氨基酸脱氢酶突变体的高通量筛选方法  

High-Throughput Screening Method for Amino Acid Dehydrogenase Mutants

作　　者：任红 谢莹莹 黄锦婷 金钰莹 范文静 黄桂东 钟先锋 REN Hong;XIE Yingying;HUANG Jinting;JIN Yuying;FAN Wenjing;HUANG Guidong;ZHONG Xianfeng(School of Food Science and Engineering,Foshan University,Foshan 528000,China;Guangdong Traditional Fermented Food Engineering Technology Research Center,Foshan 528000,China;Foshan Brewing Engineering Technology Research Center,Foshan 528000,China)

机构地区：[1]佛山科学技术学院食品科学与工程学院,广东佛山528000 [2]广东省传统发酵食品工程技术研究中心,广东佛山528000 [3]佛山市酿造工程技术研究中心,广东佛山528000

出　　处：《化学与生物工程》2024年第6期21-28,共8页Chemistry & Bioengineering

基　　金：国家自然科学基金项目(32302023);广东省基础与应用基础研究基金区域联合基金项目(2019A1515110036,2019A1515110973);广东省教育厅特色创新项目(2023KTSCX131);佛山科学技术学院高层次人才科研启动经费项目;广东省大学生创新创业训练计划项目(S202211847108)。

摘　　要：基于显色体系和破壁条件的优化,构建了酶解破壁与显色反应相结合的氨基酸脱氢酶突变体高通量筛选方法。以颜色和吸光度表征内消旋-2,6-二氨基庚二酸脱氢酶(StDAPDH)酶活,以碘硝基四唑紫(INT)-吩嗪硫酸甲酯(PMS)为显色剂,通过其与StDAPDH的偶联反应优化了显色体系;以StDAPDH酶活为评价指标,在单因素实验的基础上,采用响应面法探究了菌体量、破壁时间、溶菌酶加量对菌体破壁效果的影响。确定最优显色体系为:INT浓度0.1 mg·mL^(-1)、PMS浓度0.5μg·mL^(-1)、检测波长510 nm;确定最优菌体破壁条件为:30 mL OD 600值为1.5的菌悬液,离心后用10 mL PBS缓冲液重悬菌体沉淀,加入0.2 mg·mL^(-1)的溶菌酶,在37℃下破壁50 min。该方法简单高效,为氨基酸脱氢酶突变体的高通量筛选提供了一定参考。In this paper,based on the optimizations of color system and wall-breaking conditions,we established a high-throughput screening method for amino acid dehydrogenase mutants that combines enzymatic wall-breaking and coloring.Through the characterization of meso-2,6-diaminopimelate dehydrogenase(StDAPDH)enzyme activity by color and absorbance,using iodonitrotetrazolium chloride(INT)-phenazine methosulfate(PMS)as a chromogenic agent,we optimized the color system by coupling reaction of INT-PMS with StDAPDH.Moreover,taking StDAPDH enzyme activity as an evaluation index,based on single-factor experiments,we investigated the effects of bacteria volume,wall-breaking time,and lysozyme dosage on the wall-breaking effect by response surface methodologies.The optimal color system is determined as follows:the INT concentration is 0.1 mg·mL^(-1),the PMS concentration is 0.5μg·mL^(-1),and the detection wavelength is 510 nm.The optimal wall-breaking conditions are determined as follows:30 mL bacterial suspension with OD 600 value of 1.5 are centrifugally collected and then re-suspended with 10 mL PBS buffer,0.2 mg·mL^(-1)lysozyme is added,and the bacteria walls are broken for 50 min at 37℃.The established method is simple and efficient,which provides a certain reference for high-throughput screening of amino acid dehydrogenase mutants.

关 键 词：显色体系 破壁 高通量筛选 氨基酸脱氢酶 

分 类 号：Q5[生物学—生物化学]