title: Production and characterisation of recombinant α-L-arabinofuranosidase for production of xylan hydrogels.
abstract: A recombinant strain of the protease-deficient, non-acidifying pH mutant Aspergillus niger D15 (A. niger D15 [abfB]) strain was developed to secrete α-L-arabinofuranosidase (AbfB) free of endo-1,4-β-xylanases for selective hydrolysis of xylan into hydrogels. The A. niger D15 [abfB] strain expressed the α-L-arabinofuranosidase abfB gene under the transcriptional control of the glyceraldehyde-3-phosphate dehydrogenase promoter (gpd(P)) and glucoamylase terminator (glaA(T)) in fermentation cultures containing 10 % glucose. The yield, activity, purity, kinetics and ability of the recombinant AbfB to selectively hydrolyse xylans into hydrogels were assessed. The recombinant AbfB secreted in 125-mL shake flasks and 10-L bioreactor fermentation cultures had specific activities against ρ-nitrophenyl-α-arabinofuranoside of up to 4.4 and 2.7 U g⁻¹ (dry weight), respectively. In addition, the recombinant AbfB was present as a single protein species on silver-stained 10 % sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The recombinant AbfB had optimal activity at 40-55 °C and pH 3.0 to pH 5.0 and was stable at temperature and pH of up to 60 °C and pH 6.0, respectively. About 20 % of the available arabinose in the xylan was released by the recombinant AbfB from the hydrolysis of low viscosity wheat and oat spelt arabinoxylans and about 9 and 5 % from bagasse and bamboo arabinoglucuronoxylans, respectively, that led to the formation of the hydrogels. Therefore, the constructed A. niger D15 [abfB] strain presented a microbial system for the production of recombinant AbfB with the required purity for the modification of xylans into hydrogels.
authors.affiliation: Department of Process Engineering, University of Stellenbosch, P/Bag X1, Matieland, Stellenbosch 7602, South Africa.
authors.forename: A F A
authors.initials: AF
authors.lastname: Chimphango
authors.forename: S H
authors.initials: SH
authors.lastname: Rose
authors.forename: W H
authors.initials: WH
authors.lastname: van Zyl
authors.forename: J F
authors.initials: JF
authors.lastname: Görgens
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D019149:Bioreactors; D001709:Biotechnology / Q000379:methods*; D003470:Culture Media; D005285:Fermentation; D015966:Gene Expression Regulation, Fungal; D006026:Glycoside Hydrolases / Q000096:biosynthesis* / Q000235:genetics; D020100:Hydrogels / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D014990:Xylans / Q000378:metabolism*
chemical_list: D003470:Culture Media; D020100:Hydrogels; D011994:Recombinant Proteins; D014990:Xylans; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase