PRODUCTION OF SACCHARIFYING ENZYME USING THE WASTE-WATER OF A SHOCHU DISTILLERY

A saccharifying enzyme was produced by Aspergillus awamori var. kawachi using wastewater generated by a Shochu distillery. The production of the saccharifying enzyme was of the non-growth associated type, and 80 U of activity per ml of broth was obtained in about 6 d of flask cultivation. Since the Shochu distillery wastewater contained high concentrations of volatile fatty acids that were converted to their free forms and severely inhibited cell growth at low pH, the optimum initial pH ranged from 4.5 to 6.0. It is suggested that cell autolysis facilitated the release of the saccharifying enzyme, but a released protease digested the saccharifying enzyme with a subsequent decrease in activity. The saccharifying enzyme was easy to purify, and the purified enzyme was homogeneous when analyzed by disc electrophoresis. The molecular weight was estimated to be 54,000 Da by SDS-PAGE, and the isoelectric point was found to be pH 3.6 by isoelectric focusing. The optimum temperature and pH for the reaction ranged from 50 to 55-degrees-C and 4.5 to 5.5, respectively. The saccharifying enzyme could not digest raw starch. The hydrolyzate of soluble starch hydrolyzed by the saccharifying enzyme was composed of two to four oligosaccharides. From these results and the amino acid sequence in the N-terminal, the enzyme produced was concluded to be alpha-amylase.