Recombinant Cyanide Hydratases에 의한 시안화물 분해

국문 초록 (Abstract)

시안화물을 포름아미드로 변환시키는 nitrilase의 일종인 시안 수화효소 (cyanide hydratase, CHT) 를 진균류인 Neurospora crassa 와 Aspergillus nidulans로부터 유전자 조작을 통하여 His에 태그 또는 언태그된 형태로 대장균에 형질변환시켜 발현하였다. 발현된 효소를 고정 metal affinity chromatography로 정제하였다. 정제된 효소들의 pH 안정성, 동력학적 매개변수의 값을 검토하였다. 실험 결과 N. crassa 의 CHT가 50% 정도 더 넓은 pH 안정 범위를 가졌고 3배 가량 turnover rate도 높았다. 반면 A. nidulans CHT의 Km 값 (효소포화 용량)이 N. crassa CHT보다 더 크게 나타났다. 두 진균류에서 CHT의 유도발현은 질소성분과 상관없이 KCN에 의해 가능하였으며, 생분해 실험결과 N. crassa CHT에 의해 최대 82%/h의 시안분해가 가능하였다.

다국어 초록 (Multilingual Abstract)

The genes of cyanide hydratase(CHT), a kind of nitrilases whichhydrolyze cyanide to formamide were extracted from N. crassa and A. nidulans, the two fungal strains. The recombinant forms of the CHT originated from N. crassa and A. nidulans were prepared with N-terminal hexahistidine purificationtags or no tags, and expressed in E. coli. The enzymes were purified using immobilized metal affinity chromatography. They were compared according to their pH activity profiles, and kinetic parameters. The N. crassa CHT has the wider pH range of activity above 50% and three-fold higher turnover rate (6.6 x 10⁸ min⁻¹) than the A. nidulans, meanwhile the CHT of A. nidulans has the higher K_m value. Expression of CHT in both N. crassa and A. nidulans were induced by the presence of KCN, regardless of any presence of nitrogen sources. Max. 82% of KCN was degraded in 60 min for biological degradation tests.

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