New Technique for High Yield Production of Glucose by Using of Mutarotase

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(1) We kinetically analyzed the glucoamylase-catalyzed condensation of beta-glucose (two substrates reaction), and obtained the rate parameters and reaction rate equation which makes an accurate estimate of condensation product formation in any beta-glucose concentration. Addition of alpha-glucose to beta-glucose reaction system activated the rate of condensation. We analyzed this activation, and found that the subsite 1 of glucoamylase did not bind to alpha-glucose, meaning no inhibition to condensation, and that alpha-glucose had higher affinity to the subsite 2 than beta-glucose. We changed the composition of alpha-and beta-glucose with keeping the total glucose concentration constant, and measured the reaction rate. The velocity was reduced with decreasig in the molar fraction of beta-glucose, suggesting that the decrease of substrate (beta-glucose) is more effective than the activation by alpha-glucose and that the mutarotase suppresses the formation of condensation products.(2) I … More t was found that the mutarotase reduced the amount of disaccharides in 30% which was the products from 30% beta-glucose by glucoamylase. We investigated the effect of mutarotase on glucose production by two methods. The first was the butch-typed method which is presently used in the industrial glucose production system. The second was the immobilized enzyme technique where glucoamylase was linked to matrix, and then reaction was done in the column by running of maltodextrin with mutarotase. In both tests mutarotase gave the effective results, the increase of glucose production and the decrease of condensation products.(3) We analyzed the amino acid sequence of porcine kidney mutarotase for developing the extensive project in the cloning of its gene and over-production of enzyme. The N-terminus of mutarotase was found to be blocked. We purified the N-terminal-blocked peptide fragment which was prepared by protease digestion, and determined its amino acid sequence by tandem mass spectrometry, elucidating that the acetyl group blocked the N-terminus of mutarotase. Less