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| Optimization of Expression and the Character Ization of a G4-amylase Enzyme |
| ZHAO Yun, ZHU Bei-lin, WANG Zheng-hua, ZHOU Jie, WU Zi-rong, HUANG Jing |
| School of Life Science, East China Normal University, Shanghai 200241, China |
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Abstract G4-amylase catalyses hydrolysis of 1,4-α-D-glucosidic linkages in amylaceous polysaccharides to remove successive maltotetraose residues from the non-reducing chain ends. As a new type of exo-amylase, it is widely used in food, medical care and other fields. The enzyme’s gene sequence from Pseudomonas stutzeri was optimized and it showed 75% homology with the wild gene sequence. The optimized gene was synthesized and then cloned into expression vector pET32a(+). The recombinant protein was shown to be expressed in Escherichia coli BL21(DE3) as inclusion bodies. By protein refolding and purification, gel electrophoresis homogeneous and active protein which had a molecular weight of 57kDa (by SDS-PAGE). Hydrolysis product analysis of seven kinds sources of starch showed that only maltotetraose were presented. By DNS determination.The optimum temperature is 45℃, the optimal pH is 8.0. This research provided a new idea for heterologous expression of this particular food-enzyme using genetic engineering.
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Received: 15 October 2012
Published: 25 May 2013
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