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

(1)对葡糖淀粉酶催化的β-葡萄糖缩合反应（双底物反应）进行了动力学分析，得到了反应速率参数和反应速率方程，该方程能准确估计任意浓度β-葡萄糖下缩合产物的生成。在β-葡萄糖反应体系中加入α-葡萄糖可加快缩合速率。我们分析了这种激活，发现葡糖淀粉酶的亚位点1不与α-葡萄糖结合，这意味着对缩合没有抑制作用，并且α-葡萄糖对亚位点2的亲和力高于β-葡萄糖。我们在保持总葡萄糖浓度恒定的情况下改变α-和β-葡萄糖的组成，并测量反应速率。速率随着β-葡萄糖摩尔分数的降低而降低，表明底物（β-葡萄糖）的减少比α-葡萄糖的激活更有效，并且变旋酶抑制缩合产物的形成。(2)我 ...更多信息 发现变旋酶使30%的β-葡萄糖通过葡糖淀粉酶的产物二糖的量减少30%。我们通过两种方法研究了变旋酶对葡萄糖产生的影响。第一种是目前工业葡萄糖生产系统中使用的屠宰式方法。第二种是固定化酶技术，将葡萄糖淀粉酶连接到基质上，然后通过麦芽糊精和变旋酶的流动在柱中进行反应。在这两个测试中，变旋酶给出了有效的结果，葡萄糖产量的增加和缩合产物的减少。(3)本研究对猪肾变旋酶的氨基酸序列进行了分析，为进一步开展该酶基因的克隆和高产研究奠定了基础。发现变旋酶的N-末端被阻断。通过蛋白酶消化制备N端封闭的肽段，纯化后用串联质谱法测定其氨基酸序列，证实乙酰基封闭了变旋酶的N端。少

(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