Proteomic analysis of the biomass hydrolytic potentials of Penicillium oxalicum lignocellulolytic enzyme system

Abstract

Background: The mining of high-performance enzyme systems is necessary to develop industrial lignocellulose bioconversion. Large amounts of cellulases and hemicellulases can be produced by Penicillium oxalicum. Hence, the enzyme system of this hypercellulolytic fungus should be elucidated to help design optimum enzyme systems for effective biomass hydrolysis.

Results: The cellulolytic and xylanolytic activities of an SP enzyme system prepared from P. oxalicum JU-A10 were comparatively analyzed. Results indicated that the fungus possesses a complete cellulolytic-xylanolytic enzyme system. The cellobiohydrolase- and xylanase-specific activities of this system were higher than those of two other enzyme systems, i.e., ST from Trichoderma reesei SN1 and another commercial preparation Celluclast 1.5L. Delignified corncob residue (DCCR) could be hydrolyzed by SP to a greater extent than corncob residue (CCR). Beta-glucosidase (BG) supplemented in SP increased the ability of the system to hydrolyze DCCR and CCR, and resulted in a 64 % decrease in enzyme dosage with the same glucose yield. The behaviors of the enzyme components in the hydrolysis of CCR were further investigated by monitoring individual enzyme dynamics. The total protein concentrations and cellobiohydrolase (CBH), endoglucanase (EG), and filter paper activities in the supernatants significantly decreased during saccharification. These findings were more evident in SP than in the other enzyme systems. The comparative proteomic analysis of the enzyme systems revealed that both SP and ST were rich in carbohydrate-degrading enzymes and multiple non-hydrolytic proteins. A larger number of carbohydrate-binding modules 1 (CBM1) were also identified in SP than in ST. This difference might be linked to the greater adsorption to substrates and lower hydrolysis efficiency of SP enzymes than ST during lignocellulose saccharification, because CBM1 not only targets enzymes to insoluble cellulose but also leads to non-productive adsorption to lignin.

Conclusions: Penicillium oxalicum can be applied to the biorefinery of lignocellulosic biomass. Its ability to degrade lignocellulosic substrates could be further improved by modifying its enzyme system on the basis of enzyme activity measurement and proteomic analysis. The proposed strategy may also be applied to other lignocellulolytic enzyme systems to enhance their hydrolytic performances rationally.

Keywords: Adsorption; Cellulase; Enzymatic hydrolysis; Penicillium oxalicum; Proteomic; Trichoderma reesei.

Fig. 1

Conversion of glucan during hydrolysis of CCR and DCCR. Enzyme loadings were set at 3.6 mg protein/g glucan (a), 20 mg protein/g glucan (b), and 100 mg protein/g glucan (c), respectively. The values shown are the mean of three replicates and the error bars indicate standard deviations from the mean values

Fig. 2

Enhancement of saccharification by BG supplementation. The curves show the glucan conversion rates with DCCR at a 9 FPA/g glucan, b 14 FPA/g glucan, and c 18 FPA/g glucan, and with CCR at d 9 FPA/g glucan, e 14 FPA/g glucan, and f 18 FPA/g glucan. The values shown are the mean of three replicates and the error bars indicate standard deviations from the mean values

Fig. 3

The dynamic changes during saccharification at the enzyme loading of 20 mg protein/g glucan. a Protein contents. b–e The activities of CBH, EG, filter paper, and BG, respectively. The values shown are the mean of three replicates and the error bars indicate standard deviations from the mean values

Fig. 4

Protein profiles of saccharification supernatants analyzed by SDS-PAGE. a SP; b ST; c Celluclast 1.5L. Enzyme loadings were set at 20 mg protein/g glucan. Samples were collected after saccharification for 0, 24, 48, 72, and 96 h, respectively. Enzyme solution before saccharification was used as control (lane C)

Fig. 5

The dynamic changes during saccharification at the enzyme loading of 100 mg protein/g glucan. a Protein contents. b–e The activities of CBH, EG, filter paper, and BG, respectively. The values shown are the mean of three replicates and the error bars indicate standard deviations from the mean values

Fig. 6

A plot of calculated molecular weight against the isoelectric point (pI) of the identified proteins. Proteins annotated with different functions are indicated with different symbols

Fig. 7

Distribution of GH, CE, and PL family proteins identified in SP and ST

Fig. 8

The relative amounts of proteins with different functional classifications identified in SP and ST. Functional predictions of SP (a) and ST (b) proteins are based on the genome databases for P. oxallium and T. reesei, respectively. Chitinase and amylase in ST were not shown because of little contents

Fig. 9

The ratio changes of major components in SP and ST before and after saccharification. a SP; b ST. For each protein, the ratio in total protein indicated by color (in triplicate), accession number in GenBank, and functional annotation were shown. A significant difference (P < 0.05) was marked with asterisk