초록

최근 북극해의 축치해(Chuckchi Sea)로부터 저온지질분해 효소활성을 보이는 Psychrobacter sp. ArcL13 균주가 분리되었다. 그러나 낮은 발현 양 때문에, 이 효소의 다양한 분야에서의 활용에 제약을 받아왔다. 따라서 유전자 재조합 기술을 이용하여, 이 효소를 대량생산하는 기술개발이 필요하였다. 재조합 지질분해효소를 만들기 위해서는 우선 해당 유전자의 동정이 필요하였기 때문에, Psychrobacter sp. ArcL13 균주로부터 PCR을 이용한 gene prospecting 방법으로 새로운 지질분해효소 유전자인 ArcL13-Lip을 분리하고 전체 염기 서열을 규명하였다. 염기 서열 분석결과 ArcL13-Lip은 일부 Psychrobacter 속 박테리아 유래의 지질분해효소들과 염기 서열의 유사성은 낮지만, 84-90%의 아미노산 서열 유사성을 보였다. ArcL13-Lip 전체 유전자를 대장균에서 발현시키고 전기영동으로 분석한 결과, 재조합 ArcL13-Lip은 약 35 kDa의 분자량을 보였으며 단백질 봉입체 형태로 발현되었다. Unfolding된 ArcL13-Lip을 다양한 첨가물이 포함된 완충용액에서 refolding 시킨 결과, glucose에 의해서 refolding 효율이 가장 크게 증가하였다. Refolding된 재조합 ArcL13-Lip은 다양한 p-nitrophenyl ester 중 p-nitrophenyl caprylate과 p-nitrophenyl decanoate에 대해 가장 높은 효소활성을 보였다. 온도에 따른 효소활성을 조사한 결과 ArcL13-Lip은 $40^{\circ}C$에서 최고의 활성을 나타내었고, $10^{\circ}C$와 $20^{\circ}C$에서 각각 최고 활성 대비 약 40%와 73%의 효소활성을 나타내었다. 이와 같이 ArcL13-Lip은 전형적인 저온활성 효소의 특징을 보여주었다.

Abstract

Recently, Psychrobacter sp. ArcL13 strain showing the extracellular lipase activity was isolated from the Chuckchi Sea of the Arctic Ocean. However, due to the low expression levels of the enzyme in the natural strain, the production of recombinant lipase is crucial for various applications. Identification of the gene for the enzyme is prerequisite for the production of the recombinant protein. Therefore, in the present study, a novel lipase gene (ArcL13-Lip) was isolated from Psychrobacter sp. ArcL13 strain by gene prospecting using PCR, and its complete nucleotide sequence was determined. Sequence analysis showed that ArcL13-Lip has high amino acid sequence similarity to lipases from bacteria of some Psychrobacter genus (84-90%) despite low nucleotide sequence similarity. The lipase gene was cloned into the bacterial expression plasmid and expressed in E. coli. SDS-PAGE analysis of the cells showed that ArcL13-Lip was expressed as inclusion bodies with a molecular mass of about 35 kDa. Refolding was achieved by diluting the unfolded protein into refolding buffers containing various additives, and the highest refolding efficiency was seen in the glucose-containing buffer. Refolded ArcL13-Lip showed high hydrolytic activity toward p-nitrophenyl caprylate and p-nitrophenyl decanoate among different p-nitrophenyl esters. Recombinant ArcL13-Lip displayed maximal activity at $40^{\circ}C$ and pH 8.0 with p-nitrophenyl caprylate as a substrate. Activity assays performed at various temperatures showed that ArcL13-Lip is a cold-active lipase with about 40% and 73% of enzymatic activity at $10^{\circ}C$ and $20^{\circ}C$, respectively, compared to its maximal activity at $40^{\circ}C$.

