갯벌에서 분리된 Psychrobacter sp. S3균 유래의 저온성 리파제에 의한 트리글리세리드의 가수분해 특성
Hydrolysis of Triglycerides with Cold-Adapted Lipase of Psychrobacter sp. S3 Isolated from IntertidalFlat.
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새만금 갯벌로부터 리파제를 생산하는 균주(S3)를 분리하였다. 생리적, 발효적 특성 및 계통분류학적 특성을 통해서이 분리균이 Psychrobacter 속에 속하는 것으로 판명되어서Psychrobacter sp. S3로 명명하였다. 이 균의 온도에 따른 배양특성을 구한 결과, 30oC에서 생장속도가 가장 빨랐으나,Fig. 3. SDS-PAGE of lipase S3. Lane 1, protein size marker; lane2, sample from ammonium sulfatate precipitation; lane 3, fromDEAE-Sepharose chromatography; lane 4, from hydroxyapitiechromatography. Arrow indicates the 33,000-sized putative lipase.Fig. 4. Effects of temperature and pH on lipase S3. A. Hydrolyticactivity toward pNPC was measured at different temperatures. B.1/T-Log(v) graph was drawn to calculate activation and inactivationenergy. C. Thermostability was measured. D. Hydrolytic activitywas measured at different pHs.COLD-ADAPTED LIPASE OF PSYCHROBACTER SP. S3 33리파제 효소의 활성은 20oC에서 가장 높았다. S3 리파제의온도에 따른 p-nitrophenyl caproate 분해활성을 측정한 결과, 최적 온도가 30oC로 판명되었으며 10oC에서도 최고활성의 80% 이상의 활성을 유지하였다. 또한, 10~30oC 범위에서의 효소활성에너지가 1.5 kcal/mol로 매우 낮게 계산되었다. 이것을 통해 S3 리파제가 전형적인 저온성 효소임이확인되었다. 이 효소는 최적 pH가 9.0~9.5인 알칼리성 효소로 확인되었다. 여러 길이의 트리글리세리드 기질을 분해할수 있으며 그 중에서 C4, C14, C16 기질을 가장 빠르게 분해하였다. S3 리파제를 트리뷰티린-아가로스 젤에 가하여 온도별로 반응시킨 결과, 30oC와 40oC에서 반응이 빠르게 진행되었으나, 4oC에서도 분해가 진행되었다.
Abstract
Lipase-producing bacteria (S3) were isolated from intertidal flat at Saemanguem. A isolated strain was identified as Psychrobacter species by physiological and fermentational characterization as well as 16S rRNA analysis. The strain was then named as Psychrobacter sp. S3. P. sp. S3 grew most rapidly at 30oC, but grew well even at 10oC and its lipase activity was most high when cultivated at 20oC. Lipase S3 had optimum temperature of 30oC for the hydrolysis of p-nitrophenyl caproate and had more than 80% activity even at 10oC. The activation energy was calculated to be 1.5 kcal/mol, which showed that it was a typical cold-adapted enzyme. It was an alkaline enzyme with optimum pH of 9.0~9.5. It could hydrolyze various length of triglycerides. Among them, it hydrolyzed most rapidly C4, C14, C16-length triglycerides. When added to tributyrin-agarose gel, lipase S3 hydrolyzed tributyrin most rapidly at 30 and 40oC, but it could hydrolyze well even at 4oC.
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