Substrate specificity and thermostability of the dehairing alkaline protease from Bacillus pumilus.
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 degrees C, and lost activity so quickly above 70 degrees C. The thermostability was largely dependent on metal ion, especially Ca(2+), although other ions, like Mg(2+), Mn(2+), and Co(2+), could sustain stability at certain extent within limited time. Cu(2+), Fe(2+), as well as Al(3+), did not support the enzyme to retain activity at 60 degrees C even in 5 min. In addition, the selected metal ions could coordinate calcium in improvement or destruction of thermostability for DHAP.
Wan
Min-Yuan
MY
The Sichuan Key Laboratory of Molecular Biology and Biotechnology, College of Life Sciences, Sichuan University, Chengdu 610064, Sichuan, China.
Wang
Hai-Yan
HY
Zhang
Yi-Zheng
YZ
Feng
Hong
H
D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry*; D003094:Collagen / Q000737:chemistry*; D010450:Endopeptidases / Q000737:chemistry*; D004789:Enzyme Activation; D004795:Enzyme Stability; D006197:Hair / Q000737:chemistry*; D013379:Substrate Specificity; D013633:Tanning / Q000379:methods*
D001426:Bacterial Proteins; D003094:Collagen; D010450:Endopeptidases; C087822:alkaline protease