Klebsillae pneumoniae XJPD-Li重组甘油脱水酶特性及其复活动力学研究
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摘要
1,3-丙二醇是一种重要的化工原料,以1,3-丙二醇为单体合成的新型聚酯材料具有良好的应用前景。甘油脱水酶是利用微生物发酵甘油生产1,3-丙二醇的限速酶,催化甘油脱水生成3-羟基丙醛。本论文针对课题组获得的甘油转化率高、生产强度大的1,3-丙二醇高效合成菌株Klebsiella pneumoniae XJPD-Li,研究其重组甘油脱水酶的催化特性、结构特征及其复活特性。
从K. pneumoniae XJPD-Li总基因组中扩增得到了甘油脱水酶基因片段,与甘油脱水酶(U30903)序列进行比对,其α亚基的第193和407位,β亚基的第47和189位的非活性中心氨基酸残基存在差异。成功构建了E. coli BL21(DE3) (pET-28a(+)-dhaBCE)表达系统,诱导条件经优化(0.8 mmol·L-1IPTG,20℃,3h),可大量表达高活性的重组甘油脱水酶,其可溶/不溶形式的比例提高到1.432,比酶活达64.5 U?mg-1。通过亲和层析快速分离纯化得到了电泳纯的重组甘油脱水酶,比活力提高2倍,酶活回收率47.5%。重组甘油脱水酶具有良好的催化性能,最适催化温度为45℃,最适pH为8.0,最适底物为甘油,对辅酶的亲和能力较强,热稳定性较好,对氧环境不敏感,其热失活动力学符合一级失活动力学模型,在氧失活及辅酶类似物失活过程中,其活性随时间呈指数衰减。
K. pneumoniae XJPD-Li重组甘油脱水酶为典型的α/β型结构,具有多个柔性结构区域,α亚基407位差异氨基酸位于柔性区域,疏水性有较大改变。针对该位点构建的突变体E407A,经研究发现其酶活性降低至突变前的30%左右,Km值相对突变前增大,最适催化温度降低,突变前后甘油脱水酶的二级结构没有明显变化,荧光光谱分析发现谱图有轻微扰动,说明该位点的氨基酸残基变化对甘油脱水酶的催化活性和构象有一定影响。利用分子动力学模拟进一步研究了甘油脱水酶非活性中心氨基酸残基对酶结构的影响,发现α和β亚基非活性中心氨基酸残基的变化对甘油脱水酶RMSD值和分子内及分子间氢键有较明显的影响,特别是对活性中心的稳定性有促进作用,使得酶结构更加紧密,稳定性提高。
对重组甘油脱水酶复活特性研究发现,重组甘油脱水酶复活因子主要以包涵体形式存在,通过亲和柱上复性可一步获得高纯度活性蛋白,回收率为20.7 %,比活性为60.6 min-1·mg-1。复性液中添加比例为6: 1的氧化还原体系(GSH:GSSG),以及添加ATP有利于甘油脱水酶地快速复活。针对甘油脱水酶复活过程参考乒乓理论建立了复活动力学模型,并对参数进行了求解。考察了甘油脱水酶体外复活寿命的调控,通过对复活体系的调控将甘油脱水酶的复活寿命提高一倍。
1,3-propanediol(1,3-PDO) is one of the most important chemicals, used as a monomer to produce the desired polyester such as polytrimethylene terephthalate. Glycerol dehydratase (GDHt) is the rate limiting enzyme in the biosynthesis of 1,3- PDO, which could dehydrate glycerol into 3-hydroxylpropionaldehyde. In provious study, Klebsiella pneumoniae XJPD-Li, screened by our group, was found to consume glycerol fast and produce 1.3-PDO with high productivity and molar yield of (1, 3-PDO to glycerol) So the properties of catalysis, structure and reacticvation of glycerol dehydratase (GDHt) from K. pneumoniae XJPD-Li were investigated in this research..
Firstly, the gene of GDHt was cloned from K. pneumoniae XJPD-Li genome and the following sequence alignment with GDHt (U30903) showed the differences of non-active site amino acid residue at No.193, 407 inαsubunit and No.47, 189 inβsubunit. To study the special properties of GDHt from K. pneumoniae XJPD-Li, the overexpression system was successfully constructed and the optimization of inducing process for recombinant GDHt were carried out. The optimum inducing conditions were the IPTG concentration of 0.8 mmol·L-1, the temperature of 20℃and inducing time of 3 h. The ratio of soluble form increased to 1.432 under the optimum condition.