주제어

#Psychrobacter sp   #ArcL13   #cold-active lipase   #gene prospecting   #refolding  

참고문헌 (31)

1. Arakawa, T. and Timasheff, S.N. 1982. Stabilization of protein structure by sugars. Biochemistry 21, 6536-6544. 
2. Bakermans, C., Ayala-del-rio, H.L., Ponder, M.A., Vishnivetskaya, T., Gilichinsky, D., Thomashow, M.F., and Tiedje, J.M. 2006. Psychrobacter cryohalolentis sp. nov. and Psychrobacter arcticus sp. nov., isolated from Siberian permafrost. Int. J. Syst. Evol. Microbiol. 56, 1285-1291. 
3. Bell, P.J., Sunna, A., Gibbs, M.D., Curach, N.C., Nevalainen, H., and Bergquist, P.L. 2002. Prospecting for novel lipase genes using PCR. Microbiology 148, 2283-2291. 
4. Cardenas, F., de Castro, M.S., Sanchez-Montero, J.M., Sinisterra, J.V., Valmaseda, M., Elson, S.W., and Alvarez, E. 2001. Novel microbial lipases: catalytic activity in reactions in organic media. Enzyme Microb. Technol. 28, 145-154. 
5. Chen, R.P., Guo, L.Z., and Dang, H.Y. 2011. Gene cloning, expression and characterization of a cold-adapted lipase from a psychrophilic deep-sea bacterium Psychrobacter sp. C18. World J. Microbiol. Biotechnol. 27, 431-441. 
6. Feller, G. 2013. Psychrophilic enzymes: from folding to function and biotechnology. Scientifica 2013, 512840. 
7. Finnegan, P.M., Brumbley, S.M., O'Shea, M.G., Nevalainen, H., and Bergquist, P.L. 2005. Diverse dextranase genes from Paenibacillus species. Arch. Microbiol. 183, 140-147. 
8. Gerday, C., Aittaleb, M., Bentahir, M., Chessa, J.P., Claverie, P., Collins, T., D'Amico, S., Dumont, J., Garsoux, G., Georlette, D., et al. 2000. Cold-adapted enzymes: from fundamentals to biotechnology. Trends Biotechnol. 18, 103-107. 
9. Hartl, F.U., Bracher, A., and Hayer-Hartl, M. 2011. Molecular chaperones in protein folding and proteostasis. Nature 475, 324-332. 
10. Hottiger, T., De Virgilio, C., Hall, M.N., Boller, T., and Wiemken, A. 1994. The role of trehalose synthesis for the acquisition of thermotolerance in yeast. II. Physiological concentrations of trehalose increase the thermal stability of proteins in vitro. Eur. J. Biochem. 219, 187-193. 
11. Jaeger, K.E., Dijkstra, B.W., and Reetz, M.T. 1999. Bacterial biocatalysts: molecular biology, three-dimensional structures, and biotechnological applications of lipases. Annu. Rev. Microbiol. 53, 315-351. 
12. Jaeger, K.E., Ransac, S., Dijkstra, B.W., Colson, C., Vanheuvel, M., and Misset, O. 1994. Bacterial lipases. FEMS Microbiol. Rev. 15, 29-63. 
13. Joseph, B., Ramteke, P.W., and Thomas, G. 2008. Cold active microbial lipases: Some hot issues and recent developments. Biotechnol. Adv. 26, 457-470. 
14. Kim, S., Wi, A.R., Park, H.J., Kim, D., Kim, H.W., Yim, J.H., and Han, S.J. 2015. Enhancing extracellular lipolytic enzyme production in an arctic bacterium, Psychrobacter sp. ArcL13, by using statistical optimization and fed-batch fermentation. Prep. Biochem. Biotechnol. 45, 348-364. 
15. Kulakova, L., Galkin, A., Nakayama, T., Nishino, T., and Esaki, N. 2004. Cold-active esterase from Psychrobacter sp. Ant300: gene cloning, characterization, and the effects of Gly ${\rightarrow}$ Pro substitution near the active site on its catalytic activity and stability. Biochim. Biophys. Acta. 1696, 59-65. 
16. Lee, J.C. and Timasheff, S.N. 1981. The stabilization of proteins by sucrose. J. Biol. Chem. 256, 7193-7201. 
