Metaproteomics Analysis And Identification Of Lignocellulose Degradation-related Enzymes

Natural lignocellulosic materials are widely available for the microorganisms in their habitats. These microorganisms produce a battery of enzymes and work synergically to biodegrade lignocellulose. Therefore, to truly understand how microorganisms hydrolyze the lignocellulose, they must be studied as systems in nature rather than individual or a few at a time. In this paper, the application of metaproteomics to understand microbial lignocellulose utilization strategies was well described.Soil samples from different climate and vegetation types were collected. Cellulose and xylanase activity and the efficiency of lignocellulose degradation were determined subsequently. The result showed the soil sample had a higher xylanase activity but a much lower even zero in cellulose activity. The highest xylanase activity reached up to 192.92 u/g. After enrichment using natural lignocellulose as culture materia, the emzymes activities of most soil samples were increased. The highest cellulose and xylanase activity were 6530.99 u/g and 1275.16 u/g, respectively. Finally, HN-5 soil sample was selected as the next experimental target by considerating three indicators comprehensively.The proteomics approach was built based on a lignocellulose-degrading Aspergillus niger strain HCB-3 isolated from HN-5 soil sample. After medium optimization for strain HCB-3 to improve the production of lignocellulose-degrading enzymes, the total soluble extracellular proteins were extracted from HCB-3 broth and applied in proteomics analysis.7 hemicellulose-degrading enzymes were identificated from the 2D gel, as 3 xylanase,1 arabinoxylan degrading enzyme,1α-L-arabinofuranosidase,1α-glucuronidase and 1 feruloyl esterase. Further results about ratio of these emzymes obtained using the software of Image Master 2D Platinum. The successful analysis confirmed the feasibility of the entireproteomic strategy in identifying the enzymes involved in the lignocellulolytic processes.The soil protein extraction and concentration conditions were optimized and ultimately determined. The HN-5 soil sample was enriched on a lignocellulose materials medium for 10 days at 28℃. The enriched sample was suspended in 0.5mol/lpH6.0 PBS, then was filtered and centrifuged to collect supernatant. Proteins were precipitated by adding 80% saturated ammonium sulfate. Ultrfiltration was used to remove salts. Then proteins were separated by two-dimensional gel electrophoresis and identified by MOLDI-TOF-TOF tandem Mass Spectrometry.5 lignocellulolytic emzymes were successfully identified, including catalase, Glucoamylase-471 complexed with acarbose, Glucan 1,4-α-Glucosidase, GPI-anchcred cell wallβ-1,3-endoglucanase and 1,4-β-D-glucan cellobiohydrolase. Their ratio was 5:30:20:40:10. Our results represent the first metaproteomic study of soil lignocellulose degradation emzymes assemblages and demonstrate the potential of metaproteomic approaches to reveal the secret of lignocellulose degradation.