Overproduction and characterization of xylanase B from Aspergillus niger.
The xynB gene, which encodes endo-beta-1,4-xylanase XynB, in Aspergillus niger BRFM281 was amplified by RT-PCR using mRNA isolated from a culture containing sugar beet pulp as an inducer. The cDNA was cloned into an expression cassette under the control of the strong and constitutive glyceraldhehyde-3-phosphate dehydrogenase gene promoter. The expression system was designed to produce the recombinant enzyme XynB with a six-histidine peptide fused to the carboxy end of the protein. Homologous overproduction of XynB was successfully achieved in shake flask cultures, and the secretion yield was estimated to be 900 mg x L(-1). The recombinant XynB was purified 1.5-fold by immobilized metal affinity chromatography to homogeneity using a one-step purification protocol with 71% recovery. The purified recombinant enzyme was fully characterized and has a molecular mass of 23 kDa and an optimal activity at pH 5.5 and 50 degrees C with stability in the pH range 4.0-7.0 and temperature up to 50 degrees C. Using soluble oat spelts xylan, the determined Km and Vmax values were 7.1 mg x mL(-1) and 3881 U x mg(-1), respectively.
Levasseur
Anthony
A
UMR 1163 INRA de Biotechnologie des Chamignons Filamenteux, IFR-BAIM, Universités de Provence et de la Méditerranée, ESIL, Marseille, France. anthony.levasseur@esil.univ-mrs.fr
Asther
Marcel
M
Record
Eric
E
D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D003470:Culture Media; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature; D014170:Transformation, Genetic; D015854:Up-Regulation
D003470:Culture Media; D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases