Enantioselective hydrolysis of benzoyl 1,2-O-isopropylideneglycerol by Bacillus coagulans

Summary Hydrolytic resolution of 1,2-O-isopropylideneglycerol was investigated with Bacillus coagulans. Resting cells of this microorganism in phosphate buffer, pH 6.8, transformed benzoyl 1,2-O-isopropylideneglycerol producing the (S)-alcohol with 45% yield and 88% enantiomeric excess.     

Isolation of acetyl esterase mutants of Bacillus subtilis 168.

Five mutants of Bacillus subtilis 168 defective in an intracellular esterase activity were identified. By polyacrylamide gel electrophoresis, four of the mutants were shown to lack esterase B activity, and the fifth lacked esterase A activity. All of the back-crossed esterase mutants were able to sporulate at wild-type frequency and produce exoprotease(s) and antibiotic(s). No difference in motility could be attributed to the esterase mutation. PBS1 transduction analysis showed all the esterase B mutations to be linked to the hisA marker.     

Improved thermostability of a Bacillus subtilis esterase by domain exchange

A moderately thermostable esterase from Geobacillus stearothermophilus (BsteE) and its homolog from Bacillus subtilis (BsubE) show a high structural similarity with more than 95 % homology and 74 % amino acid identity. Interestingly, their thermal stability differs significantly by 30 °C in their melting temperature. In order to identify the positions that are responsible for this difference, most of the flexible amino acids assumed to confer instability were found to be in the cap region. For this reason, a 30 amino acid long cap domain fragment containing ten differing positions derived from BsteE was incorporated into the homologous gene encoding for the more labile BsubE by spliced overlap-extension PCR. The melting temperature of the two wild-type esterases and the mutant was evaluated by circular dichroism spectroscopy, while the kinetic parameters and the stability were determined with a photometric assay. The cap domain mutant maintained its activity, with a catalytic efficiency more similar to BsteE, while it exhibited an increase of the melting temperature by 4 °C compared to BsubE. Additional point mutations based on the differences of the parent enzymes gave a further increase of the thermostability up to 11 °C compared to BsubE; however, a significant reduction in activity was observed.     

YesT: a new rhamnogalacturonan acetyl esterase from Bacillus subtilis

YesT, a putative protein from Bacillus subtilis ATCC 6633 that has been provisionally classified as a rhamnogalacturonan acetyl esterase (RGAE) in CE-12 family, was cloned, expressed in Escherichiacoli Rosetta (DE3), and purified. The enzyme is monomeric with a molecular mass of 37 kDa and presents thermophilic properties similar to RGAE from Aspergillus aculeatus, although YesT is more alkaliphilic. The study of inhibitors confirmed the importance of the His and the nucleophilic Ser for the esterase activity, apart from the Asp from the catalytic triad. This enzyme also presents broad substrate specificity, and is active toward 7-aminocephalosporanic acid, cephalosporin C, p-nitrophenyl acetate, beta-naphthyl acetate, glucose pentaacetate, and acetylated xylan. Moreover, YesT achieves a synergistic effect together with xylanase A toward acetylated xylan. As a member of the SGNH family, it does not adopt the common alpha/beta hydrolase fold. The primary sequence analysis and multiple sequence alignment revealed the lack of a two beta-stranded antiparallel sheet, which results in a clear change in the structure together with the disappearance of one of the three 3(10)-helices presented in RGAE structure. The similarities found in this article among the topological diagrams of RGAE, YesT, and Esterase A from Streptomyces scabies, Platelet-Activating Factor AcetylHydrolase, isoform Ib, alpha subunit [PAF-AH(Ib)alpha(1)], PAF-AH(Ib)alpha(2), the esterase domain from hemagglutinin esterase fusion glycoprotein (HEF1) from Influenza C virus, the thioesterase I (TAP) from E. coli, the hypothetical protein a1r1529 from Nostoc sp., and the hypothetical YxiM precursor that all belong to the SGNH family could indicate a possible divergence of such proteins from a common ancestor.     

The Bacillus subtilis outB gene is highly homologous to an Escherichia coli ntr-like gene.

The Bacillus subtilis outB gene was found to have strong similarities to an Escherichia coli gene complementing ntr-like mutations in Rhodobacter capsulatus. The deduced gene products had 52% identical amino acids (65% similar residues). The phenotype of strains affected in the OutB function indicates that this B. subtilis gene may be involved in nitrogen utilization.     

Identification of a putative Bacillus subtilis rho gene.

Transposon Tn917 mutagenesis of Bacillus subtilis BD99 followed by selection for protonophore resistance led to the isolation of strain MS119, which contained a single Tn917 insertion in an open reading frame whose deduced amino acid sequence was 56.6% identical to that of the Escherichia coli rho gene product. The insertional site was near the beginning of the open reading frame, which was located in a region of the B. subtilis chromosome near the spoOF gene; new sequence data for several open reading frames surrounding the putative rho gene are presented. The predicted B. subtilis Rho protein would have 427 amino acids and a molecular weight of 48,628. The growth of the mutant strain was less than that of the wild type on defined medium at 30 degrees C. On yeast extract-supplemented medium, the growth of MS119 was comparable to that of the wild type on defined medium at 30 degrees C. On yeast extract-supplemented medium, the growth of MS119 was comparable to that of the wild type at 30 degrees C but was much slower at lower temperatures; sporulation occurred and competence was developed in cells of the mutant grown at 30 degrees C. To determine whether the protonophore resistance and sensitivity to low growth temperature resulted from the insertion, a chloramphenicol resistance cassette was inserted into the wild-type B. subtilis rho gene of strain BD170; the resulting derivative displayed the same phenotype as MS119.     

