최초로 구축된 $eta$-xylosidase 유전자 함유 5.0kb HindIII 절편을 포함하는 pAX278의 Insert 크기를 줄이기 위하여 subcloning을 행한 결과, 1.4kb EcoRI-XbaI 절편이 subcloning되었으며 이를 pAK 208로 명하였다. Plasmid pAX208에 의해 형질전환된 대장균은 $eta$-xylosidase 활성이 pAX278의 경우보다 약 1.3배 증가하였고 또한, $eta$-xylosidase의 활성을 증가시키기 위하여 강력한 tac-promoter를 가진 pKK223-3 plasmid vector를 이용하여 대장균에 cloning 및 발현시켰을 때, pAX278을 가진 대장균 형질전환체와 유전자 source균인 호알칼리성 Bacillus속 K-17에 비하여 각각 약 3.3배 및 1.8배의 효소활성 증가를 나타내었다. 전체 5.0kb HindIII 절편을 cloning하여 Bacillus속 K-17에 발현시킨 균주는 각각 3.7배 및 2.0배의 $eta$-xylosidase 활성증가를 보였다. 각 형질전환체로부터 정제된 $eta$-xylosidase의 효소학적 특성은 Bacillus속 K-17과 거의 일치하였다.
To reduce the size of 5.0kb HindIII fragment containing $eta$-xylosidase gene, the 5.0kb insert of pAX278 which was previously cloned was reduced by various deletions and thus 1.4kb EcoRI-Xbal fragment was subcloned into pUC19, and the recombinant plasmid was named pAK208. The $eta$-xylosidase acnivity of E. coli harboring pAK208 was higher about 1.3times than that of pAX278. For the improvement of $eta$-xylosidase activity, we cloned and expressed the $eta$-xylosidase gene in E. coli using vector pKK223-3 containing a potent tac-promoter, and enzyme activity of the transformant harboring pKHR212 was increased about 3.3 and 1.8 times than that of E. coli(pAX278) and Bacillus sp. K-17, respectively. To obtain better expression of $eta$-xylosidase gene, the whole 5.0kb HineIII fragment was recloned into pC194, and the Bacillus sp. K-17 transformant harbor-ing the recombinant plasmid pCX174 showed higher activity than that of the E. coli (pAX278) and Bacillus sp. K-17, respectively. The characteristics of enzyme purified from transformants were consistent with those front alkalophilic Bacillus sp, K-17.
