Substrate Specificity and Thermostability of the Dehairing Alkaline Protease from Bacillus pumilus

• Published in: Applied biochemistry and biotechnology Vol. 159; no. 2; pp. 394 - 403
• Main Authors: Wan, Min-Yuan, Wang, Hai-Yan, Zhang, Yi-Zheng, Feng, Hong
• Format: Journal Article
• Language: English
• Published: New York New York : Humana Press Inc 01.11.2009; Humana Press Inc; Springer Nature B.V
• Subjects: Bacillus - enzymology; Bacillus pumilus; Bacteria; Bacterial Proteins - chemistry; Biochemistry; Biotechnology; calcium; Chemistry; Chemistry and Materials Science; cobalt; Collagen - chemistry; dehairing; Endopeptidases - chemistry; enzymatic hydrolysis; Enzyme Activation; Enzyme Stability; Hair - chemistry; hides and skins; magnesium; manganese; Peptides; Proteases; proteinases; Proteins; Substrate Specificity; Tanning - methods; temperature; thermal stability; Thermostability; Dehairing; Metal ion; Substrate specificity; Alkaline protease
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-008-8497-4

An alkaline protease (DHAP) from Bacillus pumilus has shown great potential in hide dehairing. To get better insights on its catalytic properties for application, the substrate specificity and thermostability were investigated using five natural proteins and nine synthetic peptides. The results showed that DHAP could hydrolyze five proteins tested here in different specificity. Collagen, a component of animal skin, was more resistant to hydrolysis than casein, fibrin, and gelatin. Among the synthetic peptides, the enzyme showed activity mainly with tetrapeptide substrates with the catalytic efficiency in order of Phe>Leu>Ala at P1 site, although k m value for AAVA-pN is much lower than that for AAPL-pN and AAPF-pN. With tripeptide substrates, smaller side-chain group (Gly) at P1 site was not hydrolyzed by DHAP. The enzyme showed good thermostability below 60 °C, and lost activity so quickly above 70 °C. The thermostability was largely dependent on metal ion, especially Ca²⁺, although other ions, like Mg²⁺, Mn²⁺, and Co²⁺, could sustain stability at certain extent within limited time. Cu²⁺, Fe²⁺, as well as Al³⁺, did not support the enzyme to retain activity at 60 °C even in 5 min. In addition, the selected metal ions could coordinate calcium in improvement or destruction of thermostability for DHAP.