1，3-丙二醇合成途径中关键酶的研究
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摘要
本论文主要就克雷伯氏肺炎杆菌1,3-丙二醇生物合成途径动力学特性进行了研究。重点研究了甘油脱氢酶(GDH)的动力学模型。本研究首先利用大肠杆菌高效表达源于克雷伯氏肺炎杆菌的甘油脱氢酶，研究表明：大肠杆菌高效表达该酶的最佳诱导时间为6h，最佳诱导剂IPTG浓度为0.8mmol/L。通过超声波破碎，硫酸铵盐析、SephadexG-200凝胶层析和DE-23Cellulose阴离子交换层析对裂解液中的甘油脱氢酶进行提纯，提纯后酶液比酶活达到946U/mg。纯化倍数和回收率分别为2.58倍和37%。通过聚丙烯酰胺凝胶(SDS-PAGE)电泳测得该酶亚单个基的相对分子量约为39KDa。对甘油脱氢酶的性质研究表明，酶活性随pH值的升高而增大，反应温度50℃时酶活性最高。
甘油脱氢酶催化双底物可逆反应，反应机理符合顺序Bi-Bi模型，动力学模型包含11个参数：KmNAD、KmGLY、KiNAD、Vf、KmDHA、KmNADH、KiNADH、Vr、Keq、KiGLY和KiDHA。本研究结合线性回归和非线性回归方法，逐步对这些参数进行了求解。首先利用简化模型通过线型回归计算KmNAD、KmGLY、KiNAD、Vf、KmDHA、KmNADH、KiNADH和Vr的近似解，接着固定KmNAD、KmGLY、KiNAD、Vf、KmDHA、KmNADH、KiNADH和Vr，利用非线性回归计算Keq、KiGLY和KiDHA的近似解，最后利用非线性回归计算完整模型的11个参数的精确解。计算获得参数KmNAD、KmGLY、KiNAD、Vf、KmDHA、KmNADH、KiNADH、Vr、Keq、KiGLY和KiDHA的精确解分别为：1924.47μmol/L、11300.10μmol/L、294.00μmol/L、262.5μmol/(L.min)、332.50μmol/L、95.00μmol/L、383.52μmol/L、3092.27μmol/(L.min)、0.00095、27768.37μmol/L、12.57μmol/L。
本文利用PCR方法从克雷伯氏肺炎杆菌中克隆出了二羟基丙酮激酶的两个编码基因DHAKI、DHAKII，其片段大小分别是1.86kb和3.18kb。并用同样的方法从克雷伯氏肺炎杆菌中克隆出能够表达甘油脱水酶的编码基因GDHt，其大小为2.76kb。成功把三个基因片段分别连接到PDK6质粒上构建了重组质粒PDK6-DHAKI、PDK6-DHAKII、PDK_6-GDHt在大肠杆菌中进行高效表达。
The kinety model of 1,3-propanediol bio-synthese pathway was investigated in thispaper, the main work done in this paper is modelding the glycerol dehydrogenaseKlebsiella pneumoniae glycerol dehydrogenase gene was colone and overexpressed inE.coli. The cell culture process was optimizaed, it was found that the maximal specificactivity of glycerol dehydrogenase in E. coli was obtained with 0.8 mM IPTG added to themedium after 6 hours induction. Cell lysate was obtained by ultrasonic crush from the cellmush, Glycerol dehydrogenase was purified by ammonium sulfate salting-out,SephadexG-200 gel filtration and DE-23 cellulose column chromatography. Afterpurification the specific activity of the product is 946U/mg, the final enzyme obtained hasa 2.58-fold purification with the recovery ratio of 37%. The subunit molecular weight ofenzyme was 39KDa shown by SDS-PAGE. The activitie of glycerol dehydrogenase atdifferent pH buffer was tested; it was shown the activity of the enzyme is increase with pHincrease. GDH activated in a broad range of temperature, the maximal activity wasobserved at 50℃.
The model of enzyme reaction kinetic of glycerol dehydrogenase was construedfollowed the Ordered Bi-Bi model. There were 11 parameters in the model such as KmNAD,KmGLY, KiNAD, Vf, KmDHA, KmNADH, KiNADH, Vr, Keq, KiGLYand KiDHA. The approximatevalues of the first eight were identified by linear plotting with a simplified model, in thisstep the data used was the initial ratio of the reaction. Then fixed the eight parameters inthe model, the later three parameters were identified by nonlinear regression; the data usedwas the reaction progress. Finally, all the preliminary value of the 11 parameter were usedas guesses for the final parameters estimation, using the data of reaction progress vianonlinear regression, the accurate value of the parameters of KmNAD, KmGLY, KiNAD, Vf,KmDHA, KmNADH, KiNADH, Vr, Keq, KiGLYand KiDHAwere: 1924.47μmol/L, 11300.10μmol/L, 294.00μmol/L, 262.5μmol/(L.min), 332.50μmol/L, 95.00μmol/L,383.52μmol/L, 3092.27μmol/(L.min), 0.00095, 27768.37μmol/L, 12.57μmol/Lrespectively.
