Kazuo Izaki

The two Staphylococcal bi-component toxins, leukocidin and gamma-hemolysin, share one component in common

Staphylococcal bi‐component toxins, leukocidin and γ‐hemolysin, consist of two protein components, i.e. F and S for leukocidin and HγI and HγII for γ‐hemolysin. In this study we purified HγI and HγII to homogeneity from the culture medium of Staphylococcus aureus RIMD 310925 and compared their properties with those of F and S purified from the same source. The N‐terminal 59‐ and C‐terminal 2‐residue amino acid sequences, apparent molecular mass, and isoelectri point of purified HγI were the same as those of F. In an Ouchterlony double diffusion test a fused line without spur was formed between F and HγI using either anti‐F or anti‐HγI antibodies. A synergistic action of F and HγII caused hemolysis of human red blood cells, and HγI acted synergistically with S to exhibit leukocidin activity. We conclude that the two toxins share one protein component (F = HγI) in common and leukocidin‐ and γ‐hemolysin‐specific activities are determined by S and HγII, respectively. It is also reported that the N‐terminal 58‐residue sequence of HγII is 72% similar to the corresponding sequence of S.

Purification and characterization of a raw starch-digestive amylase from non-sulfur purple photosynthetic bacterium

A strain of non-sulfur purple photosynthetic bacteria could utilize raw starch from corn, potato and cassava efficiently as a source of electron donor for hydrogen production. At least two amylases were found in the culture grown under light-anaerobic conditions with raw corn starch as a carbon source. These two amylases could digest raw starch as well as soluble starch. One of them was purified as a homogeneous protein with DEAE-cellulose column chromatography, ultra filtration with a membrane of Amicon XM 300, hydroxyapatite column chromatography, Toyopearl 55 HW gel filtration and starch gel affinity chromatography. The activity of this amylase was reactivated specifically by calcium ion after the enzyme was treated by EDTA. The optimum temperature of the enzyme activity was 40°C and the activity was stable up to 45°C. The optimum pH was 6.0 and the enzyme was quite stable at alkaline pH up to 12.0. This enzyme is an amylase that can digest raw starch from corn, potato and cassava.

Comparison of the Mannan Structure from the Cell-Wall Mutant Candida sp. M-7002 and Its Wild Type

The cell-wall mutant of a hydrocarbon-assimilating yeast, Candida sp. M-7002 and its wild type have shown a significant difference in mannose content. Each mannan was isolated from the mutant and the wild-type cells by fractionation with Cetavlon and copper reagent. Both mannans contain D-mannose, D-glucose and phosphate. The mutant mannan has a relatively high content of protein (18% in weight bases) whereas the wild-type mannan has a low protein content (5.1%) with a high amount of carbohydrate (greater than 90%). Structural analyses by enzymatic and chemical methods showed that both mannans had a mannosidic (alpha 1--6)-linked back bone substituted at O-2 by side chains of varying length. The side chains of the mutant mannan were shown to consist of single mannose units and disaccharide units whose linkages were predominantly alpha 1--2, while the wild-type mannan had two additional side chains of disaccharides. These additional side chains had alpha 1--3 linkages which were scarcely found in the mutant mannan. beta-Elimination reaction demonstrated that the mannans also contain mannosyl oligosaccharides linked to protein through O-glycosidic linkage. The chemical properties of the mannan of the Candida mutant indicates that the mutation might occur not only in the side chain structure but also in the (alpha 1--6)-linked mannan back bone.

Treatment of lipid-containing wastewater using bacteria which assimilate lipids

Abstract Bacteria which grew in a medium containing olive oil as a sole source of carbon were isolated from two meat plants in the Sendai district of Japan. All of the isolates tested assimilated beef tallow, lard, olive oil and used salad oil as a sole carbon source in shaking cultures. One of the isolates, strain 351, digested lipids most efficiently, as shown by the amount of n -hexane extracts that remained. This bacterium was identified as Bacillus sp. A new and efficient laboratory-scale apparatus for the biological treatment of lipid-containing wastewater was devised using strain 351. The apparatus consisted of a water circulation system for the primary treatment of the water, in which strain 351 was inoculated, and an ordinary aeration tank using activated sludge as a secondary treatment. Lipids in the wastewater could be almost completely removed by this apparatus without physical treatment. On the other hand, an ordinary aeration system in the laboratory using an air stone and air pump resulted in the floating of lipids, and was not successful in digesting lipids even in the presence of strain 351.

