摘要
【目的】通过联苯水解酶的表达,纯化,化学修饰以及酶学性质研究,以期了解水解酶的结构和功能,为其定向改造,获得高效的和能够拓宽底物范围的新突变酶奠定基础。【方法】将水解酶基因在大肠杆菌BL21(Escherichia coli BL21)中进行异源表达,表达产物经离子交换层析以及凝胶层析后进行酶学特性研究,同时通过圆二色谱法对该酶二级结构与热稳定性之间的关系进行分析。【结果】经Q Sepharose和Sephacryl S-300两步纯化获得了电泳纯的联苯水解酶(biphenyl hydrolase,BphD),SDS-PAGE鉴定其单体为31 kDa,Sepharose 12凝胶柱层析分析其为四聚体。该酶最适反应温度和最适pH分别为80℃和9,在pH4-11的缓冲液中酶活相对稳定。该酶在60℃温浴1 h,酶活剩余90%,而在70℃温浴时,半衰期为1 h,是迄今为止在红球菌中报道的唯一一例热稳定性高的水解酶。圆二色谱显示该酶以α-helix占主导地位,当温度升高到70℃时,其二级结构发生了明显的变化,75℃和80℃时BphD的结构已经遭到严重的破坏。序列比对表明:Ser,His,Asp以及Trp残基在几种同源水解酶中相对保守。化学修饰表明Ser,His,Asp在R04水解酶催化底物水解过程中担任重要角色,Trp残基可能位于酶的活性中心,并作为关键氨基酸参与底物的水解过程。【结论】获得了电泳纯的BphD,其pH稳定性以及耐高温特性在同源水解酶中较为罕见,色氨酸关键氨基酸的地位也为进一步了解该酶转化底物的机制奠定了一定的基础。
[Objective]Biphenyl hydrolase（BphD） is an enzyme of the biphenyl biodegradation pathway in Rhodococcus sp.R04.Expression,purification and properties of BphD,together with chemical modification were investigated.[Method]Using heat-induced expression vector pBV220,we expressed bphD heterologously in E.coli BL21（DE3）.The products were purified by chromatography,and the purified enzyme was used to study its functions and properties by circular dichroism.[Results] BphD has been purified to homogeneity with a final purification fold of 5.02.With 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid（HOPDA） as a substrate,the optimum pH and temperature of BphD are 9 and 80℃,respectively and the Km is 0.44 μmol /L.The enzyme had remaining 90% of activity incubating for 1 hour at 60℃,and had a half-life of 1 hour at 70℃,making it the most thermostable biphenyl hydrolase reported in Rhodococcus.Secondary structure of BphD has undergone great changes with the temperature up to 70℃,and has been severely damaged at 80℃.The sequence alignment between BphD and other homologous hydrolases as well as chemical modification of BphD indicated that Ser,His and Asp play important roles in the hydrolysis of HOPDA.In addition,Trp residue may be near the active center of enzyme as the key amino acid involved in substrate hydrolysis,which is not previously reported in the literature.[Conclusion] BphD with pH stability and thermostability is quite rare in the homologous hydrolases in Rhodococcus.The key status of Ser,His,Asp and Trp will build a basis for more learnings of the transformation mechanism of BphD.
出处
《微生物学报》
CAS
CSCD
北大核心
2010年第12期1633-1641,共9页
Acta Microbiologica Sinica
基金
国家自然科学基金项目(30800030)
山西省青年科技基金(207021030)
山西省基金(2007031003)~~
关键词
联苯水解酶
热稳定性
二级结构
化学修饰
Biphenyl hydrolase
thermostability
secondary structure
chemical modification
