产角蛋白酶毕赤酵母工程菌的构建及酶学性质
Construction and characterization of recombinant Pichia pastoris strain with keratinase production

将Brevibacillus brevis US575角蛋白酶基因kerUS进行密码子优化，并在毕赤酵母GS115中表达，以提高角蛋白酶产量。结果表明：优化的基因可在毕赤酵母中成功表达，重组角蛋白酶的分子质量约30 ku，最佳反应条件为pH 9.5和60℃，1 mmol/L的Mn2+、Ba2+对角蛋白酶活性有促进作用。此外，重组角蛋白酶易降解干酪素和β角蛋白（羽毛粉），酶促反应最大速度vmax=0.019 g/（L·min）， Km=4.64 g/L，比活力=440 U/mg。研究获得的产角蛋白酶重组毕赤酵母具有较好的应用前景。
The codon of keratinase gene kerUS from the Brevibacillus brevis US575 was optimized and expressed in Pichia pastoris GS115 to improve keratinase production. Results showed that the gene optimized was successfully expressed in Pichia pastoris. The molecular weight of recombinant keratinase was about 30 ku. The optimal reaction conditions of keratinase were pH 9.5 and 60℃. The keratinase activity was improved by 1 mmol/L Mn2+ and Ba2+. In addition,the recombinant keratinase efficiently degraded α-keratin (keratin azure) and β-keratin (feather powder). The maximum speed of the enzyme reaction,the constant Km,and the specific activity was 0.019 g/(L·min）,4.64 g/L,and 440 U/mg,respectively. It is indicated that the novel recombinant Pichia pastoris strain with keratinase production has a good application prospect.