Characteristics of Microbial Pretense far Application to Abolished Protein Resource

폐단백자원에 이용하기 위한 미생물 Protease의 특성

To extract insoluble proteins and to improve functional properties of abolished proteins, a protease producing Aspergillus sp. MS-18 was isolated from soil. The enzyme was purified and its enzymological characteristics were investigated. It was found that production of protease reached to the maximum when the wheat brae medium containing, 3% arabinose, 0.5% polypepton, 0.1% $(NH_4)_2SO_4$ and 0.2% magnesium chloride was cultured for 3 days. Protease was purified 16.9 folds after ion exchange chromatography and gel filtration and the specific activity was 340.4 unit/mg. Purified enzyme was confirmed as a single band by the polyacrylamide gel electrophoresis. The molecular weight of protease was estimated to be 30,000. Crystalization form of purified protease was a stick shape with rounding edges. The optimum pH and temperature for the protease activity were 9.0 and $60^{\circ}C$, respectively. The enzyme was stable in pH 7.0-12.0 at $50^{\circ}C$. The activity of purified enzyme was inhibited by $Hg^{2+}$, $Cu^{2+}$, $Zn^{2+}$ and $Pb^{2+}$, whereas it was activited by $Na^+$, $Mg^{2+}$ and $Mn^{2+}$. The activity of the protease was inhibited by the treatment with ethylenediaminetetraacetic acid and phenylmethane sulfonyl fluoride. The result suggests that the purified enzyme is a serine protease with metal ion at active site. Km and Vmax of purified protease were $29.33\;{\mu}mole/L$ and $5.13\;{\mu}g/min$, respectively.

폐단백질을 활용하는 방도의 하나로 폐단백질 자원으로 부터 불용성 단백질의 분리 효율성을 높이고 기능성을 개선하기 위하여 protease를 생산하는 Aspergillus sp. MS-18 균주를 토양으로 부터 분리하고 이 균주가 생산하는 효소를 정제하여 특성을 살펴보았다. 효소 생산을 위한 최적 배양조건은 3% arabinose, 0.5% polypepton, 0.1% ammonium sulfate, 0.1% magnesium chloride 첨가로 3 일 배양이었다. 효소는 ion exchange chromatography, gel filtration 등으로 16.9 배 정제할 수 있었으며 비활성역가는 340.4 unit/mg이었다. 정제효소는 polyacryl amide gel 전기영동상 단일 밴드로 나타났으며, 분자량은 30,000 정도로 추정되었고 결정구조는 모서리가 둥그스럼한 막대 모양이었다. 정제 효소의 최적작용 pH와 온도는 9.0, $60^{\circ}C$였으며, pH 7.0-12.0까지 $50^{\circ}C$에서 안정하였다. 금속이온중 $Na^+$, $Mg^{2+}$, $Mn^{2+}$등에 의해 활성이 증대 되었으나, $Hg^{2+}$, $Cu^{2+}$, $Zn^{2+}$, $Pb^{2+}$에 의해 효소 활성이 저해되었고 저해제중 ethylenediaminetetra acetic acid와 phenyl methanesulfonyl fluoride에 의한 활성 저해가 관찰되어 금속 이온이 효소 활성에 관여하는 serine protease로 추정되었으며 정제효소의 Km, Vmax는 $29.33\;{\mu}mole/L$, $5.13\;{\mu}g/min$이었다.