Comparative Evaluation of Adsorption of Major Enzymes in a Cellulase Cocktail Obtained from Trichoderma reesei onto Different Types of Lignin

Cellulase adsorption onto lignin decreases the productivity of enzymatic hydrolysis of lignocellulosic biomass. Here, adsorption of enzymes onto different types of lignin was investigated, and the five major enzymes—cellobiohydrolases (CBHs), endoglucanase (Cel7B), β-glucosidase (Cel3A), xylanase (XYNIV), and mannanase (Man5A)—in a cellulase cocktail obtained from Trichoderma reesei were individually analyzed through SDS-PAGE and zymogram assay. Lignin was isolated from woody (oak and pine lignin) and herbaceous (rice straw and kenaf lignin) plants. The relative adsorption of CBHs compared to the control was in the range of 14.15–18.61%. The carbohydrate binding motif (CBM) of the CBHs contributed to higher adsorption levels in oak and kenaf lignin, compared to those in pine and rice lignin. The adsorption of endoglucanase (Cel7B) by herbaceous plant lignin was two times higher than that of woody lignin, whereas XYNIV showed the opposite pattern. β-glucosidase (Cel3A) displayed the highest and lowest adsorption ratios on rice straw and kenaf lignin, respectively. Mannanase (Man5A) was found to have the lowest adsorption ratio on pine lignin. Our results showed that the hydrophobic properties of CBM and the enzyme structures are key factors in adsorption onto lignin, whereas the properties of specific lignin types indirectly affect adsorption.

Identification of a Broad-Spectrum Peptidoglycan Hydrolase Associated with the Particle of Xanthomonas oryzae Phage Xop411

Virion-associated peptidoglycan hydrolases (VAPGH) in bacteriophages are potential antimicrobials. Xop411 is a syphophage infecting the Gram-negative <i>Xanthomonas oryzae</i> pv. oryzae that causes bacterial leaf blight in rice plants. The Xop411 gp21 protein was identified here as a peptidoglycan glycohydrolase by Western blotting and zymogram assay, and localized to the phage tail by immunogold-labelling electron microscopy. This protein showed an apparent molecular mass of 17 kDa in SDS-polyacrylamide gels, larger than that calculated from the amino acid sequence, 15 kDa with 130 residues. The recombinant gp21 expressed in <i>Escherichia coli</i> formed inclusion bodies, which gained enzyme activity after in-gel renaturation. In contrast, the secreted recombinant protein (s-gp21His) expressed in <i>Pichia pastoris</i> was soluble and enzymatically active. Plate assays showed that s-gp21His was capable of killing 3 species of <i>Xanthomonas</i>, a genus containing 27 closely related plant pathogenic species, as well as the opportunistic <i>Pseudomonas aeruginosa</i> and <i>Stenotrophomonas maltophilia</i> causing nosocomial infections. These results indicate that the Xop411 gp21 has possible wide applications as an antimicrobial against xanthomonads and at least 2 opportunistic bacteria. Several other VAPGH from <i>Xanthomonas</i> phages were also identified by bioinformatic analysis, with 1 being confirmed by Western blotting.