Proteomic Analysis of Compositional Differences between Exogenous Fibrolytic Enzyme Preparations that were Effective or Ineffective at Improving Forage Digestibility
The objective was to use novel proteomic tools to identify differences in proportions of key enzymes and auxiliary proteins involved in hemicellulose and lignocellulose degradation between effective and ineffective exogenous fibrolytic enzyme preparations (EFE). We recently examined effects of applying 12 EFE from 3 companies on in vitro NDF digestibility (NDFD) of bermudagrass haylage (BH). The most- (2A) and second most- (11C) effective EFE were from Trichoderma reesei and they increased the NDFD of BH from 35.6 (Control) to 40.4 and 40.0%, respectively. The least effective EFE (9C) was from T. reesei and Aspergillus spp. and the NDFD of BH treated with this EFE was 36.2% (SEM= 0.55). The relative ratios of proteins in either 11C to 2A or 9C to 2A were analyzed in triplicate using quantitative proteomics. Specifically, EFE were analyzed with isobaric tags for relative and absolute quantitation coupled with liquid chromatography-mass spectrometry (iTRAQ LC-MS/MS) . The identification and analysis of proteins were performed using ProteinPilotTM Software version 4.5. Proteins were identified using the National Center for Biotechnology Information database for T. reesei and Aspergillus spp. The unused score threshold was set to > 1.3 (equivalent to 95% confidence or better). The Student’s t-test was used to measure the significance of the relative ratio of the proteins. The degrees of freedom were the number of distinct peptides within the protein evaluated minus 1. Quantitation was based on at least three unique peptides for each protein. The 2A EFE had 10 times more endoglucanase III, 17 times more acetylxylan esterase with Cellulose Binding Module 1, 33 times more xylanase III, 25 times more β-xylosidase, 7.69 times more polysaccharide monooxygenase with Cellulose Binding Module 1, and 3 times more swollenin compared to 9C. Relative to 11C, 2A had 14.3 times more xylanase III, 14.3 times more β-xylosidase, 7.7 times more endoglucanase III, and 1.9 times more polysaccharide monooxygenase. Therefore, the efficacy of the EFE at increasing NDFD was reflected by the relative proportions of novel xylanolytic and cellulolytic enzymes and auxiliary proteins that they contained.