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Structure and Function of Multifunctional Amylase OPMA-G

Author: HaoZuo
Tutor: ZhangYingJiu
School: Jilin University
Course: Biochemistry and Molecular Biology
Keywords: Structure Function Multifunctional amylase
CLC: Q55
Type: Master's thesis
Year: 2011
Downloads: 32
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Abstract

α-amylase (EC 3.2.1) are a class of enzymes capable of degrading starch, part of which has both hydrolase activity and having a turn glycosidase activity, known multifunctional amylase. Cloned in our laboratory and expressed multifunction amylase OPMA-G, it has the α-amylase family consists of four conserved regions in the structure, function both hydrolase and Glycosyltransferase catalytic activity, the catalytic The starch degradation product as maltose, maltotriose, isomaltotriose, isobutyl maltotetraose, but does not produce glucose, wherein isobutyl the maltotriose and isobutyl maltotetraose belong isomalto-oligosaccharides. Differences the bifidus factor maltooligosaccharide efficacy relative to traditional sugar, they have to improve the function of the intestinal microflora, and be able to prevent tooth decay. Therefore, the study of the structure and function of the OPMA-G can provide theoretical guidance for the transformation of such enzyme molecules and functional optimization. In order to clarify the specific function of the C-terminal domain of OPMA-G, the papers obtained based on the room reserved of OPMA-G gene C-terminally truncated ΔOPMA-G gene recombinant expression vector pET28a the-ΔOPMA-G and effective expression in E. coli BL21 (DE3), the expression product form of inclusion body protein was 42%. Expression of inclusion bodies using urea concentration gradient dialysis method completely renaturation, refolding ΔOPMA-G detected by SDS-PAGE purity of 85% or more. By comparative analysis of the nature and function of the OPMA-G ΔOPMA-G results showed: the OPMA-G ΔOPMA-G specific activity of 8.22 U / mg, 6.53 U / mg, the optimum temperature were 50 ° C, 40 ° C The optimum pH is 7.5. The OPMA-G ΔOPMA-G are only specific recognition and hydrolysis of starch but does not act on pullulan and p-cyclodextrin, the enzymatically generated product also are maltose, maltotriose, isobutyl the maltotriose and iso maltotetraose sugar, the product of the proportion of no difference. , OPMA-G ΔOPMA-G showed a non-calcium-dependent enzyme, the Zn2 Ni2 and the K activation on both Fe3, Fe2, Cu2, and EDTA was inhibited. The non-denaturing electrophoresis showed: the ΔOPMA-G still single trimer, dimer, tri-mer multimeric state exists, the C-terminal oligomerization OPMA-G no effect. Relative the OPMA-G starch adsorption rate (8.5%), ΔOPMA-G is reduced to 5.8%, which indicated that the OPMA-G C-terminal with Substrate adsorption associated C-terminal deletion caused by the reduction in the rate of substrate adsorption thereby affecting the reduction of the enzyme activity. In order to further study the relationship of structure and function of the OPMA-G, the papers homology modeling and sequence analysis of multifunctional amylase OPMA-G, selected to the V200 conduct site-directed mutagenesis to study the impact of the the V200 site for the enzyme. Experiments using overlapping PCR method get the OPMA-G two point mutants OPMA-GM3 (V200G) and of OPMA-GM4 (V200K) gene and construct a recombinant expression vector pET28a-OPMA-GM3 and pET28a-OPMA-GM4. Two mutants were effectively expressed in E. coli BL21 (DE3), the expression rate of 37% and 38%, respectively. The expression product OPMA-GM3 OPMA-GM4 both soluble expression Ni2-NTA column purification, SDS-PAGE detected in more than 95% purity. And the specific activity of the wild-type the OPMA-G of 8.22 U / mg compared to the mutant body OPMA--GM3 OPMA--GM4 than vitality were 9.51,8.63 the U / mg. Enzymatic Properties: The results show that mutant OPMA--GM3 OPMA-GM4 same only the specific recognition and hydrolysis of starch substrate, OPMA-GM4 the OPMA-G hydrolyzed starch product as maltose, maltotriose, isomaltotriose and different maltotetraose, OPMA-GM3 product in addition to the above oligosaccharides, glucose, and maltose increased significantly, while the the OPMA-GM4 catalytic product isomaltotriose proportion increased. The optimum temperature of the enzyme of the two mutants was still 50 ° C. After 30 ℃ -70 ℃ incubated 1h maintained more than 70% viability. The OPMA-GM4 the optimum pH is not changed and remains 7.5, pH stability is also unchanged, but the OPMA-GM3, the optimum pH is reduced to 7.0, its pH stability also occurs acid shift. In addition, the impact of various ions on the mutant enzyme is the same as the wild enzyme OPMA-G. The above experimental results show that: the bit point V200 steric and charge affects both the catalytic properties of the enzyme may be a large steric hindrance and a positive charge is conducive to product formation of the branch. So far, the relationship between the structure and function of the multifunctional amylase understand yet thorough, its hydrolysis, turn glycosides catalytic mechanism has not been fully elucidated. In recent decades, the researchers continue through molecular modification techniques to study the relationship between the structure and function of the multifunctional amylase. Due to the improvement of computer simulation technology, site-directed mutagenesis molecular transformation of technology applied to multi-amylase. The topics analyzed the multifunctional amylase OPMA-G C-terminal fragment and V200 locus enzyme catalytic function, provides a theoretical basis for further optimization OPAM-G functionality, and laid the foundation for their industrial applications.

Structure and Function of Multifunctional Amylase OPMA-G

Abstract

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