Extracellular xylanases from two pathogenic races of Fusarium oxysporum f. sp. ciceris: enzyme production in culture and purification and characterization of a major isoform as an alkaline endo-beta-(1,4)-xylanase of low molecular weight.
Fusarium oxysporum f. sp. ciceris, the causal agent of Fusarium wilt of chickpea, comprises eight pathogenic races and two pathotypes. Races 0 and 5, representative of the least virulent yellowing pathotype and the most virulent wilt pathotype, respectively, produced extracellular xylanases when grown on minimal medium supplemented with either 1% commercial birchwood xylan or 0.3% chickpea cell walls. The pattern of extracellular proteins analysed by denaturing polyacrylamide gel electrophoresis in the two media presented some minor but distinctive differences between fungal races. By preparative isoelectrofocusing, the xylanase activity in cell wall-culture filtrates could be resolved into basic and neutral fractions with pI values around to 10 and 8, respectively, whereas the xylan-culture filtrates contained an additional acidic fraction of pI around 4. A common major xylanase was purified 7-fold to homogeneity by cation-exchange chromatography and chromatofocusing. The purified xylanase has a molecular weight of 21.6 kDa, optimum pH and temperature of 5.5 and 55 degrees C, respectively, pI in the range of 8.2 to 9.0, and Km and Vmax values of 2.24 mg ml(-1) (birchwood xylan as substrate) and 1200 nkat mg(-1) protein (72 U mg(-1) protein), respectively. The enzyme has an endo mode of action, hydrolysing xylan to xylobiose and higher short-chain xylooligosaccharides without forming free xylose.
Jorge
Inmaculada
I
Departamento de Bioquímica y Biología Molecular, ETSIAM, Universidad de Córdoba, Apartado 3048, 14080, Córdoba, Spain.
de la Rosa
Olga
O
Navas-Cortés
Juan A
JA
Jiménez-Díaz
Rafael M
RM
Tena
Manuel
M
D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D005670:Fusarium / Q000201:enzymology* / Q000472:pathogenicity; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight
D005656:Fungal Proteins; D007527:Isoenzymes; D043364:Endo-1,4-beta Xylanases