of the ORFs had an 85% identity to Escherichia coli glyceraldehyde-3-phosphate dehydrogenase (Gap). Both of these proteins have a higher similarity to eukaryotic than to prokaryotic Gaps. The second ORF could not be readily identified. The sequence of the putative product indicated that it was a member of the family of ATP-binding, membrane-associated proteins. The highest amino acid identity (36%) was with E. coli FtsE, a protein involved in cell division.