The C-terminal region of the S component of staphylococcal leukocidin is essential for the biological activity of the toxin

The Staphylococcal toxin leukocidin consists of two protein components, F and S. From a culture medium of Staphylococcus aureus RIMD 310925, we isolated a truncated form of S (LS2), of which the C‐terminal 17‐residue segment is missing. Unlike intact S, LS2 showed neither leukocytolytic activity in the presence of F nor affinity for monosialoganglioside GM1 (GM1). When excited at 280 nm, both S and LS2 exhibited intrinsic tryptophan fluorescence with an emission maximum at 318 nm. Upon binding to GM1, the emission maximum of S underwent a blue shift to 310 nm, whereas no change in fluorescence took place on mixing GM1 with LS2. We conclude that the C‐terminal region of S is essential for its biological activity as well as for its binding to GM1 and that this binding is accompanied by a conformational change of the S protein.

Cloning and expression of pectin lyase gene from Erwinia carotovora in Escherichia coli

A pectin lyase (PNL; EC 4.2.2.10) gene of Erwinia carotovora Er was cloned and expressed in Escherichia coli. The analysis of the nucleotide sequence of the 0.6 kb StuI-EcoRI fragment, which was hybridized with the mixed oligonucleotide probe for PNL gene, revealed the presence of an open reading frame (0RF) and correlated exactly with the known N-terminal 18 amino acid sequence of PNL. When a plasmid pTN2159, which has a BamHI-EcoRI fragment containing this ORF, was introduced into E. coli JM109, PNL was not expressed. When a tac-promoter was inserted in front of the ORF, PNL was efficiently expressed in E. coli. Synthesis of PNL by E. coli was also confirmed by immunoblot analysis.

Cloning of two protease genes fromRhodocyclus gelatinosa APR 3-2 and their expression inEscherichia coli

Two different protease genes were cloned fromRhodocyclus gelatinosa APR 3-2 inEscherichia coli HB 101/λ with pBR329 or its derivatives. The recombinant plasmids designated as pRP100 and pRP300 contained 11.2 and 10.6 kb DNA fragments, respectively. The differences of both plasmids in restriction enzyme maps indicate that these plasmids contained different protease genes. DNA fragments coding for protease, 6.4 kb and 4.5 kb from pRP100 and pRP300, were subcloned into pRP329 and designated as pRP101 and pRP301, respectively. The two cloned proteases were excreted in culture medium ofE. coli, and ß-lactamase ofE. coli, which was originally localized in periplasmic space, was also excreted in the medium.

Purification and characterization of a neutral serine protease from non-sulfur purple photosynthetic bacterium

A neutral serine protease was purified as a homogeneous protein from the culture broth of photosynthetic bacterium T-20 by sequential chromatographies on columns of DEAE-cellulose, Toyopearl HW 55F, hydroxyapatite, and CM-cellulose. The molecular weight was estimated to be approximately 44,000 by SDS-PAGE, while the value of approximately 80,000 was obtained when the Hedrick-Smith method was used; this suggested that the enzyme consists of two identical subunits. The isoelectric point was determined to be 6.3 by isoelectric focusing. The enzyme had a pH optimum at 7.8. Maximal enzyme activity was detected at 50°C, and the activity was stable up to 50°C for 5 min at pH 7.0–7.2. The substrate specificity of the protease was investigated with a series of synthetic peptidyl-p-nitroanilide. The best substrate examined was Suc-Ala-Ala-Pro-Phe-pNA. The protease activity was inhibited by various inhibitors of serine protease such as chymostatin, PMSF, and DFP. EDTA, which is an inhibitor of metal protease, also inhibited the protease activity, whereas inhibitors of thiol and aspartic proteases had no significant effect.

Enzymatic Properties of an Extracellular Quinoprotein, Enacyloxin Oxidase

Some properties of enacyloxin (ENX) oxidase from Frateuria sp. [Oyama et al., Biosci. Biotech. Biochem., 58, 1914-1917 (1994)] were studied. The enzyme catalyzed the oxidation of ENX IVa, ENX IIIa, and decarbamoyl ENX IVa, specifically. The optimum pH and temperature for the enzyme activity were pH 9.0 and 60°C, respectively. It is suggested that the enzyme is a quinoprotein but its redox cofactor is different from pyrroloquinoline quinone.

Stimulation of Pectolytic Enzyme Formation of Erwinia aroideae by an Active Factor in Carrot Extracts:Part II. Partial Purification of the Factor and the Stimulating Effect of Some Other Compounds

Formation of pectinase system in Erwinia aroideae was stimulated to a considerable extent when the cells were incubated in a pectin medium containing carrot extracts. The active factor in the extract was purified about 30 fold by ethanol precipitation, and further purification was achieved by ninhydrin treatment, charcoal adsorption, dialysis and gel filtration with Sephadex G-10. Although crude carrot extract preparation also stimulated protease formation in this organism, no stimulating activity for protease formation was found in the purified factor. Acetate and butyrate which had been shown to stimulate pectinase formation, were found to stimulated protease formation as well. Pectinase formation by this organism was also stimulated by polyamines and inorganic phosphate to a considerable extent.