High level production of tyrosinase in recombinant Escherichia coli
© Ren et al.; licensee BioMed Central Ltd. 2013
Received: 24 September 2012
Accepted: 20 February 2013
Published: 27 February 2013
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.
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.
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.
KeywordsTyrosinase Recombinant protein production High cell density Fed batch culture Bioprocess engineering Exponential feeding
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) . Tyrosinase is essential for many living organisms to carry out various functions, including melanin biosynthesis as defense against the harmful effects of UV light [2–4]. In plants, it is required for the biosynthesis of phenolic polymers such as lignin, flavinoids, and tannins . Tyrosinases also play an important role in the regulation of the oxidation-reduction potential of cell respiration and in wound healing in plants [6, 7].
Due to the ability of tyrosinases to react with phenols, these enzymes have been proposed for the uses in a variety of biotechnological, biosensor and biocatalysis applications [1, 8, 9]. For example, tyrosinases can be applied in detoxification of phenol-containing wastewater and contaminant soils , synthesis of L-3,4-dihydroxyphenylalanine (L-DOPA), one of the preferred drugs for the treatment of Parkinson‘s disease , or as additives in food processes due to their cross-linking abilities [12, 13]. Tailoring polymers, e.g. grafting of silk proteins onto chitosan via tyrosinase reactions have also been reported [14, 15]. Immobilized tyrosinase has been investigated as an electrochemical biosensor for a range of phenolic compounds . The enzyme can react with exposed tyrosyl side chains in polypeptides, and the reactive quinones formed allow for protein-protein cross-linking [17–21].
Tyrosinases have been isolated and purified from various sources such as animals, plants, fungi, and bacteria [2, 22–25]. The commercial production of tyrosinase is mostly reported from the common mushroom Agaricus bisporus. Extensive research has been carried out by using this mushroom tyrosinase because of its commercial availability. However, the use of tyrosinase from this source is problematic as the enzyme exhibits relatively low solvent and temperature stability, as compared to some bacterial tyrosinases [26–28]. Moreover, commercial tyrosinases are typically contaminated with other enzymes for example different isoforms, resulting in preparations of variable quality and activity . Almost all reported tyrosinase-producing microbial (both fungal and bacterial) strains also produce other polyphenol oxidases such as peroxidase and laccase . The presence of laccase and peroxidase along with tyrosinase imposes serious problems for commercial usage . All these enzymes can use tyrosine as substrate but produce different products, resulting in reduced yield as well as increased cost for the downstream process. Recently, it was reported that a new Actinomycetes isolate produced only tyrosinase and did not exhibit peroxidase or laccase activities . The production yield of tyrosinase was about 4.8 U mL-1 after 48 h incubation, which is still far too low to be applied for commercial purposes.
The recombinant production of tyrosinases becomes an attractive alternative to obtain large amounts of protein. Recombinant strains offer the possibility of a higher protein production level, better growth, and consequently, an improved productivity when compared to non-recombinant production systems. However, tyrosinases appear to be difficult to express in recombinant hosts and only a few examples have been reported. The human tyrosinase has been expressed mostly as insoluble protein in inclusion bodies in Escherichia coli[32, 33]. Tyrosinase from Pycnoporus sanguineus was expressed in Aspergillus niger, and the Streptomyces castaneoglobisporus tyrosinase was expressed in a complex with its “caddy” protein, ORF378, in E. coli. Tyrosinase production has also been reported for the filamentous fungus Trichoderma reesei. Overexpression of the tyrosinase gene allowed the native host T. reesei to produce about 1 g L-1 tyrosinase in laboratory-scale batch fermentation after 6 days of cultivation . Since growth of filamentous fungi is slow compared with most single-cell microorganisms, Pichia pastoris carrying the tyrosinase gene of T. reesei was tested and yielded 24 mg L-1 active recombinant tyrosinase in 3 days . The E. coli recombinant was also used as host to produce tyrosinase from Streptomyces sp. REN-21, and 54 mg L-1 of the enzyme were obtained in the cytoplasm after 16 h incubation . Recently, we have overexpressed and characterized the tyrosinase from Verrucomicrobium spinosum in E. coli. However, even though the yield of this enzyme (150 mg L-1) was higher than those reported from other bacterial tyrosinases (unpublished data), we considered this yield could be further improved.
