DESC:[001] DESCRIPTION OF THE INVENTION:
[002] The following specification particularly describes the invention and the manner in which it is to be performed
[003] PRIORITY CLAIM
[004] This application claims priority from the Provisional Application Number 2180/MUM/2012 filed with Indian patent Office, Chennai on 30th July 2012, entitled “PRODUCTION OF EXTRACELLULAR TYROSINASE ENZYME USING THE FUNGUS GLIOCEPHALOTRICHUM FOR VARIOUS APPLICATIONS”, the entirety of which is expressly incorporated herein by reference.
[005] Technical field of the invention
[006] The present invention relates to a process for production of high amounts of extracellular tyrosinase enzyme in the fungus Gliocephalotrichum sp. MTCC 5489. More specifically the invention relates to the production of extracellular tyrosinase enzyme in the Gliocephalotrichum sp. MTCC 5489 by growing the fungus in a suitable culture medium having optimal culture conditions and production of L-dihydroxyphenylalanine or melanin by conversion of tyrosine using the tyrosinase enzyme, either in vivo in culture of the fungus or under in vitro condition.
[007] Background of the invention
[008] Enzymes are proteins that function as biocatalysts in living organisms and perform many important biological functions. Intracellular enzymes are produced within the cells of organisms, while extracellular enzymes are secreted out of the cells. Extracellular enzymes produced by the microorganisms bacteria and fungi, such as proteases, lipases, cellulases and amylases are particularly useful because their yields are high, they can be harvested easily from the culture medium and the microorganisms can be grown under controlled conditions in large volumes. These enzymes are used in detergent, food, paper and biofuel industries (Wolfgang Aehle 2004 Enzymes in Industry Production and applications Chapter 5 Industrial Enzymes, Wiley-VCH Verlag GmbH & Co, Weinheim, pp. 101-262).
[009] Tyrosinase (monophenol, o-diphenol:oxygen oxidoreductase, EC:1,14,18,1) is an enzyme that is produced by plants, fungi, bacteria and mammals. Tyrosinase catalyzes phenolic compounds, including the amino acid tyrosine and therefore belongs to the class of enzymes called phenoloxidases. Tyrosinase o-hydroxylates monophenols to diphenols (monophenolase activity) and also further o-oxidizes diphenols to quinones (diphenolase activity).
[0010] Fungal tyrosinase from the mushroom Agaricus bisporus is well studied (Seo, S.-Y., Sharma, V.K, Sharma, N, 2003. Mushroom tyrosinase recent prospects. J. Agric. Food Chem. 51 (10), 2837–2853). Tyrosinase enzyme is responsible for oxidation of tyrosine, leading to the production of dihydroxyphenylalanine or DOPA, which is further converted by the enzyme to melanin by oxidation of tyrosine. L-DOPA is an important drug in treatment of Parkinson’s disease. Melanins are heteropolymeric, high molecular pigments found in animals, plants, bacteria, fungi and protists (Plonka PM and M Grabacka. 2006. Melanin synthesis in microorganisms biotechnological and medical Aspects. Acta Biochimica Polonica 53: 429-443; Hill, Z.H. 1992) Melanin protects living organisms against UV radiation, lytic enzyme attack and a number of other environmental hazards, possesses antioxidant properties and appears to protect fungi against ionizing radiations (Dadachova A and Casadevall A. 2008. Ionizing radiation how fungi cope, adapt, and exploit with the help of melanin. Current Opinion in Microbiology 11: 525–531; Schweitzer A,D, et ak, 2009. Physico-chemical evaluation of rationally designed melanins as novel nature-inspired radioprotectors. PloS ONE 4: e7229). Melanins are potentially useful in medicine, pharmacology and in cosmetic preparations (Gallas, J. and Eisner, M. 2006. Melanin polyvinyl alcohol plastic laminates for optical applications US Patent 7029758, Montefiori, D.C, and Zhou, J.Y. 1991. Selective antiviral activity of synthetic soluble 1-tyrosine and 1-dopa melanins against human immunodeficiency virus in vitro. Antiviral Research 15: 11–25).
[0011] The use of tyrosinase to produce various compounds has been described in the prior art like tyrosinase obtained commercially from mushrooms has been immobilized on various supports to produce L-DOPA from tyrosine, using L-ascorbate as the reducing agent (Seetharam G and BA Saville. 2002. L-DOPA production from tyrosinase immobilized on zeolite. 2002. Enzyme and Microbial Technology 31:747-753; Carvalho G.M.J., T.L.M. Alves and D.M.G. Freire. 2000. L-DOPA production by immobilized tyrosinase. Applied Biochemistry and Biotechnology 84-86:791-800; Norouzian D, e al. 2007. Immobilization of mushroom tyrosinase by different methods in order to transform L-tyrosine to L-3, 4dihydroxyphenylalanine (L-dopa). Biotechnology 6: 436-439). Microbial biomass producing intracellular tyrosinase and suspended in buffer has been used to convert L-tyrosine to L-DOPA.
[0012] Since tyrosinases can bring about covalent bonding between peptides and carbohydrates, they can be used in cross-linking of proteins. This property has tremendous applications in structure engineering of meat-derived products and materials science (Halaouli et al., 2006, Selinheimo et al., 2007).
