BACKGROUND Trypsin has many applications in food and pharmaceutical manufacturing. Although commercial trypsin is usually extracted from porcine pancreas, this source carries the risks of infectivity and immunogenicity. Microbial Streptomyces griseus trypsin (SGT) is a prime alternative because it possesses efficient hydrolysis activity without such risks. However, the remarkable hydrolysis efficiency of SGT causes autolysis, and five autolysis sites, R21, R32, K122, R153, and R201, were identified from its autolysate. RESULTS The tbcf (K101A, R201V) mutant was screened by a directed selection approach for improved activity in flask culture (60.85 ± 3.42 U mL-1, increased 1.5-fold). From the molecular dynamics simulation, in the K101A/R201V mutant the distance between the catalytical residues D102 and H57 was shortened to 6.5 Å vs 7.0 Å in the wild type, which afforded the improved specific activity of 1527.96 ± 62.81 U mg-1. Furthermore, the production of trypsin was increased by 302.8% (689.47 ± 6.78 U mL-1) in a 3-L bioreactor, with co-overexpression of chaperones SSO2 and UBC1 in Pichia pastoris. CONCLUSIONS SGT protein could be a good source of trypsin for insulin production. As a result of the hydrolysates analysis and direct selection, the activity of the tbcf (K101A, R201V) mutant increased 1.5-fold. Furthermore, the production of trypsin was improved threefold by overexpressing chaperone protein in Pichia pastoris. Future studies should investigate the application of SGT to insulin and pharmaceutical manufacturing.

中文翻译：

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改进灰链霉菌胰蛋白酶的生产，以酶促方式处理胰岛素前体。

背景技术胰蛋白酶在食品和药物制造中具有许多应用。尽管通常从猪胰腺中提取商业胰蛋白酶，但是这种来源具有传染性和免疫原性的风险。微生物灰链霉菌胰蛋白酶（SGT）是一种主要的替代选择，因为它具有有效的水解活性而没有此类风险。但是，SGT出色的水解效率会导致自溶，并从其自溶物中发现了五个自溶位点R21，R32，K122，R153和R201。结果通过定向选择方法筛选了tbcf（K101A，R201V）突变体，以提高烧瓶培养的活性（60.85±3.42 U mL-1，增加1.5倍）。从分子动力学模拟，在K101A / R201V突变体中，催化残基D102和H57之间的距离缩短到6.5Åvs. 7。在野生型中为0Å，比活性提高了1527.96±62.81 U mg-1。此外，在3-L生物反应器中，胰蛋白酶的产量增加了302.8％（689.47±6.78 U mL-1），并且伴有伴侣蛋白SSO2和UBC1在毕赤酵母中的过表达。结论SGT蛋白可能是胰岛素生产中胰蛋白酶的良好来源。水解产物分析和直接选择的结果是，tbcf（K101A，R201V）突变体的活性提高了1.5倍。此外，通过在巴斯德毕赤酵母中过表达伴侣蛋白，胰蛋白酶的产量提高了三倍。未来的研究应调查SGT在胰岛素和药物生产中的应用。47±6.78 U mL-1）在3-L生物反应器中，伴有伴侣蛋白SSO2和UBC1在毕赤酵母中的共表达。结论SGT蛋白可能是胰岛素生产中胰蛋白酶的良好来源。水解产物分析和直接选择的结果是，tbcf（K101A，R201V）突变体的活性提高了1.5倍。此外，通过在巴斯德毕赤酵母中过表达伴侣蛋白，胰蛋白酶的产量提高了三倍。未来的研究应调查SGT在胰岛素和药物生产中的应用。47±6.78 U mL-1）在3-L生物反应器中，伴有伴侣蛋白SSO2和UBC1在毕赤酵母中的共表达。结论SGT蛋白可能是胰岛素生产中胰蛋白酶的良好来源。水解产物分析和直接选择的结果是，tbcf（K101A，R201V）突变体的活性提高了1.5倍。此外，通过在巴斯德毕赤酵母中过表达伴侣蛋白，胰蛋白酶的产量提高了三倍。未来的研究应调查SGT在胰岛素和药物生产中的应用。此外，通过在巴斯德毕赤酵母中过表达伴侣蛋白，胰蛋白酶的产量提高了三倍。未来的研究应调查SGT在胰岛素和药物生产中的应用。此外，通过在巴斯德毕赤酵母中过表达伴侣蛋白，胰蛋白酶的产量提高了三倍。未来的研究应调查SGT在胰岛素和药物生产中的应用。