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构建一种重组中性蛋白酶I以提高其在酸性pH下的酶活性。

Engineering a recombination neutral protease I from to improve enzyme activity at acidic pH.

作者信息

Hu Yucheng, Li Tong, Tu Zhui, He Qinghua, Li Yanping, Fu Jinheng

机构信息

State Key Laboratory of Food Science and Technology, Nanchang University No. 235 Nanjing East Road Nanchang 330047 China

Sino-German Joint Research Institute, Nanchang University No. 235 Nanjing East Road Nanchang 330047 China.

出版信息

RSC Adv. 2020 Aug 19;10(51):30692-30699. doi: 10.1039/d0ra05462c. eCollection 2020 Aug 17.

DOI:10.1039/d0ra05462c
PMID:35516032
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9056373/
Abstract

Extracellular neutral proteases (NPs) in () play a role in hydrolyzing soybean proteins into smaller peptides at pH about 7.5. The optimum pH of moromi fermentation (The second stage of soy sauce fermentation.) is 4.5-5.5. NPI is acid sensitive. To decrease the pH optimum of NPI, we got a mutant NPI-Y122FK246ID382V from the error-prone PCR library that showed optimal activity at pH 5.5. The specific activity at 40 °C of the NPI-Y122FK246ID382V mutant was 1383.50 U mg, which was 2.75-fold that of wild-type (503.09 U mg). The Michaelis constants of the mutant decreased from 22.13 mM (wild-type) to 19.98 mM (NPI-Y122FK246ID382V). The residues at positions 122 and 246 are important in influencing hydrolytic activity at pH 5.5 through site-directed mutagenesis. And the pH optimum of double amino acid mutants (Y122FK246I) shifted dramatically to an acidic pH compared to those of single amino acid substitution. Molecular models and structural comparisons of native and mutant provided further insight on the basis to improve catalytic efficiency at acidic pH. These results indicated that we modified the neutral protease I of , which can effectively improve the application of the neutral protease in industrial production, and finally lay the foundation for improving the utilization rate of raw protein.

摘要

（）中的细胞外中性蛋白酶（NPs）在pH约7.5时发挥作用，将大豆蛋白水解成较小的肽。酱醪发酵（酱油发酵的第二阶段）的最适pH为4.5 - 5.5。NPI对酸敏感。为了降低NPI的最适pH，我们从易错PCR文库中获得了一个突变体NPI - Y122F K246I D382V，其在pH 5.5时表现出最佳活性。NPI - Y122F K246I D382V突变体在40℃时的比活性为1383.50 U mg，是野生型（503.09 U mg）的2.75倍。突变体的米氏常数从22.13 mM（野生型）降至19.98 mM（NPI - Y122F K246I D382V）。通过定点诱变，122位和246位的残基在影响pH 5.5时的水解活性方面很重要。与单氨基酸取代相比，双氨基酸突变体（Y122F K246I）的最适pH显著向酸性pH偏移。天然型和突变体的分子模型及结构比较为提高酸性pH下的催化效率提供了进一步的依据。这些结果表明，我们对（）的中性蛋白酶I进行了改造，可有效提高中性蛋白酶在工业生产中的应用，最终为提高原料蛋白利用率奠定基础。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/64e5/9056373/9b91e8f0363b/d0ra05462c-f3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/64e5/9056373/4a09110546ed/d0ra05462c-f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/64e5/9056373/707fddd5d230/d0ra05462c-f2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/64e5/9056373/9b91e8f0363b/d0ra05462c-f3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/64e5/9056373/4a09110546ed/d0ra05462c-f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/64e5/9056373/707fddd5d230/d0ra05462c-f2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/64e5/9056373/9b91e8f0363b/d0ra05462c-f3.jpg

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