Hidemasa Motoshima

Characterization and expression analysis of the exopolysaccharide gene cluster in Lactobacillus fermentum TDS030603.

Part of the exopolysaccharide gene cluster of Lactobacillus fermentum TDS030603 was characterized. It consists of 11,890 base pairs and is located in the chromosomal DNA, 13 open reading frames of which were encoded. Out of the 13 open reading frames, six were found to be involved in exopolysaccharide synthesis; however, five were similar to transposase genes of other lactobacilli, and two were functionally unrelated. Expression analysis revealed that the exopolysaccharide synthesis-related genes were expressed during cultivation. Southern analysis using specific primers for the exopolysaccharide genes indicated that duplication of the gene cluster did not occur. The plasmid-cured strain maintained its capacity for exopolysaccharide production, confirming that the exopolysaccharide gene cluster of this strain is located in the chromosomal DNA, similarly to thermophilic lactic acid bacteria. Our results indicate that this exopolysaccharide gene cluster is likely to be functional, although extensive gene rearrangement occurs.

Oral Administration of Freeze-Dried Kefir Reduces Intestinal Permeation of and Oral Sensitization to Ovalbumin in Mice

An increase in plasma ovalbumin concentrations after intragastric administration of ovalbumin was suppressed by concomitant freeze-dried kefir in BALB/c mice. Serum levels of ovalbumin-specific immunoglobulin G and proliferation of splenic mononuclear cells in mice immunized orally with ovalbumin were suppressed by feeding freeze-dried kefir. We propose that kefir reduces intestinal permeation of food antigen, which contributes to suppression of oral sensitization.

Content and Chemical Compositions of Cerebrosides in Lactose-assimilating Yeasts

Cerebrosides were found in ten lactose-assimilating yeasts. Representative component ceramide moieties of cerebrosides from nine of these yeasts contained 9-methyl-4-trans, 8-trans-sphingadienine, and 2-hydroxy fatty acid with carbon chain lengths of 16 or 18. The major ceramide moieties in Brettanomyces anomalus, however, differed from those in other yeasts, and were predominately moieties containing 2-hydroxymyristic acid. Thus we found that various cerebroside molecular species are present in yeasts.

Purification, Characterization, and Gene Cloning of Lysyl Aminoeptidase from Streptococcus thermophilus YRC001

We purified and characterized an aminopeptidase from Streptococcus thermophilus YRC001 to obtain an enzyme for the application of reducing bitter-defect in cheese manufacturing. The purified enzyme was a monomer, and its molecular mass was estimated to be 90-100 kDa. It had a broad substrate specificity, and mostly hydrolyzed lysyl and leucyl peptides. The optimal temperature and pH for the enzyme were 35°C and pH 6.5, respectively. EDTA, o-phenanthroline, and p-chloromercuribenzoate inhibited its activity, therefore it was considered to be a metallopeptidase. The purified enzyme efficiently reduced the bitterness of a trypsin digest of reconstituted skim milk. Therefore, we cloned a gene for the enzyme from YRC001. The nucleotide sequence of a 2,940-bp XbaI fragment containing the gene was analyzed. The gene encoded 849 amino acids, and the calculated molecular mass for the mature enzyme (initial methionine is removed) was 96,434. The deduced amino acid sequence showed high homology with the known bacterial lysyl aminopeptidase (aminopeptidase N).

Cloning and Nucleotide Sequencing of Genes Encoding Mn-Superoxide Dismutase and Class II Fumarase from Thermus aquaticus YT-1

Abstract A 6955-bp sequence of a Pst I- Hin dIII DNA fragment containing the manganese-superoxide dismutase (MnSOD) gene of Thermus aquaticus YT-1 was determined. The gene ( sod ) encoded a polypeptide consisting of 204 amino acid residues (the mature enzyme without the initiation methionine) with a calculated Mr of 22,773. The deduced amino acid sequence of the sod gene showed 92% identity to that of Thermus thermophilus HB8 determined chemically. The sod gene was well expressed in Escherichia coli and a heat-stable active enzyme was produced. An open reading frame encoding fumarase was found 91 bp upstream of the MnSOD gene in tandem form. The fum gene encoded a polypeptide consisting of 466 amino acid residues with a calculated M r of 50,950. The deduced amino acid sequence of the fum gene product has similarity to that of the fumC of E. coli (57% identity), suggesting that this gene encodes a class II type fumarase.

Bacillus Aminopeptidase I

Publisher Summary This chapter elaborates the structural chemistry and the biological aspects of bacillus aminopeptidase I (API). It is an aminopeptidase of broad specificity that releases not only neutral, but also acidic and basic amino acids including proline from the N-terminus. It is more active against peptides than against Leu-NHPhN02. API consists of 12 subunits of two different types. The activity of API as mentioned above refers to that of API hybrids. The substrate specificity of α subunit differs from that of β subunit. In experiments concerning the recombination of API subunits, it was revealed that only the a subunit is needed for degradation of neutral peptides, whereas dipeptides having an N-terminal aspartic or glutamic acid residue are substantially hydrolyzed only by forms of the enzyme containing β subunit as well. The amounts of the three aminopeptidases in Bacillus stearothermophilus vary according to the strain. Obligately thermophilic strains produce substantial amounts of API and APII but little APIII, whereas obligately mesophilic strains produce substantial amounts of APIII but very little API or APII. The enzyme activity may be regulated both by a variable Co/Zn ratio and by certain metabolites or membrane components.

Identification of Polypeptides Constituting Lactophorin by Monoclonal Antibody to Bovine Milk Lactophorin

A monoclonal antibody to lactophorin (LP) was prepared by creating hybridoma from mouse myeloma cells and spleen cells from mice immunized with PAS-4 concentrated fraction from bovine milk fat globule membrane. The prepared antibody recognized a polypeptide moiety of LP27, the major component constituting LP, but not a carbohydrate moiety. Immunoblot analysis showed that all polypeptides (LP17, LP20, LP27, LP40, and LP50) constituting LP were recognized by the antibody. The identities of LP20, LP40, and LP50 were verified by N-terminal and internal amino acid sequencing. LP20 contains hydrolysate of LP27 besides LP27 without the O-glycosyl sugar chain. These results suggest that LP40 and LP50 are homo- or heterodimers of LP20 and LP27. This is the first report to the effect that LP was constructed from several forms of polypeptides, derived from LP27.