| dc.description.abstract | Thermophilic actinomycete (Thermomonospora fusca YX) is able to secrete many different extracellular thermophilic proteins, especially the cellulose degrading enzymes. This bacterium was isolated from horse compost by Henssen et al. in Germany 1957. In March of 2007, Wilson et al. completed the genome sequence of YX with a total of 3117 ORFs; furthermore, in the same year of 2009, T. fusca YX was published for the identification of cellobiose using the study of proteomics.
In our study, we want to find the extracellular proteins secreted from T. fusca YX using cellulose as the sole carbon source grown at 55oC. The extracellular proteins are collected when bacteria are harvested at stationary phase. This study uses proteomics (1D and 2D SDS-PAGE using in-gel and in-solution digestion), followed by two different mass analysis (LC-Q-TOF-MS/MS and MALDI-Q-TOF-MS/MS). The enzymes identified are analyzed using Pfam and extracellular protein prediction tools (CELLO and PSORTb) to delicately predict the proteins in the subcellular location. The 93 proteins were identified and among them, 32 (34.4%) were extracellular proteins, 8 (8.6%) were membrane proteins, 53( 57%) were cytosolic proteins. The 32 extracellular proteins were further using COG for function classification. There were 30% of proteins related to glycolysis and most are either slightly acidophilic proteins (the pIs lower than 7 are 82%). This study also identified 12 glycoside hydrolyzing enzyme (for example, cellulase), 5 glucose binding proteins such as cellulose binding proteins, 2 were polysaccharide related degrading enzymes (pectate lyase, parallel beta-helix repeat-containing ricin B lecctin), 4 were protein protease (serine peptidase, aminopeptidase Y, hypothetical protein Tfu 0745, hypothetical protein Tfu 0746), 4 were esterase/lipase (such as acetyl xylan esterase and triacylglycerol lipase), 1 antioxidant enzymes (superoxide dismutase), 2 cell wall/solute-binding proteins (the surface protein, hypothetical protein Tfu 2615), 1 antibiotic (bacteriocin) and 1 heat shock proteins (heat shock protein DnaJ).
In 2007, the Wilson group uses only 2D SDS-PAGE to analyze extracellular proteomes and the 15 proteins identified in the literature were compared with 32 proteins found in our lab. There were 11 matched proteins with the additional 21 thermostable secretomes that were not identified in Wilson’s study. The 21 proteins are for example, the glucose degrading enzymes such as cellulose, xylanase, pectate lyase, and mannan endo-1,4-beta-mannosidase. The protease that is able to degrade large molecules of organic materials into smaller ones in the environment like serine peptidase, aminopeptidase Y, acetyl xylan esterase and triacyoglycerol lipase; proteins like superoxide dismutase that is able to act again the toxins from hydrogen peroxide or free radicals in order to protect cells from damage. And finally, antibiotic like bacteriocin for inhibit or terminate bacterial infection.
We selected a growth phase and carbon source (cellulose) that is able to secrete a great amount of secretome, which is different from Wilsons that use cellobiose as the sole carbon source and harvest bacteria at the log phase. Other than 2D SDS-PAGE for protein separation, 1D SDS-PAGE and 4 different conditions of Gel-free digestion are also used in order to increase the protein amount. The three different isolation methods can provide a full report for complex proteomic research. We use T. fusca YX to as a model and built a research platform for studying the secretome of the environmental microorganisms, especially this thermostable cellulolytic bacterium is helpful in the exploration of bioethanol research; in addition, when comparing the above tools for predicting cellular localization in terms of precision and performance, CELLO is the most suitable prediction tool for the cell localization of T. fusca YX in the proteomic study of actinomycetes.
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