[0013] Mushroom tyrosinase is often used to screen for anti-browning agents and skin whitening agents isolated from natural sources and made synthetically (Neeley E et al., 2009. Variations in IC50 Values with Purity of Mushroom Tyrosinase. Int. J Mol. Sci. 10: 3811-3823). Tyrosinase may be used to decontaminate water containing phenols (El-Shora, Hameed M. and M. Metwally. 2008. Use of tyrosinase enzyme from Bacillus thuringensis for the decontamination of water polluted with phenols. Biotechnology 7: 305-310). Mushroom tyrosinase which has been used in most biotechnology studies is expensive, since it is an intracellular enzyme that is produced in small amounts.
[0014] It will be useful to have microorganisms that produce extracellular tyrosinase. Several species of Streptomyces produce extracellular tyrosinase (Popa C and G Bahri. 2011. Streptomyces tyrosinase production and practical applications. Innovative Romanian Food Biotechnology 8: 1-7). However, the yields are low.
[0015] US Patent 5801047 provides a method for production of extracellular tyrosinase in genetically transformed microorganisms such as Streptomyces antibioticus to enable its application in production of polyphenolic polymers (Della-Cioppa, G., Garger, S.J., Holtz, R.B., McCulloch, M.J., Sverlow, G.G, 1998 Method for making stable extracellular tyrosinase and synthesis of polyphenolic polymers therefrom). The patent claims a maximum production of 60 units/ml of diphenolase activity of tyrosinase in the culture medium which converts DOPA to dopachrome. This patent does not describe monophenolase units of tyrosinase that can convert tyrosine to DOPA. Cabrera-Valladares et al. cloned the melA gene for tyrosinase production from Rhizobium etli, into the bacterium Escherichia coli in order to prepare extracellular melanin (Cabrera-Valladares, N., Martínez, A., Pinero, S., Lagunas-Munoz, V.H., Tinoco, R., de Anda, R., Vázquez-Duhalt, R., Bolívar, F. and Gosset, G. 2006. Expression of the melA gene from Rhizobium etli CFN42 in Escherichia coli and characterization of the encoded tyrosinase. Experimental and Microbial Technology 38: 772–779).These authors achieved a final MelA tyrosinase activity corresponding to 1.08 U/mL of diphenolase activity. Monophenolase activity of tyrosinase was not reported.
[0016] Halaouli et al. have reviewed various attempts for homologous and heterologous expressions of fungal tyrosinases for extracellular tyrosinases (Halaouli S, M. Asther, J.-C Sigoillot, M Hamdi and A Lomascolo. 2006. Fungal tyrosinases: new prospects in molecular characteristics, bioengineering and biotechnological applications). Extracellular fungal tyrosinases have not been reported. Selinheimo et al. (2006) detected a tyrosine gene in the fungus Trichoderma reesei and over expressed the gene in the native host extracellulary using a strong promoter. The highest amount of enzyme produced was 1 g per liter, with a specific activity of 300 nkat. mg-1, corresponding to a total of 300,000 nkat activity of o-diphenoloxidase in 1 litre culture medium. The publication does not discuss monophenolase units relating to conversion of tyrosine and it pertains to a recombinant organism. US Patent Application 20090203882 provides an invention describing the above process. Halaouli et al. produced extracellular tyrosinase using a recombinant fungus Aspergillus niger, which was transformed using a tyrosinase gene from another fungus, Pycnosporus sanguineus (Halaouli et al., 2006. Cloning and characterization of a tyrosinase gene from the white-rot fungus Pycnoporus sanguineus, and overproduction of the recombinant protein in Aspergillus niger. Applied Microbiology and Biotechnology 70: 580–589). The best transformant produced extracellular tyrosinase activities of 534 and 1,668 U per liter for monophenolase and diphenolase, respectively, estimated as µmol of MBTH-quinone adducts formed. The publication deals with a recombinant organism and monophenolase units are not related to the conversion of tyrosine to L-DOPA.
[0017] It has been attempted to produce an unlimited source of tyrosinase for further studies by cloning the human tyrosinase gene into the bacterium Escherichia coli (Kong, K. H.; Park, S. Y.; Hong, M. P.; Cho, S. H. 2000. Expression and characterization of human tyrosinase from a bacterial expression system. Comp. Biochem. Physiol.125: 563).
[0018] US Patents 5,218,079 and 5,225,435 of 1993 provide a process for melanin produced from dopachrome, 5,6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acid or by incubating 5,6-dihydroxyindole-2-carboxylic acid. These are various derivatives of tyrosine and it would be more economical to produce melanin directly from tyrosine.
[0019] US Patent 6,315,988 provides a process of using tyrosinase to produce melanin in vitro by reacting dihydroxyphenylalanine or tyrosine with the enzyme in the presence of an acidic protein and or a peptide. It is not related to the production of tyrosinase as such.
[0020] A water soluble, extracellular melanin has been reported in a mutant strain of Bacillus thuringensis that produces extracellular tyrosinase (Aghajanyan, A., A. Hambardzumyan, A. Hovsepyan, R. Asaturian, A. Vardanyan and A. Saghiyan, 2005. Isolation, purification and physicochemical characterization of water soluble Bacillus thuringenesis melanin. Pigment Cell research.18; 130-135). Melanin yields in the above are less than 3 g per liter of culture medium.
