Abstract
A new lipase from Serratia marcescens SRICI-01 (Trx-SmL) was successfully overexpressed in Escherichia coli with thioredoxin (Trx) fusion tag. Intriguingly, the concentration of potassium phosphate buffer (KPB) showed significant impact on the aggregation state of Trx-SmL during ultrasonic disruption. The proportion of inclusion bodies increased dramatically with the increase of KPB concentration from almost completely soluble in 10 mM KPB to insoluble in 200 mM KPB. Based on this new finding, a novel method for refolding and purification of recombinant Trx-SmL was developed by one-step ultrasonication. The Trx-SmL was firstly precipitated in 200 mM KPB, washed for three times, and subsequently subjected to ultrasonic process in 10 mM KPB where refolding and purification occurred simultaneously. This established method was proved to be a straightforward, economical, and efficient purification approach to facilely obtain recombinant Trx-SmL protein with high purity (> 90%) and activity recovery yield (> 80%) from cell lysates. The application potential of the purified fusion Trx-SmL was further demonstrated by kinetic bioresolution of (±)-trans-3-(4-methoxyphenyl)glycidic acid methyl ester [(±)-MPGM] producing optically pure (−)-MPGM, a key intermediate for diltiazem, with an overall yield of 41.5% and ee of 99%.
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Abbreviations
- MPGM:
-
3-(4′-Methoxyphenyl)glycidic acid methyl ester
- SmL:
-
Serratia marcescens lipase
- Trx-SmL:
-
Serratia marcescens lipase fused with thioredoxin
- KPB:
-
Potassium phosphate buffer
- IBs:
-
Inclusion bodies
- pNPA:
-
p-Nitrophenyl acetate
References
Jaeger, K. E., & Eggert, T. (2002). Lipases for biotechnology. Current Opinion in Biotechnology, 13, 390–397.
Treichel, H., Oliveira, D. D., Mazutti, M. A., Luccio, M. D., & Oliveira, J. V. (2010). A review on microbial lipases production. Food & Bioprocess Technology, 3, 182–196.
Ghanem, A., & Aboul-Enein, H. Y. (2005). Application of lipases in kinetic resolution of racemates. Chirality, 17, 1–15.
Matsumae, H., Furni, M., & Shibatani, T. (1993). Lipase-catalyzed asymmetric hydrolysis of 3-phenylglycidic acid ester, the key intermediate in the synthesis of diltiazem hydrochloride. Journal of Fermentation and Bioengineering, 75, 93–98.
Long, Z. D., Xu, J. H., Zhao, L. L., Pan, J., Yang, S., & Hua, L. (2007). Overexpression of Serratia marcescens lipase in Escherichia coli for efficient bioresolution of racemic ketoprofen. Journal of Molecular Catalysis B: Enzymatic, 47, 105–110.
Shibatani, T., Omori, K., Akatsuka, H., Kawai, E., & Matsumae, H. (2000). Enzymatic resolution of diltiazem intermediate by Serratia marcescens lipase: molecular mechanism of lipase secretion and its industrial application. Journal of Molecular Catalysis B: Enzymatic, 10, 141–149.
Li, X. Y., Tetling, S., Winkler, U. K., Jaeger, K. E., & Benedik, M. J. (1995). Gene cloning, sequence analysis, purification and secretion by Escherichia coli of an extracellular lipase from Serratia marcescens. Applied and Environmental Microbiology, 61, 2674–2680.
Jaeger, K. E., Schneidinger, B., Rosenau, F., Werner, M., Lang, D., Dijkstra, B. W., Schimossek, K., Zonta, A., & Reetz, M. T. (1997). Bacterial lipases for biotechnological applications. Journal of Molecular Catalysis B: Enzymatic, 3, 3–12.
Idei, A., Matsumae, H., Kawai, E., Yoshioka, R., Shibatani, T., Akatsuka, H., & Omori, K. (2002). Utilization of ATP-binding cassette exporter for hyperproduction of an exoprotein: construction of lipase-hyperproducing recombinant strains of Serratia marcescens. Applied Microbiology and Biotechnology, 58(3), 322–329.
