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

1.在基因组中含有2份endoinulinase genes （inuA and inuB）的副本。只有原始的B基因是可转录的、可持续的。四个距离5'-从起始密码子可见的位置-80 （A）、-72 （G）、-69 （A）和-65 （A）的终点。在94和297个基点之间，inuB mRNAs在不同的站点中被聚合化，在停止密码子下游。我们已经确定了1201-和1017-bp 5'-NeuA和NeuB基因的非编码区域，尊重地。inuB promoter region包括-116 （TATATA）中的putative TATA box。A. A. Niger Endoinulinase与溴乙酰-纤维素（BAC）、纤维素-碳酸酯（CC）和CNBr-激活的Sepharose 4 B（CAS）具有生物活性。All three immobilized enzymes increased the resistance to inactivations by p-chloromercuribenzoate or Fe D13+イエD1。在最佳温度下，在45 - 60摄氏度的情况下，胰岛素氢化物被观察到在CAS-非流动性酶中。pH值稳定性在BAC-非流动性内素酶中减少，略微减少到碱性化合物中，并转移到CC-非流动性酶中的酸性化合物。在CAS-immobilized enzyme.3中的BAC-immobilized endoinulinase and increased in the CAS-immobilized enzyme.3。一种从青霉素sp. TN-88中提取的内皮素酶基因被克隆和测序。1，545 bp的开放阅读框（ORF）没有被内含子中断，并编码了25个氨基酸信号肽和490个氨基酸成熟蛋白。成熟酶包含三个Cys驻地和十个潜在的N-连接的糖基化位点。使用过的氨基酸序列显示了72%和85%的A的身份。Niger和Penicillium purpurogenum endoinulinases， Respectively。一个邻居-结合树展示了一种真菌内素酶从另一个β-fructofuranosidases中形成一个不同的群体。这是因为真菌内素酶基因是细菌起源的产物。

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.