鼠李糖乳杆菌L-乳酸脱氢酶的生物信息学分析和基因克隆  

Bioinformatics Analysis and Gene Cloning of L-Lactate Dehydrogenase from Lactobacillus rhamnosus

作　　者：许黎明 蒋国凤[2] 伍新龄 廖秀芳 劳夏姐 牙韩春 李秀年 XU Liming;JIANG Guofeng;WU Xinling;LIAO Xiufang;LAO Xiajie;YA Hanchun;LI Xiunian(College of Medicine and Health,Industrial Technology Engineering Center for Zhuang&Yao Medicinal Organisms,Botanical Garden of Zhuang&Yao Medicinal Plants,Colaborative Innovation Center of Great Health,Guangxi Vocational&Technical Institute of Industry,Nanning 530001,China;College of Forestry,Guangxi Key Laboratory of Forest Ecology and Conservation,State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources,Guangxi University,Nanning 530004,China)

机构地区：[1]广西工业职业技术学院医药健康学院,壮瑶医药药用生物产业技术工程研究中心,壮瑶医药药用植物园,大健康协同创新中心,广西南宁530001 [2]广西大学林学院,广西森林生态与保育重点实验室,亚热带农业生物资源保护与利用国家重点实验室,广西南宁530004

出　　处：《食品工业科技》2023年第11期153-162,共10页Science and Technology of Food Industry

基　　金：广西科技计划重点研发项目(桂科AB17292001);广西高校中青年教师科研基础能力提升项目(2023KY1314);广西工业职业技术学院科研基金(GYKY2022001A)。

摘　　要：本研究以鼠李糖乳杆菌(Lactobacillus rhamnosus)L-乳酸脱氢酶(Lr-L-LDH)为研究对象,以研究较多的Lr-L-LDH1为对照,对基因组注释的两个Lr-L-LDH1和Lr-L-LDH2基因,进行异同分析。采用在线网站和专业软件对Lr-L-LDH1和Lr-L-LDH2的一级结构、基本特性、亲疏水性、二级结构进行分析和预测,对三级结构进行同源建模以及酶和底物的分子对接分析,并对酶的编码基因进行系统发育分析,克隆表达及酶活性检测。结果显示:相较于Lr-L-LDH1,Lr-L-LDH2有着相似的分子特性,二级和三级结构,但Lr-L-LDH2编码序列短,氨基酸序列同源性低(48.08%),有不同的进化地位;Lr-L-LDH2也含有乳酸脱氢酶催化活性位点序列和保守的NAD^(+)结合位点序列(GXGXXG),能够形成典型的活性三维口袋域,是NAD^(+)依赖型四聚体结构L-乳酸脱氢酶,在细胞中需要果糖1,6-二磷酸(FBP)来激活,催化丙酮酸还原为L-乳酸;体外克隆表达和酶学分析表明,Lr-L-LDH2酶活力极显著低于Lr-L-LDH1(P<0.01)。Lr-L-LDH1和Lr-L-LDH2都具有乳酸脱氢酶活性,推测二者在乳酸表达调控中相互作用,对鼠李糖乳杆菌L-乳酸脱氢酶的分子改造应同时考虑Lr-L-LDH1和Lr-L-LDH2的作用,研究对乳酸发酵工业的基因工程改造提供分子基础和科学依据。In this study,L-lactate dehydrogenase(Lr-L-LDH)from Lactobacillus rhamnosus was investigated.The well-known Lr-L-LDH1 was taken as the control,differences between Lr-L-LDH1 and Lr-L-LDH2 annotated from the genome of L.rhamnosus were analyzed.The primary structure,basic properties,hydrophobicity,secondary structure of Lr-L-LDH1 and Lr-L-LDH2 were predicted and analyzed using online websites and professional software.Homology modeling of the tertiary structure,molecular docking of enzymes and substrates,phylogenetic analysis,in vitro cloning,expression,and enzyme activity assays were further studied.The results showed that,in comparison to Lr-L-LDH1,although Lr-L-LHD2 had similar molecular characteristics,secondary and tertiary structures,Lr-L-LHD2 exhibited differences:A shorter sequences,low amino acid identities(48.08%),and a different phylogenetic status.Lr-L-LHD2 also contained a catalytically active site,a highly conserved NAD^(+)binding site sequence(GXGXXG),a three-dimensional active pocket domain,was an NAD^(+)dependent tetrameric L-lactate dehydrogenase,and be activated by fructose 1,6-diphosphate in the cytoplasm for catalyzing the reduction of pyruvate to L-lactic acid.In vitro enzyme activity was significantly lower than Lr-L-LDH1(P<0.01).In conclusion,both Lr-L-LDH1 and Lr-L-LHD2 showed enzyme activities of L-lactate dehydrogenase,indicating they may regulate the L-lactate metabolism pathway together.Considerations of both Lr-L-LDH1 and Lr-L-LHD2 should be taken for the future molecular modifications of L-LDH in L.rhamnosus,and the study provided the molecular and scientific basis for the gene engineering modifications of lactic acid fermentation industry.

关 键 词：鼠李糖乳杆菌 L-乳酸脱氢酶 生物信息学 进化分析 活性位点 酶活力 

分 类 号：Q819[生物学—生物工程]