Dissertation > Excellent graduate degree dissertation topics show

Stability of Recombinant Enzyme Preparation of α-CGTase

Author: ZhengXianLiang
Tutor: WuJing
School: Jiangnan University
Course: Biochemistry and Molecular Biology
Keywords: α- cyclodextrin glycosyltransferase Site-directed mutagenesis Chemically modified Chemical additives Thermal stability Circular dichroism
CLC: Q814
Type: Master's thesis
Year: 2010
Downloads: 102
Quote: 0
Read: Download Dissertation

Abstract

Cyclodextrin glycosyltransferase (CGT enzyme short, EC 2.4.1.19) through intramolecular transglycosylation reaction of starch and starch-like into the cyclodextrin. Since the formation of cyclodextrin with guest molecules containing complexes, guest molecules to change the physical and chemical properties, and thus in the food, pharmaceutical and cosmetic industries used more and more widely, CGT enzyme has become a hot research. Our previous research work, the successful implementation of the Paenibacillus maceransα-cyclodextrin glycosyltransferase (α-CGT enzyme) in E. coli expression exocytosis. But the study, we found that both the natural α-CGT recombinase enzymes or enzyme preparations are poor thermal stability, is not conducive to large-scale industrial applications. To improve the recombinant α-CGT enzyme stability, this study the gene level, the level of macromolecular chemical modification and changing the level of the enzyme molecule microenvironment of the three aspects of the enzyme α-CGT thermal stability studies conducted. Further, circular dichroism (CD) of the α-CGT enzyme and its natural structure of the thermal stability of the relationship between the polyhydroxy compound and the stability of the enzyme mechanism made a preliminary study. The main results are as follows: (1) purified from P. macerans natural α-CGT enzyme as well as from Escherichia coli and Bacillus subtilis recombinant enzyme found after heat treatment from P. macerans, E. coli and B. subtilis The residual activity of the enzyme α-CGT were 28%, 30% and 25%. Seen from different strains of α-CGT enzyme similar thermal stability, if the direct use, it can not achieve the requirements of industrial enzymes. According to three kinds of bacteria on α-CGT enzyme production, and ultimately determine from E. coli recombinant α-CGT enzyme as a follow-up study of this topic. (2) analysis of the recombinant α-CGT enzyme pH optimum storage and storage protein concentration. The results show pH6.5, protein content of 4 mg / mL of the enzyme α-CGT best thermal stability. The protein content of 4 mg / mL, pH 6.5, respectively, in the crude enzyme preparation 4 ℃, 25 ℃ and 37 ℃ storage. When the enzyme solution at 4 ℃ and 25 ℃ storage time, T1 / 2 was 108 d and 50 d, when stored at 37 ℃ α-CGT enzyme activity decreased rapidly, T1 / 2 of 2.9 d, a week after the activity completely lost. (3) was studied using site-directed mutagenesis salt bridge recombinant α-CGT to improve the thermal stability of the enzyme. Studies show that the mutant single mutant enzymes CGTM-1 (K192R) thermal stability than unmutated not much improved; mutated amino acid after three mutant enzyme CGTM-3 (T185S/I186Y/K192R) Thermal stability improved obviously. After 30min at 50 ℃ water bath CGTM-3 mutant enzyme residual activity was more than twice the control. The protein content of 4 mg / mL, pH 6.5 CGTM-3 mutant enzyme crude enzyme preparation, respectively 4 ℃, 25 ℃ and 37 ℃ storage, regular sampling measuring enzyme activity reached its T1 / 2 was 120 d, 80 d and 7 d. (4) using glutaraldehyde cross-linking method to study the chemical modification to improve the recombinant α-CGT enzyme stability. α-CGT enzyme glutaraldehyde crosslinked polymer molecules, after Cheng, 50 ℃ water bath for 30 min compared to the control found improved thermal stability of the enzyme, but the cross-linking process in a 50% loss of activity. Α-CGT later crude enzyme solution crosslinking, the crosslinking process although reduced loss of activity, the enzyme stability is improved, but still can not meet the requirements of industrial application. (5) using the α-CGT enzyme solution added chemical additives studied enzyme microenvironment changes to improve the recombinant α-CGT enzyme stability. When adding a variety of protective agent alone at 50 ℃ water bath for 1 h and at room temperature for 108 d after the discovery of the enzyme containing 20% ??glycerol solution best stability, with and without any additives were still 91% compared to the enzyme solution and 50 % activity in the control enzyme solution at 50 ℃ water bath for 1 h, only less than 10% of the activity has been at room temperature for 108 d after no activity. Gelatin, CaC12 and PEG400 also have some protective effect. The four kinds of protective agents were superimposed with optimal concentrations of α-CGT enzyme added to the fermentation broth 40 ℃ storage 45 d, the fermentation broth α-CGT residual enzyme activity almost unchanged at around 100%. Using circular dichroism (CD) under different conditions the thermal stability of the enzyme and protein conformational changes in the relationship between changes. Found that the enzyme protecting agent to help maintain the natural three-dimensional conformation, significantly reduced the α-CGT under high degree of denaturation of the enzyme, the enzyme at high temperatures the enzyme activity remained high. In addition, different molecular weights of PEG and glycerol protective effect of α-CGT enzyme study found hydroxyl content of polyhydroxy compound may affect the enzyme α-CGT important factor in the thermal stability.

Related Dissertations

CLC: > Biological Sciences > Bioengineering ( Biotechnology ) > Enzyme Engineering
© 2012 www.DissertationTopic.Net Mobile