E. coli is commonly used as host for the rapid and economical production of recombinant proteins. Different cultivation techniques have been developed to increase the final biomass concentration [39, 40]. However, high-level production of functional proteins in E. coli may not be a routine matter and is sometimes challenging. Not every protein can be produced efficiently due to the unique structural features of the protein, its folding pathways and its degradation by host cell proteases . Since useful recombinant proteins have to be biologically active, the objectives for production should include not only maximization of the amount of recombinant protein, but also of total enzyme activity.
The current work is based on the successful cloning of the tyrosinase gene from V. spinosum in E. coli and its purification and characterization . The objective of this work was to develop a suitable strategy for efficient production of active tyrosinase in E. coli. The effect of temperature, inducer isopropyl-beta-D-thiogalactopyranosides (IPTG) concentrations and the starting time of induction in different operation modes (batch and fed-batch) were investigated. About 3 g L-1 active tyrosinase were obtained after 28 hours of incubation under the developed conditions. To our knowledge, this is the best yield and productivity ever reported for recombinant tyrosinase.
Results and discussion
Production of tyrosinase in batch culture
In order to establish an efficient production process for tyrosinase, preliminary optimization studies were performed in shake flasks. This allowed the selection of the best production host, growth temperature, time for induction and inducer concentration by using conventional methodology with changing one variable at a time.
Influence of different host strains on tyrosinase production
Comparison of different E. coli hosts for tyrosinase production
Max. act. (U mL-1) d
0.26 ± 0.04
7.7 ± 0.4
0.44 ± 0.05
DH5α + IPTG
0.27 ± 0.03
8.0 ± 0.3
0.44 ± 0.01
0.60 ± 0.01
8.9 ± 0.4
0.03 ± 0.00
BLR + IPTG
0.57 ± 0.02
7.5 ± 0.6
0.03 ± 0.00
0.60 ± 0.02
7.4 ± 0.3
0.03 ± 0.00
JM109 + IPTG
0.59 ± 0.03
7.7 ± 0.4
0.07 ± 0.01
Influence of oxygen supply on tyrosinase production
Influence of induction stage on tyrosinase production
The influence of different concentrations of IPTG on cell growth and tyrosinase production was first tested. It was found that the biomass, the growth rate and the total tyrosinase activities were not influenced significantly by the tested IPTG concentrations between 0.1 mM and 1.0 mM (data not shown).
Comparison of induction stages for tyrosinase production
Growth stage a
Max. act. (U mL-1) d
0.39 ± 0.03
7.2 ± 0.5
0.05 ± 0.01
0.57 ± 0.02
8.4 ± 0.5
0.47 ± 0.07
0.57 ± 0.03
8.9 ± 1.0
0.76 ± 0.06
Investigation of the induction stage is an important parameter for the development of the optimized protein production, especially when a strong promoter system is used, which was the case in this study. Induction of a strong promoter often leads to a sudden burst of protein synthesis which may inhibit cell growth due to a severe metabolic burden . This can be well demonstrated by the results obtained here: early induction led to a reduced growth rate (Table 2).
Influence of temperature on tyrosinase production
Influence of culture temperature on tyrosinase production
Max. act. (U mL-1) c
0.31 ± 0.02
7.8 ± 0.9
0.12 ± 0.03
0.52 ± 0.02
8.0 ± 0.7
0.22 ± 0.03
0.60 ± 0.03
10.2 ± 0.9
0.76 ± 0.06
0.66 ± 0.02
10.8 ± 1.0
0.36 ± 0.05
Tyrosinase production using fed-batch cultivation
Different processes focusing on nutrient feeding strategies have been developed to grow cells to high cell densities and to overproduce proteins . The most important function of such strategies is to prevent overfeeding, as inhibitory concentrations of the feed components can accumulate in the fermenter, or underfeeding, by which the organism is starved for essential nutrients. The method of choice depends on many different factors, including the metabolism of the organism, the potential for production of inhibitory substrates and induction conditions. Batch , continuous , and a variety of fed-batch processes [39, 47] have been reported for growing cells to high densities. Among these, fed-batch is the most commonly used method to produce recombinant proteins .
