Burkholderia multivorans DH4를 이용한 lipase 생산

Theories and Applications of Chem. Eng., 2002, Vol. 8, No. 1

화학공학의 이론과 응용 제8권 제1호 2002년

이동환, 김영권*, 김승욱

고려대학교 화공생명공학과

고려바이오 연구소*

Production of lipase by newly isolated Burkholderia multivorans DH4

Dong Hwan Lee, Yung Kwon Kim*, Seung Wook Kim

Department of Chemical and Biological Engineering, Korea University

Institute of Korea Biotech*

Introduction

With the increase of biotechnology application for the fats and oils industry, many enzymes related with lipids have been used for a variety of purposes. Lipase is the enzyme which hydrolyzes the esters of long chain aliphatic acids from glycerol at oil/water interfaces. Recently lipases were studied intensively for the production of fatty acids and the interesterification of lipids.

In order to find microorganisms which produce lipase, screening of microorganisms from waste water and sludge of oil industry was done. Selected microorganisms were identified. By using selected microorganism lipase was produced in a batch culture. Culture conditions and medium for lipase production were optimized.

Materials and Methods

Microorganisms were isolated from sludge and waste water of oil production factory. After testing lipase activity of microorganisms, Burkholderia multivorans DH4 was finally selected.

The stock cultures were maintained by transferring the microorganism monthly on nutrient agar plates. Basal seed medium was LB (1%tryptone, 0.5% yeast extract, 1%NaCl) broth. Basal main medium was consisted of 1% bacto peptone, 0.5% yeast extract, 0.02% MgSO47H2O, 0.1% K2HPO4, 1% olive oil. When the main medium was optimized, the medium contained 2% soluble starch, 1% casamino acid, 0.5% K2HPO4, 0.02% MgSO4·7H2O, 2% olive oil. pH was adjusted to 6.0 prior to sterilization. Seed and main cultures were carried out in 250 ml Erlenmeyer flasks containing 50 ml of medium. Flask cultures were operated at 200 rpm, 30℃ on a rotary shaking incubator.

A batch culture was performed at 30℃ in 2.5 L stirred tank reactor with optimized medium. Initial pH was 6.0 and agitation speed was 200rpm. Air flow rate was 1.0 v/v·min.

Cell mass was measured by UV spectrophotometer at 600 nm. Free fatty acids produced by lipase from triglyceride were determined by observing the color developed using cupric acetate-pyridine as a color developing reagent.

Result and discussion

Optimization of culture conditions

In order to determine optimal temperature and pH for lipase production by Burkholderia multivorans DH4, main culture in the shake flask was carried out at various temperatures and pH. Optimal temperature and pH was 30℃ and pH 6 respectively. Then 0.53 U/ml of lipase activity was obtained. In order to determine optimal agitation speed for lipase production, main culture in the shake flask was carried out at various agitation speed. Optimal agitation speed was 200 rpm, then 0.54 U/ml of lipase activity was obtained.

Optimization of main culture medium

Burkholderia multivorans DH4 was cultured in seed medium of LB broth for 1day. Seed culture (4%, v/v) was used as inoculum to main culture. Main medium was cultured for 36 hr. Seed and main cultures were carried out in 250 ml Erlenmeyer flasks containing 50 ml of medium. Flask cultures were operated at 200 rpm, 30℃ on a rotary shaking incubator.

In order to optimize culture medium, various carbon, nitrogen, phosphate sources and mineral salts, lipase inducers were tested. Each source was added to basal medium at various concentration. As carbon source 2% soluble starch was determined. When 2% soluble starch was used as carbon source, lipase activity was 1.29 U/ml(Fig. 1). As nitrogen source 1% casamino acid was determined. When 1% casamino acid was used as nitrogen source, 1.99 U/ml of lipase activity was obtained(Fig. 2). As phosphate source 0.5% K2HPO4 was determined. In this case, 2.21 U/ml of lipase activity was obtained(Fig. 3). To select proper inducer, various oils and fatty acids were tested. Fatty acids were less effective to produce lipase than oils. It was thought that fatty acids inhibited the production of lipase. As inducer 2% olive oil was determined. When 2% olive oil was used, 3.09 U/ml of lipase activity was obtained(Fig. 4). As mineral salt 0.02% MgSO4·7H2O was determined. In this case, lipase activity was 2.94 U/ml(Fig. 5). After all, the optimized medium was determined to be 2% soluble starch, 1% casamino acid, 0.02% MgSO4·7H2O, 0.5% K2HPO4 and 2% olive oil.

Production of lipase in 2.5L stirred tank reactor

A batch culture of Burkholderia multivorans DH4 was performed at 30℃ in 2.5L stirred tank reactor with optimized medium. Initial pH was 6.0 and agitation speed was 200rpm. Air flow rate was 1.0 v/v·min. Cell growth, the production of lipase and pH change in the batch culture are shown in Fig. 6. After 32 hr of cultivation lipase activity was the highest, this was 18% lower lipase activity(2.54 U/ml) than that in 250 ml shake flask.

Acknowledgement

Theories and Applications of Chem. Eng., 2002, Vol. 8, No. 1

This study was partially supported by Ministry of Commerce, Industry and Energy.

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