Lipolytic activity of Itb1.1 and Lk3 thermostable lipases expressed in Escherichia coli and Pichia pastoris
Published:  Apr 23, 2022DOI: 10.7324/JAPS.2022.120905
Pichia pastoris has been reported as a better expression host compared to Escherichia coli for producing various heterologous proteins. Gene encoding thermostable lipases Itb1.1 and Lk3 were previously expressed in E. coli with low specific activity. In this study, the genes were subcloned successfully into Pichia expression vector pPICZαA and integrated into the genome of P. pastoris GS115. The yeast transformants were grown in buffered methanolcomplex medium with methanol induction to express the proteins. Meanwhile, E. coli BL21 (DE3) recombinants were regenerated in Luria Bertani with isopropyl β-d-1-thiogalactopyranoside induction to express the proteins. Purified proteins were obtained through nickel-nitrilotriacetic acid affinity chromatography. The lipolytic activity assay was conducted at pH 8 and 70°C for Itb1.1 and pH 8 and 50°C for Lk3. The specific activity of Itb1.1 expressed in E. coli cells was 1.2064 U/mg, whereas that of the enzyme expressed in P. pastoris was 7.6836 U/mg. Moreover, the specific activity of Lk3 expressed in E. coli was 0.3523 U/mg, whereas that in P. pastoris was 4.8508 U/mg. Therefore, the specific activity of Itb1.1 and Lk3 expressed in P. pastoris was 6 and 14 times higher than that expressed in E. coli, respectively. The data suggested that P. pastoris is a better host for the expression of Itb1.1 and Lk3 proteins.
Simatupang DF, Widhiastuty MP, Madayanti F, Akhmaloka A. Lipolytic activity of Itb1.1 and Lk3 thermostable lipases expressed in Escherichia coli and Pichia pastoris. J Appl Pharm Sci, 2022. doi: https://doi.org/10.7324/JAPS.2022.120905
Ahmad M, Hirz M, Pichler H, Schwab H. Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Appl Microbiol Biotechnol, 2014; 98(12):5301-17. https://doi.org/10.1007/s00253-014-5732-5
Bornscheuer U, Reif OW, Lausch R, Freitag R, Scheper T, Kolisis FN, Menge U. Lipase of Pseudomonas cepacia for biotechnological purposes: purification, crystallization and characterization. Biochim Biophys Acta, 1994; 1201(1):55-60. https://doi.org/10.1016/0304-4165(94)90151-1
Brilliantoro R, Zidny R, Widhiastuty MP, Akhmaloka A. Hydrolytic and transesterification activities of thermostable lipase ITB1.1. Biosci Biotechnol Res Asia, 2015; 12(1):1-6. https://doi.org/10.13005/bbra/1628
Cereghino JL, Cregg JM. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev, 2000; 24(1):45-66. https://doi.org/10.1111/j.1574-6976.2000.tb00532.x
Contesini FJ, Davanço MG, Borin GP, Vanegas KG, Cirino JPG, de Melo RR, Mortensen UH, Hildén K, Campos DR, Carvalho PDO. Advances in recombinant lipases: application in the pharmaceutical industry. Catalysts, 2020; 10:1-33. https://doi.org/10.3390/catal10091032
Fickers P. Pichia pastoris: a workhorse for recombinant protein production. Curr Res Microbiol Biotechnol, 2014; 2(3):354-63.
Furqan BRN, Akhmaloka A. Heterologous expression and characterization of thermostable lipase (Lk1) in Pichia pastoris GS115. Biocatal Agric Biotechnol, 2020; 23:101448. https://doi.org/10.1016/j.bcab.2019.101448
Gasser B, Prielhofer R, Marx H, Maurer M, Nocon J, Steiger M, Puxbaum V, Sauer M, Mattanovich D. Pichia pastoris: protein production host and model organism for biomedical research. Future Microbiol, 2013; 8(2):191-208. https://doi.org/10.2217/fmb.12.133
Govindappa N, Hanumanthappa M, Venkatarangaiah K, Periyasamy S, Sreenivas S, Soni R, Sastry K. A new signal sequence for recombinant protein secretion in Pichia pastoris. J Microbiol Biotechnol, 2014; 24(3):337-45. https://doi.org/10.4014/jmb.1308.08085
Hasan F, Shah AA, Hameed A. Industrial applications of microbial lipases. Enzyme Microb Technol, 2006; 39(2):235-51. https://doi.org/10.1016/j.enzmictec.2005.10.016
Invitrogen Life Technologies. User manual - EasySelectTM Pichia Expression Kit. Invitrogen Life Technologies, Carlsbad, CA, 2010.
