| Summary: | Thermostable and alkalitolerant a-amylase from Anoxybacillus sp. SK3-4 (ASKA) yields high level of maltose (69%) from starch hydrolysis. A putative raw starch binding domain was found in the C-terminal domain of ASKA. Starch binding domain (SBD) plays a role in hydrolyzing insoluble starch which enhances the amylolytic rate. The gene that encodes the ASKA is 1,518 bp. The present study involved in silico analyses and protein mutagenesis of two C-terminal truncated variants, namely mutant ?50 and ?100. The respective mutants had an elimination of 81 and 112 amino acids from C-terminal end of ASKA. Firstly mutagenesis was made in in silico structure. Bioinformatics analysis revealed that the eight ß-strand forms as an open-sided distorted ß-sandwich fold which of putative domain C. Using Neighbor-Joining (NJ) method, the phylogenetic analysis of the domain C had confirmed that starch binding region of ASKA is the non-catalytic domain C and not the typical SBD of CBM families. Actual gene truncation was constructed via protein engineering approach and the truncated genes were expressed in Escherichia coli, under the promoter of T7. However, both recombinant proteins exhibited no activity with soluble starch as a result of inclusion bodies aggregation; despite several optimization on expression conditions were made.
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