Finding Biomass Degrading Enzymes Through an Activity-Correlated Quantitative Proteomics Platform (ACPP)

Abstract

The microbial secretome, known as a pool of biomass (i.e., plant-based materials) degrading enzymes, can be utilized to discover industrial enzyme candidates for biofuel production. Proteomics approaches have been applied to discover novel enzyme candidates through comparing protein expression profiles with enzyme activity of the whole secretome under different growth conditions. However, the activity measurement of each enzyme candidate is needed for confident "active" enzyme assignments, which remains to be elucidated. To address this challenge, we have developed an Activity-Correlated Quantitative Proteomics Platform (ACPP) that systematically correlates protein-level enzymatic activity patterns and protein elution profiles using a label-free quantitative proteomics approach. The ACPP optimized a high performance anion exchange separation for efficiently fractionating complex protein samples while preserving enzymatic activities. The detected enzymatic activity patterns in sequential fractions using microplate-based assays were cross-correlated with protein elution profiles using a customized pattern-matching algorithm with a correlation R-score. The ACPP has been successfully applied to the identification of two types of "active" biomass-degrading enzymes (i.e., starch hydrolysis enzymes and cellulose hydrolysis enzymes) from Aspergillus niger secretome in a multiplexed fashion. By determining protein elution profiles of 156 proteins in A. niger secretome, we confidently identified the 1,4-α-glucosidase as the major "active" starch hydrolysis enzyme (R = 0.96) and the endoglucanase as the major "active" cellulose hydrolysis enzyme (R = 0.97). The results demonstrated that the ACPP facilitated the discovery of bioactive enzymes from complex protein samples in a high-throughput, multiplexing, and untargeted fashion. Graphical Abstract ᅟ.

Keywords: Correlation; Enzyme activity; LC-MS/MS; Peptide counting; Proteomics.

Figure 1

Schematic representation of the ACPP workflow.

Figure 2

Protein elution profiling of ACPP on the E coli. cell lysate with the spiked-in standard AAG. (A) the UV chromatogram from the mono Q based LC separation; (B) Hierarchical clustering of protein elution profiles on the basis of their similarities; (C) SDS PAGE gel image of collected LC separation fractions.

Figure 3

Two biomass hydrolysis reactions are catalyzed by different enzymes. (A) 1, 4-α-glucosidase (AAG) is a typical enzyme that can directly convert starch into glucose; (B) Cellulose is converted into glucose in a two-step reaction, and the endo-cellulase (i.e., endoglucanase) activity can be measured in the presence of β-glucosidase.

Figure 4

The ACPP on starch hydrolysis enzyme characterization from the E coli. cell lysate with the spiked-in standard AAG. (A) The overlaps between the measured starch hydrolysis activities and the AAG protein elution profiles; (B) Correlations between detected activity pattern and the filtered elution profiles from all the identified proteins (total peptide count cutoff of 10).

Figure 5

The ACPP on detecting biomass degrading enzymes from A. niger secretome. (A) the UV chromatogram from the LC separation; (B) the overlaps between the measured starch hydrolysis activities and the best matched protein elution profile 1, 4-α-glucosidase (AAG); (C) the overlaps between the measured cellulose hydrolysis activities and the best matched protein elution profile (endoglucanase, GH5); (D) the overlaps between the measured starch hydrolysis activities and the protein elution profile of β-glucosidase.

Figure 6

The cellulose activity assays on A. niger secretome. Three assays were included: Sigmacell cellulose+HPLC fractions+ standard β-glucosidase, cellubiose+HPLC fractions, and Sigmacell cellulose + HPLC fractions. The blue line represents the endoglucanase elution profile, the green line represents the β -glucosidase elution profile, and the red line evaluates if endoglucanase and β-glucosidase are co-eluted.