Lopez, J. L., & Matson, S. L. (1997). A multiphase/extractive enzyme membrane reactor for production of diltiazem chiral intermediate. Journal of Membrane Science, 125, 189–211.
Gao, L., Xu, J. H., Li, X. J., & Liu, Z. Z. (2004). Optimization of Serratia marcescens lipase production for enantioselective hydrolysis of 3-phenylglycidic acid ester. Journal of Industrial Microbiology and Biotechnology, 31, 525–530.
Long, Z. D., Xu, J. H., & Pan, J. (2007). Immobilization of Serratia marcescens lipase and catalytic resolution of trans-3-(4′-methoxyphenyl)glycidic acid methyl ester. Chinese Journal of Catalysis, 28, 175–179.
Hu, B., Pan, J., Yu, H. L., Liu, J. W., & Xu, J. H. (2009). Immobilization of Serratia marcescens lipase onto amino-functionalized magnetic nanoparticles for repeated use in enzymatic synthesis of diltiazem intermediate. Process Biochemistry, 44, 1019–1024.
Zhao, L. L., Pan, J., & Xu, J. H. (2010). Efficient production of Diltiazem chiral intermediate using immobilized lipase from Serratia marcescens. Biotechnology and Bioprocess Engineering, 15, 199–207.
Mohammadi, M., Sepehrizadeh, Z., Ebrahim-Habibi, A., Shahverdi, A. R., Faramarzi, M. A., & Setayesh, N. (2016). Enhancing activity and thermostability of lipase A from Serratia marcescens by site-directed mutagenesis. Enzyme Microbial Technology, 93-94, 18–28.
Chen, K. C., Zheng, M. M., Pan, J., Li, C. X., & Xu, J. H. (2017). Protein engineering and homologous expression of Serratia marcescens lipase for efficient synthesis of a pharmaceutically relevant epoxyester. Applied Biochemistry and Biotechnology, 183, 543–554.
Li, S. X., Pang, H. Y., Lin, K., Xu, J. H., Zhao, J., & Fan, L. Q. (2011). Refolding, purification and characterization of an organic solvent-tolerant lipase from Serratia marcescens ECU1010. Journal of Molecular Catalysis B: Enzymatic, 71, 171–176.
Su, E., Xu, J. J., & Wu, X. P. (2015). High-level soluble expression of Serratia marcescens H30 lipase in Escherichia coli. Biotechnology and Applied Biochemistry, 62, 79–86.
Hannig, G., & Makrides, S. C. (1998). Strategies for optimizing heterologous protein expression in Escherichia coli. Trends in Biotechnoogy., 16, 54–60.
Horchani, H., Ouertani, S., Gargouri, Y., & Sayari, A. (2009). The N-terminal His-tag and the recombination process affect the biochemical properties of Staphylococcus aureus lipase produced in Escherichia coli. Journal of Molecular Catalysis B: Enzymatic, 61, 194–201.
Fahnert, B., Lilie, H., & Neubauer, P. (2004). Inclusion bodies: formation and utilisation. Advanced in Biochemical Engineering/Biotechnology, 89, 93–142.
Clark, E. D. B. (2001). Protein refolding for industrial processes. Current Opinion in Biotechnology, 12, 202–207.
Crotti, P., Terretti, M., Macchia, F., & Stoppioni, A. (1986). Ring-opening reaction of cis- and trans-2,3-bis (4-metoxy-benzyl)oxirane: competition between assistance by and migration of an aryl group. Journal of Organic Chemistry, 51, 2759–2766.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.
Akatsuka, H., Kawai, E., Omori, K., Komatsubara, S., Shibatani, T., & Tosa, T. (1994). The lipA gene of Serratia marcescens which encodes an extracellular lipase having no N-terminal signal peptide. Journal of Bacteriology, 176(7), 1949–1956.
Bae, H. A., Lee, K. W., & Lee, Y. H. (2006). Enantioselective properties of extracellular lipase from Serratia marcescens ES-2 for kinetic resolution of (S)-flurbiprofen. Journal of Molecular Catalysis B: Enzymatic, 40, 24–29.
