Keratinase is capable of distinctive degradation of keratin, which provides an eco-friendly approach for keratin waste management towards sustainable development. In this study, the recombinant keratinase (KERBP) from Brevibacillus parabrevis was successfully expressed in Escherichia coli. The purified KERBP had the specific activity of 6005.3 U/mg. It showed remarkable tolerance to various surfactants and also no collagenolytic activity. However, the moderate thermal stability limited its further application. Thus, protein engineering was further adopted to improve its stability. The variants of T218S, S236C and N181D were constructed by site-directed mutagenesis and combinatorial mutagenesis. Compared with the wild type, the t_1/2 at 60 °C for the variants T218S, S236C and N181D were 3.05-, 1.18- and 1-fold increase, respectively. Moreover, the double variants N181D-T218S and N181D-S236C significantly improved thermostability with 5.1 and 2.9 °C increase of T₅₀, and prolonging t_1/2 at 60 °C with 4.09 and 1.54-fold, respectively. And the catalytic efficiency of the T218S and N181D-T218S variants was also significantly improved. Furthermore, the keratinase displayed favorable ability to dehair wool from skin within 7 h, which showed potential in leather dehairing. Our work contributes to a further insight into the thermostability of keratinase and offers a promising alternative for industrial leather application.

角蛋白酶能够使角蛋白独特地降解，从而为角蛋白废物管理实现可持续发展提供了一种生态友好的方法。在该研究中，来自副短杆菌的重组角蛋白酶（KERBP）在大肠杆菌中成功表达。纯化的KERBP具有6005.3U / mg的比活性。它显示出对各种表面活性剂的显着耐受性，也没有胶原蛋白水解活性。但是，适度的热稳定性限制了其进一步的应用。因此，进一步采用蛋白质工程来提高其稳定性。T218S，S236C和N181D的变体通过定点诱变和组合诱变构建。与野生型相比，t _1/2在60°C下，变体T218S，S236C和N181D分别增加了3.05、1.18和1倍。此外，双重变体N181D-T218S和N181D-S236C显着提高了热稳定性，T ₅₀升高了5.1和2.9°C ，在60°C时延长了t _1/2分别为4.09和1.54倍。T218S和N181D-T218S变体的催化效率也显着提高。此外，角蛋白酶显示出在7小时内从皮肤上脱毛的有利能力，这显示了皮革脱毛的潜力。我们的工作有助于进一步了解角蛋白酶的热稳定性，并为工业皮革应用提供有前途的替代方法。