Ken K. Y. Wong

Trichoderma Xylanases, Their Properties and Application

AbstractTrichoderma spp. are known to produce enzymes with high xylanolytic activity. Different xylanases and various components of their xylanolytic system have been identified and purified. Some of the xylanases have been characterized extensively with respect to their physicochemical, hydrolytic, and molecular properties. Cellulase-free xylanase preparations have been tested successfully in industrial applications such as the prebleaching of kraft pulps in the pulp and paper industry. Future work on understanding the functional significance of xylanase multiplicity, the mechanisms of xylanase prebleaching, and the structural conformation of xylanases could lead to improved or alternative applications of Trichoderma xylanases.

Purification and characterization of two d-xylanases from Trichoderma harzianum

Abstract Two endo-1,4-β- d -xylanases (1,4-β- d -xylan xylanohydrolase, EC 3.2.1.8) from Trichoderma harzianum E58 have been purified by ultrafiltration and chromatography on carboxymethyl-Sepharose, phenyl-Sepharose and Sephadex G-75. The d -xylanases were shown to be homogeneous by the criteria of dodecyl sulphate polyacrylamide gel electrophoresis and isoelectric focusing. The molecular weights were estimated to be 20 000 and 29 000, with pl values of 9.4 and 9.5, respectively. Typically, 456 mg of the 20 000 dalton and 1.9 mg of the 29 000 dalton d -xylanases were purified from 4.2 litre of culture filtrate with specific activities of 370 and 75 U mg −1 , respectively. Optimum d -xylanase activities were obtained when the enzymes were incubated at pH 5, 50°C, for the 20 000 dalton protein and pH 5, 60°C for the 29 000 dalton protein .

Evaluation of the enzymatic susceptibility of cellulosic substrates using specific hydrolysis rates and enzyme adsorption

Adsorption of cellulases to cellulose is a critical step in the hydrolysis of cellulosic substrates. However, the importance of adsorption in determining the hydrolysis rate is unclear. The accessibility to cellulases and specific hydrolysis rates were measured for various substrates. No correlation was found between the amount of enzyme adsorbed and the initial hydrolysis rate for different substrates. Specific hydrolysis rates were found to differ among substrates. Furthermore, both accessibility to cellulases and the specific hydrolysis rate of substrates were found to be changed by chemical and physical pretreatment of the substrate.

Size-exclusion chromatography of lignin- and carbohydrate-containing samples using alkaline eluents

Size-exclusion chromatography of carbohydrate- and lignin-containing samples, prepared from wood and pulp samples, was carried out using alkaline eluents. The elution of carbohydrate and lignin macromolecules can be monitored with good selectivity using a pulsed amperometric and a UV absorbance detector, respectively. However, the stability of the signals from samples stored in alkaline solutions requires careful consideration in the analysis because marked changes are observed in many cases. Furthermore, the interactions between aromatic molecules and the separation column are also dependent on the concentration of alkali in the eluent. Since there seem to be no adsorptive interactions between carbohydrate and lignin macromolecules during size-exclusion chromatography, the described method can be used to monitor chemical and enzymic modifications of the apparent molecular mass of these two classes of compounds, whether they occur in mixtures or covalently linked complexes.

The mechanism of xylanase prebleaching of kraft pulp : An examination using model pulps prepared by depositing lignin and xylan on cellulose fibers

Model pulps, made by depositing lignin and/or xylan on cellulose fibers under alkaline conditions, were subjected to a xylanase-chelation-peroxide bleaching sequence. On average, 14% (0.7g) of the added xylan had been redeposited on 20g cellulose. However, the kappa numbers of the pulps were smaller than 1, indicating that only a small amount of oxidizable compounds, e.g. lignin and sugar chromophores, had precipitated. A brightness increase occurred immediately after the xylanase stage. After peroxide bleaching a further brightness increase was observed with model pulps containing both lignin and xylan. Analysis of the filtrate obtained from the enzyme stage showed that only model pulps containing xylan released sugar. The molecular mass distribution of solubilized material was analyzed with pulsed amperometric and UV detectors. The results suggested that lignin-carbohydrate bonds are formed during the redeposition of lignin and xylan on the cellulose fibers and that xylanases can partly hydrolyze the lignin-carbohydrate complexes. This mechanism is proposed to be a major contribution to the bleach boosting effect of these enzymes. Although the formation of chromophoric xylan was also observed during alkaline cooking, the results indicate that the hydrolysis of this class of compounds has a limited role in xylanase prebleaching.

