BMC Biotechnology | |
High level production of tyrosinase in recombinant Escherichia coli | |
Qun Ren1  Bernhard Henes1  Michael Fairhead1  Linda Thöny-Meyer1  | |
[1] Laboratory for Biomaterials, Swiss Federal Laboratories for Materials Science and Technology (Empa), Lerchenfeldstrasse 5, CH-9014 St, Gallen, Switzerland | |
关键词: Exponential feeding; Bioprocess engineering; Fed batch culture; High cell density; Recombinant protein production; Tyrosinase; | |
Others : 1123236 DOI : 10.1186/1472-6750-13-18 |
|
received in 2012-09-24, accepted in 2013-02-20, 发布年份 2013 | |
【 摘 要 】
Background
Tyrosinase is a bifunctional enzyme that catalyzes both the hydroxylation of monophenols to o-diphenols (monophenolase activity) and the subsequent oxidation of the diphenols to o-quinones (diphenolase activity). Due to the potential applications of tyrosinase in biotechnology, in particular in biocatalysis and for biosensors, it is desirable to develop a suitable low-cost process for efficient production of this enzyme. So far, the best production yield reported for tyrosinase was about 1 g L-1, which was achieved by cultivating the filamentous fungus Trichoderma reesei for 6 days.
Results
In this work, tyrosinase from Verrucomicrobium spinosum was expressed in Escherichia coli and its production was studied in both batch and fed-batch cultivations. Effects of various key cultivation parameters on tyrosinase production were first examined in batch cultures to identify optimal conditions. It was found that a culture temperature of 32 °C and induction at the late growth stage were favorable, leading to a highest tyrosinase activity of 0.76 U mL-1. The fed-batch process was performed by using an exponential feeding strategy to achieve high cell density. With the fed-batch process, a final biomass concentration of 37 g L-1 (based on optical density) and a tyrosinase activity of 13 U mL-1 were obtained in 28 hours, leading to a yield of active tyrosinase of about 3 g L-1. The highest overall volumetric productivity of 103 mg of active tyrosinase per liter and hour (corresponding to 464 mU L-1 h-1) was determined, which is approximately 15 times higher than that obtained in batch cultures.
Conclusions
We have successfully expressed and produced gram quantities per liter of active tyrosinase in recombinant E. coli by optimizing the expression conditions and fed-batch cultivation strategy. Exponential feed of substrate helped to prolong the exponential phase of growth, to reduce the fermentation time and thus the cost. A specific tyrosinase production rate of 103 mg L−1 h−1 and a maximum volumetric activity of 464 mU L−1 h-1 were achieved in this study. These levels have not been reported previously.
【 授权许可】
2013 Ren et al; licensee BioMed Central Ltd.
【 预 览 】
Files | Size | Format | View |
---|---|---|---|
20150216021101629.pdf | 729KB | download | |
Figure 4. | 64KB | Image | download |
Figure 3. | 49KB | Image | download |
Figure 2. | 30KB | Image | download |
Figure 1. | 29KB | Image | download |
【 图 表 】
Figure 1.
Figure 2.
Figure 3.
Figure 4.
【 参考文献 】
- [1]Fairhead M, Thöny-Meyer L: Bacterial tyrosinases: old enzymes with new relevance to biotechnology. New Biotechnol 2012, 29(2):183-191.
- [2]Claus H, Decker H: Bacterial tyrosinases. Syst Appl Microbiol 2006, 29(1):3-14.
- [3]del Marmol V, Beermann F: Tyrosinase and related proteins in mammalian pigmentation. FEBS Lett 1996, 381(3):165-168.
- [4]Halaouli S, Asther M, Sigoillot JC, Hamdi M, Lomascolo A: Fungal tyrosinases: new prospects in molecular characteristics, bioengineering and biotechnological applications. J Appl Microbiol 2006, 100(2):219-232.
- [5]Marusek CM, Trobaugh NM, Flurkey WH, Inlow JK: Comparative analysis of polyphenol oxidase from plant and fungal species. J Inorg Biochem 2006, 100(1):108-123.
- [6]Mayer AM: Polyphenol oxidases in plants and fungi: Going places? A review. Phytochemistry 2006, 67(21):2318-2331.
- [7]Walker JRL, Ferrar PH: Diphenol oxidases, enzyme-catalysed browning and plant disease resistance. Biotechnol Genet Eng Rev 1998, 15:457-498. Edited by Tombs MP
- [8]Jus S, Kokol V, Guebitz GM: Tyrosinase-catalysed coupling of functional molecules onto protein fibres. Enzyme Microb Technol 2008, 42(7):535-542.
- [9]Jus S, Kokol V, Guebitz GM: Tyrosinase-catalysed coating of wool fibres with different protein-based biomaterials. J Biomat Sci-Polym E 2009, 20(2):253-269.
- [10]Martorell MM, Pajot HF, Rovati JI, Figueroa LIC: Optimization of culture medium composition for manganese peroxidase and tyrosinase production during Reactive Black 5 decolourization by the yeast Trichosporon akiyoshidainum. Yeast 2012, 29(3–4):137-144.
