dc.description.abstract | In this study, G. kaustophilus ATCC8005, a Gram-positive, aerobic, and thermophilic bacteria, was grown at its optimal growth conditions, 55oC, pH 7. Extracellular proteins from the culture medium (secretome) and whole intracellular proteins in cellular extracts (cytosol proteome) were collected at mid-log phase. By using 1D SDS-PAGE/in-gel digestion, 2D SDS-PAGE/in-gel digestion and in-solution digestion, proteins were identified by ESI-Q-TOF in combination with Mascot search program. The genome sequence from G. kaustophilus HTA426 allows us to accurately identify (>90%) the target proteins in a high-throughput format.
In total, there were 49 proteins identified from secretome. Among them, 5 (10%) and 11 (22%) of them were found to be extracellular and located in membrane part localization, respectively. Among the extracellular proteins, they are thermostable amino acid and oligopeptide binding proteins, nuclease related proteins and one is hypothetical protein. Plus the 517 unique proteins identified from cytosol proteome, there is ~15% ORFs (HTA426) identified in log-phase. These identified proteins will be helpful to explore the growth mechanism of the thermophiles in adverse environment such as high temperature. Futhermore, it can also be used to build a prediction tool base on the experimental data of the heat-stable proteins for searching novel applicable thermostable proteins.
By comparing to a 55oC-treated cell-free extract of Geobacillus kaustophilus ATCC8005, only 68% of proteins is heat stable in vitro. Proteins belong to amino acid transport and metabolism and enzyme category are more thermostable. Furthermore, in order to verify the method for screening the thermostable proteins, we select the enzymes with different temperature range and its importance to the industrial application from those identified proteins. We found three antioxidant enzymes from 55~100oC: superoxide dismutase (SOD), peroxiredosin (PRX) and thioredoxin (TXN). When identify two unknown function proteins from 55~85oC and 75~85 oC, and sequences are compared using BLAST, we found endo-1, 4 beta-glucanase of Bacillus halodurans is similar to the hypothetical proteins we found. But the cellulase activity is still waiting for further research. Under the temperatures of 55~75oC and 55~65oC, two thermostable enzymes, cephalosporin acylase and monoacylglycerol lipase (MGLP), respectively, were identified. We will use traditional biochemical approach to verify these enzymes’ thermostability. In this thesis, we completed the thermostability assay of two thermostable proteins, which are SOD and MGLP. The Tm of SOD is 94oC, and the Topt. and Tm of MGLP is 65oC and 82oC. Thus, we have proved past students’ platform for screening thermostable proteins. In addition, when comparing with the published paper, the SOD found in this experiment has the highest thermostability. After heating for 94oC for 30 minutes, there still has half of the activity, thus we applied the patent to protect our finding. | en_US |