J. Pazlarová

External factors involved in the regulation of synthesis of an extracellular proteinase in Bacillus megaterium: effect of temperature

SummaryWe 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.

Kinetics of α-amylase production in a batch and a fed-batch culture ofBacillus subtilis

Analysis of the kinetics of α-amylase production in a batch and a fed-batch culture ofBacillus subtilis made it possible to derive a konetic model of the process describing mutual interactions between growth and production. The specific growth rate is limited by the concentration of both corn-steep liquor and starch. Higher concentrations of reducing sugars in the medium also inhibit growth. The overall production of α-amylase is a result of an equilibrium between the rate of enzyme production and its degradation due to the effect of environment. The actual specific production rate is directly proportional to the specific production rate is directly proportional to the specific growth rate (characterizing the physiological state of the culture) and is inhibited by higher concentrations of corn-steep liquor in the medium.

Kinetics of α-amylase production in a batch and fed-batch culture of Bacillus subtilis with caseinate as nitrogen source and starch as carbon source

SummaryAnalysis of a large number of experimental data from the cultivation of Bacillus subtilis formed the basis for a kinetic model of the process explaining the effect of composition of the culture medium and of the growth rate on the rate of enzyme production. The resulting rate of formation of α-amylase (EC 3.2.1.1) reflects the sum of the rate of enzyme production and the rate of its degradation as affected by the environment. The kinetic dependence confirms the previously described mechanism of regulation of enzyme biosynthesis. The mathematical model of the process served here to determine the optimal conditions for enzyme biosynthesis which were then verified in a fed-batch cultivation. The production of the enzyme in fed-batch culture was found to be twice that found in a batch cultivation.

Isolation of the pMI 10 plasmid from the α-amylase producing strain of Bacillus subtilis

SummaryB. subtilis A 18, a producer of exocellular amylase, was found to carry covalently closed DNA plasmid molecules (pMI 10). The pMI 10 was isolated and characterized by electron microscopy, electrophoretic mobility and restriction endonuclease cleavage pattern. The pMI 10 was absent in all α-amylase low productive or nonproductive clones. The pMI 10 DNA was transformed together with pUB 110 DNA into B. subtilis RM 125 arg-leu- recipient cells, and, hence, compatibility of these plasmids could be demonstrated.

Production of an extracellular proteinase and α-amylase byBacillus subtilis in chemostat

The formation of two extracellular enzymes (α-amylase and a proteinase) ofB. subtilis was investigated in batch and continuous cultures. Differences were observed in the production of both enzymes in batch culture when studied in flasks and in a fermentor. The values of proteinase activity from continuous cultivation never reached those obtained during batch cultivation. The optimal dilution rate for proteinase synthesis was 0.1 – 0.2/h. The optimal dilution rate for the production of α-amylase was 0.2 –0.3/h.

External factors involved in the regulation of an extracellular proteinase synthesis in Bacillus megaterium

SummaryA mathematical model was formulated to describe the kinetics and stoichiometry of growth and proteinase production in Bacillus megaterium. Synthesis of the extracellular proteinase in a batch culture is repressed by amino acids. The specific rate of formation of the enzyme (rE) can be described by the formula {ie373-1}, where k2 and k3 stand for the non-repressible and repressible part of enzyme synthesis respectively, kS2 is a repression coefficient and S2 indicates the concentration of amono acids; the values of k2 and kS2 depend on the composition of the mixture of amino acids. Even in a high concentration, a single amino acid is less effective than a mixture of amino acids. The dependence of the proteinase repression on the concentration of an external amino acid (leucine) follows the same course as its rate of incorporation into proteins, approaching saturation at concentrations higher than 50 μM (half saturation approximately 10 μM). However, the total uptake of leucine did not exhibit any saturation even at 500 μM external concentration.

Use of zeolite to controlBacillus megaterium extracellular proteinase production

Synthetic and natural zeolite were used to control the nitrogen source level inBacillus megaterium cultures producing extracellular proteinase by means of reversible adsorption. The addition of biochemically inert, calcium aluminosilicates significantly increased the yield of the enzyme. A simple mathematical model explaining the mechanism has been formulated and its parameters estimated.

Regulation of extracellular proteins and α-amylase secretion by temperature inBacillus subtilis

Abstractα-Amylase was found to be the main protein secreted byBacillus subtilis, corresponding to 90, 87 and 60% of total extracellular proteins at 30, 40 and 45°C, respectively. A change in temperature can affect the pattern of proteins secreted as detected by gel electrophoresis.14C-Leucine incorporation into extracellular proteins and their proportion at the end of the growth phase was higher at 30°C than that at 40 or 45°C. The effect of temperature on α-amylase synthesis as determined by its enzymic activity and on the extracellular protein synthesis followed a similar pattern.

Effect of temperature on α-amylase formation and DNA replication inBacillus subtilis

The effect of temperature on extracellular α-amylase synthesis and chromosomal and plasrnid DNA replication inBacillus subtilis A18 carrying plasmid pMI 10 was studied. The specific growth rate μ increased with elevated temperature up to 42.5°C, while the activities of α-amylase per population dry mass decreased. No obvious quantitative changes of14C-thymidine incorporation per dry mass increase and no basic differences in plasmid copy number in the range of temperatures from 25 to 40°C were found.

Functional half life of the α-amylase mRNA ofBacillus amyloliquefaciens

The functional half life of α-amylase mRNA was investigated inBacillus amyloliquefaciens CCM 3502. The amount of mRNA was followed indirectly: the amount of protein translated by preformed mRNA was measured after transcription was terminated by rifampicin. The radioactivity of14C-leucine-labeled extracellular protein produced by drug-treated cells was a measure of the quantity of α-amylase, because this amount formed, in standard experiments, at least 90% of the total extracellular proteins. The effect of rifampicin and chloramphenicol was studied at the level of mRNA transcription and translation. Negligible radio-activity of the extracellular protein produced by cells after the addition of chloramphenicol (200 μg·ml−1) proved that α-amylase is formed de novo, and no form of preformed enzyme is secreted. From the radioactivity of extracellular proteins synthesized after the inhibition of mRNA by rifampicin, the half life of α-amylase mRNA was calculated to be 26.9 min at 30°C. The half life of mRNA for cellular proteins was calculated to be 2.9 min.