| Summary: | 碩士 === 國立中央大學 === 生命科學研究所 === 95 === The world demand for enzymes is expected to increase 7 percent per year to nearly US$5 billion in 2009. The high value-added enzymes are most needed in industries, medical and research applications. Chemical reactions often need to be performed at high temperatures to accelerate industrial processes. However, not many enzymes are stable to heat. Research is required to make proteins remain active and stable while being heated to overcome current limits on their industrial applications. Accordingly, investigations of the key features on structure and sequence from thermophilic and mesophilic proteins are crucial topic in basic research. Microorganisms have been found to grow in environments at a very wide range of temperatures. Proteins from an organism grown at high temperature are more active and stable in such temperatures. Microorganisms are grouped into different classes according to their temperature optima:
hyperthermophiles (>80℃), thermophiles (45-80℃), mesophiles (20-45℃), and psychrophiles (<20℃). This research plans to establish a high-throughput screening of thermostable proteins by using proteomic approach. Geobacillus kaustophilus ATCC8005, a thermophilic bacterium was grown at its optimal temperature (55℃) and harvested at log phase. The crude cell extracts were obtained followed by a 55-100℃ treatment for 30 min. The soluble sub-proteomes were obtained after ultracentrifugation to remove aggregated proteins. After separating by 1D and 2D-gel electrophoresis, the proteins were digested by trypsin in-gel. Another approach is the proteomes of which the proteins were digested without separation. The MALDI-Q-Tof MS and ESI-Q-Tof MS were used to identify proteins from 2D PAGE and 1D PAGE/in-solution digestion, respectively. The sequenced genome from G. kaustophilus HTA426 allows us to accurately identify the target proteins in a high-throughput format. There were a total of 330, 207, 114, 75, 44 and 32 thermostable proteins identified at 55, 65, 75, 85, 95 and 100℃, respectively. There are three methods to confirm the accuracy of our research: (1) the Tm of 17 thermostable proteins were predicted by semi-quantification of spots volume change from different heated temperatures; (2) comparison of 13 thermostable proteins from current studies to purified proteins (Geobacillus sp.): 11 thermostable proteins were shown to possess very close properties with the literature report; (3) the Tm of 17 thermostable proteins were predicted to be reasonable with the heat treatment results.
The identified proteins are further analyzed through literature and patent for their potential in applications. Among them, peroxiredoxin, thioredoxin, superoxide dismutase, acetyl-CoA acetyltransferase, branched-chain amino acid aminotransferase and hypothetical protein GK2713/GK2820 (contained cellulase conserved domain) are the candidates for our future cloning work to confirm their thermal stability.
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