Studies On Construction Of Recombinant E.coli For Thermostable α-Glucosidase Production And Fermentation Conditions Optimization

α-Glucosidase are widely found in organisms with different properties. As restriction enzyme, it can release glucose by cuttingα-1, 4 glycosidic bond from the non-reducing end of oligosaccharides, or transferr free glucose residue to the other carbohydrate formingα-1, 6 glycosidic bond, by which non-fermentative isomaltooligosaccharide, glycolipids or glycopeptides are synthesized.α-Glucosidase is paid attention for producing isomaltooligosaccharide used in the food industry. But the activity is impacted by enviroment conditions greatly. So the thermostableα-glucosidase becomes a research focus.In this experiment, a-glucosidase gene was multiplied by PCR from Thermophilic eubacteriumThermus nonproteolyticus HG102 , ligated with plasmid PET28a(+), and transferred into the E.coli(DH5a) competent cells. The recombinant plasmid was extracted and vertificated by enzyme digestion. Then, the plasmid was induced by IPTG and expressed in E.coli(BL21). After cultured in LB liquid medium, cells were broken by ultrasonic for enzyme activity detection and SDS-PAGE electrophoresis analysis.The results indicated that a-glucosidase gene acquired by PCR was about 1900kb, the activity was detected after induction and expression, and the enzyme molecular weight was approximately 65 kD. The results were consistent with the theoretical value.α-Glucosidase properties were studied preliminarily. The optimum pH was 7.0, and the enzyme was stable at pH 5.0-8.0. The optimum temperature was 85℃, With the treatment at 85℃for 3 h,α-glucosidase activity was about 60% of the control. It was showed that the enzyme had good thermal stability.The single factor optimization experiments were carried out involving carbon source, inoculum size, metal ions, IPTG added time, induction time. The results were thatα-glucosidase activity was the highest at the conditions of.adding 1% glycerol and 0.01% of the Mn2+ in the LB liquid medium, inoculation of 6 %, adding IPTG 1.2 mmol / L at 2h, induced 16 h,.The final activity of the initial 4.25 U / mL increased to 40.85 U / mL, increased 8.6 times.Baed on the results of single factor experiments, the orthogonal experiments.of three factors and three level of inoculum size, inducer adding time and induction time were designed. The influence order was inducer adding time > inoculum size > induction time. Optimum conditions were inoculation of 8%, adding IPTG at 2 h, induced time for 20 h. The finalα-glucosidase activity increased to 63.53 U / mL.