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Conversion of Inulin to Useful Substances

将菊粉转化为有用物质

基本信息

批准号：
08660401
负责人：
OHTA Kazuyoshi
金额：
$ 1.41万
依托单位：
Miyazaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

1.Simultaneous saccharification and fermentation of Jerusalem artichoke tubers were conducted batchwise at 30ﾟC using Aspergillus niger 817 and Saccharomyces cerevisiae 1200. Ethanol concentrations obtained were 10.4% (v/v) from the ground tubers after 15 h, 15.0% from the juice concentrate after 72 h, and 20.1% from the flour after 120 h.2.In a screening of inulinolytic fungi, Penicillium sp.TN-88 was selected as the best producer of inulinase. The extracellular endoinulinase P-II was purified to be homogeneous, as judged by SDS-polycrylamide gel electrophoresis, with an apparent M_r of 68.0 kDa. The specific activity was 105 U/mg. The enxyme activity was highest at pH 5.2 and 50ﾟC.The enzyme hydrolyzed inulin to the extent of 70% and liberated inulotriose as the main product, but lacked activity toward sucrose, raffinose or levan. The apparent K_m value for inulin was 0.20 mM at 40ﾟC and pH 5.0 The N-terminal amino acid sequence of the enzyme was 1-DDYRPAFHFC PAENXMNEPN GLIQIXSTXH-30.3.Two genomic genes encoding endoinulinase from Aspergillus niger 12 were cloned in E.coli and sequenced. Southern-blot analysis indicated that the endoinulinase genes, inuA and inuB,were present on separate EcoRI-digested fragments of sizes 3.9 kbp and 5.9 kbp, respectively. Each fragment contained a single open reading frame of 1,548 bp, and no intervening sequences were found within the coding region. The mature enzymes consisted of 493 amino acids and was preceded by a putative signal peptide of 23 amino acids. The enzymes contained two Cys residues and six potential sites for N-linked glycosylation. The cell-free extracts of E.coli JM109 harboring the cloned inuA and inuB genes showed inulinase activities of 0.42 and 0.59 U/mg of protein, respectively. The deduced amino acid sequences of the mature A.niger enzymes showed 73% identity with that of the Penicillium purpurogenum endoinulinase.

1.以黑曲霉817和酿酒酵母1200为原料，在30℃的温度下分批进行了菊芋块茎的糖化和发酵。15 h后块茎的乙醇浓度为10.4% (v/v)， 72 h后浓缩汁的乙醇浓度为15.0%，120 h后面粉的乙醇浓度为20.1%。在菊粉酶解真菌的筛选中，选择青霉sp.TN-88为最佳产菊粉酶菌。经sds -聚丙烯酰胺凝胶电泳鉴定，胞外内生酶P-II均相，表观M_r为68.0 kDa。比活性为105 U/mg。酶活性在pH为5.2和50cc时最高。该酶对菊糖的水解率达70%，主要产物为菊糖，但对蔗糖、棉子糖和利末酸缺乏水解活性。该酶n端氨基酸序列为1-DDYRPAFHFC PAENXMNEPN GLIQIXSTXH-30.3，其表观K_m值为0.20 mM。在大肠杆菌中克隆了两个编码黑曲霉12型内生粉酶的基因组基因并进行了测序。Southern-blot分析表明，inuA和inuB基因分别存在于ecori消化的3.9 kbp和5.9 kbp的片段上。每个片段包含一个1548 bp的开放阅读框，编码区内未发现中间序列。成熟酶由493个氨基酸组成，其前导有23个氨基酸的信号肽。这些酶含有两个半胱氨酸残基和六个潜在的n链糖基化位点。含有克隆的inuA和inuB基因的大肠杆菌JM109无细胞提取物的菊粉酶活性分别为0.42和0.59 U/mg蛋白。推导出的成熟黑曲霉酶的氨基酸序列与紫红色青霉内生酶的同源性为73%。