[0021] US Patent 5,814,495 provides a process to overproduce up to 3.3 g of melanin per liter culture medium in a genetically transformed Streptomyces strain carrying the gene for tyrosinase (Della-Cioppa, G., Garger, S.J., Sverlow, G.G., Turpen, T.H., Grill, L.K., Chedekal, M.R., 1998b. Melanin production by Streptomyces).
[0022] United States Patent 5,837,505 provides a process of producing up to 3.3 g per liter extracellular melanin by Escherichia coli which was transformed with a vector containing DNA sequences from Streptomyces spp.
[0023] Lagunas-Munoz et al. have used a recombinant Escherichia coli containing the tyrosinase coding gene from Rhizobium etli to produce up to 6.0 g extracellular melanin per liter culture (Lagunas-Munoz VH, N Cabrera-Valladares, F Bolivar, G Gosset and A Martinez. 2006. Optimum melanin production using recombinant Escherichia coli. Journal of Applied Microbiology. 101: 1002-1008).
[0024] There are major drawback in the existing prior art like tyrosinase is generally produced intracellularly and in low quantities that are not enough for large scale commercial use. They are also difficult to extract than enzymes produced extracellularly. Extracellular tyrosinases are rare in bacteria and absent in fungi (Halouli S, M. Asther, J.-C Sigoillot, M Hamdi and A Lomascolo. 2006. Fungal tyrosinases: new prospects in molecular characteristics, bioengineering and biotechnological applications). Monophenoloxidase activity of tyrosinase is the bottleneck in converting tyrosine and high levels of this activity are rare. The bottleneck in converting tyrosine to diydroxyphenylalanine and further to dopaquinone and melanin dopaquinones is the insufficient production of monophenolase activity.
[0025] Therefore, a biological process to produce copious amounts of extracellular tyrosinase with high monophenolase and diphenolase activity would be useful to develop various products and for various applications as mentioned above (Selinheimo, E., Saloheimo, M., Ahola, E., Westerholm-Parvinen, A., Kalkkinen, N., Buchert, J., Kruus, K., 2006. Production and characterization of a secreted, C-terminally processed tyrosinase from the filamentous fungus Trichoderma reesei. FEBS J. 273, 4322–4335). The present process offers the same.
[0026] Summary of the invention:
[0027] Extracellular production of the enzyme tyrosinase in copious amounts from a naturally occurring microorganism has several biotechnogical applications, such as the production of melanin, the drug dihydroxyphenylalanine, cross-linking of proteins, bioremediation, etc. It is therefore an object of present invention to disclose a process for production of high levels of extracellular microbial tyrosinase enzyme.
[0028] A further object of the invention is to disclose a process for production of high levels of extracellular microbial tyrosinase, with high levels of both monophenoloxidase and diphenoloxidase activities.
[0029] Yet another object of the invention is to disclose a process to produce high levels of extracellular tyrosinase from a naturally occurring strain of the fungus Gliocephalotrichum sp. MTCC 5489.
[0030] A further object of this invention is to generate high levels of extracellular melanin by the fungus Gliocephalotrichum sp. MTCC 5489 in vivo by providing tyrosine to the culture medium that will be converted by the tyrosinase enzyme into melanin.
[0031] A further object of this invention is to use the extracellular tyrosinase from the fungus Gliocephalotrichum sp. MTCC 5489 to convert tyrosine to melanin in vitro.
[0032] Yet another object of the present invention is to use the high levels of extracellular tyrosinase obtained as above for in vitro production of L-dihydroxyphenylalanine or L-DOPA, useful in treating Parkinson’s disease.
[0033] A further object of the invention is to use the tyrosinase enzyme obtained as above for cross-linking proteins and bioremediation.
[0034] Brief description of the drawings
[0035] The foregoing and other features of embodiments will become more apparent from the following detailed description of embodiments when read in conjunction with the accompanying drawings. In the drawings, like reference numerals refer to like elements.
[0036] FIG 1 shows a graph depicting monophenolase activity of tyrosinase from Gliocephalotrichum sp. MTCC 5489 assayed at different pHs in accordance to one or more embodiments of the invention.
[0037] FIG 2 shows a graph depicting monophenolase activity of tyrosinase from Gliocephalotrichum sp. MTCC 5489 assayed at different ages of the culture in accordance to one or more embodiments of the invention.
[0038] FIG 3 shows a graph depicting monophenolase activity of tyrosinase from Gliocephalotrichum sp. MTCC 5489 at different temperatures in accordance to one or more embodiments of the invention.
[0039] FIG 4 shows a graph depicting monophenolase activity of tyrosinase at different dilutions from Gliocephalotrichum sp. MTCC 5489 incubated for varying lengths of time with tyrosine in accordance to one or more embodiments of the invention.
[0040] FIG 5 shows a graph depicting production of L-DOPA by incubating tyrosine with tyrosinase from Gliocephalotrichum sp. MTCC 5489 for different time intervals in accordance to one or more embodiments of the invention.
