Purification of Glucoamylase Produced by Rhizopus oryzae

Rhizopus oryzae가 생산(生産)하는 Glucoamylase의 정제(精製)

These experiments were conducted to purify the glucoamylase produced by Rhizopus oryzae. Two forms of glucoamylase (GI and GII) from Phizopus oryzae were purified by $(NH_2)_2SO_4$ fractionation, acetone fractionation and successive column chromatography on DEAE-cellulose and CM-cellulose. The specific activities of GI and GII toward soluble starch were 157.6 U/㎎. protein (37.5 fold of crude extract), and 164.7 U/㎎. protein (39.2 fold of curde extract), respectively, and the yields of them were 4.3% and 3.8%, respectively. The two purified enzymes have shown a single band by polyacrylamide disc gel electrophoresis and SDS-polyacrylamide gel electrophoresis. The protein bands of their electrophoresis gel were revealed to have glucoamylase activity by iodine staining and were proved to be glycoprotein by periodic acid Schiff's staining.

Pizopus oryzae가 생산(生産)하는 glucoamylase를 유안(硫安)및 acetone 분획(分劃)과 이온교환수지의 column chromatography에 의하여 정제(精製)하였다. 즉(卽) 조효소액(粗酵素液)을 유안분획(硫安分劃) acetone 분획(分劃), DEAE-cellulose column chromatography, CM-cellulose column chromatography에 의(依)하여 두가지형(型)의 glucoamylase를 분리정제(分離精製)하였으며 이들을 각각(各各) glucoamylase I과 II라고 하였다. Glucoamylase I과 II의 Specific activity는 각각(各各) 157.6U/mg protein (조효소액(粗酵素液)의 37.5배(倍)) 164.7U/mg protein (조효소액(粗酵素液)의 39.2배(倍)) 이었고 수율(收率)은 각각(各各) 4.3%, 3.8%이었다. Glucoamylase I과 II는 polyacrylamide disc gel electrophoresis와 SDS-polyacrylamide gel electrophoresis에 의(依)하여 각각(各各) 단일(單一)한 band를 나타내었고 이 단백질(蛋白質) band는 옥도염색(沃度染色)에 의(依)해 glucoamylase 활성(活性)을, PAS염색(染色)에 의(依)해 glycoprotein임을 확인(確認)하였다.