Vavious kinds of microorganisms have been employed to produce various type of amylolytic enzymes. Generally, some differences in amylolytic properties have been found between the microbial amylase preparations. Accordingly, it may be considered that they were used according to each characteristic property, for example, active pH range, pH stability. Some amylase preparation possessing acid activity and acid stability of dextrinogenic and saccharifying functions are suitable for various uses under acidic conditions, being especially suitable as digestive enzyme which functions against dietary provides a strongly acidic conditions.
Acid amylases from $\underline{Paecilomyces}$ $\underline{subglobosum}$ were purified with the methods of Sephadex G-150 gel filtration and Sepharose-glycogen affinity chromatography and the enzymes were characterized.
Maximum activity of amylase was shown for 5 days culture on solid media at 30℃. Concentration of enzyme was not satisfied with either $(NH_4)_2SO_4$ fractionation or alcohol and acetone precipitation. Purification of amylase was carried out by Sephadex G-150 gel filtration and amylases were separated to A, B fractions. Fraction A and Fraction B were purified 7.7, 0.94 folds respectively. Affinity chromatography on the A, B fraction of gel filtration and crude extract showed 18 folds purification of fraction B and 10.4 folds of crude extract as glucoamylase activity. Glucoamylase gained by affinity chromatography was showed a single protein band on the gel electrophoresis.
However α-amylase did not showed any affinity on Sepharose4B glycogen affinity chromatographic colum and therefore fraction A by gel filtration showed a mode of action α-amylase and the enzyme purified by affinity chromatography, a glucoamylase.
This was confined by determination of the products of enzyme incubation mixtures by thin layer chromatography.
Optimum pH of α-amylase and glucoamylase were 4.5 and 4 respecitively, while both activities were stable between pH 3.5 and pH 9. Optimum temperature of α-amylase and glucoamylase were both stable at 37℃ for several days.
Km of glucoamylase for starch and glycogen were 0.154% and 0.125%.
전분 분해력을 가진 효소들은 여러종류의 미생물에서 얻고 있으며, 효소들의 특성은 미생물 종류에 따라 서로 다르다는 것이 알려져있다. 이런 사실을 이용하여 특정조건에 알맞는 효소를 찾아내어 공업적으로 이용하고 있으며 예를들어 산성조건에서 효소의 안정도와 효소역가를 보여주는 내산성 아밀라제는 강한 산성을 보여주고있는 위속에서 전분을 분해할수 있어 소화효소제로서 이용가능성이 크다.
본 실험에서는 $\underline{Paecilomyces}$ $\underline{subglobosum}$ 이 분비하는 내산성 아밀라제를 Sephadex G-150 gel filtration 과 Sepharose-glycogen affinity chromatography 를 이용하여 정제하였고, 효소특성을 조사하였다.
고체배지에서 5일간 30℃로 배양한 배지가 효소역가를 가장 많이 가지고 있었으며 $(NH_4)_2SO_4$ fractionation과 alcohol, acetone 침전에 의한 효소농축은 사용할수 없었다.
Sephadex G-150 gel filtration 으로 효소를 A, B fraction으로 정제하였으며, 각각 7.7배, 0.94배 정제했다.
A, B fraction과 효소추출액을 affinity chromatography column 에 통과시킨 결과 glucoamylase 만이 B fraction에서 18배 정제되었고, 조효소에서는 10.4배 정제되었다.
Affinity chromatography에 의해 얻은 glucoamylase 는 전기영동에서 단일 단백질 band 로 나타났으며 α-아밀라제는 affinity chromatography 에 친화력이 없었음을 확인하였다.
Gel filtration 으로 얻은 A fraction 의 효소는 α-아밀라제의 작용기작을 보였고, affinity chromatography 에 의해 얻은 효소는 glucoamylase 작용은 보이고 있음을 thin layer chromatography에 의한 효소반응 생성물의 확인으로 알았다.
α - 아밀라제와 glycoamylase 는 최적 pH 가 각각 4.5의 4이며 안정도는 모두 pH3.5-9사이이고 최적온도는 40℃와 50℃ 이고, 37℃ 에서 모두 수일간 안정하다.
glycoamylase 의 Km 값은 전분과 glycogen 에대해 각각 0.154%와 0.125%이다.
