抄録
Rice a-amylase isozymes, AmylA and Amy3D, were expressed by Saccharomyces cerevisiae. Reaction properties of those isozymes purified by immuno-affinity chromatography were characterized to elucidate their functional roles. The pH optima of AmylA (pH 4 .2) and Amy3D (pH 5.5) correlate with the pH value of the endosperm tissue at times in rice seedling development when these isozymes are expressed. AmylA showed higher reactivities to soluble starch and starch granules than Amy3D. On the other hand, Amy3D showed much higher reactivity to maltoheptaose than AmylA. These results suggest that the individual isozymes play different functional roles during the seedling development process. A mutant enzyme, [N240Q]AmylA, which does not have an N-linked carbohydrate chain was created by site-directed mutagenesis . Thermostability of the mutant was much lower than the wild-type enzyme, which suggests that the carbohydrate chain of AmylA is important for the enzyme stability. The differences in reaction properties between the wild type enzyme and the mutant suggest that the carbohydrate chain of AmylA significantly affects the hydrolysis efficiency and the substrate recognition of AmylA for soluble starch . From the comparison of amino acid sequence of AmylA to those of other α-amylases whose 3D structures were clarified, the carbohydrate chain of A mylA is suggested to be positioned on the surface near the active cleft. Therefore, direct interactions between the carbohydrate chain and the substrate might affect the hydrolysis efficiency and substrate recognition.
