Ebrahim Valipour, Burhan Arikan


The bacterial enzyme tyrosinase, with its high oxidizing capacity, can be applied in phenolic biotransformation, pharmaceutical, cosmetics and textile industries. In this research, a native Bacillus sp.-producing tyrosinase was isolated from a soil sample. The strain was identified by morphological, biochemical and molecular tests using bioinformatics analysis, and was named Bacillus megaterium strain M36. According to the blast analysis of 16S rDNA (1434 bp), the strain showed 99% identity with Bacillus megaterium DSM319. The production of tyrosinase from the isolated strain was optimized by classic and response surface methods (RSM). The optimal conditions for tyrosinase production by the strain were determined to be as follow: growth temperature 36°C, pH of medium 7.0, incubation time 16 h, with medium containing 0.4 mg/mL  L-tyrosine, 0.05% yeast extract, 0.423% tryptone, 3.4% NaCl and 148.4 µM CuSO4. Results of experiments performed under the optimized condition showed an actual yield of 0.522 IU of enzyme, while the result under the initial conditions using basal medium (before optimization) gave 0.0312 IU of enzyme (16.7-fold increase). SDS-PAGE analysis showed that the tyrosinase enzyme from Bacillus megaterium strain M36 is about 34 kDa.


Key words: Bacillus megaterium; tyrosinase; 16S rDNA; RSM; optimization


Received: October 2, 2015; Revised: October 22, 2015; Accepted: October 23, 2015; Published online: June 6, 2016

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Claus H, Decker H. Bacterial tyrosinases. Syst Appl Microbiol. 2006;29(1):3-14.

Decker H, Tuczek F. Tyrosinase/catecholoxidase activity of hemocyanins: structural basis and molecular mechanism. Trends Biochem Sci. 2000;25(8):392-7.

Seo SY, Sharma VK, Sharma N. Mushroom tyrosinase: Recent prospects. J Agric Food Chem. 2003;51(10):2837-53.

Bell AA, Wheeler MH. Biosynthesis and functions of fungal melanins. Annu Rev Phytopathol. 1986;24:411-51.

Marino SM, Fogal S, Bisaglia M, Moro S, Scartabelli G, De Gioia L, Spada A, Monzani E, Casella L, Mammi S, Bubacco L. Investigation of Streptomyces antibioticus tyrosinase reactivity toward chlorophenols. Arch Biochem Biophys. 2011;505(1):67-74.

Franciscon E, Grossman MJ, Paschoal JA, Reyes FG, Durrant LR. Decolorization and biodegradation of reactive sulfonated azo dyes by a newly isolated Brevibacterium sp. strain VN-15. Springerplus. 2012;1(1):37.

Saratale RG, Saratale GD, Chang JS, Govindwar SP. Bacterials decolorization and degradation of azo dyes: A review. J Taiwan Inst Chem Eng. 2011;42(1):138-57.

Xu DY, Chen JY, Yang Z. Use of cross-linked tyrosinase aggregates as catalyst for synthesis of L-DOPA. Biochem Eng J. 2012;63:88-94.

Allouche N, Damak A, Ellouz R, Sayadi S. Use of whole cells of Pseudomonas aeruginosa for synthesis of the antioxidant hydroxytyrosol via conversion of tyrosol. Appl Environ Microbiol. 2004;70(4):2105-9.

Pandey G, Muralikrishna C, Bhalerao UT. Mushroom tyrosinase catalyzed synthesis of coumestans, benzofuran derivatives and related heterocyclic-compounds. Tetrahedron. 1989;45(21):6867-74.

Berliner DL, Erwin RL, McGee DR. Therapeutic uses of melanin. United States Patent, US5703051 A. 1997 Dec 30.

Michalik J, Emilianowicz-Czerska W, Switalski L, Raczynska-Bojanowska K. Monophenol monooxygenase and lincomycin biosynthesis in Streptomyces lincolnensis. Antimicrob Agents Chemother. 1975;8(5):526-31.

Herlihy WC. Skin tanning composition and method. United States Patent. 2002; US4968497 A.

Warner JC, Stoler EJ. Coloring composition containing an aromatic compound and an initiator. United States Patent US8231689 B2, 2012 Jul 31.

Tu YY, Xu XQ, Xia HL, Watanabe N. Optimization of theaflavin biosynthesis from tea polyphenols using an immobilized enzyme system and response surface methodology. Biotechnol Lett. 2005;27(4):269-74.

Aghaie-Khouzani M, Forootanfar H, Moshfegh M, Khoshayand M, Faramarzi M. Decolorization of some synthetic dyes using optimized culture broth of laccase producing ascomycete Paraconiothyrium variabile. Biochem Eng J. 2012;60:9-15.

Dalfard AB, Khajeh K, Soudi MR, Naderi-Manesh H, Ranjbar B, Sajedi RH. Isolation and biochemical characterization of laccase and tyrosinase activities in a novel melanogenic soil bacterium. Enzyme Microb Technol. 2006;39(7):1409-16.

Sambasiva Rao KR, Tripathy N, Rao S, Prakasham R. Production, Characterization, Catalytic and Inhibitory activities of Tyrosinase. Res J Biotechnol.Vol. 2013;8(1):83-98.

