Expression of an Aspergillus niger Glucose Oxidase in Saccharomyces cerevisiae and Its Use to Optimize Fructo-oligosaccharides Synthesis

Abstract

Fructo-oligosaccharides (FOS) represent the most abundantly supplied and utilized group of nondigestible oligosaccharides as food ingredients. These prebiotics can be produced from sucrose using the transglycosylating activity of -fructofuranosidases (EC 3.2.1.26) at high concentrations of the starting material. The main problem during FOS synthesis is that the activity of the enzyme is inhibited by the glucose generated during the reaction, and therefore the maximum FOS content in commercial products reaches up to 60% on a dry substance basis. The glucose oxidase (gox) gene from Aspergillus niger BT18 was cloned and integrated, as part of an expression cassette, into the ribosomal DNA of a Saccharomyces cereVisiae host strain. One of the recombinant strains with a high copy number of the gox gene and showing a high GOX specific activity was used to produce the enzyme. Addition of the extracellular glucose oxidase to the FOS synthesis reaction helped to remove the glucose generated, avoiding the inhibition of the fungal -fructofuranosidase. As a result, a final syrup containing up to 90% of FOS was obtained.

High-level expression of the Penicillium notatum glucose oxidase gene in Pichia pastoris using codon optimization

“…GOD activity assay GOD activity was determined by the method described previously (Valdivieso-Ugarte et al 2006). Briefly, reaction systems containing 260 lM o-dianisidine in 2.5 ml 100 mM citric acid/Na 2 HPO 4 buffer (pH 6.2), 300 ll 18% (w/v) D-glucose, and 100 ll horseradish peroxidase (90 U ml -1 ) were preheated at 35°C for 5 min.…”

Preparation of High-Purity Fructo-oligosaccharides by Aspergillus japonicus β-Fructofuranosidase and Successive Cultivation with Yeast

“…As the content of glucose decreases gradually and its feedback effect disappears quickly, the activity of β-fructofuranosidase is recovered; thus, sucrose is again converted into FOSs (Figure 2), which is similar to previous reports (2)(3)(4). As a result, the content of sucrose decreases gradually from 9.99% at the beginning to 1.32% at 16 h and to almost zero at 20 h. What is more interesting is that the content of both mono-and disaccharides decreases significantly from 40.98% at the beginning to 5.54% at 16 h and to 1.84% at 24 h; this huge and quick utilization especially for mono sugars is much more helpful to remove the feedback effect of glucose and to recover the activity of β-fructofuranosidase (5)(6)(7)(8)19).…”

Enhanced Thermostability of Glucose Oxidase through Computer-Aided Molecular Design

“…The mixed plasmids were linearised with Bgl II, and then transformed into P. pastoris GS115 competent cells by electroporation. Transformants were cultivated on RDB plates at 28 °C for 48 h, and then inoculated and incubated on MM plates for another 24 h. The positive transformants were screened by chromogenic reaction for 30 min at room temperature [ 30 ]. Individual positive transformants were placed in 48-well microtiter plates containing 500 μL of buffered glycerol complex (BMGY) medium, and then incubated at 28 °C for 48 h with 200 r/min of shaking.…”