[0041] FIG 6 shows a graph depicting in vitro melanin formation with as judged by absorbance with respect to time in accordance to one or more embodiments of the invention.
[0042] FIG 7 shows in vitro synthesis of melanin in accordance to one or more embodiments of the invention.
[0043] Detailed description of the invention
[0044] In order to more clearly and concisely describe and point out the subject matter of the claimed invention, the following definitions are provided for specific terms which are used in the following written description.
[0045] By the term “Monophenolase activity” we mean that the enzyme being capable of hydroxylation of monophenols such as tyrosine, as context requires.
[0046] By the term “Diphenolase activity” we mean that the enzyme being capable of oxidation of diphenols such as dihydroxyphenylalanine and catechol, as context requires.
[0047] By the term “in vitro” we mean production taking place in an artificial environment outside the living organism, as context requires.
[0048] By the term “in vivo” we mean production occurring within a living organism or in a natural setting in this case it is fungus, as context requires.
[0049] By the term “Extracellular enzyme” we mean an enzyme that is secreted by a cell and that works outside of that cell, as context requires.
[0050] The present invention overcomes the drawback of the existing technology by providing a process for production of extracellular tyrosinase enzyme from fungus Gliocephalotrichum sp. MTCC 5489 in a high yield with high level of monophenolase and diphenolase activity. The obtained tyrosinase enzyme can be further used for production of many biotechnologically useful products like L-DOPA and melanin.
[0051] The fungus Gliocephalotrichum sp. MTCC 5489 was grown in a culture medium with the following composition. Glucose: 1%; Peptone: 1%; Yeast Extract: 0.1%; CuSO4: 0.0005 %; MgSO4 0.02%; FeSO4: 0.01 %; KH2PO4: 0.05 %; Tyrosine: 1.0 %; Water: 100 ml. The fungus was grown on a shaker at 200 rpm at a room temperature of ~25 to 30?C. Culture filtrate containing the enzyme was harvested by filtration and removal of the biomass at 36 hours.
[0052] In order that this invention to be more fully understood the following preparative and testing examples are set forth. These examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention in any way.
[0053] Example 1. Effect of pH on monophenolase activity of tyrosinase
Fig 1 shows the monophenolase activity of the tyrosinase enzyme obtained from the fungus Gliocephalotrichum sp. MTCC 5489 at different pH. The results are shown as enzyme units per Liter. The activity was analyzed as given in literature (Munjal, N. and S.K. Sawhney. 2002. Stability and properties of mushroom tyrosinase entrapped in alginate, polyacrylamide and elatin gels. Enzyme and Microbial Technology 30: 613-619.). Thus, 1 ml of enzyme was added to 2 ml of 0.1M buffers at 5 different pHs, namely 4.0, 5.0, 6.0, 7.0 and 8.0, each containing 0.005 g tyrosine and 0.0024 g ascorbic acid. Heat deactivated enzyme was used as control blank. The reaction mixture was incubated for 15 minutes at room temperature of 25 to 30°C. Subsequently 1 ml of 0.5 N HCl was added, followed by 1 ml nitrite-molybdate reagent, when a yellow colour appears. This was followed by the addition of 1 ml 1N NaOH, which yields a reddish colour. The volume was then made up to 4 ml and the absorbance was read at 460 nm. The difference between the reaction and control was read against a L-DOPA standard to determine the amount of L-DOPA formed. One Unit of monophenolase is defined as 1 µ mole of L-DOPA formed per minute by 1 ml of enzyme. The enzyme was active at pH of 4.0 to 7.0.The best activity was achieved at a pH of 5.0, the enzyme activity being up to nearly 45 Units of monophenolase per Liter of enzyme, yielding 8,865 ?mole or 8.9 mg of L-DOPA per minute.
[0054] Example 2. Effect of age of culture on monophenolase activity of tyrosinase of Gliocephalotrichum simplex MTCC 5489
The fungus Gliocephalotrichum sp. MTCC 5489 was grown in a culture medium, the enzyme extracted and the monophenolase activity of the tyrosinase was analyzed as given above. The enzyme extracted at different ages of culture, namely 12, 24, 36, 48 and 60 hours was assayed at pH 5.0. One Unit of monophenolase is defined as 1 ?mole of L-DOPA formed per minute by 1 ml of enzyme. The results, as enzyme units per Liter are given in Fig. 2. The enzyme was produced throughout from 12 to 48 hours of the culture. The best monophenolase activity was obtained at 36 hours.
[0055] Example 3. Effect of temperature on monophenolase activity of tyrosinase
The fungus Gliocephalotrichum sp. MTCC 5489 was grown as given in a culture medium with optimum condition, the enzyme was harvested after 36 hours and the monophenolase activity at the optimum pH of 5.0 was assayed at different temperatures, namely 20, 30 and 40?C. One Unit of monophenolase is defined as 1 ?mole of L-DOPA formed per minute by 1 ml of enzyme. The results, as enzyme units per Liter are given in Fig. 3.The enzyme was active between 20?C and 40?C. The best activity of 20 Units was obtained when assayed at a temperature of 30 and 40?C.

