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

1.Aspergillus niger12 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 between94 and 297bp downstream of the stop codon.We have determined the nucleotide sequences of the 1201-and 1017-bp5‘-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 Sepharose4B(CAS)。All three immobilized enzymes increased the resistance to inactivations by p-chloromercuribenzoate or Fe I D 13+I D 1.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 I D2m ii D2 value decreased in the BAC-immobilized endoinulinase and increased in the CAS-immobilized enzyme.3。An endoinulinase gene from Penicillium sp.Strain TN-88was cloned and sequenced.The open reading frame(ORF)of 1，545bp 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 showed72and85%identities to those of A.niger and Penicillium purpurogenum endoinulinases，respectively。A neighbor-joining tree showed that fungal endoinulinases form a distinct group from otherβ-fructofuranosidases。It is postulated that the fungal endoinulinase genes are of bacterial origin.

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.