title: Molecular cloning and over-expression of a fructosyltransferase from Aspergillus niger QU10.
abstract: The main commercial production of fructooligosaccharides (FOS) comes from enzymatic transformation using sucrose as substrate by microbial enzyme fructosyltransferase. A fructosyltransferase genomic DNA was isolated from Aspergillus niger QU10 by PCR. The nucleotide sequence showed a 1 941 bp size, and has been submitted to GenBank (KF699529). The cDNA of the fructosyltransferase, containing an open reading frame of 1 887 bp, was further cloned by RT-PCR. The fructosyltransferase gene from Aspergillus niger was functionally expressed both in Escherichia coli and Pichia pastoris GS 115. The highest activity value for the construction with the α-factor signal peptide reached 431 U/mL after 3 days of incubation. The recombinant enzyme is extensively glycosylated, and the active form is probably represented by a homodimer with an apparent molecular mass of 200 kDa as judged from mobility in seminative PAGE gels. The extracellular recombinant enzyme converted sucrose mostly to FOS, mainly 1-kestose and nystose, liberating glucose. FOS reached a maximal value and represented about 58% of total sugars present in the reaction mixture after 4 h reaction. The results suggest that the availability of recombinant Pichia pastoris as a new source of a FOS-producing enzyme might result of biotechnology interest for industrial application.
authors.forename: Guoqing
authors.initials: G
authors.lastname: Zhang
authors.forename: Jing
authors.initials: J
authors.lastname: Yang
authors.forename: Jiaji
authors.initials: J
authors.lastname: Shi
authors.forename: Shijun
authors.initials: S
authors.lastname: Qian
authors.forename: Yapeng
authors.initials: Y
authors.lastname: Chao
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006602:Hexosyltransferases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010843:Pichia; D013395:Sucrose / Q000378:metabolism; D014312:Trisaccharides / Q000378:metabolism; D001426:Bacterial Proteins
chemical_list: D018076:DNA, Complementary; D005656:Fungal Proteins; D006602:Hexosyltransferases; D013395:Sucrose; D014312:Trisaccharides; C072433:1-kestose; C021451:inulosucrase; D001426:Bacterial Proteins