Abstract
BackgroundProteases are important for hydrolysis of proteins to generate peptides with many bioactivities. Thus, the development of novel proteases with high activities is meaningful to discover bioactive peptides. Because natural isolation from animal, plant and microbial sources is impractical to produce large quantities of proteases, gene cloning and expression of target protease are preferred.ResultsIn this study, an alkaline serine protease gene (GsProS8) from Geobacillus stearothermophilus was successfully cloned and expressed in Bacillus subtilis. The recombinant GsProS8 was produced with high protease activity of 3807 U/mL after high cell density fermentation. GsProS8 was then purified through ammonium sulfate precipitation and a two-step chromatographic method to obtain the homogeneous protease. The molecular mass of GsProS8 was estimated to be 27.2 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and 28.3 kDa by gel filtration. The optimal activity of GsProS8 was found to be pH 8.5 and 50 °C, respectively. The protease exhibited a broad substrate specificity and different kinetic parameters to casein and whey protein. Furthermore, the hydrolysis of whey protein using GsProS8 resulted in a large amount of peptides with high angiotensin-I-converting enzyme (ACE) inhibitory activity (IC50 of 0.129 mg/mL).ConclusionsGsProS8 could be a potential candidate for industrial applications, especially the preparation of antihypertensive peptides.
Highlights
Proteases are important for hydrolysis of proteins to generate peptides with many bioactivities
They are composed of serine residue forming a catalytic triad with aspartic acid and histidine in the active site, which can be inactivated by phenylmethylsulfonylfluoride (PMSF), diodopropyl fluorophosphate (DFP) and chymostatin [2]
Thereafter, Pichia pastoris was used for secretory expression of the protease gene from G. stearothermophilus F1, to solve the formation of inclusion bodies and incorrect protein folding induced by E. coli expression system, but the expression level was still low [10]
Summary
Proteases are important for hydrolysis of proteins to generate peptides with many bioactivities. Microbial serine proteases (EC 3.4.21.14) are one of most important proteases to be applied in different food fields, such as meat tenderization, cheese ripening, flavor development, baking, and preparation of bioactive peptides [1]. They are composed of serine residue forming a catalytic triad with aspartic acid and histidine in the active site, which can be inactivated by phenylmethylsulfonylfluoride (PMSF), diodopropyl fluorophosphate (DFP) and chymostatin [2]. As high protease activity and pH stability are important for industrial applications, a proper expression system will be essential to improve the expression level of the target protease genes from G. stearothermophilus [1]
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