生物技术通报 ›› 2022, Vol. 38 ›› Issue (8): 252-260.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1422

• 研究报告 • 上一篇    下一篇

耐热漆酶ba4基因鉴定与酶学性质分析

王雨辰1,2(), 丁尊丹2, 关菲菲2, 田健2, 刘国安1(), 伍宁丰2()   

  1. 1.西北师范大学生命科学学院,兰州 730070
    2.中国农业科学院生物技术研究所,北京 100081
  • 收稿日期:2021-11-13 出版日期:2022-08-26 发布日期:2022-09-14
  • 作者简介:王雨辰,女,硕士研究生,研究方向:细胞生物学和环保酶工程;E-mail: 13519669461@163.com
  • 基金资助:
    国家重点研发计划(2021YFC2100300)

Identification of the Thermostable Laccase Gene ba4 and Characterization of Its Enzymatic Properties

WANG Yu-chen1,2(), DING Zun-dan2, GUAN Fei-fei2, TIAN Jian2, LIU Guo-an1(), WU Ning-feng2()   

  1. 1. College of Life Science,Northwest Normal University,Lanzhou 730070
    2. Biotechnology Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100081
  • Received:2021-11-13 Published:2022-08-26 Online:2022-09-14

摘要:

漆酶(EC 1.10.3.2)是一种氧化还原酶,在有毒和致癌化合物的氧化降解方面具有应用价值。通过序列分析,从UniParc数据库中筛选到耐热的漆酶基因ba4,其全长1 860 bp,编码620个氨基酸。通过最适反应温度回归预测模型(PMT)预测出BA4是耐热的漆酶,并在NCBI蛋白数据库中进行比对分析,其与来源于Klebsiella michiganensis的铜抗性系统多铜氧化酶(STW26195.1)相似性为58.75%,证明漆酶ba4是Copper_res_A超家族新的漆酶基因。将其全序列合成并在大肠杆菌BL21(DE3)中异源表达并纯化,性质测定结果表明该酶在温度45-65℃之间均有较高的酶活,最适温度为50℃,最适pH为5.5。以ABTS为底物测定米氏常数(Km)(2 144.5±358.5)μmol/L,kcat为(44.06±3.14)min-1,最大反应速率(Vmax)为623.2 μmol/(min·g),kcat/Km为(0.020 9±0.002)L/(μmol·min)。漆酶BA4(60-70 U/L)在50℃条件下与玉米赤霉烯酮(0.1 mg/mL)反应2 h,降解率达到了90%以上;漆酶BA4(70-80 U/L)在40℃和50℃条件下与棉酚反应(1 mg/mL)1 h,降解率均为30%。漆酶BA4良好的酶学性质以及对玉米赤霉烯酮和棉酚有效降解为酶的应用奠定了良好的基础。

关键词: 漆酶, 酶学性质, 玉米赤霉烯酮, 棉酚

Abstract:

Laccase(EC 1.10.3.2)is an oxidoreductase,and it has application value in the oxidative degradation of toxic and carcinogenic compounds. Through sequence analysis,thermostable laccase gene ba4 was screened from the UniParc database. The gene full length was 1,860 bp,encoding 620 amino acids. By optimal reaction temperature regression prediction model(PMT),it was predicted that BA4 laccase was a thermostable enzyme. Comparison analysis in the NCBI protein database showed 58.75% similarity with the multicopper oxidase of copper-resistance system from Klebsiella michiganensis(STW26195.1),proving that laccase ba4 was new laccase gene in the Copper_res_A superfamily. The entire sequence was synthesized and heterologously expressed in Escherichia coli BL21(DE3)and purified. The results of characterization demonstrated that the enzyme had high activity at a temperature of 45-65℃,the optimal temperature and pH were 50℃ and 5.5. The Michaelis constant Km measured with ABTS as the substrate was(2 144.5±358.5)μmol/L,the kcat was(44.06±3.14)min-1,the maximum reaction rate Vmax was 623.2 μmol/(min·g),and the kcat/Km was(0.020 9±0.002)L/(μmol·min). The degradation rate reached > 90% while laccase BA4(60-70 U/L)reacted with zearalenone(0.1 mg/mL)at 50℃ for 2 h. The degradation rate was 30% when laccase BA4(70-80 U/L)reacted with gossypol(1 mg/mL)at 40 and 50℃ for 1 h. The promising enzymatic properties of laccase BA4 and the effective degradation of zearalenone and gossypol have laid a good foundation for the application of the enzyme.

Key words: laccase, enzymatic property, zearalenone, gossypol