Institute of Animal Nutrition, Sichuan Agricultural University, Ya'an, Sichuan, PR China.
PLoS One. 2013;8(3):e58393. doi: 10.1371/journal.pone.0058393. Epub 2013 Mar 5.
The main keratinase (kerA) gene from the Bacillus licheniformis S90 was optimized by two codon optimization strategies and expressed in Pichia pastoris in order to improve the enzyme production compared to the preparations with the native kerA gene. The results showed that the corresponding mutations (synonymous codons) according to the codon bias in Pichia pastoris were successfully introduced into keratinase gene. The highest keratinase activity produced by P. pastoris pPICZαA-kerAwt, pPICZαA-kerAopti1 and pPICZαA-kerAopti2 was 195 U/ml, 324 U/ml and 293 U/ml respectively. In addition, there was no significant difference in biomass concentration, target gene copy numbers and relative mRNA expression levels of every positive strain. The molecular weight of keratinase secreted by recombinant P. pastori was approx. 39 kDa. It was optimally active at pH 7.5 and 50°C. The recombinant keratinase could efficiently degrade both α-keratin (keratin azure) and β-keratin (chicken feather meal). These properties make the P. pastoris pPICZαA-kerAopti1 a suitable candidate for industrial production of keratinases.
为了提高酶的产量，与使用天然 kerA 基因的制剂相比，我们对来自地衣芽孢杆菌 S90 的主要角蛋白酶（kerA）基因进行了两种密码子优化策略的优化，并在毕赤酵母中进行了表达。结果表明，成功将根据毕赤酵母密码子偏性的相应突变（同义密码子）引入角蛋白酶基因中。毕赤酵母 pPICZαA-kerAwt、pPICZαA-kerAopti1 和 pPICZαA-kerAopti2 产生的角蛋白酶活性最高分别为 195 U/ml、324 U/ml 和 293 U/ml。此外，每个阳性菌株的生物量浓度、靶基因拷贝数和相对 mRNA 表达水平均无显著差异。重组毕赤酵母分泌的角蛋白酶的分子量约为 39 kDa。它在 pH7.5 和 50°C 时具有最佳活性。重组角蛋白酶能有效降解α-角蛋白（角蛋白azure）和β-角蛋白（鸡毛粉）。这些特性使毕赤酵母 pPICZαA-kerAopti1 成为角蛋白酶工业生产的合适候选者。
