Extremely thermophilic bacterium, Thermus caldophilus GK24 is a strain belonging to genus Thermus. It seems likely an ancient line that predates the development of distinct Gram-positive and Gram-negative microorganisms. Because enzymes from thermophilic organisms are designed to "take the heat", many researchers believe they may one day be useful as industrial catalysts for the production of fuels, pharmaceuticals, foods industry, and specialty chemicals, among other applications. Especially in carbohydrates field, thermostability gives advantages in substrates solubility by increasing the reaction temperatures as well as no need of cooling. Thermus caldophilus GK24 produces xylose isomerase. Activity screening for xylose isomerase was carried out with other enzyme using glucose and crude extract. As it was difficult to detect the isomerization process, analysis of isomerization reaction was achieved by HPAEC. Reaction mixture with fructose was also analyzed and the peak pattern was found to be identical regardless of kind of substrate or concentration of substrate. The gene encoding xylose isomerase (xylA) was cloned from Thermus caldophilus GK24 and the DNA sequence was determined. The gene xylA encodes the enzyme xylose isomerase (XI) consisting of 387 amino acids (calculated Mr of 44, 749). There was a partial xylulose kinase gene overlapping 4 bp at the end of xylose isomerase gene. The xylose isomerase gene was stably expressed in E. coli and purified by a procedure of heat treatment at 90℃ for 10 min followed by a column chromatography of DEAE-Sephacel. The molecular mass of the purified enzyme was estimated to be 45 KDa on the SDS-polyacrylamide gel electrophoresis, suggesting that molecular mass of the cloned xylose isomerase is absolutely the same as that purified from the donor bacterium. However, molecular mass of the cloned xylose isomerase was 185 KDa in native condition, indicating that the xylose isomerase was consisted as homermeric tetramer. The optimum temperature for the maximum enzyme activity was determined at 90℃. Thermostability tests revealed that half life at 85℃ was 2 months and at 95℃ was 2 hours. The optimum pH was approximately 7.0, a value near the minimal by-product formation was reported. The isomerization yields of the cloned xylose isomerase was about 55% from glucose. Thus far this efficiency of isomerization is the highest yield among those reported by other known enzymes. The Km values for the various sugar substrates were calculated as 106 mM for glucose, 180 mM for xylose. Divalent cations such as $Mn^{2+}$, $Co^{2+}$, and $Mg^{2+}$ were also required for the enzyme activity and the treatment of 100 mM EDTA completely inhibted the enzyme capacity.
A thermostable β-D-xylosidase was also isolated from a thermostable strain of Thermus caldophilus GK24. The purified enzyme exhibited both activities of β-D-xylosidase and β-D-glucosidase with 43,000 Dalton of molecular mass and an isoelectric point of 5.2. Maximum enzyme activity was seen at pH 7.5 in 80℃. The Km values of β-D-xylosidase were a relatively high. Km values of 8.2 and 18.3 mM was determined for p-nitrophenyl xylopyranoside (pNP-X) and for xylobiose respectively. The heat stability of the enzyme was also evident showing that only 20 % of the initial activity was lost after a 12 hours incubation at 80℃. When cultures were grown on 0.1 % cellobiose and maltose each, after 48 hours, cellular β-D-xylosidase level was elevated approximately by two folds.
내열성 탄수화물 효소 xylose isomerase와 β-xylosidase를 Thermus caldophilus GK24를 중심으로 연구하였다. T. caldophilus GK24는 75℃ 이상의 고온에서 자라는 초호열성 박테리아로 그람 양성과 음성 박테리아의 특성을 모두 가지고 있다. 탄수화물을 매개로 하는 분야에서는 재료의 용해도와 반응온도 상승에 따른 반응산물의 생산효율 증가로 내열성과 고온반응성이 큰 장점이 된다. 따라서 T. caldophilus GK24가 내재하고 있는 탄수화물 효소들은 많은 응용가능성을 가지고 있다. T. caldophilus GK24에서 xylose isomerase의 역가를 확인하고 이 효소를 T. caldophilus GK24는 물론이고 T. flavus AT62와 T. thermophilus HB8에서도 PCR과 hybridization을 통해서 유전자를 클로닝 하였다. 이 유전자들의 염기서열을 확인하고 다른 보고된 효소들과 비교하고 또 아미노산으로 번역하여서도 비교해 본 결과 xylose isomerase 임을 확인하였다. 이 유전자들은 1164 염기쌍과 387개의 아미노산으로 이루어져 있었으며 전자개시 codon은 세가지 모두 GTG였다. 이들을 대장균 내에서 발현시키기 위해서 전사개시 codon을 ATG로 바꾸고 lac, tac, 그리고 T7 promoter를 이용하여 β-galactosidase의 N-terminal 아미노산이나 pelB leader 펩타이드와 융합시킨 형태, 그리고 순수한 형태 등으로 다양하게 발현을 시도하여 보았다. 발현된 결과를 통해서 볼때 높은 G+C 함량이나 다른 codon 사용도는 물론이고 융합된 형태에서도 효소능에는 영향이 거의 없었다. 분자량 측정으로 본 결과 분자량 18만 5천 dalton의 homomeric tetramer로서 최적반응 조건은 pH 7.5, 85℃로 상당히 높은 온도에서 최적조건이 나타났다. 이 효소는 80℃에서 두 달 이상 효소능이 반이상 유지될 정도로 높은 열안정성을 보였지만 β-xylosidase와 마찬가지로 얼거나 말리면 효소능이 극도로 감소됨을 알 수 있었다. Km으로 친화도를 조사하여 보니 포도당에 대해 106 mM, 그리고 xylose에 대해 183 mM로 오히려 포도당 쪽에 높은 친화력을 보였다. 효소능을 위해 $Mn^{2+}$나 $Co^{2+}$, 그리고 $Mg^{2+}$ 등을 필요로 하고 없을 때는 거의 효소능이 없었다. 이 효소들의 아미노산을 비교하여 본 결과 아미노산이 상온(mesophile) 미생물 보다 소수성 아미노산을 많이 바뀌어 있으나 active site 부근은 거의 유지가 되고 있었다. T. caldophilus GK24로부터 β-xylosidase의 생산은 기질이나 기질 유사체에 의해서만 증가 되었고 정제 후 분자량을 측정하여 본 결과 분자량 43,000 dalton의 단일분자구조(monomer)로 이루어져 있었다. pI값은 pH 5.2 였고 최적반응 조건은 pH 7.5에서 80℃였으나 1시간 이상의 반응 시에는 75℃가 더 좋았다. 이 효소는 4℃에서 부터 65℃까지 pH 6.0에서 8.5까지 12시간 이상 안정하였으나 얼거나 말리면 효소능의 거의 전부를 상실하였다. Km은 xylose에 대해 8.2 mM 이고 xylobiose에 대해 18.3 mM로 다른 보고된 효소와 비슷하거나 약간 더 컸다.
