Abstract
Manufactured steroid compounds have many applications in the pharmaceutical industry. Due to the chemical complexity and chirality of steroids, there is an increasing demand for enzyme-based bioconversion processes to replace those based on chemical synthesis. In this context, thermostability of the involved enzymes is a highly desirable property as both the increased half-life of the enzyme and the enhanced solubility of substrates and products will improve the yield of the reactions. Metagenomic libraries from thermal environments are potential sources of thermostable enzymes of prokaryotic origin, but the number of expected hits could be quite low for enzymes handling substrates such as steroids, rarely found in prokaryotes. An alternative to metagenome screening is the selection of thermostable variants of well-known steroid-processing enzymes. Here we review and detail a protocol for such selection, where error-prone PCR (epPCR) is used to introduce random mutations into a gene to create a variants library for further selection of thermostable variants in the thermophile Thermus thermophilus. The method involves the use of folding interference vectors where the proper folding of the enzyme of interest at high temperature is linked to the folding of a reporter encoding a selectable property such as thermostable resistance to kanamycin, leading to a life-or-death selection of variants of reinforced folding independently of the activity of the enzyme.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
García JL, Uhía I, Galán B (2012) Catabolism and biotechnological applications of cholesterol degrading bacteria. Microb Biotechnol 5:679–699
Plessis-Rosloniec KZD (2011) Steroid transformation by Rhodococcus strains and bacterial cytochrome P450 enzymes. Dissertation, University of Groningen
Fernandes P, Cruz A, Angelova B, Pinheiro HM, Cabral JMS (2003) Microbial conversion of steroid compounds: recent developments. Enzym Microb Technol 32:688–705
Diane W, Stephan R, Wolfgang Z (1997) Application of thermostable enzymes for carbohydrate modification. In: Praznik W, Huber A (eds) Carbohydrates as raw materials IV : proceedings of the fourth international workshop on carbohydrates as organic raw materials. WUV-Universitatverlag, Vienna
Fredrich A, Antrakian G (1996) Keratin degradation by Fervidobacterium pennavorans, a novel thermophilic anaerobic species of the order Thermotogales. Appl Environ Microbiol 62:2875–2882
Leuschner C, Antranikan G (1995) Heat stable enzymes from extremely thermophilic and hyperthermophilic microorganisms. World J Microbiol Biotechnol 11:95–114
Zeikus JG, Vieille C, Savchenko A (1998) Thermozymes: biotechnology and structure-function relationships. Extremophiles 2:179–183
Goetschel R, Bar R (1992) Formation of mixed crystals in microbial conversion of sterols and steroids. Enzym Microb Technol 14:462–469
Fernández-Arrojo L, Guazzaroni ME, López-Cortés N et al (2010) Metagenomic era for biocatalyst identification. Curr Opin Biotechnol 21:725–733
Lorenz P, Liebeton K, Niehaus F, Eck J (2002) Screening for novel enzymes for biocatalytic processes: accessing the metagenome as a resource of novel functional sequence space. Curr Opin Biotechnol 13:572–577
Bastien G, Arnal G, Bozonnet S et al (2013) Mining for hemicellulases in the fungus-growing termite Pseudacanthotermes militaris using functional metagenomics. Biotechnol Biofuels 6:78–92
Rashamuse K, Sanyika W, Ronneburg T et al (2012) A feruloyl esterase derived from a leachate metagenome library. BMB Rep 45:14–19
Chen IC, Lin WD, Hsu SK et al (2009) Isolation and characterization of a novel lysine racemase from a soil metagenomic library. Appl Environ Microbiol 75:5161–5166
Guazzaroni ME, Silva-Rocha R, Ward RJ (2015) Synthetic biology approaches to improve biocatalyst identification in metagenomic library screening. Microb Biotechnol 8:52–64
Kumwenda B, Litthauer D, Bishop OT et al (2013) Analysis of protein thermostability enhancing factors in industrially important thermus bacteria species. Evol Bioinformatics Online 9:327–342
Zhou XX, Wang YB, Pan YJ, Li WF (2008) Differences in amino acids composition and coupling patterns between mesophilic and thermophilic proteins. Amino Acids 34:25–33