The optimal setting of the value for the feeding rate plays an important role in obtaining long-lasting fed-batch growth of E. coli cells. The feeding rate should maintain optimum cellular health, which is required to overcome the metabolic stress associated with recombinant protein expression . A too high feeding rate would lead to accumulation of glycerol very early during the fed-batch, a premature slowdown of growth and, therefore, a short cultivation time. A too low feeding rate may lead to prolonged cultivation time before reaching high cell density, thus resulting in a low process productivity. It has been reported that a specific growth rate of 0.3 h−1 prevents several negative effects, such as increased cell lysis, higher levels of endotoxin accumulation and membrane stiffness, which are characteristics of cells at low specific growth rates [49, 50]. An exponential feeding strategy to maintain a specific growth rate of 0.3 h−1 was therefore used in this study. A feeding rate of 0.4 h−1 was also tested and resulted in overfeeding and foaming problems (data not shown).
Comparison of tyrosinase production in batch and fed-batch cultures
Cultivation time (h)
Vol. activity of tyrosinase (U mL-1)
Yield of tyrosinase (g L-1)
Vol. productivity of tyrosinase (mg L-1 h-1)
Vol. act. of tyrosinase per time (mU mL-1 h-1)
Shake flask (Batch)
10.0 ± 0.9
0.76 ± 0.06
0.17 ± 0.01
7.1 ± 0.4
31.7 ± 2.5
102.0 ± 2.0
13.00 ± 2.18
2.89 ± 0.48
103.2 ± 17.1
464.3 ± 77.8
No significant change in activity was observed even 12 h after the highest cell density in the fed batch experiment was reached (Figure 3B). Previously it has been observed that loss of tyrosinase activity due to proteolytic activity in recombinant Streptomyces was detected during all phases of batch culture, especially in stationary phase . When the tyrosinase of S. antibioticus was expressed in E. coli, the activity of intracellular tyrosinase decreased with time . The results obtained in this study suggest that V. spinosum tyrosinase produced in E. coli is stable, at least during the 40 hour cultivation tested here. This will undoubtedly simplify the purification of this enzyme.
Stability of tyrosinase expression
Summary of the optimized process used in this study for tyrosinase production
Minimal medium containing glycerol and NZ-amine
Dissolved oxygen (%)
Fed-batch culture with exponential feeding
Glycerol and NZ-amine
Feeding rate (h-1)
At OD600 value of 30-40
End of the exponential growth
Since tyrosinase has potential for a broad range of applications, it is expected that an efficient production process will facilitate its actual usage. This work demonstrated an effective process of a bacterial tyrosinase production in the laboratory scale. Further improvement such as in the yield of active enzyme and validation of the scalability of the process are still needed.
Bacterial strains and plasmid
E. coli DH5α (E. coli Genetic Stock Center), BLR (Novagen) and JM109 (New England BioLabs) were tested as hosts for tyrosinase production. The plasmid pMFvpt, which contains the gene encoding the cytoplasmic full-length tyrosinase (53.5 kDa) of V. spinosum, was used to produce the recombinant tyrosinase .
All chemicals used were purchased from Sigma–Aldrich (Buchs Switzerland) unless otherwise stated.
Luria broth (LB), 5 g yeast extract, 10 g tryptone, and 5 g NaCl per liter, was used for pre-inoculum cultures. It was supplemented with ampicillin to a final concentration of 0.1 mg mL-1.
The medium for batch cultures in shake flasks contained (g L-1): glycerol 10, NZ-amine 5, (NH4)2HPO4 4, KH2PO4 5, K2HPO4 7.4, MgSO4 * 7H2O 1.2, thiamine HCl 0.015, ampicillin 0.1 and 10 mL of trace element solution (TES). TES contained (g l-1): CaCl2 * 2H2O 5, FeCl3*4H2O 7, Zn(CH3COO)2 * 2H2O 1.3, MnCl2 * 4H2O 1.5, CoCl2 * 6H2O 0.25, H3BO3 0.3, Na2MoO4 * 2H2O 0.25, ethylenediaminetetraacetic acid (EDTA) 1.25 and 10 mL of concentrated HCl. Cells were induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) under the conditions described in the Results section. Thiamine, ampicillin, TES and IPTG were filter-sterilized (0.2 μm, Millipore).
