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

变旋酶高产生产葡萄糖新技术

基本信息

批准号：
05556011
负责人：
CHIBA Seiya
金额：
$ 6.53万
依托单位：
HOKKAIDO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1995
项目状态：
已结题

项目摘要

(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

(1)我们对葡糖淀粉酶催化的β-葡萄糖缩合反应（两种底物反应）进行了动力学分析，得到了速率参数和反应速率方程，可以准确估计任何β-葡萄糖浓度下缩合产物的形成。将α-葡萄糖添加到β-葡萄糖反应体系中激活了缩合速率。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了α-和β-葡萄糖的组成，并测量了反应速率。随着β-葡萄糖摩尔分数的降低，速度降低，这表明底物（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）我发现变旋酶减少了30％的二糖量，这是30％β-葡萄糖的产物通过葡糖淀粉酶。我们通过两种方法研究了变旋酶对葡萄糖生产的影响。第一种是目前在工业葡萄糖生产系统中使用的butch型方法。第二种是固定化酶技术，其中葡糖淀粉酶与基质连接，然后通过麦芽糖糊精与变旋酶的运行在柱中完成反应。在这两项试验中，变旋酶都取得了有效的结果，增加了葡萄糖的产量，减少了缩合产物。(3)对猪肾变旋酶的氨基酸序列进行了分析，以开展其基因克隆和酶过量生产的广泛计划。发现变旋酶的 N 末端被阻断。我们纯化了蛋白酶消化制备的N端封闭的肽片段，并通过串联质谱测定了其氨基酸序列，说明乙酰基封闭了变旋酶的N端。较少的