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124 2020, V ol.41, No.14食品科学※生物工程
关于友情的故事烟曲霉单宁酶Kex2位点突变及酶学特性表征
卢海强，陈伟，黄蕾，谷新晰，田洪涛*
（河北农业大学食品科技学院，河北保定071000）
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K m和V max分别为1.149 mmol/L和
10.427 mmol/（L·min），AfTanΔKR的动力学参数K m和V max分别为1.46 mmol/L和35.84 mmol/（L·m
折星星in）。此外，
涉密文件管理办法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
Mutational Analysis of Kex2 Sites and Characterization of Tanna from Aspergillus fumigatus
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LU Haiqiang, CHEN Wei, HUANG Lei, GU Xinxi, TIAN Hongtao*
(College of Food Science and Technology, Hebei Agricultural University, Baoding 071000, China) Abstract: In this work, we investigated the effect of the Kex2 cleavage sites (K315-R316) on the enzymatic properties of tanna (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 proteas.
Compared with the wild-type tanna (AfTanA), the mutants exhibited lower catalytic temperature and stronger stability. The optimum reaction temperatures of the mutants and the wild-type tanna
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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 tanna at low temperatures and its production and storage. The K m and V max values of AfTanR316A were determined as 1.149 mmol/L and
10.427 mmol/(L·min). The K m and V max 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 parately with AfTanA and the mutants showed a 50% increa in the total phenolic concentration. In conclusion, the Kex2 protea cleavage sites (K315-R316) of AfTanA have a significant effect on the stability and catalytic efficiency, and AfTanΔKR could potentially be ud in the food industry in the future.
Keywords: tanna; site-directed mutation; Kex2 protea; stability; food enzymes大岛爱衣留
DOI:10.7506/spkx1002-6630-20190611-111
中图分类号：Q816 文献标志码：A 文章编号：1002-6630（2020）14-0124-07
收稿日期：2019-06-11王安石资料
基金项目：国家自然科学基金青年科学基金项目（31501417）；河北省食品科学与工程学科“双一流”建设资金项目（2016SPGCA18）第一作者简介：卢海强（1981—）（ORCID: 0000-0001-7011-4423），男，副教授，博士，研究方向为食品酶资源开发与利用。
E-mail:********************
*通信作者简介：田洪涛（1963—）（ORCID: 0000-0001-5750-8321），男，教授，博士，研究方向为微生物资源开发与利用。
E-mail:*****************
