烟曲霉单宁酶Kex2位点突变及酶学特性表征

doi:10.7506/spkx1002-6630-20190611-111

摘要/Abstract

摘要： 研究单宁酶AfTanA中Kex2蛋白酶切割位点（K315-R316）对酶学性质的影响。通过对单宁酶AfTanA中Kex2蛋白酶切割位点（K315-R316）进行定点突变，构建单点突变体AfTanR316A和缺失突变体AfTanΔKR。经十二烷基硫酸钠-聚丙烯酰氨凝胶电泳检测发现两突变体Kex2蛋白酶抗性明显增强。经对酶学性质对比发现，两突变体具有较低的催化反应温度和较强的稳定性。单宁酶的最适反应温度由40 ℃（野生型）降低到20 ℃（两突变体）。突变体AfTanΔKR和突变体AfTanR316A在pH 12.0条件下处理1 h后，剩余酶活力由野生型AfTanA的0%提高到85%和60%；而在50 ℃条件下处理1 h后，剩余酶活力由野生型AfTanA的2%提高到40%和21%，上述性质的改变有利于单宁酶在低温条件的应用和酶制剂的生产贮存。突变体AfTanR316A动力学参数Km和Vmax分别为1.149 mmol/L和10.427 mmol/（L·min），AfTanΔKR的动力学参数Km和Vmax分别为1.46 mmol/L和35.84 mmol/（L·min）。此外，柿子汁经野生型单宁酶AfTanA和突变体处理后，多酚含量均提高50%左右。综上所述，单宁酶AfTanA中的Kex2蛋白酶切割位点（K315-R316）对酶的稳定性和催化效率影响显著，改造后的单宁酶AfTanΔKR能够更好地应用在食品工业中。

关键词: 单宁酶；定点突变；Kex2蛋白酶；稳定性；食品酶

Abstract: In this work, we investigated the effect of the Kex2 cleavage sites (K315-R316) on the enzymatic properties of tannase (AfTanA) from Aspergillus fumigates. We constructed a single amino acid mutant (AfTanR316A) and a double amino acid deletion mutant (AfTanΔKR). The two mutants were significantly enhanced for?resistance?to Kex2 proteases. Compared with the wild-type tannase (AfTanA), the mutants exhibited lower catalytic temperature and stronger stability. The optimum reaction temperatures of the mutants and the wild-type tannase were 20 and 40 ℃, respectively. After being treated for 1 h at pH 12.0, AfTanΔKR, AfTanR316A and AfTanA remained 85%, 60% and 0% of their original activity, respectively. After being treated for 1 h at 50 ℃, AfTanΔKR, AfTanR316A and AfTanA remained 40%, 21% and 2% of their original activity, respectively. The improved stability against pH and temperature were beneficial to the application of tannase at low temperatures and its production and storage. The Km and Vmax values of AfTanR316A were determined as 1.149 mmol/L and 10.427 mmol/(L·min). The Km and Vmax values of AfTanΔKR were determined as 1.46 mmol/L and 35.84 mmol/(L·min), respectively. In addition, compared with natural persimmon juice, juices treated separately with AfTanA and the mutants showed a 50% increase in the total phenolic concentration.?In?conclusion, the Kex2 protease cleavage sites (K315-R316) of AfTanA have a significant effect on the stability and catalytic efficiency, and AfTanΔKR could potentially be used in the food industry in the future.

Key words: tannase; site-directed mutation; Kex2 protease; stability; food enzymes

中图分类号:

Q816

卢海强，陈伟，黄蕾，谷新晰，田洪涛. 烟曲霉单宁酶Kex2位点突变及酶学特性表征[J]. 食品科学, 2020, 41(14): 124-130.

LU Haiqiang, CHEN Wei, HUANG Lei, GU Xinxi, TIAN Hongtao. Mutational Analysis of Kex2 Sites and Characterization of Tannase from Aspergillus fumigatus[J]. FOOD SCIENCE, 2020, 41(14): 124-130.

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烟曲霉单宁酶Kex2位点突变及酶学特性表征

Mutational Analysis of Kex2 Sites and Characterization of Tannase from Aspergillus fumigatus

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