We studied the effect of temperature on the production of an extracellular neutral metalloproteinase of Bacillus megaterium in a laboratory fermentor under constant aeration and pH. The optimal temperature for growth (35-38° C) was higher than that for the synthesis of proteinase during exponential growth (below 31° C). The critical biomass concentration at which the exponential growth terminated decreased with increase in cultivation temperature. The specific rate of proteinase synthesis decreased when the critical biomass concentration was achieved. The observed decrease in proteinase synthesis was related to the cultivation temperature. The temperature also influenced the level of mRNA coding for proteinase. We formulated a mathematical model of cultivation describing the dependence of growth and proteinase synthesis on dissolved oxygen and temperature. The parameters of the model were identified for temperature intervals from 21 to 41° C using a computer. The optimum temperature for the enzyme production was 21° C. The productivity (enzyme activity/time) was maximal at 24-28° C. When optimizing the temperature profile of cultivation, we designed a suboptimal solution represented by a linear temperature profile. We have found that under conditions of continuous decrease in temperature, the maximal production of the proteinase was achieved at a broad range of temperature (26-34° C) when the rate of temperature decrease was 0.2-0.8° C/h. The initial optimal temperature for the enzyme productivity was in the range of 32-34° C. The optimum temperature decrease was 0.8° C/h.

• Publication type
• Journal Article MeSH

Intracellular proteolytic activities of B. megaterium KM occur soluble in the cytoplasm and periplasm and insoluble in the membrane. Two proteolytic enzymes were found in the cytoplasmic fraction by gel filtration on Sephadex G 150 and by polyacrylamide gel electrophoresis. The first enzyme called CI was stable, had a relative molecular mass of Mr = 105,000 (M = 105 kg/mol) and was inhibited by EDTA and PMSF, whereas the second, designated CII, was labile and had a relative molecular mass of Mr = 46,000 (M = 46 kg/mol). Because of its lability it could not be characterized in detail. In the "periplasm" only a single proteolytic enzyme P (Mr = 28,000; M = 28 kg/mol) inhibited by EDTA could be demonstrated. The extracellular enzyme exhibited similar properties. The membrane proteolytic activity was sensitive to PMSF and EDTA. The membrane enzymes have not yet been solubilized. In cells of the mutant KM 12 that does not produce the extracellular proteinase, only one type of proteinase, in all its properties identical with the cytoplasmic proteinase CI, could be demonstrated.

• MeSH
• Bacillus megaterium enzymology   genetics   MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Endopeptidases analysis   MeSH
• Chromatography, Gel MeSH
• Mutation MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Endopeptidases MeSH

The synthesis of exocellular proteinase decreases with increasing concentration of amino acids in the growth medium. After removal of amino acids the enzyme synthesis is gradually restored to normal values. The presence of inhibitors of transcription (actinomycin D) or translation (chloramphenicol) blocks the restoration of enzyme synthesis. No active or inactive precursors of the exocellular enzyme could be detected in the cell. It is likely that the enzyme synthesis is regulated by amino acids at the level of specific mRNA synthesis rather than at the translation level or at the level of secretion. The activity of the enzyme that has already been secreted to the external medium is partially inhibited by amino acids. The periplasmic proteinase is repressed by amino acids to the same extent as the exocellular enzyme. The content of the enzyme(s) inside the protoplast is also decreased during growth in the presence of amino acids.

• MeSH
• Enzyme Activation MeSH
• Amino Acids pharmacology   MeSH
• Bacillus megaterium drug effects   enzymology   MeSH
• Chloramphenicol pharmacology   MeSH
• Dactinomycin pharmacology   MeSH
• Enzyme Repression MeSH
• Protease Inhibitors pharmacology   MeSH
• Kinetics MeSH
• Peptide Hydrolases biosynthesis   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Amino Acids MeSH
• Chloramphenicol MeSH
• Dactinomycin MeSH
• Protease Inhibitors MeSH
• Peptide Hydrolases MeSH

Protein turnover was followed in populations of Bacillus megaterium growing in temperature range of 17-48 degrees C in different media. Higher temperature stimulated the protein turnover (expressed as the amount of protein degraded during 3.3 h) in all the media tested up to the optimal growth temperature (40-42 degrees C). Protein turnover in a medium containing amino acids continued to be stimulated by temperature even above this point; no further significant increase of turnover was found in the other media.

