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.
Subject(s)
Aspergillus niger , 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 , MetabolismABSTRACT
In this study, we investigated the mechanism of transformation by Bacillus subtilis strain 168 by proteomic analysis. B. subtilis strain 168 was able to stereoselectively transform cis-propenylphosphonic acid (cPPA) to fosfomycin. The maximal fosfomycin production was 816.6 microg/mL after two days cultivation, with a conversion rate of 36.05%. We separated the whole cellular proteins by two-dimensional gel electrophoresis (2-DE) method, and 562 protein spots were detected in the presence of cPPA in the medium, while 527 protein spots were detected in the absence of cPPA. Of them, 98 differentially expressed protein spots were found. Among them, 52 proteins were up-regulated whereas 20 were down-regulated in the presence of cPPA in the medium, and 26 induced at the presence of cPPA. The differentially expressed proteins were analyzed by combined MS and MS/MS methods. Eighty protein spots, including 45 up-regulated proteins, 17 down-regulated proteins, and 18 induced by cPPA were identified. Based on the results of proteomic analysis, we postulated two steps of transformation: in the first step, cPPA was hydrated to 2-hydroxypropylphosphonic acid; in the second step, 2-hydroxypropylphosphonic acid was transformed to fosfomycin via a dehydrogenation reaction.