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Phylogenetic analysis of inuin-degrading enzymes and efficient expression

菊因降解酶的系统发育分析及高效表达

基本信息

批准号：
12660297
负责人：
OHTA Kazuyoshi
金额：
$ 2.3万
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：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

1. An endoinulinase gene of Penicillium sp. strain TN-88 was cloned and sequenced. An open reading frame of the gene was not interrupted by introns, and it encoded a 25 amino acid signal peptide and a 490 amino acid mature protein. The mature protein contained three Cys residues and ten potential N-linked glycosylation sites. The deduced amino acid sequence showed 72 and 85 % identities with those of Aspergillus niger and Penicillium purpurogenum endoinulinase genes, respectively. A neighbor-joining tree showed that fungal endoinulinases are closely related to bacterial levanases.2. Endoinulinase was partially purified from the culture filtrate of a filamentous fungus A. niger mutant 817. The enzyme preparation was immobilized covalently onto a porous cellulose derivative, Amino-Cellulofine. A 5 % (w/v) solution (pH 5.0) of inulin was continuously hydrolyzed in a packed-bed column reactor containing the immobilized enzyme. The majority of the hydrolysis products were inulo-oligosaccharides with a degree of polymerization of 3 to 5. In vtro studies indicated that both the F_3 and F_4 was preferentially utilized in vitro by Bifidobacterium spp.3. Intracellular exo- and endoinulinases were extracted from mycelia of A. niger strain 12 and purified by DEAE-Cellulofine A-500, Sephadex G-100, and Sephadex G-200 chromatographies. The purified exoinulinase P-II had specific activities of 6.6 U/mg toward inulin and 22 U/mg toward sucrose. The endoinulinase P-III showed 108 U/mg toward inulin, but no activity toward sucrose. M__-_rs of exoinulinase P-II and endoinulinase P-III were determined by gel filtration using Sephadex G-200 as 47 and 56 kDa, respectively. Optimal pH and temperature for enzyme activity were pH 5.0 and 55 ℃ for P-II, and pH 5.3 and 45 ℃, for P-III. Both the enzymes were activated by Mn^<2+>, and inactivated by Ag^+, Hg^<2+> or p__--chloromercuribenzoate. Inulinases P-II and P-III exhibited apparent K__-_m values of 5.8 and 0.80 mM, respectively.

1.克隆了青霉TN-88菌株的内切菊粉酶基因。该基因的开放阅读框不被内含子打断，编码一个25个氨基酸的信号肽和一个490个氨基酸的成熟蛋白。成熟蛋白含有3个Cys残基和10个潜在的N-连接糖基化位点。推导的氨基酸序列与尼日尔曲霉和产紫青霉内切菊粉酶基因的同源性分别为72%和85%。邻接树显示真菌内切菊粉酶与细菌果聚糖酶的亲缘关系较近.从丝状真菌A. 817号尼日尔突变体。将酶制剂共价固定在多孔纤维素衍生物Amino-Cellulofine上。在含有固定化酶的填充床柱反应器中连续水解菊糖的5%（w/v）溶液（pH 5.0）。大部分水解产物是聚合度为3至5的菊粉寡糖。体外研究表明，双歧杆菌3号在体外对F_3和F_4都有优先利用。从A.经DEAE-Cellulofine A-500、Sephadex G-100和Sephadex G-200层析纯化。纯化的外切菊粉酶P-II对菊粉的比活力为6.6U/mg，对蔗糖的比活力为22 U/mg。内切菊粉酶P-III对菊粉的活性为108 U/mg，但对蔗糖没有活性。用SephadexG-200凝胶过滤法测得外切菊粉酶P-II和内切菊粉酶P-III的分子量分别为47和56 kDa。P-II的最适pH为5.0，最适温度为55 ℃; P-III的最适pH为5.3，最适温度为45 ℃。这两种酶都能被Mn^<2+>激活，而被Ag^+、Hg^<2+>和对氯汞苯甲酸盐灭活。菊糖酶P-II和P-III的表观K__-_m值分别为5.8和0.80 mM。