title: Cloning, overexpression, and characterization of a highly active endoinulinase gene from Aspergillus fumigatus Cl1 for production of inulo-oligosaccharides.
abstract: In this study, an endoinulinase gene from Aspergillus fumigatus was cloned and overexpressed in Pichia pastoris. A maximum activity, 3860 U/ml, of the recombinant endoinulinase was obtained by using a high cell-density fermentation approach. The recombinant endoinulinase with a molecular weight of 58 kDa was purified for further enzymatic investigation. The pH and temperature optima were pH 6.0 and 55 °C, and the K m and V max values toward inulin were 2.18 mM and 1590 μmol min(-1) mg(-1), respectively. The degree of polymerization (DP) for inulo-oligosaccharides product 3, 4, 5, and >5 was determined by thin-layer chromatography (TLC) and high-performance liquid chromatograph (HPLC). Immobilized endoinulinases were prepared and characterized, which showed higher stability than the free endoinulinase under various temperature levels. A residual activity of 81.2 % could be still obtained after ten reaction cycles. Thus, the present recombinant endoinulinase exhibited great potential for scale-up production of inulo-oligosaccharides.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, Department of Food Science and Technology, School of Biotechnology, East China University of Science and Technology, 130 # Meilong Rd, P. O. Box 283, Shanghai, 200237, People's Republic of China.
authors.forename: Min
authors.initials: M
authors.lastname: Chen
authors.forename: Xiao
authors.initials: X
authors.lastname: Lei
authors.forename: Chuze
authors.initials: C
authors.lastname: Chen
authors.forename: Shiyang
authors.initials: S
authors.lastname: Zhang
authors.forename: Jingli
authors.initials: J
authors.lastname: Xie
authors.forename: Dongzhi
authors.initials: D
authors.lastname: Wei
mesh_terms: D000595:Amino Acid Sequence; D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000235:genetics*; D019149:Bioreactors / Q000382:microbiology; D002452:Cell Count; D003001:Cloning, Molecular; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005285:Fermentation; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007444:Inulin / Q000737:chemistry*; D008670:Metals / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis; D013696:Temperature
chemical_list: D004800:Enzymes, Immobilized; D008670:Metals; D009844:Oligosaccharides; D011994:Recombinant Proteins; D007444:Inulin; D006026:Glycoside Hydrolases