[0056] Example 4. Effect of enzyme concentration and incubation time on monophenolase activity of Gliocephalotrichum sp. MTCC 5489
The fungus was grown as shown in culture medium in optimum condition, the enzyme was harvested after 36 hours and the monophenolase activity at the optimum pH of 5.0 and a temperature of 25?C was assayed using different enzyme dilutions of 1, 5 and 10. One Unit of monophenolase is defined as 1 ?mole of L-DOPA formed per minute by 1 ml of enzyme. The results, as enzyme units per Liter are given in Fig. 4. Enzyme diluted 5 times was optimal. The enzyme activity reached a steady state by 60 minutes. A maximum of approximately 45 Units L-1 of monophenolase activity was achieved. This corresponds to the formation of 45?mole of DOPA formed per minute by 1 L of enzyme, or 7.88 mg of DOPA formed per L of enzyme in 1 minute.

Example 5. Diphenolase activity of tyrosinase from Gliocephalotrichum sp. MTCC 5489
The fungus was grown as shown in culture medium in optimum condition, the enzyme was harvested after 36 hours and the diphenolase activity assayed as given in literature (Surwase, S.N., S.A. Patil, O.A. Apine and J.P Jadhav. 2012. Efficient microbial conversion of L-tyrosine to L-DOPA by Brevundinmonas sp. SGJ. Applied Biochemistry and Biotechnology 167: 1015-1028). Thus, 0.1 ml of of enzyme was added to a mixture of 2.6 ml of 50 mM potassium phosphate buffer of pH 6.5, 0.1 ml 5mM catechol, 0.1 ml of 2.1 mM ascorbic acid and 0.1 ml of 0.065 mM EDTA. Absorbance at 265 nm was recorded at the beginning of the reaction and after 1 min and decrease in absorbance was noted. One Unit of diphenolase activity corresponded to a decrease of 0.001 in absorbance per ml per minute. The fungus showed an average catecholase or diphenolase activity of 453 U/ml/min.

