RESUMO
Biochemical properties of Bacillus intermedius subtilisin-like proteinase (AprBi) secreted by a B. subtilis recombinant strain in the early and late stationary phases of growth have been determined. Protein structure was analyzed and its stability estimated. It was noted that the enzyme corresponding to different phases of bacterial growth retains activity in the presence of reducing and oxidizing agents (C2H5OH and H2O2). Different effects of bivalent metal ions on activity of two proteinase fractions were found. Calcium ions more efficiently activate proteinase secreted in the late stationary phase. Unlike the first enzyme fraction, the second forms catalytically active dimers.
Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/metabolismo , Proteínas Recombinantes/metabolismo , Subtilisina/metabolismo , Sequência de Aminoácidos , Bacillus/genética , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Cálcio/farmacologia , Catálise/efeitos dos fármacos , Cobre/farmacologia , Etanol/farmacologia , Peróxido de Hidrogênio/farmacologia , Concentração de Íons de Hidrogênio , Magnésio/farmacologia , Manganês/farmacologia , Dados de Sequência Molecular , Proteínas Recombinantes/isolamento & purificação , Homologia de Sequência de Aminoácidos , Subtilisina/genética , TemperaturaRESUMO
Two subtilisin-like serine proteinases of Bacillus intermedius secreted by the Bacillus subtilis recombinant strain AJ73 (pCS9) on the 28th and 48th h of culture growth (early and late proteinase, respectively) have been isolated by ion-exchange chromatography on CM-cellulose and by FPLC. Molecular weights of both proteinases were determined. The N-terminal sequences of the recombinant protein and mature proteinases of the original strain were compared. Kinetic parameters and substrate specificities of the early and late proteinase were analyzed. Physicochemical properties of the enzymes were studied.
Assuntos
Bacillus subtilis/enzimologia , Bacillus/enzimologia , Proteínas Recombinantes , Serina Endopeptidases , Subtilisina , Sequência de Aminoácidos , Bacillus/genética , Bacillus/crescimento & desenvolvimento , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Cromatografia por Troca Iônica , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/isolamento & purificação , Serina Endopeptidases/metabolismo , Especificidade por Substrato , Subtilisina/química , Subtilisina/genética , Subtilisina/isolamento & purificação , Subtilisina/metabolismo , TemperaturaRESUMO
The effect of the components of the nutrient medium on growth and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant strain Bacillus subtilis AJ73(pCS9) was studied. The production of proteinase was found to be dependent on the composition of the nutrient medium and showed two peaks, at the 28th and 48th h of growth. The concentrations of the main components of the nutrient medium (peptone and inorganic phosphate) optimal for the biosynthesis of subtilisin-like serine proteinase at the 28th and 48th h of growth were determined in factorial experiments. Complex organic substances, casein at concentrations of 0.5-1%, gelatin at concentrations of 0.5-1%, and yeast extract at a concentration of 0.5%, stimulated the production of subtilisin-like serine proteinase by the recombinant strain. The study of the sporulation dynamics in this strain showed that the proteinase peaks at the 28th and 48th h of growth correspond, respectively, to the initial stage of sporulation and to the terminal stages of endospore formation (V-VII stages of sporulation).
Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Bacillus/enzimologia , Proteínas Recombinantes/biossíntese , Serina Endopeptidases/biossíntese , Subtilisina/biossíntese , Bacillus/genética , Bacillus subtilis/genética , Bacillus subtilis/fisiologia , Meios de Cultura/química , Meios de Cultura/metabolismo , Serina Endopeptidases/isolamento & purificação , Esporos Bacterianos/enzimologia , Esporos Bacterianos/crescimento & desenvolvimento , Subtilisina/isolamento & purificaçãoRESUMO
The effect of certain nutrients on the growth and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant strain Bacillus subtilis AJ73(pCS9) was studied. Glucose was found to inhibit the synthesis of proteinase in the early (28 h of growth) but not in the late stationary phase (48 h of growth). The inhibitory effect of the other mono- and disaccharides studied was less pronounced. Casamino acids added to the medium at concentrations of 0.1-1% as an additional carbon and nitrogen source stimulated enzyme biosynthesis. Individual amino acids (cysteine, asparagine, glutamine, tryptophan, histidine, and glutamate) also stimulated enzyme biosynthesis in the early stationary phase by 25-30%, whereas other amino acids (valine, leucine, alanine, and aspartate) were ineffective or even slightly inhibitory to enzyme production. The stimulatory effect of the first group of amino acids on the synthesis of proteinase in the late stationary phase was negligible. In contrast, the bivalent ions Ca2+, Mg2+, and Mn2+ stimulated biosynthesis of proteinase in the late stationary phase (by 20-60%) and not in the early stationary phase. The data indicate that there are differences in the biosyntheses of proteinase by the recombinant B. subtilis strain during the early and late periods of the stationary phases.
