Conversion of Inulin to Useful Substances

1.以黑曲霉817和酿酒酵母1200为菌种，在30ﾟC的条件下对菊芋块茎同时糖化发酵进行了研究。在菊粉水解菌的筛选中，青霉TN-88是菊粉酶的最佳产生菌。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法测定，胞外酶P-II为均一酶，表观相对分子质量为68.0 kDa。比活力为105U/mg。在pH值为5.2、温度为50ﾟ时，酶活最高。该酶对菊粉的水解率可达70%，主要产物为菊粉三糖，但对蔗糖、棉子糖和乳糖缺乏活性。菊粉的表观K_m值为0.2 mM，温度为40ﾟ，pH为5.0时，酶的N端氨基酸序列为1-DDYRPAFFFC PAENXMNEPN GLIQIXSTXH-30.3。Southern-Blot分析表明，内切菊粉酶基因inuA和inuB分别存在于3.9kbp和5.9kbp的EcoRI酶切片段上。每个片段含有1,548个碱基的单一开放阅读框，在编码区内没有发现插入序列。成熟的酶由493个氨基酸组成，前面有一个由23个氨基酸组成的信号肽。这些酶含有两个半胱氨酸残基和六个潜在的N-糖基化位点。含有克隆的inuA和inuB基因的大肠杆菌JM109的无细胞提取物的菊粉酶活性分别为0.42和0.59U/mg蛋白质。其推导的氨基酸序列与紫青霉内切菊粉酶有73%的同源性。

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.