논문 상세보기
pp.158-168
에너지 요구 반응인 NADP 전환 반응계에 acetate kinase에 의한 ATP 생성계를 도입하여 NAD의 NADP로의 효율적인 전환을 시도하였다. 전환 균체는 세포막 투과성 향상을 위하여 toluene 처리 후 동결 건조한 B. ammoniagenes Cells를 사용하였으며, 전환 균체의 고정 화 담체로는 3% k-carrageenan을 사용하였다. 한편 ATP 생성계 효소인 acetate kinase는 E. coli로부터 추출 정제하여 사용하였다. 전환 반응의 최적 온도 및 pH는 각각 37℃, pH 7.5였다. 또한 10mM acetyl phosphate, 5mM ADF, 200mM inorganic phosphate, 10mM MgCl_2, 고정화 균체 250㎎/㎖ , acetate kinase 49.5mUnit/㎖에서 가장 높은 전환율을 나타내었다. 상기 최적 조건하에서 12시간 전환 반응시 5mM (340㎎/㎖)NAD가 3.6mM NADP로 전환되어 약 72%의 전환율을 나타내었다.
For the conversion of NAD to NADP, immobilized Brevibacterium ammoniagenes cells with NAD kinase was coupled with ATP generating system by acetate kinase. The membrane permeability of B. ammoniagenes was improved by toluene treatment of cells. The toluene treated B. ammoniagenes cells were immobilized for stable enzyme activity. Partially purified acetate kinase was used in the reaction system. The optimum conditions for the efficient conversion of NAD to NADP by energy-coupled system were investigated. B. ammoniagenes cells treated with toluene for the improvement of membrane permeability showed 4.5 fold improved permeability in the conversion of NAD to NADP compared with intact cells. 3% K-carrageenan as the immobilization matrix of B. ammoniagenes showed the best efficiency for the conversion of NAD to NADP. The optimum conditions for the NAD to NADP conversion reaction coupled with ATP-generating system were 10mM acetylphosphate, 5mM ADP, 200mM inorganic phosphate, 10mM MgCl_2, 250㎎/㎖ immobilized cells, 49.3mUnit/㎖ acetate kinase, pH 7.5 and 37℃. Under the optimum conditions, 72% of 5mM(340㎎/㎖) NAD was converted to NADP in 12 hours.
