/pdf/purification-and-characterization-2y44lhntkf.pdf

Purification and Characterization

http://protocol-online.org/forums/uploads/monthly_09_2010/msg-6750-042877300 1283321951.ipb

Interlaboratory testing of methods for assay of xylanase activity

Gel electrophoresis of proteins: A practical approach

Basic features of regulation of expression of the genes encoding the cellulases of the filamentous fungus Trichoderma reesei QM9414, the genes cbh1 and cbh2 encoding cellobiohydrolases and the genes egl1, egl2 and egl5 encoding endoglucanases, were studied at the mRNA level. The cellulase genes were coordinately expressed under all conditions studied, with the steady-state mRNA levels of cbh1 being the highest. Solka floc cellulose and the disaccharide sophorose induced expression to almost the same level. Moderate expression was observed when cellobiose or lactose was used as the carbon source. It was found that glycerol and sorbitol do not promote expression but, unlike glucose, do not inhibit it either, because the addition of 1 to 2 mM sophorose to glycerol or sorbitol cultures provokes high cellulase expression levels. These carbon sources thus provide a useful means to study cellulase regulation without significantly affecting the growth of the fungus. RNA slot blot experiments showed that no expression could be observed on glucose-containing medium and that high glucose levels abolish the inducing effect of sophorose. The results clearly show that distinct and clear-cut mechanisms of induction and glucose repression regulate cellulase expression in an actively growing fungus. However, derepression of cellulase expression occurs without apparent addition of an inducer once glucose has been depleted from the medium. This expression seems not to arise simply from starvation, since the lack of carbon or nitrogen as such is not sufficient to trigger significant expression.

Regulation of cellulase gene expression in the filamentous fungus Trichoderma reesei.

Production of fungal xylanases

Xylanase production by Trichoderma longibrachiatum

Xylose oligomers rapidly induced xylanase activity of Trichoderma longibrachiatum, whereas induction was delayed in the presence of glucose. Cellobiose, cellopentaose, and xylobiose did not induce detectable levels of cellulase activity. However, mixtures of xylobiose with cellobiose or cellopentaose rapidly induced cellulase activity. In addition, mixtures of xylobiose with cellopentaose or cellobiose induced xylanase activity more effectively than xylobiose alone. Both xylanase and cellulase activity were detected after a lag period in the presence of lactose.

Interrelationship of Xylanase Induction and Cellulase Induction of Trichoderma longibrachiatum.

A laminarinase [endo-(1,3)-β-d-glucanase] has been purified from Trichoderma longibrachiatum cultivated with d-glucose as the growth substrate. The enzyme was found to hydrolyze laminarin to oligosaccharides varying in size from glucose to pentaose and to lesser amounts of larger oligosaccharides. The enzyme was unable to cleave laminaribiose but hydrolyzed triose to laminaribiose and glucose. The enzyme cleaved laminaritetraose, yielding laminaritriose, laminaribiose, and glucose, and similarly cleaved laminaripentaose, yielding laminaritetraose, laminaritriose, laminaribiose, and glucose. The enzyme cleaved only glucans containing β-1,3 linkages. The pH and temperature optima were 4.8 and 55°C, respectively. Stability in the absence of a substrate was observed at temperatures up to 50°C and at pH values between 4.9 and 9.3. The molecular mass was determined to be 70 kilodaltons by sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis, and the pI was 7.2. Enzyme activity was significantly inhibited in the presence of HgCl2, MnCl2, KMnO4, and N-bromosuccinimide. The Km of the enzyme on laminarin was 0.0016%, and the Vmax on laminarin was 3,170 μmol of glucose equivalents per mg of the pure enzyme per min.

Purification and Characterization of an Endo-(1,3)-β-d-Glucanase from Trichoderma longibrachiatum

Two endoxylanases were purified from the culture medium of Trichoderma longibrachiatum. Both enzymes were highly basic, and lacked activity on carboxymethyl-cellulose. An enzyme of 21.5 kDa (xylanase A) had a specific activity of 510 U/mg protein, a Km of 0.15 mg soluble xylan/ml, possessed transglycosidase activity and generated xylobiose and xylotriose as the major endproducts from xylan or xylose oligomers. A larger enzyme of 33 kDa (xylanase B) had a specific activity of 131 U/mg protein, a Km of 0.19 mg soluble xylan/ml, lacked detectable transglycosidase activity and generated xylobiose and xylose as major endproducts from xylan and xylose oligomers. Xylotriose was the smallest oligomer attacked by both enzymes. In addition, xylotriose inhibited hydrolysis of xylopentanose by both enzymes, while xylobiose appeared to inhibit xylanase B, but not xylanase A.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1991.tb16404.x

Purification and characterization of two xylanases from Trichoderma longibrachiatum.

A method capable of detecting as little as 0.11 U of xylanase activity in polyacrylamide gels was developed. The method entails incubation of protein gels in contact with substrate gels containing unmodified xylan, followed by immersion of substrate gels in 95% ethanol. Resulting zymograms contain transparent bands corresponding to enzymatic activity against an opaque background. Images

J. C. Royer

Papers

Xylanase production by Trichoderma longibrachiatum

Interrelationship of Xylanase Induction and Cellulase Induction of Trichoderma longibrachiatum.

Purification and Characterization of an Endo-(1,3)-β-d-Glucanase from Trichoderma longibrachiatum

Purification and characterization of two xylanases from Trichoderma longibrachiatum.

Simple, sensitive zymogram technique for detection of xylanase activity in polyacrylamide gels.