通过同源建模分析选取对Lactobacillus fermentum CGMCC2921来源的L-阿拉伯糖异构酶(简称L-AI酶)催化D-半乳糖生产D-塔格糖起重要作用的氨基酸位点进行突变,发现当Q16,M311,K423和Q438位点的氨基酸突变为丙氨酸时,突变酶Km值降低,其中突变酶M311A降至本体的51.6％,对D-半乳糖的转化率提高了18.7％.当K423位点的氨基酸残基分别突变为丙氨酸、天冬酰胺或精氨酸时,突变酶与底物的亲和力以及D-半乳糖的转化率随着423位点突变氨基酸侧链长度的增加而降低.运用计算机分子模拟技术分析表明,当M311位点氨基酸突变为丙氨酸以后,催化位点氨基酸残基与底物D-半乳糖之间的氢键作用增强,导致与底物亲和力增大,从而提高了酶活力.
参考文献
| [1] | Patrick J W;Lee N .[J].Journal of Biological Chemistry,1968,243:4312. |
| [2] | Levin G V .[J].Journal of Medicinal Food,2002,5:23. |
| [3] | Lu Y;Levin GV;Donner TW .Tagatose, a new antidiabetic and obesity control drug.[J].Diabetes, obesity & metabolism,2008(2):109-134. |
| [4] | Kim P .[J].Applied Microbiology and Biotechnology,2004,65:243. |
| [5] | Oh DK .Tagatose: properties, applications, and biotechnological processes[J].Applied Microbiology and Biotechnology,2007(1):1-8. |
| [6] | Manjasetty BA;Chance MR .Crystal structure of Escherichia coli L-arabinose isomerase (ECAI), the putative target of biological tagatose production[J].Journal of Molecular Biology,2006(2):297-309. |
| [7] | Kim HJ;Kim JH;Oh HJ;Oh DK .Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase that increases the production rate of D-tagatose[J].Journal of applied microbiology,2006(1):213-221. |
| [8] | Oh DK;Oh HJ;Kim HJ;Cheon J;Kim P .Modification of optimal pH in L-arabinose isomerase from Geobacillus stearothermophilus for D-galactose isomerization[J].Journal of molecular catalysis, B. Enzymatic,2006(1/4):108-112. |
| [9] | 江波;沐万孟;程丽芳 .[P].CN 101 768 581,2010. |
| [10] | Lee SJ;Lee DW;Choe EA;Hong YH;Kim SB;Kim BC;Pyun YR .Characterization of a thermoacidophilic L-arabinose isomerase from Alicyclobacillus acidocaldarius: role of Lys-269 in pH optimum[J].Applied and Environmental Microbiology,2005(12):7888-7896. |
| [11] | Rhimi M;Bajic G;Ilhammami R;Boudebbouze S Maguin E Haser R Aghajari N .[J].Microbial Cell Factories,2011,10:96. |
| [12] | Oh H J;Kim H J;Oh D K .[J].Biotehnol Lett,2006,28:145. |
| [13] | Rhimi M;Aghajari N;Juy M;Chouayekh H Maguin E Haser R Bejar S .[J].Biochimie,2009,91:650. |
| [14] | Rhimi M;Juy M;Aghajari N;Haser R;Bejar S .Probing the essential catalytic residues and substrate affinity in the thermoactive Bacillus stearothermophilus US100 L-arabinose isomerase by site-directed mutagenesis.[J].Journal of bacteriology,2007(9):3556-3563. |
| [15] | Prabhu, P;Jeya, M;Lee, JK .Probing the Molecular Determinant for the Catalytic Efficiency of L-Arabinose Isomerase from Bacillus licheniformis[J].Applied and Environmental Microbiology,2010(5):1653-1660. |
| [16] | Xu Zh;Qing Y J;Li Sh;Feng X H Xu H Ouyang P K .[J].Journal of Molecular Catalysis B:Enzymatic,2011,70:1. |
| [17] | Bradford M M .[J].Analytical Biochemistry,1976,72:248. |
| [18] | Dische Z;Borenfreund E .[J].Journal of Biological Chemistry,1951,192:583. |
| [19] | Wu GS.;Robertson DH.;Brooks CL.;Vieth M. .Detailed analysis of grid-based molecular docking: A case study of CDOCKER - A CHARMm-based MD docking algorithm[J].Journal of Computational Chemistry: Organic, Inorganic, Physical, Biological,2003(13):1549-1562. |
| [20] | Brooks B R;Bruccoleri R E;Olafson B D;States D J Swaminathan S Karplus M .[J].Journal of Computational Chemistry,1983,4:187. |
| [21] | Lovell SC;Davis IW;Arendall-WB 3rd;de-Bakker PI;Word JM;Prisant MG;Richardson JS;Richardson DC .Structure validation by Calpha geometry: phi,psi and Cbeta deviation.[J].Proteins: Structure, Function, and Genetics,2003(3):437-450. |
上一张
下一张
上一张
下一张
计量
- 下载量()
- 访问量()
文章评分
- 您的评分：
-
10%
-
20%
-
30%
-
40%
-
50%