在中异源表达细胞外蛋白酶 pAsPs。

Heterologous Expression of Extracellular Proteinase pAsPs of in .

机构信息

Laboratory of Molecular Biotechnology, The Institute of Cytology and Genetics, SB RAS, 630090 Novosibirsk, Russia.

Kurchatov Genomic Center of the Institute of Cytology and Genetics, SB RAS, 630090 Novosibirsk, Russia.

出版信息

Int J Mol Sci. 2022 Nov 30;23(23):15035. doi: 10.3390/ijms232315035.

DOI:10.3390/ijms232315035

PMID:36499360

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9739362/

Abstract

Neutral protease pAsPs gene was obtained by sequence optimization of NpI protease from pAsPs was for the first time integrated in the genome of yeast strain T07, and then produced in a 5 L bioreactor with an enzyme yield of 150,800 U/mL of culture liquid towards casein. The specific activity of the pAsPs was 7,657,000 U/mg toward casein, 2320 U/mg toward hemoglobin, and 25,344 U/mg toward azocasein per 1 mg of the protein. The enzyme was found to be inhibited by Cu. Optimal activity pH was shown in the range of pH 6.5-8.0, and optimal temperature-50-60 °C. The molecular mass of the recombinant protease pAsPs was shown to be 67.5 kDa. Mass-spectrometric analysis confirmed the identity of the amino acid sequence of the obtained pAsPs preparation with the predicted sequence, with 17% coverage and protein score 288. Thus, the novel neutral protease pAsPs is a promising candidate for large-scale use in manufacturing, including the food industry.

摘要

中性蛋白酶 pAsPs 基因是通过对 NpI 蛋白酶的序列优化得到的，首次将 pAsPs 整合到酵母菌株 T07 的基因组中，然后在 5L 生物反应器中生产，酶活达到 150800U/mL 对酪蛋白。pAsPs 的比活为 7657000U/mg 对酪蛋白，2320U/mg 对血红蛋白，25344U/mg 对偶氮酪蛋白，每毫克蛋白。该酶被发现被 Cu 抑制。最佳活性 pH 范围为 pH6.5-8.0，最佳温度为 50-60°C。重组蛋白酶 pAsPs 的分子量为 67.5kDa。质谱分析证实了所获得的 pAsPs 制剂的氨基酸序列与预测序列的同一性，覆盖率为 17%，蛋白评分 288。因此，新型中性蛋白酶 pAsPs 是大规模应用于制造的有前途的候选者，包括食品工业。