A substantial overproduction and secretion of endo-β-1,4-glucanase via a shuttle vector, pCK98, carrying a $\underline{Bacillus}$ $\underline{subtilis}$ gene coding for this enzyme was allowed in $\underline{Bacillus}$ $\underline{subtilis}$ and $\underline{subtilis}$. $\underline{megaterium}$. Activities of the exocellular enzymes produced by $\underline{subtilis}$. $\underline{subtilis}$ RM125 (pCK98) and $\underline{subtilis}$. $\underline{megaterium}$ (pCK98) were found to be higher than that produced by the original gene donor $\underline{subtilis}$. $\underline{subtilis}$ strain by 52- and 43-fold, respectively. Most of the enzyme was produced during the exponential growth period and the production was not repressed by glucose or cellobiose. The plasmid was stable in $\underline{subtilis}$. $\underline{megaterium}$ but not in $\underline{subtilis}$. $\underline{subtilis}$.
The production of β-glucosidase via new shuttle vectors having a $\underline{Alcaligenes}$ $\underline{faecalis}$ gene encoding this enzyme was less effectively allowed in $\underline{Bacillus}$ and $\underline{E}$. $\underline{coli}$ cells even though the gene was put on amplifiable plasmids. Contrary to the endo-β-1, 4-glucanase gene, the β-glucosidase gene was comparatively unstable in $\underline{Bacillus}$ and $\underline{E}$. $\underline{coli}$ cells. On the analysis of plasmids isolated from resultant transformed cells, intactness, deletion, rearrangement or modification of β-glucosidase gene portion in shuttle plasmids were all found. The concomitant production of glucose from carboxymethyl cullulose (CMC) with β-glucosidase and endo- β-1,4-glucansase complex produced by $\underline{Bacillus}$ or $\underline{E}$. $\underline{coli}$ transformants was observed depending on the comparatively long incubation period.
Additionally, a novel procedure using polysaccharides and dyes for the rapid detection and comparison of various polysaccharase activities was established. The starch, CMC, lichenan, laminarin, or pustulan as substrates, together Congo Red, Trypan Blue, or Iodine as dyes was combinedly used in agar plates for the selective detection and comparison of various polysaccharase activities in microorganisms.
Endo-β-1, 4-glucanase를 coding하는 $\underline{Bacillus}$ $\underline{subtilis}$유전자를 가진 shuttle vector, pCK98 을 통하여 $\underline{Bacillus}$ $\underline{subtilis}$와 $\underline{B}$. $\underline{megaterium}$ 세포에서 이 효소의 실질적 대량 생산 및 분비를 이루었다. $\underline{B}$. $\underline{subtilis}$와 $\underline{B}$. $\underline{megaterium}$ 형질 전환주가 생산 및 분비한 효소의 양은 모균 $\underline{B}$. $\underline{subtilis}$ 균주가 생산한 것에 비하여 각각 52배와 43배만큼 높았다. 대부분의 효소는 대수 증식기 동안에 생산되었고 포도당이나 cellobiose 에 의해 생산이 억제되지않았다. $\underline{B}$. $\underline{megaterium}$ 세포에서는 plasmid 가 안정하였으나 $\underline{B}$.$\underline{subtilis}$에서는 불안정하였다.
β-Glucosidase를 coding하는 $\underline{Alcaligenes}$ $\underline{faecalis}$유전자를 가진 새로운 shuttle vector들을 통한 $\underline{Bacillus}$와 대장균 세포에서의 이 효소의 생산은 비록 이들 유전자가 multicopy의 plasmid에 실려있더라도 효과적으로 허용되지 않았다. Endoglucanase 유전자와는 대조적으로, β-glucosidase 유전자는 $\underline{Bacillus}$와 대장균 세포에서 비교적 불안정하였다. 형질 전환주로부터 분리한 plasmid들을 분석해 본 결과 shuttle plasmid내의 β-glucosidase유전자 부위에서 온전함, 결실, 재배열, 혹은 변이 등이 모두 나타났다. $\underline{Bacillus}$ 혹은 대장균 형질 전환주에 의하여 생산된 β-glucosidase와 endoglucanase 복합체에 의하여 CMC로부터 포도당의 부수적인 생산이비교적 긴 반응 시간 동안에 따라 관찰되었다.
부가적으로, 여러 가지 다당류 분해능의 신속한 검출과 비교를 위한 다당류들과 dye들을 사용한 새로운 방법이 확립되었다. Starch, CMC, lichenan, laminarin 혹은 pustulan들을 기질로 하고, 더불어 Congo Red, Trypan Blue 혹은 Iodine 을 dye 들로 하여 이들을 조합하여 agar배지 상에서 사용함으로써, 미생물의 여러 가지 다당류 분해능을 선택적으로 검출 및 비교할 수 있게 되었다.