17. Novototskaya-Vlasova, K., Petrovskaya, L., Kryukova, E., Rivkina, E., Dolgikh, D., and Kirpichnikov, M. 2013a. Expression and chaperone-assisted refolding of a new cold-active lipase from Psychrobacter cryohalolentis $K5^T$. Protein Expr. Purif. 91, 96-103. 
18. Novototskaya-Vlasova, K.A., Petrovskaya, L.E., Rivkina, E.M., Dolgikh, D.A., and Kirpichnikov, M.P. 2013b. Characterization of a cold-active lipase from Psychrobacter cryohalolentis $K5^T$ and its deletion mutants. Biochemistry (Mosc) 78, 385-394. 
19. Novototskaya-Vlasova, K., Petrovskaya, L., Yakimov, S., and Gilichinsky, D. 2012. Cloning, purification, and characterization of a cold-adapted esterase produced by Psychrobacter cryohalolentis K5T from Siberian cryopeg. FEMS Microbiol. Ecol. 82, 367-375. 
20. Park, I.H., Kim, S.H., Lee, Y.S., Lee, S.C., Zhou, Y., Kim, C.M., Ahn, S.C., and Choi, Y.L. 2009. Gene cloning, purification, and characterization of a cold-adapted lipase produced by Acinetobacter baumannii BD5. J. Microbiol. Biotechnol. 19, 128-135. 
21. Salameh, M.A. and Wiegel, J. 2007. Purification and characterization of two highly thermophilic alkaline lipases from Thermosyntropha lipolytica. Appl. Environ. Microbiol. 73, 7725-7731. 
22. Sarkar, P., Yamasaki, S., Basak, S., Bera, A., and Bag, P.K. 2012. Purification and characterization of a new alkali-thermostable lipase from Staphylococcus aureus isolated from Arachis hypogaea rhizosphere. Process Biochem. 47, 858-866. 
23. Shandilya, H., Griffiths, K., Flynn, E.K., Astatke, M., Shih, P.J., Lee, J.E., Gerard, G.F., Gibbs, M.D., and Bergquist, P.L. 2004. Thermophilic bacterial DNA polymerases with reverse-transcriptase activity. Extremophiles 8, 243-251. 
24. Singer, M.A. and Lindquist, S. 1998. Multiple effects of trehalose on protein folding in vitro and in vivo. Mol. Cell 1, 639-648. 
25. Smalas, A.O., Leiros, H.K., Os, V., and Willassen, N.P. 2000. Cold adapted enzymes. Biotechnol. Annu. Rev. 6, 1-57. 
26. Sunna, A. and Bergquist, P.L. 2003. A gene encoding a novel extremely thermostable 1,4-beta-xylanase isolated directly from an environmental DNA sample. Extremophiles 7, 63-70. 
27. Suzuki, T., Nakayama, T., Kurihara, T., Nishino, T., and Esaki, N. 2002. Primary structure and catalytic properties of a cold-active esterase from a psychrotroph, Acinetobacter sp. strain no. 6. isolated from Siberian soil. Biosci. Biotechnol. Biochem. 66, 1682-1690. 
28. Vallejo, L.F. and Rinas, U. 2004. Strategies for the recovery of active proteins through refolding of bacterial inclusion body proteins. Microb. Cell Fact. 3, 11. 
29. Zhang, A.J., Gao, R.J., Diao, N.B., Xie, G.Q., Gao, G., and Cao, S.G. 2009. Cloning, expression and characterization of an organic solvent tolerant lipase from Pseudomonas fluorescens JCM5963. J. Mol. Catal. B Enzym. 56, 78-84. 
30. Zhang, J., Lin, S., and Zeng, R.Y. 2007. Cloning, expression, and characterization of a cold-adapted lipase gene from an Antarctic deep-sea psychrotrophic bacterium, Psychrobacter sp. 7195. J. Microbiol. Biotechnol. 17, 604-610. 
31. Zhao, J.C., Zhao, Z.D., Wang, W., and Gao, X.M. 2005. Prokaryotic expression, refolding, and purification of fragment 450-650 of the spike protein of SARS-coronavirus. Protein Expr. Purif. 39, 169-174. 

이 논문을 인용한 문헌 (0)

1. 이 논문을 인용한 문헌 없음