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Received March 31, 2008
Accepted May 21, 2008
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소의 히아론산 분해효소(PH-20)의 Pichia pastoris에서의 생산 최적화
Optimization of Bovine Testicular PH-20 hyaluronidase Production in Pichia pastoris
인하대학교 생물공학과, 402-701 인천시 남구 용현동 253
Department of Biological Engineering, Inha University, 253 Yonghyun-dong, Nam-gu, Inchon 402-751, Korea
ekkim@inha.ac.kr
Korean Chemical Engineering Research, August 2008, 46(4), 764-768(5), NONE Epub 10 September 2008
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Abstract
소 고환 유래의 hyaluronidase 효소 PH-20을 pPIC9 expression vector를 사용하여 Pichia pastoris에서 발현하였다. 생산된 재조합 단백질 rPH-20b는 75 kDa의 분자량을 보였고 7460 units/L의 효소활성을 보였다. 세포내보다 세포외의 효소활성이 두배 높았다. pH 의 경우 buffer를 사용 안 한 경우가, 또한 30 ℃ 배양 조건에서 높은 활성을 보였다. 1M sorbitol 삼투압조건과 0.3% 메탄올의 생산유도인자를 사용시 성장과 생산에 유리하였으며 0.4M 아르기닌을 첨가시 재조합 단백질의 분해가 감소하였다.
Bovine testicular hyaluronidase PH-20 was cloned into pPIC9 vector and expressed in Pichia pastoris. Recombinant PH-20 was 75 kDa MW and 7460 units/L activity. Extracellular hyaluronidase activity was two times higher than that of intracellular activity. Non-buffered medium and 30 ℃ cultivation was favorable for PH-20 production. 1M sorbitol as an osmotic pressure and 0.3% methanol inducer increased cell growth and enzyme activity. 0.4 M arginine augmentation decreased the proteolytic degradation of recombinant hyaluronidase.
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