Purification, Characterization and Application of .ALPHA.-Amylase from Pseudozyma aphidis I-8

A yeast strain I-8 was isolated as an alpha-amylase producer from digestive juice of Nepenthes bicalarate. The yeast was identified as Pseudozyma aphidis by the morphological test and comparative 26S rDNA-D1/D2 and ITS-5.8S rDNA gene sequence analysis. The alpha-amylase was purified from the culture filtrate by (NH sub(4)) sub(2)SO sub(4) precipitation, DEAE-TOYOPEARL 650M, Butyl-TOYOPEARL 650M, Hydroxylapatite and TOYOPEARL HW-55 chromatography. The purified enzyme was shown as a single band and the molecular mass was 55 kDa by SDS-PAGE. The specific activity was 1679 U/mg protein. The optimum temperature and pH were around 60C and 5.0, respectively. The enzyme was stable in a pH range from 5.0 to 9.0 and at below 60C. The enzyme hydrolyzed soluble starch and released glucose, maltose and oligosaccharides. Maltooligosaccharides (G3-G5) were also favorable substrate but it showed no activity toward maltose, isomaltose or pullulan. On the hydrolysis of soluble starch, the iodine color of the reaction mixture disappeared at almost 10% of reducing sugar formation and the hydrolysis limit was about 70% of soluble starch. From these results, the alpha-amylase was recognized as a unique alpha-amylase. The alpha-amylase was applied to the bread making process. Addition of the alpha-amylase to the bread making process presented no effect toward the crumb softness or color but the improved taste of the bread by sensory evaluation.