The Study Of Thermostable Alkaline Phophatase (TAP) From Bacteria Of Thermus Sp. Isolated From Hot Springs Of Yunnan And The Classification Of The Strain For TAP

Alkaline phosphatase (AP) is a kind of hydrolase, which can catalyze the hydrolysis of monophosphoester to produce phosphate or transfer phosphate. AP exists widely in microbial and animal kingdom, but hasn't been found in advanced frond. AP plays a vital important role in phosphate cycle of world creatures, and has been used widely in the fields of diagnostics, biochemistry and molecular biology. The commercial alkaline phosphatases are CIAP (calf intestine alkaline phosphatase ) and E. coll AP, which have high specific activity and have been studied intensively. Those two kinds of enzymes are only fit for using at room temperature and easy to loss activity at a relatively high temperature. Thermostable alkaline phosphatase (TAP) is one kind of alkaline phosphatase, which has the same catalysis but can keep stable at high temperature. This kind of enzyme has been isolated from themophile bacteria, and its applications are much more widely than CIAP and E. coli AP.In the study of the thesis, we had isolated thermophile bacteria without endospore from the environmental samples, which were collected from the hot springs of Eryuan and Tengchong county, Yunnan province. 31 strains of alkaline phosphatase positive were screened from those strains, and 6 strains were taken out for further study for the relatively high enzyme activity. According to the criteria of resuming screen, the strain RHY12-2 was finally chosen for the highest special activity and thermostablity in a wide temperature range. The bacterium RHY12-2 might be a novel species of Thermus sp. according to the morphological, physiological, biochemical and genie characteristics, and be proposed to name as Thermus yunnanensis (AY557603).The specific activity of RHY12-2 TAP was increased from 0.56 u/mg to 16.7 u/mg for 30-fold after optimize experiments, which was projected in accordance with the growth curve and orthogonal design for the fermentation ingredients.Following the steps of NH4SO4 treatment, n-butanol extraction, ion exchange chromatograph and affinity chromatograph, the cell extract of strain RHY12-2 was purified for 193-fold and the purified enzyme has 99 percent purity with the final special activity of 3223u/mg. The purified RHY12-2 TAP showed a single band in theSDS-PAGE gel. RHY12-2 TAP was a homodimer, and the molecular weights of the protein and its subunit are 100 KDa and 52 KDa, respectively. In the comparison of N-termininal amino acid sequences of thermostable alkaline phosphatases from Thermus sp., the similarity of RHY12-2 TAP is 80 percent.The kinetics parameters were determined in the hydrolysis reaction of pNPP. The enzyme activity and thermostability can remain relatively high in a broad temperature and pH ranges. It was quite active and stable in 65-80*C, but quite unstable beyond 90 C. Mg (II) and Co (II) ions can increase the thermostability of the enzyme, and the enzyme can be activated in the incubation with Co (II) at high temperature. The enzyme can endure 2mM EDTA for 18 hours and remain half of its activity. These characteristics give a way to the enzyme for a wide foreground of researches and practices.