生物技术通报 ›› 2022, Vol. 38 ›› Issue (8): 252-260.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1422
王雨辰1,2(), 丁尊丹2, 关菲菲2, 田健2, 刘国安1(), 伍宁丰2()
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: 基金资助:
WANG Yu-chen1,2(), DING Zun-dan2, GUAN Fei-fei2, TIAN Jian2, LIU Guo-an1(), WU Ning-feng2()
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。
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.
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 |
No. | 1 | 2 | 3 | 4 | 5 |
---|---|---|---|---|---|
棉酚Gossypol/(mg·mL-1) | 0.00 | 0.05 | 0.1 | 0.15 | 0.2 |
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 |
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 |
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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