Raffinose 배지에서 배양한 E. coli세포로 부터 $alpha$-galactopyrosyl linkage를 가수분해시키는 $alpha$-galactosidase를 순수 분리 정제하였으며, 얻어진 효소는 disc gel 전기영동 실험에서 단일 단백질 임이 밝혀졌다. 이 효소의 비 활성도는 63.0 units/mg protein으로서 비교적 높은 활성도를 갖고 있었으며 SDS-gel에 의한 전기영동 실험과 침강 원심분리 방법에 의한 침강 속도값의 측청 결과에 의한 분자량 값은 각각 55,000과 240,000 dalton이였는바 이로부터 이 효소의 구조가 tetramer 임을 추측할 수 있었다. 이 효소는 식물의 씨앗이나 동물조직으로 부터 얻은 동종의 효소와 비교해 볼때 상이한 기질 특이성을 갖고 있을뿐 아니라 특히 2-mercaptoethanol과 $NAD^+$ 존재하에서 효소 활성도가 크게 증가된다는 점이 특이하며, 효소 희석에 의한 불활성화 현상도 이 효소의 특이성이라 할 수 있다. 한편 이 효소는 $alpha$-galactoside의 가수분해 반응뿐 아니라 galactosyl group을 cellobiose나 maltose등에 이전시킬 수 있는 transgalatosylation 반응도 촉진시킴을 알 수 있었다.
The $alpha$-galactosidase($alpha$-D-galactohydrolase, EC 3.2.1.22) was isolated from the cells of E. coli and purified to physical homogeneity by a combination of ammonium sulfate fractionation, DEAE-cellulose ion exchange chromatography, and molecular sieve gel filtration. The final purified enzyme showed a single band on polyacrylamide disc gel electrophoresis and had a specific activity of 63.0 units per mg protein based on p-nitrophenyl-$alpha$-D-galactoside (PNPGaI) as the substrate. The apparent molecular weight of the enzyme was estimated to be about 55,000 and 240,000 based on the electrophoretical mobility on dodecyl sulfate gel and she $S_{20,w}$ value of 10.7 S, respectively. This suggests the enzyme has a tetrameric structure in its active form. Its optimum pH was 8.3. The enzyme showed a reversible inactivation by dilution, but such an inactivation could be prevented by the presence of $NAD^+$ and mercaptoethanol. In addition, unlike other known glycosidases, this enzyme required $NAD^+$ and mercaptoethanol as the cofactors for its full activity. The substrate specificity of the enzyme was limited toward $alpha$-galactopyranosides, and the estimated $K_m$ values for PNPGal, melibiose and raffinose were 2.5 mM, 35.6 mM and 26.2 mM, respectively. It was also demonstrated that the enzyme is capable of transgalactosylation from PNPGal to a limited number of galactosyl acceptor sugars such as cellobiose, maltose lactose and sucrose.