Purification was carried out by affinity chromatography. Homogeneity of recombinant GDHt was obtained conveniently resulted in 2.11-fold purification and an overall yield of 47.5%. The optimum pH and reaction temperature of putified recombinant GDHt were pH 8.0 and 45℃, respectively. The Km of GDHt for different substrates showed that glycerol was the optimum substance and the affinity was strong between coenzyme B12 and GDHt. It showed that the recombinant GDHt was relative thermostability, and a bit blunt to oxygen. The thermo inactivation kinetics fit the first order kinetics. Oxygen and coenzyme analogue inactivation kinetics was agreed with exponential regression.
Analyzing results by biosoftware described the typicalα/βstructure of K. pneumoniae GDHt,including multiple soft regions. The No. 407 ofαsubunit was sited in soft region, whose hydrophobic character was different with that ofαsubunit in GDHt (U30903). The mutant E407A of GDHt showed lower special activity and higher Km than that of previous mutation. The circle dichromism (CD) spectra of GDHt showed the second structure of GDHt has little change, while fluorescence spectra some disturbances before and after mutation. That is the No 407 ofαsubunit could influence the conformation and activity of GDHt. Molecular Dynamic Simulation was applied to determine the relationship between structure and function. The RMSD,Hydrogen bonds inter and intro moleculars were changed obviously. It is proved that non-active site amino acid residues, No.193, 407 inαsubunit and No.47, 189 inβsubunit, were important to the stability of recombinant GDHt, especially its active site, which provided the structure of GDHt more compacted.
The reactivation properties of recombinant GDHt was determined by GDHt reactivation factor (GDHt-Rf) from K. pneumoniae XJPD-Li, which was founded existing mainly in the form of inclusion body. Renaturation was carried out by the method of one step purification and renaturation. The renaturation ratios and special activity of GDHt-Rf were 20.7%, 60.6 min-1?mg-1. Addition of GSH to GSSG ( 6:1 ) and ATP in the renaturation solution was suitable to correctly renaturate the inclusion body of GDHt-Rf. The in vitro reactivation kinetic model was established according to Pingpang BiBi mechanism and the parameters were calculated. The simulated values can fit the experimental values. The reactivation life of GDHt in vitro was doubled when the reactivation system was modified.
从K. pneumoniae XJPD-Li总基因组中扩增得到了甘油脱水酶基因片段,与甘油脱水酶(U30903)序列进行比对,其α亚基的第193和407位,β亚基的第47和189位的非活性中心氨基酸残基存在差异。成功构建了E. coli BL21(DE3) (pET-28a(+)-dhaBCE)表达系统,诱导条件经优化(0.8 mmol·L-1IPTG,20℃,3h),可大量表达高活性的重组甘油脱水酶,其可溶/不溶形式的比例提高到1.432,比酶活达64.5 U?mg-1。通过亲和层析快速分离纯化得到了电泳纯的重组甘油脱水酶,比活力提高2倍,酶活回收率47.5%。重组甘油脱水酶具有良好的催化性能,最适催化温度为45℃,最适pH为8.0,最适底物为甘油,对辅酶的亲和能力较强,热稳定性较好,对氧环境不敏感,其热失活动力学符合一级失活动力学模型,在氧失活及辅酶类似物失活过程中,其活性随时间呈指数衰减。
K. pneumoniae XJPD-Li重组甘油脱水酶为典型的α/β型结构,具有多个柔性结构区域,α亚基407位差异氨基酸位于柔性区域,疏水性有较大改变。针对该位点构建的突变体E407A,经研究发现其酶活性降低至突变前的30%左右,Km值相对突变前增大,最适催化温度降低,突变前后甘油脱水酶的二级结构没有明显变化,荧光光谱分析发现谱图有轻微扰动,说明该位点的氨基酸残基变化对甘油脱水酶的催化活性和构象有一定影响。利用分子动力学模拟进一步研究了甘油脱水酶非活性中心氨基酸残基对酶结构的影响,发现α和β亚基非活性中心氨基酸残基的变化对甘油脱水酶RMSD值和分子内及分子间氢键有较明显的影响,特别是对活性中心的稳定性有促进作用,使得酶结构更加紧密,稳定性提高。
对重组甘油脱水酶复活特性研究发现,重组甘油脱水酶复活因子主要以包涵体形式存在,通过亲和柱上复性可一步获得高纯度活性蛋白,回收率为20.7 %,比活性为60.6 min-1·mg-1。复性液中添加比例为6: 1的氧化还原体系(GSH:GSSG),以及添加ATP有利于甘油脱水酶地快速复活。针对甘油脱水酶复活过程参考乒乓理论建立了复活动力学模型,并对参数进行了求解。考察了甘油脱水酶体外复活寿命的调控,通过对复活体系的调控将甘油脱水酶的复活寿命提高一倍。
