Purification of Bacillus mesentericus proteolytic enzymes by affinity chromatography

By means of affinity chromatography on Ovomucoid-Sepharose two proteinases hydrolyzing casein and elastin were isolated from the supernatant of the Bacillus mesentericus culture medium. The activity yield of proteinases was 100%. The characteristics of the purified enzymes were studied. It is demonstrated that B. mesentericus possesses several proteinases.     

Effect of inhibitors and cations on the proteolytic activity of Bacillus mesentericus

The effect of some inhibitors and bivalent metal cations (Mn2+, Ca2+, Fe2+, Zn2+, Mg2+, Co2+ and Cu2+) on the proteolytic activity of two Bacillus mesentericus strains (strain 8 and strain 64 M-variant) was comparatively studied. The both enzymes were shown to be serine proteinases, but the proteinase of strain 64 was also a metal-dependent enzyme. Metal ions exerted no essential effect on the proteinase of strain 8. Ca2+ and Mg2+ ions stimulated the proteinase activity of strain 64 whereas Fe2+ and Zn2+ ions inhibited it in the case of three substrates. Therefore, the two proteinases are different.     

Effect of exogenous factors on extracellular alkaline ribonuclease synthesis in Bacillus mesentericus

Bacillus mesentericus was found to assimilate nucleic acids as a source of nitrogen and phosphorus. Nucleic acids added to the medium as a source of nitrogen or phosphorus stimulated synthesis of ribonuclease. When washed bacterial cells were incubated for a short period of time in a fresh nutrient medium containing RNA, synthesis of RNAase was also induced. Synthesis of the enzyme was inhibited by high concentrations of chloramphenicol and actinomycin D, and stimulated by low concentrations of actinomycin D. Therefore, alkaline RNAase is an inducible enzyme which participates in the nutrition processes of bacteria.     

Inactivation of Bacillus piliformis spores by heat and certain chemical disinfectants

The inactivation of Tyzzer's organism (Bacillus piliformis) spore isolated from rats by heat and various chemical disinfectants was studied. The spores were from B. piliformis-infected rat liver tissues. The spore suspension (10(4) 50% of rat liver lesion producing dose with prednisolone treatment/ml) was treated with heart or disinfectants. Inactivation of the spores was examined in experimentally infected rats. Rats were inoculated perorally with a treated spore suspension and injected subcutaneously with prednisolone. On the sixth day after inoculation, rats were examined grossly for liver lesions. Spores were inactivated at 80 degrees C for 15 min but not at 60 degrees C for 30 min. Spores were inactivated by 0.4% peracetic acid, 0.015% sodium hypochrolite, 1% iodophol, 5% phenol. Alcide and 0.37% formaldehyde solution, but not by 0.037% formaldehyde solution, 70% ethanol, 0.3% benzethonium chloride solution, 3% cresol and soap solution, or 4% chlorhexidine digluconate. These findings suggest that B. piliformis spores are relatively sensitive to heat and certain chemical disinfectants.     

Effect of etiroxate on LCAT activity and on certain energy metabolism enzymes in the rat

Etiroxate (Skleronorm Grünenthal R) was administered 42 days to male Wistar rats and their serum and liver cholesterol and triglyceride levels, the rate of esterification of free cholesterol in their plasma by lecithin cholesterol acyltransferase (LCAT) (EC 2.3.1.43) and thriosephosphate dehydrogenase (TPDH) (EC 1.2.1.12), lactate dehydrogenase (LDH) (EC 1.1.1.27), hexokinase (HK) (EC 2.7.1.1), c-glycerol-3-phosphate dehydrogenase (GPDH) (EC 1.1.1.8), malate dehydrogenase (MDH) (EC 1.1.1.37) citrate synthase (CS) (EC 4.1.3.7) and hydroxyacylcoenzyme A dehydrogenase (HOADH) (EC 1.1.1.35) activity were determined in their liver. After 14 and 28 days, animals given etiroxate (600 micrograms/kg) had smaller weight increments than the controls and a significantly lower plasma free and esterified cholesterol level, but a significantly higher liver cholesterol concentration. Their final plasma and liver cholesterol concentrations did not differ significantly from the control values. Plasma triglyceride levels were significantly raised in treated animals at all the given intervals. LCAT activity was significantly higher throughout the whole time of treatment, with the maximum increase in the last phase. Glycolytic and oxidative enzyme activities were significantly raised, whereas GPDH activity was the same as in the controls. The results show that etiroxate accelerates cholesterol turnover in the endogenous pool by activating LCAT and stimulating energy metabolism.     

Effect of castanospermine on the structure and secretion of glycoprotein enzymes in Aspergillus fumigatus.

Aspergillus fumigatus secretes a number of glycosidases into the culture medium when the cells are grown in a mineral salts medium containing guar flour (a galactomannan) as the carbon source. At least some of these glycosidases have been reported to be glycoproteins having N-linked oligosaccharides. In this study, we examined the effect of the glycoprotein processing inhibitor, castanospermine, on the structures of the N-linked oligosaccharides and on the secretion of various glycosidases. Cells were grown in the presence of various amounts of castanospermine; at different times of growth, samples of the media were removed for the measurement of enzymatic activity. Of the three glycosidases assayed, beta-hexosaminidase was most sensitive to castanospermine; and its activity was depressed 30 to 40% at 100 micrograms of alkaloid per ml and even more at higher alkaloid concentrations. On the other hand, beta-galactosidase activity was hardly diminished at castanospermine levels of up to 1 mg/ml, but significant inhibition was observed at 2 mg/ml. beta-Galactosidase was intermediate in sensitivity. Cells were grown in the presence or absence of castanospermine and labeled with [2-3H]mannose, [6-3H]glucosamine, or [1-3H]galactose to label the sugar portion of the glycoproteins. The secreted glycoproteins were digested with pronase to obtain glycopeptides, and these were identified on Bio-Gel P-4 (Bio-Rad Laboratories). The glycopeptides were then digested with endoglucosaminidase H to release the peptide portion of susceptible structures, and the released oligosaccharides were reisolated and identified on Bio-Gel P-4. The oligosaccharides from control and castanospermine-grown cells were identified by a combination of enzymatic and chemical studies. In control cells, the oligosaccharide appeared to be mostly Man8GlcNAc and Man9GlcNAc, whereas in the presence of alkaloid, the major structures were Glc3Man7GlcNAc and Glc3Man8GlcNAc. These data fit previous observations that castanospermine inhibits glucosidase I.     

Hydrolase biosynthesis by morphological variants of Bacillus mesentericus as dependent on the storage conditions

A method used to prepare the inoculum (a strain of Bacillus mesentericus, a producer of a complex of hydrolytic enzymes) has been studied for its effect on the activity of proteinases and amylases under submerged cultivation in fermenters. Optimal conditions for the culture storage and inoculum cultivation are developed to obtain standard enzymic preparations.     

Structure and antitumor activity of polysaccharides from the micelles of Aspergillus oryzae

Three polysaccharide fractions active against mammary gland adenocarcinoma Ca-755 in mice but inactive against sarcoma C-180 were isolated from Aspergillus oryzae strain 5214. The main fraction, extracted from the mycelium by cold dilute alkali in the presence of sodium borohydride, was shown to be linear (1----3)-a-D-glucopyranan (pseudonigeran) according to 13C NMR spectroscopy, partial acid hydrolysis and periodate oxidation data.