Dihydroxyacetone kinase genes DHAKI and DHAKII were cloned from theKlebsiella pneumonia TUAC01 to by PCR, the size of the two genes were 1.86kb and3.18kb. The gene of glycerol dehydratase was cloned from the Klebsiella pneumoniaTUAC01 ues the same method, and the size of thegene was 2.76kb. The three genes wereconnected whin PDK6 plasmid to construct express plasmid PDK6- DHAKI,PDK6-DHAKII and PDK6-GDHt.
甘油脱氢酶催化双底物可逆反应，反应机理符合顺序Bi-Bi模型，动力学模型包含11个参数：KmNAD、KmGLY、KiNAD、Vf、KmDHA、KmNADH、KiNADH、Vr、Keq、KiGLY和KiDHA。本研究结合线性回归和非线性回归方法，逐步对这些参数进行了求解。首先利用简化模型通过线型回归计算KmNAD、KmGLY、KiNAD、Vf、KmDHA、KmNADH、KiNADH和Vr的近似解，接着固定KmNAD、KmGLY、KiNAD、Vf、KmDHA、KmNADH、KiNADH和Vr，利用非线性回归计算Keq、KiGLY和KiDHA的近似解，最后利用非线性回归计算完整模型的11个参数的精确解。计算获得参数KmNAD、KmGLY、KiNAD、Vf、KmDHA、KmNADH、KiNADH、Vr、Keq、KiGLY和KiDHA的精确解分别为：1924.47μmol/L、11300.10μmol/L、294.00μmol/L、262.5μmol/(L.min)、332.50μmol/L、95.00μmol/L、383.52μmol/L、3092.27μmol/(L.min)、0.00095、27768.37μmol/L、12.57μmol/L。
本文利用PCR方法从克雷伯氏肺炎杆菌中克隆出了二羟基丙酮激酶的两个编码基因DHAKI、DHAKII，其片段大小分别是1.86kb和3.18kb。并用同样的方法从克雷伯氏肺炎杆菌中克隆出能够表达甘油脱水酶的编码基因GDHt，其大小为2.76kb。成功把三个基因片段分别连接到PDK6质粒上构建了重组质粒PDK6-DHAKI、PDK6-DHAKII、PDK_6-GDHt在大肠杆菌中进行高效表达。
The kinety model of 1,3-propanediol bio-synthese pathway was investigated in thispaper, the main work done in this paper is modelding the glycerol dehydrogenaseKlebsiella pneumoniae glycerol dehydrogenase gene was colone and overexpressed inE.coli. The cell culture process was optimizaed, it was found that the maximal specificactivity of glycerol dehydrogenase in E. coli was obtained with 0.8 mM IPTG added to themedium after 6 hours induction. Cell lysate was obtained by ultrasonic crush from the cellmush, Glycerol dehydrogenase was purified by ammonium sulfate salting-out,SephadexG-200 gel filtration and DE-23 cellulose column chromatography. Afterpurification the specific activity of the product is 946U/mg, the final enzyme obtained hasa 2.58-fold purification with the recovery ratio of 37%. The subunit molecular weight ofenzyme was 39KDa shown by SDS-PAGE. The activitie of glycerol dehydrogenase atdifferent pH buffer was tested; it was shown the activity of the enzyme is increase with pHincrease. GDH activated in a broad range of temperature, the maximal activity wasobserved at 50℃.
The model of enzyme reaction kinetic of glycerol dehydrogenase was construedfollowed the Ordered Bi-Bi model. There were 11 parameters in the model such as KmNAD,KmGLY, KiNAD, Vf, KmDHA, KmNADH, KiNADH, Vr, Keq, KiGLYand KiDHA. The approximatevalues of the first eight were identified by linear plotting with a simplified model, in thisstep the data used was the initial ratio of the reaction. Then fixed the eight parameters inthe model, the later three parameters were identified by nonlinear regression; the data usedwas the reaction progress. Finally, all the preliminary value of the 11 parameter were usedas guesses for the final parameters estimation, using the data of reaction progress vianonlinear regression, the accurate value of the parameters of KmNAD, KmGLY, KiNAD, Vf,KmDHA, KmNADH, KiNADH, Vr, Keq, KiGLYand KiDHAwere: 1924.47μmol/L, 11300.10μmol/L, 294.00μmol/L, 262.5μmol/(L.min), 332.50μmol/L, 95.00μmol/L,383.52μmol/L, 3092.27μmol/(L.min), 0.00095, 27768.37μmol/L, 12.57μmol/Lrespectively.
