Purification and Characterization of Class III Lipase from a White-Rot Fungus Pleurotus ostreatus.

Pleurotus ostreatus is an edible white-rot fungus with lignocellulosic biomass degrading enzymes that have been studied extensively. However, until now, lipolytic enzymes from P. ostreatus, which degrade extractives in lignocellulosic biomass, have not been purified and characterized. In this study, P. ostreatus was inoculated into the rapeseed oil containing culture to induce lipase. The lipase in the culture broth was successfully purified to homogeneity by chromatographic methods. The molecular weight of the purified lipase was 27 kDa, and its optimal pH and temperature were 5.0 and 30 °C, respectively. The purified lipase showed high activity with the substrates 4-methylumbelliferyl (4-MU) decanoate (C10:0) and 4-MU oleate (C18:1), and no activity with 4-MU acetate (C2:0) and 4-MU butyrate (C4:0). The amino acid sequences and substrate specificities of the purified lipase suggested that it belonged to class III. Kinetic parameters measurements (Km and Vmax) showed that 4-MU palmitate had a high affinity for the purified lipase, and it was the substrate most efficiently hydrolyzed by the purified lipase.

The isolation, characterization and effect of lignin isolated from steam pretreated Douglas-fir on the enzymatic hydrolysis of cellulose.

Douglas-fir was SO(2)-steam pretreated at different severities (190, 200, and 210°C) to assess the possible negative effect of the residual and isolated lignins on the enzymatic hydrolysis of the steam pretreated substrates. When various isolated lignins were added to the Avicel hydrolysis reactions, the decrease in glucose yields ranged from 15.2% to 29.0% after 72 h. It was apparent that the better hydrolysis yields obtained at higher pretreatment severities were more a result of the greater accessibly of the cellulose rather than any specific change in the non-productive binding of the lignin to the enzymes. FTIR and (13)C NMR characterization indicated that the lignin in the steam pretreated substrates became more condensed with increasing severity, suggesting that the cellulases were adsorbed to the lignin by hydrophobic interactions. Electrostatic interactions were also involved as the positively charged cellulase components were preferentially adsorbed to the lignins.

Enhancing the enzymatic hydrolysis of lignocellulosic biomass by increasing the carboxylic acid content of the associated lignin.

To assess the effects that the physical and chemical properties of lignin might have on the enzymatic hydrolysis of pretreated lignocellulosic substrates, protease treated lignin (PTL) and cellulolytic enzyme lignin (CEL) fractions, isolated from steam and organosolv pretreated corn stover, poplar, and lodgepole pine, were prepared and characterized. The adsorption of cellulases to the isolated lignin preparations corresponded to a Langmuir adsorption isotherm. It was apparent that, rather than the physical properties of the isolated lignin, the carboxylic acid functionality of the isolated lignin, as determined by FTIR and NMR spectroscopy, had much more of an influence when lignin was added to typical hydrolysis of pure cellulose (Avicel). An increase in the carboxylic content of the lignin preparation resulted in an increased hydrolysis yield. These results suggested that the carboxylic acids within the lignin partially alleviate non-productive binding of cellulases to lignin. To try to confirm this possible mechanism, dehydrogenative polymers (DHP) of monolignols were synthesized from coniferyl alcohol (CA) and ferulic acid (FA), and these model compounds were added to a typical enzymatic hydrolysis of Avicel. The DHP from FA, which was enriched in carboxylic acid groups compared with the DHP from CA, adsorbed a lower mount of cellulases and did not decrease hydrolysis yields when compared to the DHP from CA, which decreased the hydrolysis of Avicel by 8.4%. Thus, increasing the carboxylic acid content of the lignin seemed to significantly decrease the non-productive binding of cellulases and consequently increased the enzymatic hydrolysis of the cellulose.

The effect of isolated lignins, obtained from a range of pretreated lignocellulosic substrates, on enzymatic hydrolysis.

The influence of the residual lignin remaining in the cellulosic rich component of pretreated lignocellulosic substrates on subsequent enzymatic hydrolysis was assessed. Twelve lignin preparations were isolated by two isolation methods (protease treated lignin (PTL) and cellulolytic enzymatic lignin (CEL)) from three types of biomass (corn stover, poplar, and lodgepole pine) that had been pretreated by two processes (steam and organosolv pretreatments). Comparative analysis of the isolated lignin showed that the CEL contained lower amounts of carbohydrates and protein than did the PTL and that the isolated lignin from corn stover contained more carbohydrates than did the lignin derived from the poplar and lodgepole pine. The lower yields of acid insoluble lignin (AIL) obtained from the corn stover when using the PTL method indicated that the lignin from the corn stover had a higher hydrophilicity than did the lignin from the poplar and lodgepole pine. The isolated lignin preparations were added to the reaction mixture containing crystalline cellulose (Avicel) and their possible effects on enzymatic hydrolysis were assessed. It was apparent that the lignin isolated from lodgepole pine and steam pretreated poplar decreased the hydrolysis yields of Avicel, whereas the other isolated lignins did not appear to decrease the hydrolysis yields significantly. The hydrolysis yields of the pretreated lignocellulose and those of Avicel containing the PTL showed good correlation, indicating that the nature of the residual lignin obtained after pretreatment significantly influenced hydrolysis.

Purification and characterization of cellobiose dehydrogenase from white-rot basidiomycete Trametes hirsuta.

In order to save energy during the pulp making process, we tried to use white-rot basidiomycete, Trametes hirsuta, which degrades lignin efficiently. But a decrease in paper strength caused by cellulolytic activity ruled this out for practical application. Since the cellulolytic activity of the fungus must be decreased, we purified and characterized a cellobiose dehydrogenase (CDH) that was reported to damage pulp fiber. The CDH in the culture filtrate of C. hirsutus was purified by freeze-thawing and chromatographic methods. The pI of the enzyme was 4.2 and its molecular weight was 92 kDa. The optimal temperature and pH of the enzyme were 60-70 degrees C and 5.0 respectively. Since the purified CDH decreased the viscosity of pulp in the presence of Fe(III) and cellobiose, it was shown that the suppression of CDH should be an effective way to reduce cellulose damage.