• Applied Biological Chemistry
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• 제38권5호
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• pp.376-381
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• 1995

내열성 alkaline phosphatase의 탐색을 위해 극도호열균중에서 Thermus caldophilus GK24 균주를 선정하였다. 이 균주를 이용하여 basal salts에 sodium glutamate, bactotryptone, glucose 및 yeast extract를 첨가시킨 배지에서 alkaline phosphatase 생산을 검토하였다. 그 결과 sodium glutamate가 alkaline phosphatase 유도에 효과적인 것으로 판명되었다. Alkaline phosphatase 생산을 위한 최적유도용 배지는 basal salts에 0.3% sodium glutamate, 0.2% bactotryptone, 0.5% glucose를 첨가한 것으로 효소활성은 기본배지보다 약 6배, 표준배지 보다는 약 27.5배 증가하였다. T. caldophilus GK24 alkaline phosphatase는 유도효소로 판명되었다. 무기인산 결핍시에 효소가 생산되며, 생육배지에 무기인산을 첨가하면 효소합성에 저해효과가 있었다.

• Journal of Microbiology and Biotechnology
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• 제13권5호
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• pp.660-665
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• 2003

High-level expression of Thermus caldophilus GK24 alkaline phosphatase (Tca APase) was achieved in Escherichia coli using the pET-based expression plasmids, pEAP1 and pEAP2. In the case of plasmid pEAP2, the signal peptide region of Tca APase was replaced by the PelB leader peptide of expression vector pET-22b(+). Furthermore, the expression level was somewhat higher than that of plasmid pEAPl. A rapid purification procedure of Tca APase overproduced in E. coli was developed which involved heating to denature E. coli proteins followed by HiTrap Heparin HP column chromatography. Optimal temperature and pH and $Mg^{2+}$ dependence of the recombinant Tca APase were similar to those of native enzyme isolated from T. caldophilus GK24.

• BMB Reports
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• 제30권4호
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• pp.262-268
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• 1997

The thermophilic and thermostable alkaline phosphatase was purified to near homogeneity from the osmotic lysis of Thermus caldophilus GK24, The purified enzyme had an apparent molecular mass of 108, 000 Da and consisted of two subunits of 54,000 Da. lsoelectric-focusing analysis of the purified enzyme showed a pi of 7.3. The enzyme contained two Cys residues, and its amino acids composition was quite different from that of Thermus aquaticus YT-1 alkaline phosphatase and Escherichia coli alkaline phosphatase, The optimum pH and temperature of the enzyme were 11.0-11.5 and $80^{\circ}C$ respectively. The enzyme was stable in the pH range of 9.0-12.0 at $25^{\circ}C$ for 36 h. and the half-life at $80^{\circ}C$ (pH 11.0) was 6 h. The enzyme was activated by $MgCl_2$ and inhibited by EDTA. With ${\rho}-nitrophenyl\;phosphate\;({\rho}NPP)$ as the substrate, the enzyme had a Michaelis constant $(K_m) $of $3.6{\times}10^{-5}M$, The enzyme preferentially hydrolyzed the phosphomonoester bond of AMP in ribonucleotides and glycerophosphate.