Characterization of the starch binding domain of Lipomyces kononenkoae alpha-amylase LKA1.
Ricardo Cordero Otero (1), Sakkie Pretorius (2), Nivetha Ramachandran (1)
(1) Inst. for Wine Biotechnol., University of Stellenbosch, Victoria St., Stellenbosch, 7600, South Africa; (2) IWBT, and The Australian Wine Research Institute, Adelaide, Australia
The LKA1 - and LKA2 -encoded alpha-amylases from Lipomyces kononenkoae have unique alpha-1,4 and alpha-1,6 activity on starch. Lka1p has a comparable activity on pullulan while Lka2p has a significant dextrinase activity. Here, we report the possible role of N-terminal regions of LKA1 and LKA2 on raw starch adsorption, specificity and thermal stability. Taking into account, computer simulations and structure data, functional N terminal deletion mutants of Lka1p and Lka2p (Lka1pdeltaN and Lka2pdeltaN, respectively) were created. The full-length and the mutant amylolytic enzymes were purified and analysed for their raw starch binding function, thermal stability, effect of calcium ion on activity, and its contribution towards substrate specificity. Lka1pdeltaN shows a 95% reduction of raw starch substrate adsorption, 20% less hydrolytic activity in complex starches, and 30% less thermal stability than the wild-type enzyme. In contrast, Lka2pdeltaN did not show significant variations in raw starch adsorption or thermal stability. Furthermore, the K cat for dextrin of both truncated amylases was around 40% lower than the catalytic rate constant for their wild-type counterparts, respectively. The results are discussed in terms of possible role of N-terminal regions of Lka1p and Lka2p in raw starch adsorption, substrate specificity and thermal stability.