Substrate Specificity and Thermostability of the Dehairing Alkaline Protease from Bacillus pumilus
Substrate Specificity and Thermostability of the Dehairing Alkaline Protease from Bacillus pumilus
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DOI：
10.1007/s12010-008-8497-4
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发表时间：
2009-01
影响因子：
3
通讯作者：
Min Wan;Hong Wang;Yi-Zheng Zhang;H. Feng
中科院分区：
文献类型：
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作者：
Min Wan;Hong Wang;Yi-Zheng Zhang;H. Feng
短小芽孢杆菌（Bacilluspumilus）的碱性蛋白酶（DHAP）在皮革脱毛中显示出很好的应用前景。为了更好地了解其催化性能的应用，底物特异性和热稳定性进行了研究，使用五种天然蛋白质和九种合成肽。结果表明，DHAP对5种蛋白质均有不同程度的水解作用。胶原蛋白是动物皮肤的一种成分，比酪蛋白、纤维蛋白和明胶更耐水解。在合成肽中，该酶主要对四肽底物表现出活性，P1位点的催化效率为Phe>Leu>Ala，但AAVA-pN的Km值远低于AAPL-pN和AAPF-pN。对于三肽底物，P1位点的较小侧链基团（Gly）不被DHAP水解。该酶在60 °C以下显示出良好的热稳定性，而在70 °C以上很快失去活性。热稳定性很大程度上取决于金属离子，特别是Ca 2+，虽然其他离子，如Mg 2+，Mn 2+，Co 2+可以在有限的时间内保持一定的稳定性。Cu 2+、Fe 2+以及Al 3+并不支持酶在60 °C下保持活性，即使在5分钟内也是如此。此外，所选择的金属离子可与钙离子配位，提高或破坏DHAP的热稳定性。
An alkaline protease (DHAP) fromBacillus pumilushas 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, althoughkmvalue 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.