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Purification and characterization of an endoxylanase from Trichoderma koningii G-39.

ABSTRACT: Trichoderma koningii G-39 produced xylanases in submerged culture using oat spelt xylan or crystalline cellulose, Avicel, as the sole carbon source. A low-Mr xylanase was purified from the culture filtrate by ion-exchange chromatography on SP-Trisacryl-M and gel filtration on Fractogel TSK HW-50F. It was homogeneous on SDS/PAGE and isoelectric focusing. A typical procedure provided about 11-fold purification with 4.5% protein yield and 50% activity recovery. The purified enzyme has an Mr value of about 21,500 and a pI of 8.9. Its specific activity was 6100 units/mg of protein, with optimal activity towards 0.5% xylan at about pH 5.5 and 60 degrees C. The purified enzyme had no activity against CM-cellulose with a degree of substitution of 0.63. It also showed no beta-xylosidase activity. The Km and Vmax. values, as determined with the soluble fraction of oat spelt xylan as substrate, were 0.70 mg/ml and 1.85 x 10(6) mumol/min per mg of enzyme respectively. Hg2+ (1 mM) and SDS (10 mM) completely inhibited xylanase activity, whereas Ca2+ showed no significant effect on the enzyme activity at 1 mM, but gave 80% inhibition at 10 mM. The enzyme contained about 4.4% carbohydrate and showed an immunological relationship to a cellobiohydrolase from the same fungal strain.

SUBMITTER: Huang L

PROVIDER: S-EPMC1151344 | biostudies-other | 1991 Sep

REPOSITORIES: biostudies-other

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Project description:A GH (glycoside hydrolase) family 54 alpha-L-arabinofuranosidase from Trichoderma koningii G-39 (termed Abf) was successfully expressed in Pichia pastoris and purified to near homogeneity by cation-exchange chromatography. To determine the amino acid residues essential for the catalytic activity of Abf, extensive mutagenesis of 24 conserved glutamate and aspartate residues was performed. Among the mutants, D221N, E223Q and D299N were found to decrease catalytic activity significantly. The kcat values of the D221N and D299N mutants were 7000- and 1300-fold lower respectively, than that of the wild-type Abf. E223Q was nearly inactive. These results are consistent with observations obtained from the Aspergillus kawachii alpha-L-arabinofuranosidase three-dimensional structure. This structure indicates that Asp221 of T. koningii Abf is significant for substrate binding and that Glu223 as well as Asp299 function as a nucleophile and a general acid/base catalyst for the enzymatic reaction respectively. The catalytic mechanism of wild-type Abf was further investigated by NMR spectroscopy and kinetic analysis. The results showed that Abf is a retaining enzyme. It catalyses the hydrolysis of various substrates via the formation of a common intermediate that is probably an arabinosyl-enzyme intermediate. A two-step, double-displacement mechanism involving first the formation, and then the breakdown, of an arabinosyl-enzyme intermediate was proposed. Based on the kcat values of a series of aryl-alpha-L-arabinofuranosides catalytically hydrolysed by wild-type Abf, a relatively small Brønsted constant, beta(lg)=-0.18, was obtained, suggesting that the rate-limiting step of the enzymatic reaction is the dearabinosylation step. Further kinetic studies with the D299G mutant revealed that the catalytic activity of this mutant depended largely on the pK(a) values (>6) of leaving phenols, with beta(lg)=-1.3, indicating that the rate-limiting step of the reaction becomes the arabinosylation step. This kinetic outcome supports the idea that Asp299 is the general acid/base residue. The pH activity profile of D299N provided further evidence strengthening this suggestion.

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Purification and characterization of an endoxylanase from Trichoderma koningii G-39.

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