Wang, Y., Zhao, J., Xu, J. H., Fan, L. Q., Li, S. X., Zhao, L. L., & Mao, X. B. (2010). Significantly improved expression and biochemical properties of recombinant Serratia marcescens lipase as robust biocatalyst for kinetic resolution of chiral ester. Applied Biochemistry and Biotechnology, 162, 2387–2399.
Kiefhaber, T., Rudolph, R., Kohler, H.-H., & Buchner, J. (1991). Protein aggregation in vitro and in vivo: a quantitative model of the kinetic competition between folding and aggregation. Nature Biotechnology, 9, 825–829.
Satheeshkumar, K. S., & Jayakumar, R. (2002). Sonication induced sheet formation at the air–water interface. Chemical Communications, 19, 2244–2245.
Hawkins, C. L., & Davies, M. J. (2001). Generation and propagation of radical reactions on proteins. Biochimica et Biophysica Acta, 1504(2-3), 196–219.
Upadhyay, A. K., Murmu, A., Singh, A., & Panda, A. K. (2012). Kinetics of inclusion body formation and its correlation with the characteristics of protein aggregates in Escherichia coli. PLoS One, 7, e33951.
Patra, A. K., Mukhopadhyay, R., Mukhija, R., Krishnan, A., Garg, L. C., & Panda, A. K. (2000). Optimization of inclusion body solubilization and renaturation of recombinant human growth hormone from Escherichia coli. Protein Expression and Purification, 18, 182–192.
Vemula, S., Thunuguntla, R., Dedaniya, A., Kokkiligadda, S., Palle, C., & Ronda, S. R. (2015). Improved production and characterization of recombinant human granulocyte colony stimulating factor from E. coli under optimized downstream processes. Protein Expression and Purification, 108, 62–72.
Dyson, M. R., Shadbolt, S. P., Vincent, K. J., Perera, R. L., & McCafferty, J. (2004). Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression. BMC Biotechnology, 4, 32.
Rudolph, R., & Lilie, H. (1996). In vitro folding of inclusion body proteins. The FASEB Journal, 10, 49–56.
Burgess, R. R. (2009). Refolding solubilized inclusion body proteins. Methods in Enzymology, 463, 259–282.
Yamaguchi, S., Yamamoto, E., Mannen, T., & Nagamune, T. (2013). Protein refolding using chemical refolding additives. Biotechnology Journal, 8, 17–31.
Stempfer, G., Höll-Neugebauer, B., & Rudolph, R. (1996). Improved refolding of an immobilized fusion protein. Nature Biotechnology, 14, 329–334.
Buswell, A. M., Ebtinger, M., Vertes, A. A., & Middelberg, A. P. J. (2002). Effect of operating variables on the yield of recombinant trypsinogen for a pulse-fed dilution refolding reactor. Biotechnology and Bioengineering, 77, 435–444.
Kohyama, K., Matsumoto, T., & Imoto, T. (2010). Refolding of an unstable lysozyme by gradient removal of a solubilizer and gradient addition of a stabilizer. Journal of Biochemistry, 147, 427–431.
West, S. M., Chaudhuri, J. B., & Howell, J. A. (1998). Improved protein refolding using hollow-fibre membrane dialysis. Biotechnology and Bioengineering, 57(5), 590–599.
Batas, B., & Chaudhuri, J. B. (1996). Protein refolding at high concentration using size exclusion chromatography. Biotechnology and Bioengineering, 50, 16–23.
Li, J. J., Wang, A. Q., Janson, J. C., Ballagi, A., Chen, J., Liu, Y. D., Ma, G. H., & Su, Z. G. (2009). Immobilized Triton X-100-assisted refolding of green fluorescent protein tobacco etch virus protease fusion protein using β-cyclodextrin as the eluent. Process Biochemistry, 44, 277–282.
Acknowledgments
Thanks are due to Prof. Xu Yi, School of Chemical and Environmental Engineering Shanghai Institute of Technology, for providing the organism Serratia marcescens.
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Yin, YC., Li, HQ. & Wu, XS. Refolding with Simultaneous Purification of Recombinant Serratia marcescens Lipase by One-Step Ultrasonication Process. Appl Biochem Biotechnol 191, 1670–1683 (2020). https://doi.org/10.1007/s12010-019-03172-1
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DOI: https://doi.org/10.1007/s12010-019-03172-1