Journal of the Korean Society of Food Science and Nutrition (한국식품영양과학회지)

The Korean Society of Food Science and Nutrition (한국식품영양과학회)
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Immobilization of Burkholderia cepacia Lipase on Weak Base Styrene Resin Using Polyethyleneimine with Cross-linking

PEI(Polyethyleneimine)를 이용하여 음이온계 레진에 고정화된 Lipase AH 제조 및 효소적 Interesterification을 통한 반응 특성 연구

  • Lee, Chi Woo (Dept. of Food Science and Technology, Chungnam National University) ;
  • Lee, Ki Teak (Dept. of Food Science and Technology, Chungnam National University)
  • Received : 2014.03.05
  • Accepted : 2014.06.22
  • Published : 2014.07.31
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Abstract

This study assessed the effect of immobilized lipase on weak base styrene resin using polyethyleneimine (PEI) with cross-linking. Two procedures were used in this study. The first one, "mono-layer" lipase immobilization, involves washing PEI after adsorption. The second procedure, "multi-layer" lipase immobilization, has no washing before the cross-linking step. Treverlite XS-100200 (weak base styrene resin) was immersed with PEI solution (2.2 mg/mL). Lipase AH (from Burkholderia cepacia) was adsorbed onto the support coated with PEI before cross-linking with glutaraldehyde. Structured lipid was synthesized by immobilized lipase-catalyzed interesterification using canola oil, palmitic ethyl ester (PEE), and stearic ethyl ester (StEE). Total fatty acid contents of triacylglycerol (TAG) in structured lipids were analyzed to investigate activity, properties, and reusability of immobilized lipases. Activities of immobilized lipases on the multi-layer and mono-layer increased at a high concentration (8 mg/mL) of lipase solution used for immobilization. The results show that immobilized lipase with the mono-layer method at pH 8.0 on resin had the highest total saturated fatty acid content (26.17 area%). Activity of immobilized lipase with the multi-layer method at pH 7.5 on support was lower than that of the mono-layer, but total saturated fatty acid content was 16.79 area% higher than that of lipase AH (15.01 area%).

본 연구는 polyethyleneimine(PEI)을 이용하여 음이온계 레진에 비 고정화 효소인 lipase AH(Burkholderia cepacia)를 고정화한 후 효소적 interesterification을 통해 고정화 효소의 반응 특성을 확인하고자 하였다. 효소적 interesterification에 canola oil, palmitic ethyl ester와 stearic ethyl ester를 기질로 사용하였으며 합성물의 TAG 조성 분석을 통해 특성 연구를 진행하였다. 또한 두 가지 고정화방법(multi layer, mono layer)으로 고정화를 진행하였으며 사용된 lipase solution의 농도와 pH를 달리하여 실시하였다. 먼저 효소와 support 간의 친화력을 확인하기 위하여 단백질 함량을 측정하였다. 그 결과 고정화에 사용된 lipase solution의 농도가 8 mg/mL일 때 친화력이 좋았으며 MUIM의 경우 pH 7.5에서, MOIM의 경우 pH 8.0에서 고정화 효율이 가장 좋았다. 또한 고정화 효소의 활성을 확인하기 위하여 interesterification을 진행하였고 TAG와 sn-2 position 분석을 통하여 활성을 비교하였다. 이러한 결과를 참고하여 가장 친화력이 좋았던 조건에서 고정화를 진행하였다. 그 결과 MUIM의 경우 total saturated fatty acid(${\Sigma}SFA$)의 함량이 16.79 area%였으며 MOIM의 경우는 ${\Sigma}SFA$의 함량이 26.17 area%로 나타났다. 반면에 sn-2 position 분석결과 MUIM과 MOIM 모두 palmitic acid와 stearic acid의 함량이 증가한 경향을 나타내었는데, 이는 기존의 sn-1, 3 특이성을 가지는 lipase AH가 고정화를 통해 무작위 특이성으로 변화한 것으로 생각되었다. 또한 RP-HPLC를 이용하여 TAG의 조성을 확인한 결과, 기질은 canola oil이 주로 OOO와 LOO로 조성되었으나 고정화 효소의 interesterification 반응 후 합성물은 주로 POO/SLO, LPO/LOP와 SOO, SOS 등으로 이루어진 것으로 확인되었다. 위와 같은 결과를 바탕으로 최종 생산된 MUIM과 MOIM을 이용하여 interesterification 반응을 온도, 시간을 달리하여 진행하였다. 전체적으로 MOIM의 활성이 가장 높았으며 lipase AH, MOIM과 MUIM 모두 반응시간이 증가할수록 ${\Sigma}SFA$의 함량은 증가하였으나 12시간 이후로는 시간대비 효율은 크게 증가하지 않았다. 또한 시간이 증가함에 따라 sn-2 position의 ${\Sigma}SFA$ 함량이 높아졌다. 한편 반응온도가 높을수록 ${\Sigma}SFA$의 함량도 증가하였는데, 이는 상온인 $25^{\circ}C$보다 높은 melting point를 가지는 stearic ethyl ester와 palmitic ethyl ester가 semi solid 형태로 존재하여 활성이 저해된 것으로 생각된다. 또한 lipase AH는 재사용이 불가능하였지만 고정화 효소인 MUIM과 MOIM은 5번을 재사용하여도 그 효소의 활성이 유지되는 것을 확인할 수 있었다. 이러한 결과를 토대로 음이온계 레진에 PEI를 이용하여 lipase AH를 고정화하였을 경우 비 고정화 효소인 lipase AH보다 MUIM과 MOIM의 활성이 더 높아 산업적으로 이용 가능할 것으로 생각된다.