1,3-propanediol(1,3-PDO) is one of the most important chemicals, used as a monomer to produce the desired polyester such as polytrimethylene terephthalate. Glycerol dehydratase (GDHt) is the rate limiting enzyme in the biosynthesis of 1,3- PDO, which could dehydrate glycerol into 3-hydroxylpropionaldehyde. In provious study, Klebsiella pneumoniae XJPD-Li, screened by our group, was found to consume glycerol fast and produce 1.3-PDO with high productivity and molar yield of (1, 3-PDO to glycerol) So the properties of catalysis, structure and reacticvation of glycerol dehydratase (GDHt) from K. pneumoniae XJPD-Li were investigated in this research..
Firstly, the gene of GDHt was cloned from K. pneumoniae XJPD-Li genome and the following sequence alignment with GDHt (U30903) showed the differences of non-active site amino acid residue at No.193, 407 inαsubunit and No.47, 189 inβsubunit. To study the special properties of GDHt from K. pneumoniae XJPD-Li, the overexpression system was successfully constructed and the optimization of inducing process for recombinant GDHt were carried out. The optimum inducing conditions were the IPTG concentration of 0.8 mmol·L-1, the temperature of 20℃and inducing time of 3 h. The ratio of soluble form increased to 1.432 under the optimum condition.
Purification was carried out by affinity chromatography. Homogeneity of recombinant GDHt was obtained conveniently resulted in 2.11-fold purification and an overall yield of 47.5%. The optimum pH and reaction temperature of putified recombinant GDHt were pH 8.0 and 45℃, respectively. The Km of GDHt for different substrates showed that glycerol was the optimum substance and the affinity was strong between coenzyme B12 and GDHt. It showed that the recombinant GDHt was relative thermostability, and a bit blunt to oxygen. The thermo inactivation kinetics fit the first order kinetics. Oxygen and coenzyme analogue inactivation kinetics was agreed with exponential regression.
Analyzing results by biosoftware described the typicalα/βstructure of K. pneumoniae GDHt,including multiple soft regions. The No. 407 ofαsubunit was sited in soft region, whose hydrophobic character was different with that ofαsubunit in GDHt (U30903). The mutant E407A of GDHt showed lower special activity and higher Km than that of previous mutation. The circle dichromism (CD) spectra of GDHt showed the second structure of GDHt has little change, while fluorescence spectra some disturbances before and after mutation. That is the No 407 ofαsubunit could influence the conformation and activity of GDHt. Molecular Dynamic Simulation was applied to determine the relationship between structure and function. The RMSD,Hydrogen bonds inter and intro moleculars were changed obviously. It is proved that non-active site amino acid residues, No.193, 407 inαsubunit and No.47, 189 inβsubunit, were important to the stability of recombinant GDHt, especially its active site, which provided the structure of GDHt more compacted.
The reactivation properties of recombinant GDHt was determined by GDHt reactivation factor (GDHt-Rf) from K. pneumoniae XJPD-Li, which was founded existing mainly in the form of inclusion body. Renaturation was carried out by the method of one step purification and renaturation. The renaturation ratios and special activity of GDHt-Rf were 20.7%, 60.6 min-1?mg-1. Addition of GSH to GSSG ( 6:1 ) and ATP in the renaturation solution was suitable to correctly renaturate the inclusion body of GDHt-Rf. The in vitro reactivation kinetic model was established according to Pingpang BiBi mechanism and the parameters were calculated. The simulated values can fit the experimental values. The reactivation life of GDHt in vitro was doubled when the reactivation system was modified.
引文
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