Functional characterization of a special thermophilic multifunctional amylase OPMA-N and its N-terminal domain

A gene encoding a special thermophilic multifunctional amylase OPMA-N was cloned from Bacillus sp. ZW2531-1. OPMA-N has an additional 124-residue N-terminal domain compared with typical amylases and forms a relatively independent domain with a beta-pleated sheet and random coil structure. Here we reported an unusual substrate and product specificities of OPMA-N and the impact of the additional N-terminal domain (1-124 aa) on the function and properties of OPMA-N. Both OPMA-N (12.82 U/mg) and its N-terminal domain-truncated Delta OPMA-N (12.55 U/mg) only degraded starch to produce oligosaccharides including maltose, maltotriose, isomaltotriose, and isomaltotetraose, but not to produce glucose. Therefore, the N-terminal domain did not determine its substrate and product specificities that were probably regulated by its C-terminal beta-pleated sheet structure. However, the N-terminal domain of OPMA-N seemed to modulate its catalytic feature, leading to the production of more isomaltotriose and less maltose, and it seemed to contribute to OPMA-N's thermostability since OPMA-N showed higher activity than Delta OPMA-N in a temperature range from 40 to 80 degrees C and the half-life (t(1/2)) was 5 h for OPMA-N and 2 h for Delta OPMA-N at 60 degrees C. Both OPMA-N and Delta OPMA-N were Ca2+-independent, but their activities could be influenced by Cu2+, Ni2+, Zn2+, EDTA, SDS (1 mM), or Triton-X100 (1%). Kinetic analysis and starch-adsorption assay indicated that the N-terminal domain of OPMA-N could increase the OPMA-N-starch binding and subsequently increase the catalytic efficiency of OPMA-N for starch. In particular, the N-terminal domain of OPMA-N did not determine its oligomerization, because both OPMA-N and Delta OPMA-N could exist in the forms of monomer, homodimer, and homooligomer at the same time.