Sambasiva Rao KR, Tripathy NK, Mahalaxmi Y, Prakasham RS. Laccase- and peroxidase-free tyrosinase production by isolated microbial strain. J Microbiol Biotechnol. 2012;22(2):207-14.

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.

Caf Y, Maaşoğlu Y, Valipour E, Arikan B. Production and characterization of novel cold-active, pH tolerant and detergent-stable: α-amylase from a psychrotrophic bacterium from soil samples. N Biotechnol. 2012;29:S82.

Burhan A, Nisa U, Gökhan C, Ömer C, Ashabil A, Osman G. Enzymatic properties of a novel thermostable, thermophilic, alkaline and chelator resistant amylase from an alkaliphilic Bacillus sp. isolate ANT-6. Process Biochem. 2003;38(10):1397-403.

Radjasa OK, Martens T, Grossart H-P, Brinkhoff T, Sabdono A, Simon M. Antagonistic activity of a marine bacterium Pseudoalteromonas luteoviolacea TAB4. 2 Associated with Coral Acropora sp. J Biol Sci. (Faisalabad). 2007;7(2):239-246.

Saitou N, Nei M. The Neighbor-Joining Method - a New Method for Reconstructing Phylogenetic Trees. Mol Biol Evol. 1987;4(4):406-25.

Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013;30(12):2725-9.

Tamura K, Nei M. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol. 1993;10(3):512-26.

Anupama A, Jayaraman G. Detergent stable, halotolerant α-amylase from Bacillus aquimaris vitp4 exhibits reversible unfolding. IJABPT. 2011;2:366-76.

Bisswanger H. Practical enzymology. John Wiley & Sons; 2013. p. 387.

Lopez-Serrano D, Sanchez-Amat A, Solano F. Cloning and molecular characterization of a SDS-activated tyrosinase from Marinomonas mediterranea. Pigment Cell Res. 2002;15(2):104-11.

McMahon AM, Doyle EM, Brooks S, O’Connor KE. Biochemical characterisation of the coexisting tyrosinase and laccase in the soil bacterium Pseudomonas putida F6. Enzyme Microb Technol. 2007;40(5):1435-41.

El-Shora HM, Metwally M. Use of tyrosinase enzyme from Bacillus thuringiensis for the decontamination of water polluted with phenols. Biotechnology. 2008;7(2):305-10.

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):202-7.

Arikan B. Highly thermostable, thermophilic, and alkaline, SDS and chelator resistant amylase from a thermophilic Bacillus sp. isolate A3-15. Bioresour Technol. 2008;99(8):3071-6.

Cai H, Archambault M, Prescott JF. 16S Ribosomal RNA sequence-based identification of veterinary clinical bacteria. J Vet Diagn Invest. 2003;15(5):465-9.

Deb P, Talukdar SA, Mohsina K, Sarker PK, Sayem SA. Production and partial characterization of extracellular amylase enzyme from Bacillus amyloliquefaciens P-001. Springerplus. 2013;2(1):154.

Gupta R, Gigras P, Mohapatra H, Goswami VK, Chauhan B. Microbial alpha-amylases: a biotechnological perspective. Process Biochem. 2003;38(11):1599-616.

Park GT, Son HJ. Keratinolytic activity of Bacillus megaterium F7-1, a feather-degrading mesophilic bacterium. Microbiol Res. 2009;164(4):478-85.

Surwase SN, Jadhav JP. Bioconversion of L-tyrosine to L-DOPA by a novel bacterium Bacillus sp. JPJ. Amino Acids. 2011;41(2):495-506.

Kumar CG, Takagi H. Microbial alkaline proteases: From a bioindustrial viewpoint. Biotechnol Adv. 1999;17(7):561-94.

Surwase SN, Patil SA, Jadhav SB, Jadhav JP. Optimization of l‐DOPA production by Brevundimonas sp. SGJ using response surface methodology. Microb Biotechnol. 2012;5(6):731-7.

THOMAS M, Priest F, Stark J. Characterization of an extracellular β-amylase from Bacillus megaterium sensu stricto. J Gen Microbiol. 1980;118(1):67-72.

Chen YH, Deng YY, Wang JH, Cai J, Ren GX. Characterization of melanin produced by a wild-type strain of Bacillus thuringiensis. J Gen Appl Microbiol. 2004;50(4):183-8.

Zhang J, Cai J, Deng Y, Chen Y, Ren G. Characterization of melanin produced by a wild-type strain of Bacillus cereus. Front Biol (Beijing). 2007;2(1):26-9.

Krishnaveni R, Rathod V, Thakur M, Neelgund Y. Transformation of L-tyrosine to L-DOPA by a novel fungus, Acremonium rutilum, under submerged fermentation. Curr Microbiol. 2009;58(2):122-8.

Rani N, Joy B, Abraham TE. Cell suspension cultures of Portulaca grandiflora as potent catalysts for biotransformation of L-tyrosine into L-DOPA, an anti-Parkinson's drug. Pharm Biol. 2007;45(1):48-53.

Liu N, Zhang T, Wang Y, Huang Y, Ou J, Shen P. A heat inducible tyrosinase with distinct properties from Bacillus thuringiensis. Lett Appl Microbiol. 2004;39(5):407-12.


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