Overexpression and Phylogenetic Analysis of a Thermostable α-Glucosidase from Thermus thermophilus

Abstract:

The α-glucosidase gene agl from Thermus thermophilus HB8 was cloned into expression vector pBV220. The phylogenetic trees of α-glucosidases were constructed using Neighbor-Joining (NJ) and Maximum-Parsimony (MP) methods. Evolution analysis indicated the α-glucosidase from T. thermophile HB8 was distant from the other glycoside hydrolases 4 and 31 α-glucosidases. By weakening the mRNA secondary structure and replacing the rare codons for the N-terminal amino acids of the target protein, the expression level of the agl was increased 30-fold. The recombinant AGL was purified by the heat treatment, and had a molecular mass of 61 kDa. The optimal activity was at pH 7.8 and 95°C over a 10 min assay. The purified enzyme was stable over a pH range of 5.4-8.6, and had a 1-h half life at 85°C. Kinetic experiments at 90°C with p-nitrophenyl-α-D-glucoside as substrate gave a K_m, and V_max of 0.072 mM and 400 U/mg. Thus, this report provides an industrial means to produce the recombinant α-glucosidase in E. coli.

• Periodical: Advanced Materials Research (Volumes 1004-1005)
• Pages: 841-848
• DOI: https://doi.org/10.4028/www.scientific.net/AMR.1004-1005.841
• Online since: August 2014
• Authors: Xun Li, Hua Xiang Gu, Hao Shi, Fei Wang*
• Keywords: Overexpression, Phylogenetic Analysis, Thermus thermophilus, α-Glucosidase

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