[0057] Example 6. Production of in vitro L-DOPA using tyrosinase from Gliocephalotrichum sp. MTCC 5489
The fungus was grown then the enzyme was harvested after 36 hours. One ml of the enzyme, containing 0.037 Units of monophenolase per ml was added to 2 ml of 1M acetate buffer at pH 5, containing 0.0009 g tyrosine and 0.0024 g ascorbic acid. The reaction was carried out for 150 minutes, and L-DOPA was assayed at different time intervals, as shown in Fig 5. L-DOPA was produced in amounts equaling 1.09 g per L of enzyme used in 150 minutes.

[0058] Example 7. Production of in vitro melanin using tyrosinase from Gliocephalotrichum simplex MTCC 5489
The fungus was grown in the culture medium and the enzyme was harvested after 36 hours. A total of 1.8 mg of tyrosine was added to 4 ml of the enzyme and incubated. Absorbance at 460 nm was taken at regular intervals to check for melanin formation as shown in Fig 6. The absorbance was compared with standard melanin. One litre of enzyme could synthesize approximately a total of 2 g melanin in 150 minutes. By 3 hours, the reaction mixture as shown in the tube on the left of the Fig. 7 turned black, whereas the control treatment without the enzyme as shown in the tube on the right side of the Fig. 7 remained light in color.
,CLAIMS:What is claimed is:
1. A process of producing tyrosinase enzyme from the fungus Gliocepahlotrichum with high level of monophenolase and diphenolase activity.

2. The process of claim 1 wherein said tyrosinase enzyme is extracellular.

3. The process of Claim 1 wherein said tyrosinase enzyme has at least 40 Units of monphenolase activity and at least 450,000 units of diphenolase activity per Liter.

4. The process of Claim 1 wherein said extracellular tyrosinase enzyme produces L-dihydroxyphenylalanine under in vitro conditions.

5. The process of Claim 1 wherein said one litre of the tyrosinase enzyme produces at least 1 g per Litre of L-diydroxypheynylalanine from L-tyrosine.

6. A process of producing melanin under in vitro condition from tyrosinase enzyme derived from the fungus Gliocephalotrichum in high yield.

7. The process of claim 6 wherein said melanin production takes place using tyrosinase enzyme under in vitro condition.

8. The process of claim 6 wherein said melanin is used for various pharmaceutical, cosmetic and industrial applications.

9. The process of claim 6 wherein said melanin production yield is enhanced by immobilizing the enzyme on to a surface and used repeatedly to convert tyrosine to result in enhanced levels of melanin