Aspergillus niger에 의한 α-Galactosidase의 생산조건 및 효소적 특성

Abstract

본 연구는 대두중에 존재하는 장내 가스발생인자( flatulence factor )인 raffinose 와 stachyose를 제거하기 위하여 Aspergillus niger로 부터 생성된 효소의 이용가능성을 뒷받침하기 위해 시도되었다. 그리하여 Asp. niger에 의한 α-galactosidase의 생산조건을 알아보고, 생성된 조효소용액의 효소적 특성을 조사하였으며, 몇가지 기질에 대한 가수분해 산물을 thin-layer chromatography로 살펴 보았다. 그 결과를 요약하면 다음과 같다. 1) α-Galactosidase의 생산조건을 알아본 결과, Czapeck-Dox 액체배지에서는 pH 6.0, 120시간 배양하였을 때, 밀기울 고체배지에서도 120시간 배양하였을 때 효소생성이 제일 높았다. Czapeck-Dox 액체배지에서 배양할 때 탄소원은 raffinose, 질소원은 NaNo_(3)가 가장 효과적이었으며, 밀기울에 고체배양할 때는 질소원, 탄소원을 첨가하여도 효과가 없었다. 2) 생성된 조효소 용액의 특성을 조사한 결과 작용최적 pH는 4.0-5.0, 안정도 최저 pH는 3.5 - 6.5, 작용과 안정도에 대한 최적온도는 40 ∼ 45℃ 였다. 합성기질인 p-nitrophenyl-α- D -galactoside ( PNPG )에 대한 Michaelis constant는 0.42mM, maximum velocity는 152 μ moles substrate/minute/㎏ solid media 이었다. 저해제의 실험결과 HgCl_(3)는 강력한 저해작용을 보여 noncompetitive inhibitor로 작용하였고 PCMB는 낮은 농도에서 저해작용을 보여, competitive inhibitor로 작용했다. 3) 조효소 용액은 raffinose, sucrose, PNPG를 가수분해 하였으며 raffinose 와 stachyose의 분해과정을 thin-layer chromatography로 조사한 결과 3시간 후에는 완전 분해되어 단당류의 spot 만이 나타났다. 따라서 대두식품중의 가스발생인자를 제거하기 위해, Asp. niger에 의해 생성된 α-galactosidase가 이용될 수 있을 것으로 본다.;This study was carried out to show the applicability of α-galactosidase produced from Aspergillus niger for removal of flatulence factors such as raffinose and stachyose in soybean. The condition of enzyme production from Asp. niger was investigated, and enzymatic characteristics of α-galactosidase were examined. In addition, the extracts of hydrolysis of several substrates were measured, and hydrolysates of raffinose and stachyose were investigated by means of thin-layer chromatography. The results are summarized as follows: 1) Investigation on the production condition showed that the highest α-galactosidase activity was produced in 120 hours in either Czapeck-Dox liquid media with pH 6.0 or wheat bran solid media. In case of Czapeck-Dox liquid media, the most efficient carbon and nitrogen sources were raffinose and NaNO_(3), respectively. But the addition of carbon and nitrogen sources showed negative effects in case of wheat bran solid media. 2) Examination of enzymatic characteristics showed that optimum pH for its activity and stability were 4.0-5.0 and 3.5-6.5, respectively, and optimum temperature was 40˚-50˚C. Upon reaction on a synthetic substrate, p-nitrophenyl-α-D-galactoside, Michaelis constant was 0.42mM and maximum velocity was 152 μmoles substrate/minute/kg solid media. It was also investigated that HgCl_(2) had a strong inhibitory effect, acting as a noncompetitive inhibitor, and PCMB, even in low concentration, had an inhibitory efect, acting as a competitive inhibitor. 3) Crude α-galactosidase hydrolyzed raffinose and stachyose completely in 3 hours, giving spots of monosaccharides only on thin-layer chromatogram. In conclusion, it is supposed that α-galactosidase produced from Asp. niger can be utilized to remove the flatulence factor in soybean.