- [11]Seetharam G, Saville BA: L-DOPA production from tyrosinase immobilized on zeolite. Enzyme Microb Technol 2002, 31(6):747-753.
- [12]Lantto R, Puolanne E, Kruus K, Buchert J, Autio K: Tyrosinase-aided protein cross-linking: Effects on gel formation of chicken breast myofibrils and texture and water-holding of chicken breast meat homogenate gels. J Agric Food Chem 2007, 55(4):1248-1255.
- [13]Selinheimo E, Autio K, Krijus K, Buchert J: Elucidating the mechanism of laccase and tyrosinase in wheat bread making. J Agric Food Chem 2007, 55(15):6357-6365.
- [14]Anghileri A, Lantto R, Kruus K, Arosio C, Freddi G: Tyrosinase-catalyzed grafting of sericin peptides onto chitosan and production of protein-polysaccharide bioconjugates. J Biotechnol 2007, 127(3):508-519.
- [15]Freddi G, Anghileri A, Sampaio S, Buchert J, Monti P, Taddei P: Tyrosinase-catalyzed modification of Bombyx mori silk fibroin: Grafting of chitosan under heterogeneous reaction conditions. J Biotechnol 2006, 125(2):281-294.
- [16]Gu BX, Xu CX, Zhu GP, Liu SQ, Chen LY, Li XS: Tyrosinase immobilization on ZnO Nanorods for phenol detection. J Phys Chem B 2009, 113(1):377-381.
- [17]Fairhead M, Thöny-Meyer L: Cross-linking and immobilisation of different proteins with recombinant Verrucomicrobium spinosum tyrosinase. J Biotechnol 2010, 150(4):546-551.
- [18]Lewandowski AT, Small DA, Chen TH, Payne GF, Bentley WE: Tyrosine-based “activatable pro-tag”: Enzyme-catalyzed protein capture and release. Biotechnol Bioeng 2006, 93(6):1207-1215.
- [19]Thalmann CR, Lotzbeyer T: Enzymatic cross-linking of proteins with tyrosinase. Eur Food Res Technol 2002, 214(4):276-281.
- [20]Monogioudi E, Creusot N, Kruus K, Gruppen H, Buchert J, Mattinen M-L: Cross-linking of beta-casein by Trichoderma reesei tyrosinase and Streptoverticillium mobaraense transglutaminase followed by SEC-MALLS. Food Hydrocolloid 2009, 23(7):2008-2015.
- [21]Jus S, Stachel I, Schloegl W, Pretzler M, Friess W, Meyer M, Birner-Gruenberger R, Guebitz GM: Cross-linking of collagen with laccases and tyrosinases. Mater Sci Eng C-Mater Biol Appl 2011, 31(5):1068-1077.
- [22]Halaouli S, Asther M, Kruus K, Guo L, Hamdi M, Sigoillot JC, Lomascolo A: Characterization of a new tyrosinase from Pycnoporus species with high potential for food technological applications. J Appl Microbiol 2005, 98(2):332-343.
- [23]Hearing VJ: Mammalian monophenol monooxygenase (tyrosinase) - purification, properties, and reactions catalyzed. Methods Enzymol 1987, 142:154-165.
- [24]Lerch KLE: Purification and characterization of a tyrosinase from Streptomyces glaucescens. Eur J Biochem 1972, 31(3):427-437.
- [25]Gasparetti C, Faccio G, Arvas M, Buchert J, Saloheimo M, Kruus K: Discovery of a new tyrosinase-like enzyme family lacking a C-terminally processed domain: production and characterization of an Aspergillus oryzae catechol oxidase. Appl Microbiol Biotechnol 2010, 86(1):213-226.
- [26]Ito M, Inouye K: Catalytic properties of an organic solvent-resistant tyrosinase from Streptomyces sp REN-21 and its high-level production in E. coli. J Biochem 2005, 138(4):355-362.
- [27]Liu N, Zhang T, Wang YJ, Huang YP, Ou JH, Shen P: A heat inducible tyrosinase with distinct properties from Bacillus thuringiensis. Lett Appl Microbiol 2004, 39(5):407-412.
- [28]Shuster V, Fishman A: Isolation, cloning and characterization of a tyrosinase with improved activity in organic solvents from Bacillus megaterium. J Mol Microbiol Biotechnol 2009, 17(4):188-200.
- [29]Flurkey A, Cooksey J, Reddy A, Spoonmore K, Rescigno A, Inlow J, Flurkey WH: Enzyme, protein, carbohydrate, and phenolic contaminants in commercial tyrosinase preparations: Potential problems affecting tyrosinase activity and inhibition studies. J Agric Food Chem 2008, 56(12):4760-4768.
- [30]Sambasiva Rao KRS, Tripathy NK, Mahalaxmi Y, Prakasham RS: Laccase- and peroxidase-free tyrosinase production by isolated microbial strain. J Microbiol Biotechnol 2012, 22(2):207-214.
- [31]Kumar M, Flurkey WH: Activity, isoenzymes and purity of mushroom tyrosinase in commercial preparations. Phytochemistry 1991, 30(12):3899-3902.
