产漆酶真菌筛选、鉴定及酶促去除苯酚的条件优化  

Screening and identification of laccase-producing fungi and optimization of the enzymatic conditions for phenol removal

作　　者：岑庆静 万辉 刘薇 李绪 洪妮 付桂明[2,3] 郑洪立[1,3] 刘玉环[1,2] 巫小丹[1,3] CEN Qingjing;WAN Hui;LIU Wei;LI Xu;HONG Ni;FU Guiming;ZHENG Hongli;LIU Yuhuan;WU Xiaodan(Engineering Research Center for Biomass Conversion,Ministry of Education,Nanchang University,Nanchang 330047,China;State Key Laboratory of Food Science and Technology,Nanchang University,Nanchang University,Nanchang 330047,China;International Institute of Food Innovation,Nanchang University,Nanchang 330047,China;Agricultural Products Testing Sub Center of Nanchang Inspection and Testing Center,Nanchang 330005,China)

机构地区：[1]南昌大学生物质转化教育部工程研究中心,江西南昌330047 [2]南昌大学食品科学与技术国家重点实验室,江西南昌330047 [3]南昌大学国际食品创新研究院,江西南昌330047 [4]南昌市检验检测中心农产品检测分中心,江西南昌330005

出　　处：《南昌大学学报（理科版）》2022年第6期621-629,共9页Journal of Nanchang University(Natural Science)

基　　金：江西省博士后项目(2020RC10);江西省主要学科学术和技术带头人培养计划青年人才项目(20212BCJ23042);教育部产学合作协同育人项目(202102152021,202102083005,202101292013)。

摘　　要：以愈创木酚为底物,从自然环境中成功筛选到一株高产漆酶的粗毛革孔菌(Coriolopsis gallica)NCULAC F1。对其漆酶诱导表达条件进行探究,结果表明,9种芳香化合物对NCULAC F1表达漆酶均无显著诱导作用(P>0.05);而硫酸铜具有显著诱导作用(P<0.05),且硫酸铜与2,6-二甲基苯酚具有显著的协同诱导作用(P<0.05);最佳协同诱导条件下,摇瓶发酵5 d漆酶活力达11.29 U·mL-1,比未添加任何诱导剂的对照组显著提高了138.69%(P<0.05)。提取纯化漆酶,进一步探究漆酶对苯酚的去除效果,结果表明,酶促去除苯酚的过程遵循Michaelis-Menten动力学模型,R2为0.9843,km为52.45 mg·L-1,Vmax为181.82 mg·L-1·h-1;最佳催化条件为温度70℃,pH 6.0,加酶量5 U·mL-1,介体2’2-联氨-双(3-乙基苯并噻唑啉-6-磺酸)二胺盐(ABTS)浓度0.6 mmol·L-1,在此条件下,10 min内苯酚去除率达100%。研究结果为粗毛革孔菌漆酶的高效生产和在酚类污染物控制方面的应用提供了理论参考。A laccase producing strain, Coriolopsis gallica NCULAC F1,was successfully screened from natural environment using guaiacol as substrate.The induced expression conditions of laccase from NCULAC F1 were investigated.The results showed that 9 aromatic compounds had no significant induction effect on NCULAC F1 laccase expression(P>0.05);copper sulfate had a significant induction effect(P<0.05),and a significant synergistic induction effect with 2,6-dimethylphenol(P<0.05).Under the optimal synergistic induction condition, the laccase activity reached 11.29 U·mL-1after 5-d shaking flask fermentation, which was significantly increased by 138.69% compared with the control group without any inducer(P<0.05).Laccase was extracted and purified to further explore the removal effect of phenol.The results showed that the removal process of phenol by enzyme followed Michaelis-Menten kinetic model, with R~2=0.98,km=52.45 mg·L-1,and Vmax=181.82 mg·L-1·h-1.The optimized catalytic conditions for laccase to remove phenol were as follows: temperature 70 ℃,pH 6.0,enzyme dosage 5 U·mL-1and 2’2-hydrazine bis(3-ethylbenzothiazoline-6-sulfonic acid) diamine salt(ABTS) concentration 0.6 mmol·L-1.Under these conditions, the removal rate of phenol reached 100% within 10 min.The results provide a theoretical reference for the efficient production of laccase and its application in the control of phenolic pollutants.

关 键 词：粗毛革孔菌 漆酶 协同诱导 酚类污染物 

分 类 号：Q935[生物学—微生物学]