Abstract
BackgroundNeocallimastix patriciarum is one of the common anaerobic fungi in the digestive tracts of ruminants that can actively digest cellulosic materials, and its cellulases have great potential for hydrolyzing cellulosic feedstocks. Due to the difficulty in culture and lack of a genome database, it is not easy to gain a global understanding of the glycosyl hydrolases (GHs) produced by this anaerobic fungus.ResultsWe have developed an efficient platform that uses a combination of transcriptomic and proteomic approaches to N. patriciarum to accelerate gene identification, enzyme classification and application in rice straw degradation. By conducting complementary studies of transcriptome (Roche 454 GS and Illumina GA IIx) and secretome (ESI-Trap LC-MS/MS), we identified 219 putative GH contigs and classified them into 25 GH families. The secretome analysis identified four major enzymes involved in rice straw degradation: β-glucosidase, endo-1,4-β-xylanase, xylanase B and Cel48A exoglucanase. From the sequences of assembled contigs, we cloned 19 putative cellulase genes, including the GH1, GH3, GH5, GH6, GH9, GH18, GH43 and GH48 gene families, which were highly expressed in N. patriciarum cultures grown on different feedstocks.ConclusionsThese GH genes were expressed in Pichia pastoris and/or Saccharomyces cerevisiae for functional characterization. At least five novel cellulases displayed cellulytic activity for glucose production. One β-glucosidase (W5-16143) and one exocellulase (W5-CAT26) showed strong activities and could potentially be developed into commercial enzymes.
Highlights
Neocallimastix patriciarum is one of the common anaerobic fungi in the digestive tracts of ruminants that can actively digest cellulosic materials, and its cellulases have great potential for hydrolyzing cellulosic feedstocks
Extracellular cellulases of N. patriciarum W5 To stimulate the N. patriciarum W5 strain to produce cellulosic enzymes, we used powders of rice straw, napiergrass and sugarcane bagasse
We assayed the cellulase activities using 4-MUC, p-nitrophenyl-b-D-glucopyranoside and dye CM-cellulose as substrates and performed cross-analyses to determine the levels of exoglucanase (EXG), endoglucanase (EG) and BGLU activities in the total extracellular protein extracts
Summary
Neocallimastix patriciarum is one of the common anaerobic fungi in the digestive tracts of ruminants that can actively digest cellulosic materials, and its cellulases have great potential for hydrolyzing cellulosic feedstocks. A key step in cellulosic ethanol production is to break down cellulose into glucose and used for the production of industrial materials from plant biomass. These enzymes may improve the fiber properties of cotton for manufacturing or clothes. Pai et al [8] reported the cloning from N. patriciarum of a bifunctional xylanolytic enzyme with acetylxylan esterase and xylanase activities. This enzyme contains a double-dockerin domain, suggesting that it is a cellulosomal component and may bind tightly to the cellulosome [8]
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