The medium for batch cultures in the bioreactor contained (g L-1): glycerol 20, NZ-Amine 5, (NH4)2HPO4 4, KH2PO4 13.3, (NH4)2SO4 1, MgSO4 * 7H2O 1.2, thiamine HCl 0.015, ampicillin 0.1 and 10 ml of TES containing additional 0.15 g l-1 CuCl2 * 4H2O. The feed medium contained (g L-1): glycerol 500, NZ-Amine 100, MgSO4 * 7H2O 13.5 and (NH4)2SO4 50.
Plasmid pMFvpt was transformed into E. coli competent cells by chemical CaCl2 method . The freshly transformed cells were used to inoculate a 10 mL LB pre-culture in a 50 mL flask. The cells were incubated at 37°C and 150 rpm overnight. The pre-culture was then used to inoculate 200 mL of batch medium in a 1 L shake flask with a dilution of 1:20 (v/v). It was incubated at 150 rpm and different temperatures as described in the Results section. Growth and product formation were monitored by periodically taking samples, from which cells were harvested by centrifugation at 12’000 g for 2 min at 4°C. A final OD600 of 7–8 was routinely achieved.
For the experiment of testing oxygen influence, 1 L baffled (with 4 baffles indented into the base, Schott Duran) and non-baffled Erlenmeyer flasks (Schott Duran) were used.
For bioreactor experiments, the cells freshly transformed with plasmid pMFvpt were grown in shake flasks in LB medium for 12 h at 37°C and 150 rpm. The bioreactors were inoculated with a 1:20 (v/v) dilution of the pre-culture. The cells were grown in a 600 mL (total volume 1.4 L) computer controlled bioreactor (Infors AG, Bottmingen, CH) equipped with standard control units. Tyrosinase expression was induced by 1.25 mM IPTG. The pH was maintained at 6.90 ± 0.05 by proportional–integral (PI) controlled addition of 4 M NaOH / 28% NH4OH 1 / 1 (v/v), and the temperature was set to 32.0°C ± 0.5°C. In order to avoid oxygen limitation the dissolved oxygen (DO) level was stabilized to above 30% saturation by stirrer speed and aeration rate control. If necessary, 1’200 rpm (rotation per minute) and 1–2 vvm (volume per volume per minute) of air enriched with pure oxygen by a PI controller were applied to keep the DO above 30%. Foaming was suppressed by addition of 10–100 μl antifoam suspension (PPG 2000). Feeding was started after the batch ended, as indicated by a sudden increase of the DO-signal. The feed medium was fed exponentially into the fermenter using a variable speed peristaltic pump.
Samples were diluted to OD600 = 1, and 1 ml was centrifuged at 12’000 g for 2 min. The pellet was washed once with 1 ml and resuspended in 0.5 ml 0.1 M Tris–HCl (pH 8). The suspension was sonicated for 10 s with 10% power (Branson Ultrasonics Corp., Danbury, CT, USA), and centrifuged at 12’000 g for 2 min. The supernatant was analyzed for tyrosinase activity by measuring dopachrome formation [38, 54]. One unit is defined as 1 μmol dopachrome formed per minute. The protein concentrations in the supernatant were quantified using the Bradford method.
SDS-PAGE analysis was performed using standard methods  and gels were subsequently stained with Coomassie brilliant blue. The PageRuler plus prestained protein ladder (Fermentas, GmbH) was used as a marker in all gels.
The cell growth was followed by measuring the optical density at 600 nm (Spectronic Genesys 6, Thermo Electron Corp., UK) and correlated to cell dry weight (cdw) with a ratio cdw / OD = 0.36.