• MeSH
• Amino Acids pharmacology   MeSH
• Bacillus megaterium growth & development   metabolism   MeSH
• Bacterial Proteins metabolism   MeSH
• Kinetics MeSH
• Culture Media MeSH
• Temperature MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Amino Acids MeSH
• Bacterial Proteins MeSH
• Culture Media MeSH

• MeSH
• Bacillus megaterium growth & development   metabolism   MeSH
• Bacterial Proteins metabolism   MeSH
• Kinetics MeSH
• Culture Media MeSH
• Mutation MeSH
• Peptide Hydrolases metabolism   MeSH
• Spores, Bacterial MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Culture Media MeSH
• Peptide Hydrolases MeSH

The rate of protein turnover in asporogenic Bacillus megaterium decreases continuously during incubation in a sporulation medium. The capability of equilibration of external amino acids with amino acids in the metabolic pool of non-growing cells was retained for at least 5 h. Leucine, while repressing the synthesis of the exocellular protease, does not significantly influence the course of protein degradation in vivo. Transfer of non-growing cells after 4 h to a fresh sporulation medium does not influence the rate of protein degradation. The gradual decrease of the rate of protein turnover in non-growing cells of the asporogenic variant is thus not an artifact caused by a decreased uptake of amino acids by cells or by conditions under which the protein turnover is determined.

• MeSH
• Amino Acids metabolism   MeSH
• Bacillus megaterium growth & development   metabolism   MeSH
• Bacterial Proteins metabolism   MeSH
• Kinetics MeSH
• Culture Media MeSH
• Leucine metabolism   MeSH
• Spores, Bacterial MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Amino Acids MeSH
• Bacterial Proteins MeSH
• Culture Media MeSH
• Leucine MeSH

Functional half-life of the exocellular protease mRNA was determined in in exponentially growing and stationary cells of the asporogenic strain of Bacillus megaterium KM and in the sporogenic strain of B. megaterium 27 during sporulation. No reserve of the protease mRNA could be detected in the cells and the half-lives were determined to be 6--7 min in the exponential and stationary cells of B. megaterium KM and 7.5--8.5 min in B. megaterium 27. The mean half-life of mRNA for cell proteins was determined to be 3.5--4.5 min. Thus, as compared with the mean half-life of mRNA for cell proteins that of mRNA for the exocellular protease is slightly longer.

• MeSH
• Bacillus megaterium metabolism   physiology   MeSH
• Bacterial Proteins biosynthesis   MeSH
• RNA, Bacterial biosynthesis   metabolism   MeSH
• Dactinomycin pharmacology   MeSH
• Transcription, Genetic drug effects   MeSH
• RNA, Messenger metabolism   MeSH
• Half-Life MeSH
• Peptide Hydrolases biosynthesis   MeSH
• Spores, Bacterial MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins MeSH
• RNA, Bacterial MeSH
• Dactinomycin MeSH
• RNA, Messenger MeSH
• Peptide Hydrolases MeSH

Intracellular proteolytic activity increased during incubation of the sporogenic strain of Bacillus megaterium KM in a sporulation medium together with excretion of an extracellular metalloprotease. The exocellular protease activity in a constant volume of the medium reached a 100-fold value with respect to the intracellular activity. Maximal values of the activity of both the extracellular and intracellular enzyme were reached after 3-5 h of incubation. After 7 h 20-50% cells formed refractile spores. The intracellular proteolytic system hydrolyzed denatured proteins in vitro at a rate up to 150 mug mg-1 h-1 and native proteins at a rate up to 70 mug mg-1 h-1. Degradation of proteins in vivo proceeded from the beginning of transfer to the sporulation medium at a constant rate of 40 mug mg-1 h-1 and the inactivation of beta-galactosidase at a rate of 70 mug mg-1 h-1. The intracellular proteolytic activity was inhibited to 65-88% by EDTA, to 23-76% by PMSF. Proteolysis of denatured proteins was inhibited both by EDTA and PMSF more pronouncedly than proteolysis of native proteins; 50-65% of the activity were localized in protoplasts. Another strain of Bacillus megaterium (J) characterized by a high (up to 90%) and synchronous sporulation activity was found to behave in a similar way, but the rate of protein turnover in this strain was almost twice as high. The asporogenic strain of Bacillus megaterium KM synthesized the exocellular protease in the sporulation medium, but its protein turnover was found to decrease substantially after 3-4 h. The intracellular proteolytic system of the sporogenic strain J and the asporogenic strain KM were also inhibited by EDTA and PMSF.

• MeSH
• Bacillus megaterium enzymology   growth & development   MeSH
• Bacterial Proteins metabolism   MeSH
• Edetic Acid pharmacology   MeSH
• Endopeptidases metabolism   MeSH
• Phenylmethylsulfonyl Fluoride pharmacology   MeSH
• Mutation MeSH
• Protoplasts enzymology   MeSH
• Spores, Bacterial enzymology   growth & development   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Edetic Acid MeSH
• Endopeptidases MeSH
• Phenylmethylsulfonyl Fluoride MeSH