An α-amylase of Scytalidium sp. was produced in shake flask cultures. The enzyme was purified to a homogeneous state on polyacrylamide gel electrophoresis by acetone precipitation, DEAE-cellulose column chromatography and Sephadex G-75 gel filtration. The molecular weight of the enzyme was about 87, 000 dalton. Metal ions such as Fe2+, Na+ and Cu2+ activated the enzyme activity while Hg2+ and Zn2+ were strongly inhibitory. The α-amylase was immobilized on DEAE-cellulose by adsorption. The optimum pH of the immobilized enzyme was 6.0, as compared with 8.0 for the free enzyme. The immobilized enzyme was optimally active at 50°C and retained 50% activity at 100°C (30min) as against 40°C and 40% (100°C, 30min) for the free enzyme.
In order to obtain appropriate vectors to transform several strains of Bacillus subtilis, antifungal iturin producers, plasmids have been isolated as covalently closed circular deoxyribonucleic acid from antibiotic-resistant bacteria inhabited in composts. Eight plasmids coding antibiotic resistance transformed both competent cells of a derivative of B. subtilis Marburg 168 and protoplasts of B. subtilis NB22, an antifungal-antibiotic iturin producer, in the presence of polyethylene glycol. However, transformation efficiency was not high enough as a cloning host-vector system. To improve transformation efficiency, KCl-treatment method and electroporation method were applied to four iturin producers, and electroporation method was most effective for transformation with newly-isolated plasmids with great efficiency.
In C. murisepticum, alpha-galactosidase (EC 188.8.131.52) was induced during growth in the presence of melibiose or raffinose, and to a much lesser extent in the presence of galactose, as a single carbon source. Evidence was obtained to show that the enzymes induced by any of the sugars were identical with one another. The principal inducer of C. murisepticum alpha-galactosidase is melibiose. Raffinose elicits induction only indirectly by prior extracellular hydrolysis to melibiose. The enzyme was purified to homogeneity from melibiose-grown cells by column chromatographic fractionation of cell-free extract on Sephadex G-200, followed by that on QAE-Sephadex A-25, and finally by ultracentrifugation in a 5-20% sucrose density gradient. The purified C. murisepticum alpha-galactosidase is a homotetramer of 320 kDa molecular mass, with a Km for melibiose, 2mM; for p-nitrophenyl-alpha-D-galactopyranoside (PNPG), 0.17mM; and pH optimum of 7.5. Under optimal conditions of induction, 2% of total protein synthesized by C. murisepticum cells was alpha-galactosidase (as determined by selective immunoprecipitation of radiolabelled enzyme protein).
Accumulation of polyols and sugars in 7 strains of sugar-tolerant yeasts grown in the presence of high concentrations of glucose, sucrose, and sodium chloride was compared. Primary intracellular solutes accumulated in 4 species, Debaryomyces hansenii, Hansenula anomala, Zygosaccharomycesrouxii, and Candida tropicalis were glycerol and D-arabitol, and in Aureobasidium sp. erythritol was accumulated instead of D-arabitol. Saccharomyces cerevisiae and Torulaspora delbrueckii accumulated trehalose as well as glycerol (and D-arabitol). The fact that significant differences were observed in the amounts of the intracellular polyols, depending on the kind of media solute suggested that there were differences between the yeasts' glucose-tolerance and sucrose-tolerance as well as between sugar-tolerance and salt-tolerance. In most strains, larger amounts of polyols accumulated intracellularly in high-solute media as compared to low-solute medium. But their total concentration was at most about 1M, 30-40% of the extracellular solute concentrations. The intracellular polyols accumulated in high-solute media released extracellularly. These results suggested that intracellular polyols such as glycerol and D-arabitol could contribute to the osmoregulation of sugar-tolerant yeasts, although they might not always be produced for it. Glycerol was shown to be an important intracellular solute for osmoregulation.
Chromosomal DNAs from four species of genus Pleurotus were analyzed by the clamped homogeneous electric field (CHEF) electrophoresis. The analyses were carried out on protoplasts obtained from monokaryotic mycelia of the following species: P. pulmonarius, P. salmoneostramineus, P. ostreatus and P. cornucopiae. For P. pulmonarius, 6 DNA bands of sizes ranging from 4.9 to 1.6 megabase pairs (Mb) were clearly separated on the gel. For the other three species 6 to 8 bands ranging from 6 to 2 Mb were found. The banding patterns obtained for these four species were found to be different from each other, while those observed for six strains within the same species, P. ostreatus, showed a relatively similar pattern.
The murG gene located in the mra region at 2min on the Escherichia coli chromosome map was found to be responsible for UDP-GlcNAc: undecaprenyl-pyrophosphoryl-N-acetylmuramoyl-pentapeptide N-acetylglucosaminyl transferase activity in lipid cycle reactions of cell wall peptidoglycan synthesis. murG was concluded to be the structural gene for this enzyme from the following findings: (1) Expression of GlcNAc-transferase activity by the murG gene seemed to be thermosensitive when the chromosome carried an amber mutation in murG and a thermosensitive sup mutation. (2) Overproduction of the murG product caused an increase of GlcNAc-transferase activity. Incidentally, chemically phosphorylated polyprenol was shown to function as a carrier in lipid cycle reactions in peptidoglycan synthesis in E. coli.
Surface protein layer (S layer) of Clostridium difficile GAI 1152 was a tetragonal array with each side of 7.8nm and interior angle of 77.8°, which was composed of the two protein subunits with respective molecular weights of 38kDa and 42kDa. The S layer protein subunits were directly isolated from the whole cells by simple procedures involving extraction of the whole cells with 6M GHCl and precipitation by 50% saturation with ammonium sulfate and purified by using DEAE-Sepharose CL-6B column chromatography followed by DEAE-5 PW equipped to HPLC. Amino acid analysis showed that both the subunits were acidic polypeptide and neither proline nor cysteine were present. The 38kDa subunit exhibited a single isoelectric form (pI 4.0), whereas the 42kDa subunit showed multiple isoelectric forms ranging from 5.5 to 6.3 of pI. Limited proteolysis of the both subunits with Staphylococcusaureus V8 enzyme showed quite dissimilar peptide maps between them, suggesting marked differences in primary structures.