耐热漆酶ba4基因鉴定与酶学性质分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-1422

摘要/Abstract

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（K_m）（2 144.5±358.5）μmol/L,k_cat为（44.06±3.14）min^-1,最大反应速率（V_max）为623.2 μmol/（min·g）,k_cat/K_m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 K_m measured with ABTS as the substrate was（2 144.5±358.5）μmol/L,the k_catwas（44.06±3.14）min^-1,the maximum reaction rate V_max was 623.2 μmol/（min·g）,and the k_cat/K_mwas（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

王雨辰, 丁尊丹, 关菲菲, 田健, 刘国安, 伍宁丰. 耐热漆酶ba4基因鉴定与酶学性质分析[J]. 生物技术通报, 2022, 38(8): 252-260.

WANG Yu-chen, DING Zun-dan, GUAN Fei-fei, TIAN Jian, LIU Guo-an, WU Ning-feng. Identification of the Thermostable Laccase Gene ba4 and Characterization of Its Enzymatic Properties[J]. Biotechnology Bulletin, 2022, 38(8): 252-260.

图/表 9

表1 漆酶酶活测定体系

Table 1 Determination system for laccase enzyme activity

溶液 Solution	加入量 Added amount/μL
A液：0.2 mol/L Na₂HPO₄-0.1 mol/L 柠檬酸（pH 6.0）	750
B液：5 mmol/L ABTS	200
BA4蛋白	50

表2 标定棉酚所用浓度

Table 2 Concentrations of gossypol in marking the standa-rd curve

No.	1	2	3	4	5
棉酚Gossypol/（mg·mL^-1）	0.00	0.05	0.1	0.15	0.2

图1 棉酚标准曲线

Fig. 1 Gossypol standard curve

表3 标定玉米赤霉烯酮所用浓度

Table 3 Concentrations of ZEN in marking the standard curve

No.	1	2	3	4	5	6
玉米赤霉烯酮浓度 ZEN/（mg·mL^-1）	0.0000	0.0625	0.1250	0.2500	0.5000	1.0000

图2 玉米赤霉烯酮标准曲线

Fig. 2 ZEN standard curve

图3 漆酶BA4微好氧发酵20 h检测 M：蛋白质分子质量标准;1：IPTG诱导漆酶BA4破碎上清;2：IPTG诱导漆酶BA4破碎沉淀;3：NTA-200洗脱液

Fig. 3 Expression of BA4 after micro aerobic fermentation for 20 h M：Protein marker;1：supernatant of crushed IPTG-induced laccase BA4;2：precipitation of crushed IPTG-induced laccase BA4;3：NTA-200 eluant

图4 温度和pH对漆酶BA4的影响

Fig. 4 Effects of temperarure and pH on BA4

图5 漆酶 BA4 的米氏方程曲线

Fig. 5 Michaelis-menten equation curve of laccase BA4

图6 漆酶BA4对ZEN和棉酚的降解作用 A：色谱图,CK：ZEN对照品;处理组：漆酶BA4分别在 40℃和50℃条件下处理ZEN;B：ZEN相对降解率,漆酶BA4分别在40℃和50℃条件下处理ZEN以色谱图峰面积计算相对降解率;C：色谱图,CK：棉酚对照品;处理组：漆酶BA4分别在 40℃和50℃条件下处理棉酚;D：棉酚相对降解率,漆酶BA4分别在40℃和50℃条件下处理棉酚以色谱图峰面积计算相对降解率

Fig. 6 Degradation of ZEN and gossypol by laccase BA4 A：Chromatogram. CK：ZEN reference substance;treatment group：laccase BA4 treating ZEN at 40℃ and 50℃,respectively. B：Relative degradation rate of ZEN. Treating ZEN with laccase BA4 at 40℃ and 50℃,respectively,and the relative degradation rate was calculated based on the peak area of the chromatogram. C：Chromatogram. CK：Gossypol reference substance. Treatment group：Laccase BA4 treated gossypol at 40℃ and 50℃,respectively. D：Relative degradation rate of gossypol. Laccase BA4 was treated with gossypol at 40℃ and 50℃,respectively,and the relative degradation rate was calculated based on the peak area of the chromatogram

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