title: Cloning, characterization and heterelogous expression of the INU1 gene from Cryptococcus aureus HYA.
abstract: The INU1 gene (Accession number: JX073660) encoding exo-inulinase from Cryptococcus aureus HYA was cloned and characterized. The gene had an open reading frame (ORF) of 1653 bp long encoding an inulinase. The coding region of the gene was not interrupted by any intron. It encoded 551 amino acid residues of a protein with a putative signal peptide of 23 amino acids and the calculated molecular mass of 59.5 kDa. The protein sequence deduced from the inulinase structural gene contained the inulinase consensus sequences (WMNDPNGL), (RDP), ECP, FS and Q. It also had two conserved putative N-glycosylation sites. The inulinase from C. aureus HYA was found to be closely related to that from Kluyveromyces marxianus and Pichia guilliermondii. The inulinase gene without the signal sequence was subcloned into pPICZaA expression vector and expressed in Pichia pastoris X-33. The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 60 kDa was found. Enzyme activity assay verified the recombinant protein as an inulinase. A maximum inulinase activity of 16.3±0.24 U/ml was obtained from the culture supernatant of P. pastoris X-33 harboring the inulinase gene. The optimal temperature and pH for action of the enzyme were 50 °C and 5.0, respectively. A large amount of monosaccharides were detected after the hydrolysis of inulin with the purified recombinant inulinase.
authors.affiliation: Unesco Chinese Center of Marine Biotechnology, Ocean University of China, Yushan Road, No. 5, Qingdao, Qingdao 266003, China.
authors.forename: Tian-Shu
authors.initials: TS
authors.lastname: Cao
authors.forename: Guang-Yuan
authors.initials: GY
authors.lastname: Wang
authors.forename: Zhe
authors.initials: Z
authors.lastname: Chi
authors.forename: Zhi-Peng
authors.initials: ZP
authors.lastname: Wang
authors.forename: Zhen-Ming
authors.initials: ZM
authors.lastname: Chi
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D003454:Cryptococcus / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D015971:Gene Expression Regulation, Enzymologic; D005800:Genes, Fungal; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D014162:Transfection
chemical_list: D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C023086:inulinase