Synthesis of Alcohol-oxidase in Pichia pastoris on Various Carbon Sources

여러가지 탄소원에 의한 Pichia pastoris의 Alcohol-oxidase 생성

  • Lee, Myung-Suk (Dept of Microbiology, National Fisheries University of Pusan) ;
  • Hur, Sung-Ho (Dept. of Food Processing Dong-Eui Technical Junior College of Pusan)
  • 이명숙 (부산수산대학 미생물학과) ;
  • 허성호 (부산동의공업전문대학 식품공업과)
  • Published : 1989.12.30

Abstract

The regulation of the synthesis of alcohol-oxidase(E. C. 1. 1. 3. 13) was investigated in the methanol-utilizing yeasts during growth on different carbon sources. For this experiment, Pichia pastoris CBS 2612 and Pichia pastoris CBM 10 were cultured in mineral salt medium by changing its carbon sources. The production of alcohol-oxidase was varied by the carbon sources. For example, alcohol-oxidase was undetectable in all strains submitted to the test in the medium with glucose, but its production was rapidely increased when the carbon source was changed from glucose to methanol after 48hrs of incubation. Moreover, this enzyme was not synthesized during growth on the primary aliphatic alcohols alone(ethanol, propanol, butanol or pentanol) or on the mixed substrates(0.5% methanol+0.5% primary aliphatic alcohols). When cells were grown on the various carbon sources(glucose, xylose, lactose, glycerol, galactose, saccharose, sorbose, lactic acid or acetic acid), The alcohol-oxidase activity was detected a very little amounts. These carbon sources together with methnol yieled far better synthesis of alcohol-oxidase than in case of carbon sources alone. Especially, the alcohol-oxidase activity of the cells grown on sorbose, lactose or lactic acid together with methanol was far better or similar than that of cells grown on methanol alone. The apparent Km values for the methanol of Pichia pastoris CBS 2612 and Pichia pastoris CBM 10 enzymes were 1.92 and 210 mM, respectively. It is also active towards alcohols of shorter alkyl-chain length than $C_7$, insaturated alcohols(allylalcohol, crotyl-alcohol) and secondary alcohols (iso-amylacohol, iso-butylalcohol). The affinity of alcohol-oxidase for this alcohols decreased with the increasing length of the alkyl-chain.

Pichia pastoris의 배양조건과 배지의 탄소원에 따른 alcohol-oxidase의 생성기능과 alcohol-oxidase의 기질특이성을 실험하기 위하여 P. pastoris CBS 2612와 P. pastoris CBM 10의 2균주에 대하여 실험하였다. 배양조건에 따른 효소생성은 glucose를 탄소원으로 첨가한 mineral salt medium에서 균체를 정상기까지 배양하여 methanol이 함유된 배지에서 재배양하는 2단계 배양법의 경우가 균체를 직접 methanol배지에 배양한 경우보다 효율적이었으며 생성된 총효소량도 전자의 경우가 후자에 비해 약 1.7배 정도 많았다. 다음 탄소원에 따른 효소생성능을 비교하였다. 먼저 직쇄alcohol을 탄소원으로 사용한 경우 methanol을 제외한 ethanol, propanol, butanol 그리고 pentanol에서는 효소가 생성되지 않았고 또한 이 직쇄alcohol을 methanol과 혼합한 경우에도 극미량의 생성에 그쳤다. 여러가지 탄소화합물(glucose, xylose, lactose, glycerol, galactose, saccharose, sorbose, lactic acid, acetic acid)을 탄소원으로 사용하면 methanol에 의해 생성된 효소량보다 훨씬 작았으나 이들을 methanol과 혼합 사용하면 효소생성량은 급격히 증가하였고, 특히 xylose, lactose 그리고 lactic acid의 경우는 methanol 단독 사용시보다 오히려 다량 생성되었다. 시험 2균주 중에서도 어떤 경우에서든지 P. pastoris CBS 2612가 alcohol-oxidase 생성능이 더 좋은 것으로 나타났다. 효소의 기질특이성은 효소를 기질에 반응시킬때는 교반을, 그리고 효소 활성측정은 alcohol-oxidase가 methanol을 분해하여 생성한 formaldehyde를 직접 정량 하였을 경우 탄소쇄가 7개 이하인 직쇄alcohols과 불포화alcohols, 그리고 일부2급alcohols(isobutyl alcohol, isoamyl alcohol)에도 특이성이 있었다.

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