All measurements for growth and tyrosinase activity were performed at least in duplicates. The data presented in this report are the average values.
We thank Dr. Renate Reiss for critically reading the manuscript.
- Fairhead M, Thöny-Meyer L: Bacterial tyrosinases: old enzymes with new relevance to biotechnology. New Biotechnol. 2012, 29 (2): 183-191. 10.1016/j.nbt.2011.05.007.View ArticleGoogle Scholar
- Claus H, Decker H: Bacterial tyrosinases. Syst Appl Microbiol. 2006, 29 (1): 3-14. 10.1016/j.syapm.2005.07.012.View ArticleGoogle Scholar
- del Marmol V, Beermann F: Tyrosinase and related proteins in mammalian pigmentation. FEBS Lett. 1996, 381 (3): 165-168. 10.1016/0014-5793(96)00109-3.View ArticleGoogle Scholar
- 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. 10.1111/j.1365-2672.2006.02866.x.View ArticleGoogle Scholar
- 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. 10.1016/j.jinorgbio.2005.10.008.View ArticleGoogle Scholar
- Mayer AM: Polyphenol oxidases in plants and fungi: Going places? A review. Phytochemistry. 2006, 67 (21): 2318-2331. 10.1016/j.phytochem.2006.08.006.View ArticleGoogle Scholar
- Walker JRL, Ferrar PH: Diphenol oxidases, enzyme-catalysed browning and plant disease resistance. Biotechnol Genet Eng Rev. 1998, 15: 457-498. Edited by Tombs MPView ArticleGoogle Scholar
- Jus S, Kokol V, Guebitz GM: Tyrosinase-catalysed coupling of functional molecules onto protein fibres. Enzyme Microb Technol. 2008, 42 (7): 535-542. 10.1016/j.enzmictec.2008.02.012.View ArticleGoogle Scholar
- 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. 10.1163/156856209X404523.View ArticleGoogle Scholar
- 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.View ArticleGoogle Scholar
- Seetharam G, Saville BA: L-DOPA production from tyrosinase immobilized on zeolite. Enzyme Microb Technol. 2002, 31 (6): 747-753. 10.1016/S0141-0229(02)00182-5.View ArticleGoogle Scholar
- 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. 10.1021/jf0623485.View ArticleGoogle Scholar
- 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. 10.1021/jf0703349.View ArticleGoogle Scholar
- 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. 10.1016/j.jbiotec.2006.07.021.View ArticleGoogle Scholar
- 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. 10.1016/j.jbiotec.2006.03.003.View ArticleGoogle Scholar
- 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. 10.1021/jp808001c.View ArticleGoogle Scholar
- Fairhead M, Thöny-Meyer L: Cross-linking and immobilisation of different proteins with recombinant Verrucomicrobium spinosum tyrosinase. J Biotechnol. 2010, 150 (4): 546-551. 10.1016/j.jbiotec.2010.10.068.View ArticleGoogle Scholar
- 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. 10.1002/bit.20840.View ArticleGoogle Scholar
- Thalmann CR, Lotzbeyer T: Enzymatic cross-linking of proteins with tyrosinase. Eur Food Res Technol. 2002, 214 (4): 276-281. 10.1007/s00217-001-0455-0.View ArticleGoogle Scholar
- 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. 10.1016/j.foodhyd.2009.03.011.View ArticleGoogle Scholar
- 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. 10.1016/j.msec.2011.03.007.View ArticleGoogle Scholar
- 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. 10.1111/j.1365-2672.2004.02481.x.View ArticleGoogle Scholar
- Hearing VJ: Mammalian monophenol monooxygenase (tyrosinase) - purification, properties, and reactions catalyzed. Methods Enzymol. 1987, 142: 154-165.View ArticleGoogle Scholar
- Lerch KLE: Purification and characterization of a tyrosinase from Streptomyces glaucescens. Eur J Biochem. 1972, 31 (3): 427-437. 10.1111/j.1432-1033.1972.tb02549.x.View ArticleGoogle Scholar
- 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. 10.1007/s00253-009-2258-3.View ArticleGoogle Scholar
- 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. 10.1093/jb/mvi150.View ArticleGoogle Scholar