- [32]Chen G-H, Chen W-M, Huang Y-C, Jiang S-T: Expression of recombinant mature human tyrosinase from Escherichia coli and exhibition of its activity without phosphorylation or glycosylation. J Agric Food Chem 2012, 60(11):2838-2843.
- [33]Kong KH, Park SY, Hong MP, Cho SH: Expression and characterization of human tyrosinase from a bacterial expression system. Comp Biochem Physiol B Biochem Mol Biol 2000, 125(4):563-569.
- [34]Halaouli S, Record E, Casalot L, Hamdi M, Sigoillot JC, Asther M, Lomascolo A: Cloning and characterization of a tyrosinase gene from the white-rot fungus Pycnoporus sanguineus, and overproduction of the recombinant protein in Aspergillus niger. Appl Microbiol Biotechnol 2006, 70(5):580-589.
- [35]Kohashi PY, Kumagai T, Matoba Y, Yamamoto A, Maruyama M, Sugiyama M: An efficient method for the overexpression and purification of active tyrosinase from Streptomyces castaneoglobisporus. Protein Expr Purif 2004, 34(2):202-207.
- [36]Selinheimo E, Saloheimo M, Ahola E, Westerholm-Parvinen A, Kalkkinen N, Buchert J, Kruus K: Production and characterization of a secreted, C-terminally processed tyrosinase from the filamentous fungus Trichoderma reesei. FEBS J 2006, 273(18):4322-4335.
- [37]Westerholm-Parvinen A, Selinheimo E, Boer H, Kalkkinen N, Mattinen M, Saloheimo M: Expression of the Trichoderma reesei tyrosinase 2 in Pichia pastoris: Isotopic labeling and physicochemical characterization. Protein Expr Purif 2007, 55(1):147-158.
- [38]Fairhead M, Thöny-Meyer L: Role of the C-terminal extension in a bacterial tyrosinase. FEBS J 2010, 277(9):2083-2095.
- [39]Riesenberg D, Guthke R: High-cell-density cultivation of microorganisms. Appl Microbiol Biotechnol 1999, 51(4):422-430.
- [40]Shiloach J, Fass R: Growing E. coli to high cell density - A historical perspective on method development. Biotechnol Adv 2005, 23(5):345-357.
- [41]Makrides SC: Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Mol Biol Rev 1996, 60(3):512.
- [42]Shojaosadati SA, Kolaei SMV, Babaeipour V, Farnoud AM: Recent advances in high cell density cultivation for production of recombinant protein. Iranian J Biotechnol 2008, 6:63-84.
- [43]Hoffmann F, Weber J, Rinas U: Metabolic adaptation of Escherichia coli during temperature-induced recombinant protein production: 1. Readjustment of metabolic enzyme synthesis. Biotechnol Bioeng 2002, 80(3):313-319.
- [44]Jana S, Deb JK: Strategies for efficient production of heterologous proteins in Escherichia coli. Appl Microbiol Biotechnol 2005, 67(3):289-298.
- [45]Castrillo JI, Kaliterna J, Weusthuis RA, VanDijken JP, Pronk JT: High-cell-density cultivation of yeasts on disaccharides in oxygen-limited batch cultures. Biotechnol Bioeng 1996, 49(6):621-628.
- [46]Domingues L, Lima N, Teixeira JA: Contamination of a high-cell-density continuous bioreactor. Biotechnol Bioeng 2000, 68(5):584-587.
- [47]Lee SY: High cell-density culture of Escherichia coli. Trends Biotechnol 1996, 14(3):98-105.
- [48]Sanden AM, Prytz I, Tubulekas I, Forberg C, Le H, Hektor A, Neubauer P, Pragai Z, Harwood C, Ward A: Limiting factors in Escherichia coli fed-batch production of recombinant proteins. Biotechnol Bioeng 2003, 81(2):158-166.
- [49]Shokri A, Sanden AM, Larsson G: Growth rate-dependent changes in Escherichia coli membrane structure and protein leakage. Appl Microbiol Biotechnol 2002, 58(3):386-392.
- [50]Shokri A, Sanden AM, Larsson G: Cell and process design for targeting of recombinant protein into the culture medium of Escherichia coli. Appl Microbiol Biotechnol 2003, 60(6):654-664.
- [51]Gardner AR, Cadman TW: Product deactivation in recombinant streptomyces. Biotechnol Bioeng 1990, 36(3):243-251.
- [52]Han K, Hong J, Lim HC, Kim CH, Park Y, Cho JM: Tyrosinase production in recombinant E. coli containing trp promoter and ubiquitin sequence. Ann NY Acad Sci 1994, 721:30-42.
- [53]Sambrook J, Russel DW: Molecular Cloning: A Laboratory Manual. 3rd edition. New York: Cold Spring Harbor Laboratory Press; 2001.
- [54]Fling M, Horowitz NH, Heinemann SF: The isolation and properties of crystalline tyrosinase from Neurospora. J Biol Chem 1963, 238:2045-2053.
- [55]Laemmli UK: Cleavage of structural proteins during assembly of head of bacteriophage-T4. Nature 1970, 227(5259):680.