Structure of a Bacillus subtilis endo-beta-1,4-glucanase gene.

The nucleotide sequence of the portion of a Bacillus subtilis (strain PAP115) 3 kb Pst I fragment which contains an endo-beta-1, 4-glucanase gene has been determined. This gene encodes a protein of 499 amino acid residues (Mr = 55,234) with a typical B. subtilis signal peptide. Escherichia coli which has been transformed with this gene produces an extracellular endoglucanase with an amino-terminus corresponding to the thirtieth encoded amino acid residue. The gene is preceded by a cryptic reading frame with a rho-independent terminator structure, and itself has such a structure in the immediate 3'-flanking region. We have also identified, in the 5'-flanking region, nucleotide sequences which resemble promoter elements recognized by Bacillus RNA polymerase E sigma 43. Comparison of the encoded amino acid sequence to other known beta-glucanases reveals a small region of similarity to the encoded protein of the Clostridium thermocellum celB gene. These similar regions may contain substrate-binding and/or catalytic sites.     

A Bacillus subtilis malate dehydrogenase gene.

A Bacillus subtilis gene for malate dehydrogenase (citH) was found downstream of genes for citrate synthase and isocitrate dehydrogenase. Disruption of citH caused partial auxotrophy for aspartate and a requirement for aspartate during sporulation. In the absence of aspartate, citH mutant cells were blocked at a late stage of spore formation.     

Mutational analysis of the ribC gene of Bacillus subtilis

The nucleotide sequence of the ribC gene encoding the synthesis ofbifunctional flavokinase/flavine adenine nucleotide (FAD) synthetase in Bacillus subtilis have been determined in a family of riboflavin-constitutive mutants. Two mutations have been found in the proximal region of the gene, which controls the transferase (FAD synthase) activity. Three point mutations and one double mutation have been found (in addition to the two mutations that were detected earlier) in the distal region of the gene, which controls the flavokinase (flavin mononucleotide (FMN) synthase) activity. On the basis of all data known to date, it has been concluded that the identified mutations affect riboflavin and ATP binding sites. No mutations have been found in the PTAN conserved sequence, which forms the magnesium and ATP common binding site and is identical for organisms of all organizational levels, from bacteria too humans.     

A novel extracellular esterase from Bacillus subtilis and its conversion to a monoacylglycerol hydrolase.

A novel gene lipB, which encodes an extracellular lipolytic enzyme, was identified in the Bacillus subtilis genomic DNA sequence. We have cloned and overexpressed lipB in B. subtilis and Escherichia coli and have also purified the enzyme from a B. subtilis culture supernatant to electrophoretic homogeneity. Four different lipase assays were used to determine its catalytic activity: pH-stat, spectrophotometry, fluorimetry and the monomolecular film technique. LipB preferentially hydrolysed triacylglycerol-esters and p-nitrophenyl-esters of fatty acids with short chain lengths of 相似文献   

Cloning the gyrA gene of Bacillus subtilis.

We have isolated an eight kilobase fragment of Bacillus subtilis DNA by specific integration and excision of a plasmid containing a sequence adjacent to ribosomal operon rrn O. The genetic locus of the cloned fragment was verified by linkage of the integrated vector to nearby genetic markers using both transduction and transformation. Functional gyrA activity encoded by this fragment complements E. coli gyrA mutants. Recombination between the Bacillus sequences and the E. coli chromosome did not occur. The Bacillus wild type gyrA gene, which confers sensitivity to nalidixic acid, is dominant in E. coli as is the E. coli gene. The cloned DNA precisely defines the physical location of the gyrA mutation on the B. subtilis chromosome. Since an analogous fragment from a nalidixic acid resistant strain has also been isolated, and shown to transform B. subtilis to nalidixic acid resistance, both alleles have been cloned.     

Cloning of the Bacillus subtilis sulfanilamide resistance gene in Bacillus subtilis

A recombinant plasmid was constructed by ligation of chromosomal DNA from a sulfanilamide-resistant strain of Bacillus subtilis to the plasmid vector pUB110 which specifies neomycin resistance. Recombinant molecules generated in vitro were introduced into a B. subtilis recipient strain which carried the recE4 mutation, and selection was for neomycin-sulfanilamide-resistant transformants. A single colony was isolated containing the recombinant plasmid pKO101. This 6.3-megadalton plasmid simultaneously conferred resistance to neomycin and sulfanilamide when transferred into sensitive Rec+ or Rec- cells by either transduction or transformation.     

Translational coupling in Bacillus subtilis of a heterologous Bacillus subtilis-Escherichia coli gene fusion.

Translational coupling was demonstrated in a gene fusion in which the promoter and the N-terminal region of the Bacillus subtilis subtilisin (aprA) gene were fused to a promoterless Tn9-derived chloramphenicol acetyltransferase (CAT; EC 2.3.1.28) gene. Expression of this gene fusion results in the production of a native-sized CAT product, whereas the Tn9-derived CAT gene is usually not translated from its own ribosome binding site in B. subtilis (D. S. Goldfarb, R. L. Rodriguez, and R. H. Doi, Proc. Natl. Acad. Sci. USA 79:5886-5890, 1982). A 178-base-pair deletion, which removed part of the signal peptide and the propeptide of the aprA gene and created a translational stop codon 230 base pairs upstream of the CAT gene ribosome binding site, reduced expression of the CAT gene. A BamHI 10-mer linker insertion into this deletion site, which restored the reading frame and simultaneously removed the translation stop codon, restored CAT gene expression. The data indicate that expression of the CAT gene was dependent on translation of the truncated aprA gene into the ribosome binding site of the CAT gene.