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Genetic analysis of inuin-degrading enzymes and their application to oligosaccharide production

菊因降解酶的遗传分析及其在寡糖生产中的应用

基本信息

批准号：
10660312
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：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

1. Aspergillus niger 12 contained two copies of endoinulinase genes (inuA and inuB) in the genome. Only the inuB gene was transcribed constitutively. Four distinct 5'-ends of the transcripts were observed at positions -80 (A), -72 (G), -69 (A), and -65 (A) from the start codon. The inuB mRNAs were polyadenylated at various sites between 94 and 297 bp downstream of the stop codon. We have determined the nucleotide sequences of the 1201- and 1017-bp 5'-noncoding regions of the inuA and inuB genes, respectively. The inuB promoter region included a putative TATA box at -116 (TATATA).2. An A. niger endoinulinase has been immobilized covalently onto bromoacetyl-cellulose (BAC), cellulose-carbonate (CC) and CNBr-activated Sepharose 4B (CAS). All three immobilized enzymes increased the resistance to inactivations by p-chloromercuribenzoate or FeィイD13+ィエD1. The marked rise from 45 to 60℃ in the optimal temperature for inulin hydrolysis was observed in the CAS-immobilized enzyme. The pH stability decreased on the acidic side and increased slightly to alkaline side in the BAC-immobilized endoinulinase, and shifted to acidic side in the CC-immobilized enzyme. The KィイD2mィエD2 value decreased in the BAC-immobilized endoinulinase and increased in the CAS-immobilized enzyme.3. An endoinulinase gene from Penicillium sp. Strain TN-88 was cloned and sequenced. The open reading frame (ORF) of 1,545 bp was not interrupted by introns, and it encoded 25 amino acid signal peptide and 490 amino acid mature protein. The mature enzyme contained three Cys residues and ten potential N-linked glycosylation sites. The deduced amino acid sequence showed 72 and 85% identities to those of A. niger and Penicillium purpurogenum endoinulinases, respectively. A neighbor-joining tree showed that fungal endoinulinases form a distinct group from other β-fructofuranosidases. It is postulated that the fungal endoinulinase genes are of bacterial origin.

1.尼日尔曲霉12基因组中含有两个内切菊粉酶基因拷贝（inuA和inuB）。只有inuB基因组成型转录。在起始密码子的位置-80（A）、-72（G）、-69（A）和-65（A）处观察到转录物的四个不同的5 '末端。inuB mRNA在终止密码子下游94和297 bp之间的多个位点处被聚腺苷酸化。我们已经确定了inuA和inuB基因的1201-和1017-bp的5 '-非编码区的核苷酸序列。inuB启动子区在-116（TATATA）处包含推定的TATA盒。了全境以溴乙酰纤维素（BAC）、纤维素碳酸酯（CC）和溴化氰活化的琼脂糖4 B（CAS）为载体，共价固定了黑尼日尔内切菊粉酶。所有三种固定化酶都增加了对氯汞苯甲酸盐或铁汞D13+铁汞D1灭活的抗性。固定化酶水解菊粉的最适温度从45 ℃显著提高到60℃。固定化酶的pH稳定性在酸性侧降低，在碱性侧略有增加，在CC固定化酶的pH稳定性向酸性侧移动。BAC固定化菊粉内切酶的K值D2 m值D2降低，而CAS固定化酶的K值D2升高.从青霉TN-88菌株中克隆了一个内切菊粉酶基因。该基因的开放阅读框（ORF）长1,545 bp，不被内含子打断，编码25个氨基酸的信号肽和490个氨基酸的成熟蛋白。成熟酶含有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A.尼日尔和产紫青霉内切菊粉酶。邻接树显示真菌内切菊粉酶与其他β-呋喃果糖苷酶形成不同的组。推测真菌内切菊粉酶基因是细菌来源的。