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Cloning, Expression, Characterization and Site Mutagenesis of a β-Glucosidase from a Deep-Sea Bacterium, Aeromonas sp.HC11e-3

Author: HuangXiaoLuo
Tutor: LiuZiZuo
School: Huazhong Agricultural University
Course: Biochemistry and Molecular Biology
Keywords: BglA Aeromonas sp. HC11e-3 β - glucosidase Structural simulation
CLC: Q93
Type: Master's thesis
Year: 2011
Downloads: 55
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Abstract


p-glucosidase can be widely used in various industrial fields, such as in the degradation of cellulose, the auxiliary outer cutting cellulase and endo-cellulase degradation of cellulose to glucose monosaccharides; in the food industry, hydrolyzed juice aryl glycosides tea, to play natural flavoring effect; hydrolysis TCM active ingredients in the pharmaceutical industry, glycosidic precursors, the preparation of the active ingredients of traditional Chinese medicine; transglycosylation of p-glucosidase activity makes it has a very good application prospects in the active material of the synthetic glycosides, such as the use of p-glucosidase synthetic surfactant alkyl glycoside and prebiotics gentian chitosan, etc.. Thus the new p-glucosidase Cloning and development for the field of industrial applications of great significance. The research from laboratory stocks of marine bacteria screened a high-yield β-glucosidase marine Aeromonas bacteria of Aeromonas sp.-3 HClle. Construct a genomic library, the first time a p-glucosidase gene was cloned from the bacteria, the total length of 2382bp, encoding 793 amino acids, named Bg1A shotgun. Homologous than found that the enzyme belonging to the glycoside hydrolases third family derived from the new Apollo gowns bacterium Thermotoga neapolitana, Thermus, the homology of 45% have been reported with other biochemical characteristics of glycosidase homology not high 30%. The enzyme was cloned into the pGEX-6p-1 vector, transformed into the E. coli expression host, the use of IPTG induction enzyme, and found that the enzyme in 0.1mM IPTG induction agent, the culture temperature of 18 degrees, in the E. coli BL21 (DE3) expression. GST affinity purification system, purified enzyme, and determination of its enzymatic properties, and found that the enzyme showed the highest activity at pH6.0 and 55 ° C conditions. The enzyme hydrolysis of specific aromatic glycoside, nitrophenols glucoside, synthetic and plant origin glucosidase esculin and arbutin shows the highest activity, nitrophenols xylosidase and nitro phenol semi galactosidase also have weak activity. BglA have a high tolerance to a wide range of metal ions and chemical reagents. Ca2, Mn2, Zn2, Ba2, Pb2, Sr2 the enzyme activation, SDS, EDTA, DTT slight inhibition of the enzyme. While in the high glucose concentration of 0.5M, BglA still showed a certain activity, the enzyme glucose tolerance have a strong function. Overall, the enzyme is an easy to recombinant expression, low homology, substrate specificity, the tolerability of strong chemical reagents p-glucosidase. These characteristics make the glucosidase can be used in appropriate industrial areas. By homology alignment and structural simulation, the estimation of the enzyme may be the active sites aspartic acid 288 and glutamic acid 495. By overlap extension site-directed mutagenesis of the enzyme technology aspartate 288 and glutamate 495 mutated to alanine, the results of the activity of the mutant D288A and E495A were reduced by 820-fold and 4920-fold, confirming the Asp288 and Glu495 participate in The activity of the enzyme hydrolysis. Functional analysis of these results for the family of another glucosidase enzyme active site to provide a certain basis.

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