Molecular cloning and over-expression of a fructosyltransferase from Aspergillus niger QU10.
- Author:
Guoqing ZHANG
;
Jing YANG
;
Jiaji SHI
;
Shijun QIAN
;
Yapeng CHAO
- Publication Type:Journal Article
- MeSH:
Aspergillus niger;
enzymology;
genetics;
Base Sequence;
Cloning, Molecular;
DNA, Complementary;
Escherichia coli;
Fungal Proteins;
genetics;
metabolism;
Glycosylation;
Hexosyltransferases;
genetics;
metabolism;
Molecular Sequence Data;
Molecular Weight;
Pichia;
Sucrose;
metabolism;
Trisaccharides;
metabolism
- From:
Chinese Journal of Biotechnology
2015;31(4):512-522
- CountryChina
- Language:English
-
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.
