Identification of a novel alkaline serine protease from gazami crab (Portunus trituberculatus) hepatopancreas and its hydrolysis of myofibrillar protein

Abstract Background Proteases 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. Results In 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). Conclusions GsProS8 could be a potential candidate for industrial applications, especially the preparation of antihypertensive peptides.

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Molecular Cloning, Nucleotide Sequence, and Expression of the Structural Gene for Alkaline Serine Protease from AlkaliphilicBacillussp. 221

Bioscience Biotechnology and Biochemistry ◽

10.1271/bbb.56.1455 ◽

1992 ◽

Vol 56 (9) ◽

pp. 1455-1460 ◽

Cited By ~ 12

Author(s):

Hideto Takami ◽

Tetsuo Kobayashi ◽

Masato Kobayashi ◽

Mami Yamamoto ◽

Satoshi Nakamura ◽

...

Keyword(s):

Nucleotide Sequence ◽

Molecular Cloning ◽

Serine Protease ◽

Structural Gene ◽

Alkaline Serine Protease

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Production, purification and characterization of thermomycolase, the extracellular serine protease of the thermophilic fungus Malbranchea pulchella var. sulfurea

Canadian Journal of Microbiology ◽

10.1139/m76-023 ◽

1976 ◽

Vol 22 (2) ◽

pp. 165-176 ◽

Cited By ~ 34

Author(s):

Poh Seng Ong ◽

G. Maurice Gaucher

Keyword(s):

Serine Protease ◽

Thermophilic Fungus ◽

Amino Acid Residues ◽

Submerged Cultures ◽

Ph Optimum ◽

Alkaline Serine Protease ◽

Associated Production ◽

Specificity Study ◽

Malbranchea Pulchella ◽

The Stability

The thermophilic fungus Malbranchea pulchella produces a single extracellular, alkaline, serine protease when grown at 45 °C, on 2% casein as sole carbon source. The growth-associated production of protease in submerged cultures was inhibited by addition of glucose, amino acids, or yeast extract. A simple four-step purification which yields homogeneous protease in 78% yield is described. The protease has an isoelectric point of 6.0, a pH optimum of 8.5, and is completely inhibited by serine protease inhibitors. A specificity study with small synthetic ester substrates indicated that the protease preferentially hydrolyzed bonds situated on the carboxyl side of aromatic or apolar amino acid residues which are not β-branched, positively charged or of the D configuration. Peptidase substrates and others such as N-acetyl-L-tyrosine-ethyl ester were not hydrolyzed. The protease was stable over a broad range of pH (6.5–9.5 at 30 °C, 20 h), and was particularly thermostable (t1/2 = 110 min at 73 °C, pH 7.4) in the presence of Ca2+ (10 mM). Macromolecules and Ca2+ also provide protection against the significant autolysis which occurs at pure protease concentrations greater than 0.01 mg/ml, as well as against surface denaturation which is enhanced by the presence of a silicone antifoam agent. Hence the stability of protease in submerged cultures is rationalized.

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