Vieille C, Zeikus GJ (1996) Thermozymes: identifying molecular determinants of protein structural and functional stability. Trends Biotechnol 14:183–190
Robinson-Rechavi M, Alibes A, Godzik A (2006) Contribution of electrostatic interactions, compactness and quaternary structure to protein thermostability: lessons from structural genomics of Thermotoga maritima. J Mol Biol 356:547–557
Szilágyi A, Závodszky P (2000) Structural differences between mesophilic, moderately thermophilic and extremely thermophilic protein subunits: results of a comprehensive survey. Structure 8:493–504
Vogt G, Argos P (1997) Protein thermal stability: hydrogen bonds or internal packing? Fold Des 2:S40–S46
Jaenicke R, Bohm G (1998) The stability of proteins in extreme environments. Curr Opin Struct Biol 8:738–748
Jaenicke R (1991) Protein stability and molecular adaptation to extreme conditions. Eur J Biochem 202:715–728
Trivedi S, Gehlot HS, Rao SR (2006) Protein thermostability in archaea and bacteria. Genetics and molecular research. Genet Mol Res 5:816–827
Bornscheuer U, Kazlauskas RJ (2011) Survey of protein engineering strategies. Curr Protoc Protein Sci Chapter 26(Unit26):7
Rubingh DN, Grayling RA (2009) Protein engineering. In: Encyclopedia of life support systems. EOLSS, pp 140–165
Van Rossum T, Kengen SW, Van Der Oost J (2013) Reporter-based screening and selection of enzymes. FEBS J 280:2979–2996
Chautard H, Blas-Galindo E, Menguy T et al (2007) An activity-independent selection system of thermostable protein variants. Nat Methods 4:919–921
Mate DM, Rivera N, Sanchez-Freire E et al (2020) Thermostability enhancement of the Pseudomonas fluorescens esterase I by in vivo folding selection in Thermus thermophilus. Biotechnol Bioeng 117:30–38
Swarts DC, Makarova K, Wang Y et al (2014) The evolutionary journey of Argonaute proteins. Nat Struct Mol Biol 21:743–753
Lasa I, Castón JR, Fernández-Herrero LA et al (1992) Insertional mutagenesis in the extreme thermophilic eubacteria Thermus thermophilus HB8. Mol Microbiol 6:1555–1564
Cirino PC, Mayer KM, Umeno D (2003) Generating mutant libraries using error-prone PCR. Methods Mol Biol 231:3–9
Wilson DS, Keefe AD (2001) Random mutagenesis by PCR. Curr Protoc Mol Biol Chapter 8:Unit8 3
Zhao H, Moore JC, Volkov AA, Arnold FH (1999) Methods for optimizing industrial enzymes by directed evolution. In: Demain AL, Davies JE (eds) Manual of industrial microbiology and biotechnology, 2nd edn. ASM, Washington, DC, pp 597–604
Hanson-Manful P, Patrick WM (2013) Construction and analysis of randomized protein-encoding libraries using error-prone PCR. Methods Mol Biol 996:251–267
Otte KB, Hauer B (2015) Enzyme engineering in the context of novel pathways and products. Curr Opin Biotechnol 35:16–22
Rubin-Pitel SB, Cho CMH, Chen W, Zhao H (2007) Directed evolution tools in bioproduct and bioprocess development. In: Yang ST (ed) Bioprocessing for value-added products from renewable resources. New technologies and applications. Elsevier B.V, pp 49–72
Packer MS, Liu DR (2015) Methods for the directed evolution of proteins. Nat Rev Genet 16:379–394
Widmann M, Pleiss J, Samland AK (2012) Computational tools for rational protein engineering of aldolases. Comput Struct Biotechnol J 2:e201209016
Lee D, Redfern O, Orengo C (2007) Predicting protein function from sequence and structure. Nat Rev Mol Cell Biol 8:995–1005
Sobti M, Mabbutt BC (2013) Rational-based protein engineering: tips and tools. Methods Mol Biol 996:233–250
Khoury GA, Smadbeck J, Kieslich CA et al (2014) Protein folding and de novo protein design for biotechnological applications. Trends Biotechnol 32:99–109
Acknowledgments
This work has been supported by grant RTC-2014-1439-1 from the Spanish Ministry of Economy and Competitiveness. An institutional grant from Fundación Ramón Areces to CBMSO is also acknowledged.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Ribeiro, AL., Sánchez, M., Bosch, S., Berenguer, J., Hidalgo, A. (2023). Stabilization of Enzymes by Using Thermophiles. In: Barreiro, C., Barredo, JL. (eds) Microbial Steroids. Methods in Molecular Biology, vol 2704. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3385-4_19
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3385-4_19
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3384-7
Online ISBN: 978-1-0716-3385-4
eBook Packages: Springer Protocols