Keywords

References

  1. Kim JK, Park JK, Kim HK. 2004. Synthesis and characterization of nanoporous silica support for enzyme immobilization. Colloids Surf, A 241: 113-117. https://doi.org/10.1016/j.colsurfa.2004.04.048
  2. Lee KT, Akoh CC. 1998. Immobilization of lipases on clay, Celite 545, diethylaminoethyl-, and carboxymethyl-Sephadex and their interesterification activity. Biotechnol Tech 12: 381-384. https://doi.org/10.1023/A:1008826431721
  3. Malcata FX, Reyes HR, Garcia HS, Hill CG, Amundson CH. 1992. Kinetics and mechanism of reactions catalyzed by immobilized lipases. Enzyme Microb Technol 12: 426-446.
  4. Balco VM, Paiva AL, Malcata FX. 1996. Bioreactors with immobilized lipases: state of the art. Enzyme Microb Technol 18: 392-416. https://doi.org/10.1016/0141-0229(95)00125-5
  5. Ye P, Jiang J, Xu ZK. 2007. Adsorption and activity of lipase from candida rugosa on the chitosan-modified poly (acrylonitrile-co-maleic acid) membrane surface. Colloids Surf, B 60: 62-67. https://doi.org/10.1016/j.colsurfb.2007.05.022
  6. Wang Y, Caruso F. 2005. Mesoporous silica spheres as supports for enzyme immobilization and encapsulation. Chem Mater 17: 953-961. https://doi.org/10.1021/cm0483137
  7. Bai YX, Li YF, Yang Y, Yi LX. 2006. Covalent immobilization of triacylglycerol lipase onto functionalized novel mesoporous silica supports. J Biotechnol 125: 574-582. https://doi.org/10.1016/j.jbiotec.2006.04.003
  8. Yu HW, Chen H, Wang X, Yang YY, Ching CB. 2006. Cross-linked enzyme aggregates (CLEAs) with controlled particles: Application to Candida rugosa lipase. J Mol Catal B: Enzym 43: 124-127. https://doi.org/10.1016/j.molcatb.2006.07.001
  9. Wang HX, Wu H, Ho CT, Weng XC. 2006. Cocoa butter equivalent from enzymatic interesterification of tea seed oil and fatty acid methyl esters. Food Chem 97: 661-665. https://doi.org/10.1016/j.foodchem.2005.04.029
  10. Lee KT, Akoh CC. 1998. Structured lipids: synthesis and applications. Food Rev Int 14: 17-34. https://doi.org/10.1080/87559129809541148
  11. Lopez-Gallego F, Betancor L, Hidalgo A, Alonso N, Fernandez-Lafuente R, Guisan JM. 2005. Coaggregation of enzymes and polyethyleneimine: a simple method to prepare stable and immobilized derivatives of glutaryl acylase. Biomacromolecules 6: 1839-1842. https://doi.org/10.1021/bm050088e
  12. Albayrak N, Yang ST. 2002. Immobilization of $\beta$-galactosidase on fibrous matrix by polyethyleneimine for production of galacto-oligosaccharides from lactose. Biotechnol Prog 18: 240-251. https://doi.org/10.1021/bp010167b