Korona B, Korona D, Bielecki S. Efficient expression and secretion of two co-produced xylanases from Aspergillus niger in Pichia pastoris directed by their native signal peptides and the Saccharomyces cerevisiae α-mating factor. Enzyme Microb Technol, 2006; 39(4):683-9. https://doi.org/10.1016/j.enzmictec.2005.12.003
Lan D, Qu M, Yang B, Wang Y. Enhancing production of lipase MAS1 frommarine Streptomyces sp. strain in Pichia pastoris by chaperones co-expression. Electron J Biotechnol, 2016; 22:62-7. https://doi.org/10.1016/j.ejbt.2016.06.003
Li H, Zhang X. Characterization of thermostable lipase from thermophilic Geobacillus sp. TW1. Protein Expr Purif, 2005; 42(1):153-9. https://doi.org/10.1016/j.pep.2005.03.011
Maciver B, McHale RH, Saul DJ, Bergquist PL. Cloning and sequencing of a serine proteinase gene from a thermophilic Bacillus species and its expression in Escherichia coli. Appl Environ Microbiol, 1994; 60(11):3981-8. https://doi.org/10.1128/aem.60.11.3981-3988.1994
Nurhasanah, Nurbaiti S, Madayanti F, Akhmaloka A. Heterologous expression of gene encoded thermostable lipase and lipolytic activity. J Pure Appl Microbiol, 2017; 11(1):135-9. https://doi.org/10.22207/JPAM.11.1.18
Nurhasanah, Nurbaiti S, Warganegara FM, Akhmaloka A. Diversity of gene encoding thermostable lipase from compost based on metagenome analysis. Int J Integr Biol, 2015; 16(1):7-12.
Ol?dzka G, Da? browski S, Kur J. High-level expression, secretion, and purification of the thermostable aqualysin I from Thermus aquaticus YT-1 in Pichia pastoris. Protein Expr Purif, 2003; 29(2):223-9. https://doi.org/10.1016/S1046-5928(03)00060-3
Oslan SN, Salleh AB, Rahman RNZRAR, Leow TC, Basri M. Pichia pastoris as a host to overexpress the thermostable T1 lipase from Geobacillus zalihae. GSTF Int J Biosci, 2013; 3(1):7-13. https://doi.org/10.5176/2251-3140_3.1.45
Permana AH, Warganegara FM, Wahyuningrum D, Widhiastuty MP, Akhmaloka A. Heterologous expression and characterization of thermostable lipases from Geobacillus thermoleovorans PPD2 through Escherichia coli. Biosci Biotechnol Res Asia, 2017; 14(3):1081-8. https://doi.org/10.13005/bbra/2545
Quyen DT, Schmidt-Dannert C, Schmid RD. High-level expression of a lipase from Bacillus thermocatenulatus BTL2 in Pichia pastoris and some properties of the recombinant lipase. Protein Expr Purif, 2003; 28(1):102-10. https://doi.org/10.1016/S1046-5928(02)00679-4
Robert JM, Lattari FS, Machado AC, de Castro AM, Almeida RV, Torres FAG, Valero F, Freire DM. Production of recombinant lipase B from Candida antarctica in Pichia pastoris under control of the promoter PGK using crude glycerol from biodiesel production as carbon source. Biochem Eng J, 2017; 118:123-31. https://doi.org/10.1016/j.bej.2016.11.018
Sabri S, Rahman RNZRA, Leow TC, Basri M, Salleh AB. Secretory expression and characterization of a highly Ca2+-activated thermostable L2 lipase. Protein Expr Purif, 2009; 68(2):161-6. https://doi.org/10.1016/j.pep.2009.08.002
Soliman NA, Knoll M, Abdel-Fattah YR, Schmid RD, Lange S. Molecular cloning and characterization of thermostable esterase and lipase from Geobacillus thermoleovorans YN isolated from desert soil in Egypt. Process Biochem, 2007; 42(7):1090-100. https://doi.org/10.1016/j.procbio.2007.05.005
Widhiastuty MP, Febriani, Moeis MR, Akhmaloka, Madayanti F. Cloning, homological analysis and expression of lipase gene from hot spring isolate. Int J Integr Biol, 2011; 11(1):8-13.
Yamada R, Kimoto Y, Ogino H. Combinatorial library strategy for strong overexpression of the lipase from Geobacillus thermocatenulatus on the cell surface of yeast Pichia pastoris. Biochem Eng J, 2016; 113:7-11. https://doi.org/10.1016/j.bej.2016.05.005
Yang J, Zhang B, Yan Y. Cloning and expression of pseudomonas fluorescens 26-2 lipase gene in Pichia pastoris and characterizing for transesterification. Appl Biochem Biotechnol, 2009; 159(2):355-65. https://doi.org/10.1007/s12010-008-8419-5
Yurimoto H, Oku M, Sakai Y. Yeast methylotrophy: metabolism, gene regulation and peroxisome homeostasis. Int J Microbiol, 2011; 2011:1-8. https://doi.org/10.1155/2011/101298
4 Absract views 0 PDF Downloads 4 Total views