Functional expression of the raw starch-binding domain of Bacillus sp strain TS-23 α-amylase in recombinant Escherichia coli

Two DNA fragments encoding the starch-binding domain (SBD) of Bacillus sp. strain TS-23 alpha-amylase were prepared by polymerase chain reaction and cloned into the Escherichia coli expression vector, pQE-30, to generate pQE-N428/C607 and pQE-N465/C607. In isopropyl-beta-D-thiogalactopyranoside (IPTG)-induced E. coli strain M15 harboring these expression plasmids, the recombinant SBDs (N428/C607 and N465/C607) could comprise up to 20% of the total soluble proteins. The His-tag/SBD fusion proteins were purified to homogeneity with a His-bind affinity column and had molecular masses of approximately 22.6 and 16.5 kDa, respectively. Starch-binding assays revealed that about 7.1 and 8.3 mug, respectively, of N428/C607 and N465/C607 were bound by 1 mg of raw corn starch, indicating that the SBD of Bacillus sp. strain TS-23 alpha-amylase retain sufficient function in the absence of a catalytic center.