Cloning and characterization of chitobiase from Aeromonas caviae D6

Abstract

N-acetyl-D-glucosamine (GlcNAc) has an important role for the treatment of osteoarthritis. Consequently, GlcNAc production has been studied and developed. In this research, Chitinase (chiA) and N-acetylglucosaminidase (agd97) genes from Bacillus licheniformis SK-1 and Aeromonas caviae D6 respectively were cloned and gene cassettes containing both genes were constructed. Attempts were made to co-express both genes in E. coli BL21 (DE3) and XL-1 blue by using pET-17b and chi60 promoter from Serratia sp. TU09 in the pBSSK- vector. The expression of agd97 gene from pETAgd97-ChiA in E. coli BL21 (DE3) gave the activity of 15.656 U/ml, higher than expression of pBSK60-Agd97 by chi60 promoter in E. coli XL-1 blue, which gave the activity of 0.207 U/ml. Our attempts to construct gene cassettes were unsuccessful. However, when chiA gene was placed in a reverse orientation in front of agd97 gene, the activity of agd97 increased 76 fold compared to the activity observed from chi60 promoter in E. coli XL-1 blue. Hence, the expressed E. coli BL21 (DE3) /pETAgd97-ChiA was purified using DEAE-cellulose and sephadex G-100 column chromatography. Agd97 from E. coli BL21 (DE3) was purified 2.4 fold with a 1.4% yield. The optimum pH and temperature of the purified enzyme was 6 and 45[degree Celcius], respectively. The enzyme has the highest stability over the pH rang from 6 to 10 and at temperature below 40 [degree Celcius].