Dihydroxyacetone kinase genes DHAKI and DHAKII were cloned from theKlebsiella pneumonia TUAC01 to by PCR, the size of the two genes were 1.86kb and3.18kb. The gene of glycerol dehydratase was cloned from the Klebsiella pneumoniaTUAC01 ues the same method, and the size of thegene was 2.76kb. The three genes wereconnected whin PDK6 plasmid to construct express plasmid PDK6- DHAKI,PDK6-DHAKII and PDK6-GDHt.
引文
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[2] Yamada H Nagao A, Nishise H , et al Glycerol Dehydrogenase from Cellulomonas s p .NT3060:Purfication and Characterization[J]. Agric Biol Chem, 1982, 46(9) : 2333- 2339.
[3] Lin E C C, Magasanik B. The Activation of Glycerol Dehydrogenase from Aerobacter Aerogenesby Mono-valent cations[J]. J Biol Chem, 1960, 235(6) : 1820-1823.
[4] Deckwer W D.Microbial Conversion of Glycerol to 1,3-Propanediol[J]. Fems Microbiol Rev, 1995,16:143-149.
[5] Witt U,Müeller RJ,Augusta J,et al. Properties and Biodegradability of Polyesters Based on1,3-Propanediol.Macromol Chem Phys,1994,195:793-802.
[6] Kawashima K,Itoh H,Chgate J.Nonenzymatic Browning Reactions of Dihydroxyacetone withAminoacids or their esters. Agri Biol Chem, 1980, 44(7):1595-1599.
[7] Yamada S, Nabe K,Izuo N.Fermentation Production of Dihydroxyacetone by AcetobacterSuboxydans ATCC621[J].Ferment Technol,1979,57(3):215-220.
[8] Overchnko MB,Yanson,VA,Dobrolinskaya GM.Effect of some Ingredients of the Nutrient Mediumon Glycerol Oxidation to Dihydroxyacetone by Acetobacter Suboxydans[J].BiokhimMikrobiol.1981, 17(1):81-84.
[9] Keiko YO,Akira N,Yoshiki T.Purification,Characteriza-tion,and Gene Cloning of GlycerolDehydrogenase from Hansenula of Unaensis,and its Expression for Production of Optically Activediol[J].The Society for Biotechnology,2006,102(6):545-551.
[10] Zheng YY;Cao Y;Fang BS.Clonging and Sequence Analysis of the dhaT gene of the1,3-Propanediol Regulon from Klebsiella Pneumonia[J].Biotchnol Lett,2004,26(3):251-255.
[11] Li WJ,Fang BS,Hong Y,et al.Cloning and Expression of the Genes Encoding GlycerolDehydratase Reactivase and Identification of its Biological Activity[J].Chinese Journal ofBiotechnology, 2006,22(6):950-955.
[12] Zeng A P, BiebLl H, Deckwer W D. Microbial Conversion of Glycerol to 1,3–PropanediolRecentprogress.Fuels and Chemicals from Biomass[M]. SAHA B C, Woodward J. New York:American Chemical Society, 1997.265-279.
[13] Mccoy M. Chemical Makers Try Biotech Paths [J]. Chem Eng News, 1998, 76(25):13-19.
[14]李吉春.1,3-丙二醇的合成方法及技术进展[J].石化技术与应用,2004,221):4-11.
[15]崔小明.国内外1,3-丙二醇生产技术研究进展[J].精细化工原料及中间体,2006,3:33-37.
[16] Menzel K, Zeng A P, Deckwer W D. High Concentration and Productivity of 1,3 -Propa-nediol from Continuous Fermentation of Glycerol by Klebsiella Pneumonia [J]. Enzyme Micro-biol Technol, 1997, 20:82-86.
[17] Zeng A P, Deckwer W D. A Kinetic Model for Substrate and Energy Consumption of MicrobialGrowth under Substrate-Sufficient Conditions[J]. Biotechnol Prog, 1995, 11:71-79.
[18] Zeng A P. A Kinetic Model for Product Formation of Microbial and Mammalian Cells[J].Biotechnol Bioeng,1995,46:314-324.
[19] Ruzheiniko v S N, Bur ke J, Sedelniko va S, et al.Glycerol dehydrogenase Structure, Specificity,and Mechanism of a Family III Polyol Dehydrogenase[J]. Structure.2001, 9: 789-802.
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