Kouichi Akiyama

Purification and gene cloning of a chitosanase from Bacillus ehimensis EAG1

Bacillus ehimensis EAG1 (IFO15659) produced and secreted chitosanase in the presence of exogenous chitosan. The chitosanase was purified from the culture filtrate of the bacterium to apparent homogeneity in SDS-polyacrylamide gel electrophoresis. The purified enzyme had a molecular weight of approximately 31,000. A 1.9-kbp DNA fragment containing the chitosanase gene was cloned and the complete nucleotide sequence was determined. The sequence was found to contain a single open reading frame encoding a protein of 302 amino acids. The deduced amino acid sequence showed significant homology with the chitosanase from Bacillus circulans MH-K1.

Isolation and characterization of a collagenolytic enzyme from Bacillus licheniformis N22

A bacterium having collagenolytic activity was isolated from soil and identified as Bacillus licheniformis. The collagenolytic enzyme was obtained in a medium containing gelatin and purified to apparent homogeneity on the basis of SDS-polyacrylamide gel electrophoresis. The purified enzyme had a molecular weight of approximately 29,000, and hydrolyzed native, insoluble collagen. In addition, the enzyme hydrolyzed soluble collagen, gelatin, hemoglobin and casein, but not synthetic oligopeptides, usually used as substrates for collagenase assay.

Isolation and characterization of Pz-peptidase from Bacillus licheniformis N22

Abstract Pz-peptidase is an endopeptidase that cleaves the synthetic substrate, 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg (Pz-peptide), which was originally developed for the assay of Clostridium histolyticum collagenase (Wunsch and Heidrich, Hoppe-Seylers Z. Physiol. Chem., 333, 149–151, 1963; Morales and Woessner, J. Biol. Chem., 252, 4855–4860, 1977). Pz-peptidase was purified from the culture filtrate of Bacillus licheniformis N22. The purified Pz-peptidase showed a molecular weight of 70,000 in SDS-polyacryl-amide gel electrophoresis and 150,000 in gel filtration. Optimal pH for cleavage of Pz-peptide was 7.8. The Pz-peptidase activity was strongly inhibited by metal chelators such as EDTA and O -phenanthroline. Substrate specificity studies indicated that Pz-peptidase cleaved oligopeptides at the Xaa-Gly site in Xaa-Gly-Pro. However, Pz-peptidase failed to hydrolyze native collage, denatured collagen, hemoglobin and casein.

Cloning and sequencing of the Pz-peptidase gene from Bacillus licheniformis N22

Pz-peptidase is an endopeptidase that cleaves the synthetic substrate Pz-peptide (4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg), which was originally developed for the assay of collagenase. The Pz-peptidase gene of Bacillus licheniformis N22 was cloned and sequenced. The gene consists of 628 amino acids with a motif for zinc-dependent metalloprotease, and shares 42% amino acid identity with the oligoendopeptidase of Lactococcus lactis. This is the first report on the gene structure of a Pz-peptidase.

Targeted disruption of sti35, a stress-responsive gene in phytopathogenic fungus Fusarium oxysporum.

Abstractsti35 is one of the heat-shock genes in Fusarium oxysporum, which is a fungal pathogen for wilt disease in plants. We have isolated a genomic clone of sti35 and used it to create disruption mutations. Disruption of the sti35 coding region resulted in the loss of a 32-kDa protein present in heat-shocked cells. The disruption had no detectable effect on growth and development at various temperatures, nor on the ability to acquire thermotolerance in nutrient medium. But the sti35 disruptants showed increased thermotolerance, relative to the wild-type strain, when incubated in minimal medium after heat treatment.

Detection and cloning of the gene encoding a protein produced by nonpathogenic mutants of Fusarium oxysporum.

Treatment of Fusarium oxysporum with 5-azacytidine, a potent inhibitor of DNA methylation, induced nonpathogenic mutants. Analysis of the protein expression pattern by two-dimensional gel electrophoresis revealed a protein that is present in yeast-form cells of the mutants but absent in those of the wild-type strain. N-terminal amino acid analysis indicated that this protein is identical to a region of a polypeptide encoded by a cDNA clone, sti35, previously identified as a heat shock gene in F. oxysporum. A genomic clone for sti35 was isolated and sequence analysis revealed an intron and two heat shock elements upstream of sti35. The analysis also revealed the presence of a leader sequence composed of 27 amino acid residues, which shares a common amino acid composition with leader sequences of the proteins located in the mitochondrial matrix. Different expression patterns of sti35 in the mutants and wild-type strain were demonstrated.

Isolation of Serratia marcescens mutants which could overproduce and excrete Escherichia coli alkaline phosphatase and beta-lactamase.

Serratia marcescens mutants, which excrete Escherichia coli alkaline phosphatase (APase) encoded by the plasmid-bearing phoA gene, were isolated after mutagenesis by N-methyl-Ń-nitro-N-nitrosoguanidine. These mutants produced two to four times as much APase as did the parent strain under a phosphate-limiting condition, and more than 70% of the enzyme was released into the culture medium. In addition, overproduction and excretion of beta-lactamase was observed in these mutants.