- 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. 10.1111/j.1472-765X.2004.01599.x.View ArticleGoogle Scholar
- 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. 10.1159/000233506.View ArticleGoogle Scholar
- 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. 10.1021/jf800109a.View ArticleGoogle Scholar
- 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. 10.4014/jmb.1106.06031.View ArticleGoogle Scholar
- Kumar M, Flurkey WH: Activity, isoenzymes and purity of mushroom tyrosinase in commercial preparations. Phytochemistry. 1991, 30 (12): 3899-3902. 10.1016/0031-9422(91)83430-S.View ArticleGoogle Scholar
- 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. 10.1021/jf205021g.View ArticleGoogle Scholar
- 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. 10.1016/S0305-0491(00)00163-2.View ArticleGoogle Scholar
- 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. 10.1007/s00253-005-0109-4.View ArticleGoogle Scholar
- 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. 10.1016/j.pep.2003.11.015.View ArticleGoogle Scholar
- 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. 10.1111/j.1742-4658.2006.05429.x.View ArticleGoogle Scholar
- 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. 10.1016/j.pep.2007.04.014.View ArticleGoogle Scholar
- Fairhead M, Thöny-Meyer L: Role of the C-terminal extension in a bacterial tyrosinase. FEBS J. 2010, 277 (9): 2083-2095. 10.1111/j.1742-4658.2010.07621.x.View ArticleGoogle Scholar
- Riesenberg D, Guthke R: High-cell-density cultivation of microorganisms. Appl Microbiol Biotechnol. 1999, 51 (4): 422-430. 10.1007/s002530051412.View ArticleGoogle Scholar
- Shiloach J, Fass R: Growing E. coli to high cell density - A historical perspective on method development. Biotechnol Adv. 2005, 23 (5): 345-357. 10.1016/j.biotechadv.2005.04.004.View ArticleGoogle Scholar
- Makrides SC: Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Mol Biol Rev. 1996, 60 (3): 512-Google Scholar
- 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.Google Scholar
- 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. 10.1002/bit.10379.View ArticleGoogle Scholar
- Jana S, Deb JK: Strategies for efficient production of heterologous proteins in Escherichia coli. Appl Microbiol Biotechnol. 2005, 67 (3): 289-298. 10.1007/s00253-004-1814-0.View ArticleGoogle Scholar
- 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. 10.1002/(SICI)1097-0290(19960320)49:6<621::AID-BIT3>3.3.CO;2-V.View ArticleGoogle Scholar
- Domingues L, Lima N, Teixeira JA: Contamination of a high-cell-density continuous bioreactor. Biotechnol Bioeng. 2000, 68 (5): 584-587. 10.1002/(SICI)1097-0290(20000605)68:5<584::AID-BIT14>3.0.CO;2-1.View ArticleGoogle Scholar
- Lee SY: High cell-density culture of Escherichia coli. Trends Biotechnol. 1996, 14 (3): 98-105. 10.1016/0167-7799(96)80930-9.View ArticleGoogle Scholar
- 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. 10.1002/bit.10457.View ArticleGoogle Scholar
- 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. 10.1007/s00253-001-0889-0.View ArticleGoogle Scholar
- 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.View ArticleGoogle Scholar
- Gardner AR, Cadman TW: Product deactivation in recombinant streptomyces. Biotechnol Bioeng. 1990, 36 (3): 243-251. 10.1002/bit.260360305.View ArticleGoogle Scholar
- 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. 10.1111/j.1749-6632.1994.tb47374.x.View ArticleGoogle Scholar
- Sambrook J, Russel DW: Molecular Cloning: A Laboratory Manual. 2001, New York: Cold Spring Harbor Laboratory Press, 3Google Scholar
- Fling M, Horowitz NH, Heinemann SF: The isolation and properties of crystalline tyrosinase from Neurospora. J Biol Chem. 1963, 238: 2045-2053.Google Scholar
- Laemmli UK: Cleavage of structural proteins during assembly of head of bacteriophage-T4. Nature. 1970, 227 (5259): 680-10.1038/227680a0.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.