Xylanase treatment for the peroxide bleaching of oxygen delignified kraft pulps derived from three softwood species

Abstract Three commercial enzymes were used to evaluate the use of xylanase treatment for enhancing the peroxide bleaching of three oxygen delignified kraft pulps derived from the softwoods red cedar, Douglas fir, and western hemlock. Two bleaching sequences were examined, where the enzyme stage was placed before or between the two peroxide stages. The brightness gain achieved was dependent on the enzyme preparation, the origin of the pulp and the position of the enzyme stage in the bleaching sequence. In general, xylanase was found to enhance the pulp brightness achieved with two peroxide stages by 1–1.5% ISO. It could directly brighten oxygen delignified pulps before and after the first peroxide stage. Direct brightening of the oxygen delignified pulps, before peroxide bleaching, appeared to be correlated with the amount of UV-absorbing material solubilized during the enzyme stage.

Functional characteristics of two d-xylanases purified from Trichoderma harzianum

Two endo-1,4-β-d-xylanases (1,4-β-d-xylan xylanohydrolase, EC 3.2.1.8) were purified from Trichoderma harzianum culture filtrates. From kinetic analyses, apparent Vmax and Km values of 580 U mg−1 protein and 0.16% d-xylan were obtained for the 20 000 dalton endo-1,4-β-d-xylanase, while values of 100 U mg−1 protein and 0.066% d-xylan were obtained for the 29 000 dalton endo-1,4-β-d-xylanase. Substrate levels >1% (w/v) d-xylan were found to be inhibitory to both enzymes. Both d-xylanases were highly active against d-xylans obtained from various sources. Of the polymeric sugars tested, carboxymethyl cellulose was the only substrate which was hydrolysed to any extent. Little or no activity was observed against cellulose. Analyses by h.p.l.c. demonstrated the absence of hydrolytic activity by both d-xylanases on d-xylobiose. d-Xylotriose was cleaved to a limited extent by the 29 000 dalton d-xylanase only, while d-xylotetraose was hydrolysed by both. In the presence of d-xylotetraose, the 20 000 dalton d-xylanase had an associated transxylosidase activity which was not observed with the 29 000 dalton enzyme. When the solubilization assay was used, neither of the d-xylanases was inhibited by high concentrations of d-xylose and xylobiose.

Functional interactions among three xylanases from Trichoderma harzianum

Abstract The functional importance of xylanase multiplicity in Trichoderma harzianum has been examined by studying the hydrolysis of different forms of xylan. The hydrolyses achieved by xylanase combinations were compared with that achieved by the xylanases individually. Complementation among three purified xylanases was observed in the substrate limiting hydrolysis of isolated aspen xylans although it was not observed with oat arabinoxylan. The three enzymes together provided the greatest hydrolysis of aspen xylans, enhancing the hydrolysis of acetylated and deacetylated aspen xylans by 27 and 18%, respectively, when compared with hydrolyses obtained with the individual enzymes. The degree of complementation was much higher in the hydrolysis of aspen holocellulose. Combinations of two or three xylanases improved holocellulose hydrolysis by 26–114%. All three enzymes were required to achieve the greatest degree of hydrolysis. Xylanase multiplicity in this organism is required for effective hydrolysis of xylan in complex substrates. It appears that the characterized xylanases are not redundant enzymes since each contributes significantly and uniquely to the xylanolytic system of the fungus .

Bleach boosting and direct brightening by multiple xylanase treatments during peroxide bleaching of kraft pulps.

The effects of multiple xylanase treatments were assessed during the peroxide bleaching of three pulps: Douglas-fir (kraft); Western hemlock (oxygen delignified kraft); and trembling Aspen (kraft). The addition of a xylanase treatment stage, either before or after the peroxide bleaching stage(s), resulted in the enhanced brightening of all pulps. A higher brightness was achieved using two enzyme treatments, one before and one after the peroxide stage(s). Both bleach boosting and direct brightening seemed to contribute to the enhancement of the peroxide bleaching. Compared to xylanase prebleaching, xylanase posttreatment of peroxide bleached pulps solubilized less lignin and chromophores and made smaller amounts of these materials alkaline soluble. Nevertheless, the final brightness achieved by xylanase posttreatment was similar or superior to that achieved with xylanase prebleaching of the corresponding unbleached pulps. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 312-318, 1997.

Enzymic Hydrolysis Of Lignin—Carbohydrate Complexes Isolated From Kraft Pulp

Four carbohydrate samples extracted from kraft pulps are used as model substrates for studying the mechanism by which xylanase enhances subsequent bleaching of kraft pulp. Fourier transform infrared spectroscopy confirms that small amounts of aromatic molecules, probably lignin, remain associated with these carbohydrate samples. When the extracts are hydrolyzed with xylanase or acid, size exclusion chromatography shows a decrease in the molecular mass of their UV-absorbing constituents, as well as their carbohydrate constituents as determined by pulsed amperometric detection. The results are consistent with the hypothesis that xylanase prebleaching hydrolyzes the xylan portion of lignin-carbohydrate complexes to leave smaller lignin-containing macromolecules in pulp fiber, thus facilitating the removal of lignin components by bleaching chemicals.