  13. Andersson MM, Hatti-Kaul R. 1999. Protein stabilising effect of polyethyleneimine. J Biotechnol 72: 21-31. https://doi.org/10.1016/S0168-1656(99)00050-4
  14. Jaeger KE, Ransacb S, Dijkstrab BW, Colson C, Van Heuvel M, Misset O. 1994. Bacterial lipases. FEMS Microbiol Rev 151: 29-63.
  15. Lee KT, Foglia TA, Lee JH. 2005. Low-calorie fat substitutes:synthesis and analysis. In Handbook of Industrial Biocatalysis. Hou C, ed. CRC press, Boca Raton, FL, USA. Vol 16, p 1-19.
  16. Lee KT, Foglia TA. 2000. Synthesis, purification and characterization of structured lipids produced from chicken fat. J Am Oil Chem Soc 77: 1027-1034. https://doi.org/10.1007/s11746-000-0163-9
  17. Macrae AR. 1983. Lipase-catalyzed interesterification of oils and fats. J Am Oil Chem Soc 60: 291-294. https://doi.org/10.1007/BF02543502
  18. Posorske LH. 1984. Industrial-scale application of enzymes to the fats and oil industry. J Am Oil Chem Soc 61: 1758-1760. https://doi.org/10.1007/BF02582143
  19. Cho EJ, Lee JH, Lee KT. 2004. Optimization of enzymatic synthesis condition of structured lipids by response surface methodology. Korean J Food Sci Technol 36: 531-536.
  20. Jang YS. 2002. Prospect and situation of quality improvement in oil seed rape. Korean J Crop Sci 47: 175-185.
  21. Yamazaki H, Cheok RKH, Fraser ADE. 1984. Immobilization of invertase on polyethylenimine-coated cotton cloth. Biotechnol Lett 6: 165-170. https://doi.org/10.1007/BF00127033
  22. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. 1951. Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275.
  23. AOAC. 2000. Official methods of analysis. 17th ed. Association of Official Analytical Chemists, Washington, DC, USA. p 20-24.
  24. Lee KT, Akoh CC. 1996. Immobilized lipase-catalyzed production of structured lipids with eicosapentaenoic acid at specific positions. J Am Oil Chem Soc 73: 611-615. https://doi.org/10.1007/BF02518116
  25. Moon JH, Lee JH, Shin JA, Hong ST, Lee KT. 2011. Optimization of lipase-catalyzed production of structured lipids from canola oil containing similar composition of triacylglycerols to cocoa butter. J Korean Soc Food Sci Nutr 40: 1430-1437. https://doi.org/10.3746/jkfn.2011.40.10.1430
  26. Lee KT, Jones KC, Foglia TA. 2002. Separation of structured lipids by high performance liquid chromatography. Chromatographia 55: 197-201. https://doi.org/10.1007/BF02492142
  27. SAS. 2002. SAS/STAT User's Guide release 9.01. Statistical Analysis Systems Institute, Inc., Cary, NC, USA.
  28. Knothe G. 2005. Dependence of biodiesel fuel properties on the structure of fatty acid alkyl esters. Fuel Process Technol 86: 1059-1070. https://doi.org/10.1016/j.fuproc.2004.11.002