In vitro production of L-dihydroxyphenylalanine (L-DOPA), a drug for Parkinson’s disease, using extracellular tyrosinase from a fungus.

Summary:
L-DOPA is produced in the human body by oxidation of tyrosine by the enzyme tyrosinase. Tyrosinase is an expensive enzyme. Myko Tech’s original process uses extracellular tyrosinase produced abundantly by a microorganism and which can then be used to synthesize L-DOPA in an economical manner from tyrosine.

Full Description:
L-DOPA is produced in the human body by oxidation of tyrosine by the enzyme tyrosinase. Tyrosinase is an expensive enzyme. Myko Tech’s original process uses extracellular tyrosinase produced abundantly by a microorganism and which can then be used to synthesize L-DOPA in an economical manner from tyrosine.

*Full Description of Idea/Invention (max 2000 words, no links or contact info):

Sources of DOPA, the market and the price:
• Enzymatic synthesis: Nearly half of the total production of about 250 tons of L-DOPA is produced enzymatically (Koyanagi et al., 2009). In this method, catechol is converted to L-DOPA in the presence of pyruvate, ammonium and a chemical catalyst by the enzyme tyrosine phenol-lyase. Cells of Erwinia herbicola are used for this production. This method results in a mixture of L-DOPA and the beginning substrates, thereby requiring subsequent separation of the L-DOPA from catechol. Besides, the L-DOPA can be reversed to catechol, thus reducing yields to less than 50 %. Catechol is also a toxic compound, the pyruvate is expensive, the yield is low and separation costs of L-DOPA are expensive.
o The preset industrial production rate from the above method is 0.375 g L-1h-1.
• Chemical synthesis: The chemical process of Monsanto employs an asymmetric hydrogenation process for synthesis of L-DOPA (Knowles, 1986, Lennon, 2007). Chemical synthesis is harsh, expensive and involved several chemical steps.
• It is also conventionally extracted from the seeds of Mucuna pruriens and Vicia faba. However, the DOPA is a mixture of L- and D isomers.

L-DOPA is presently an expensive compound. More economical ways of producing L-DOPA are highly desirable.


Status of present research
o Plant sources are presently investigated for improved yields of L-DOPA.
• Portulaca grandiflora callus cultures were used to produce L-DOPA from tyrosine (Rani et al., 2007).
o Up to 48.8 mg L-1 h-1 of L-DOPA was produced at an optimal rate.
• Cell cultures of Mucuna pruriens L. and Mucuna prurita H. have been used to enhance yields of L-DOPA production (Kumar et al., 2012).
However, plant cell cultures are time-consuming and complex.
o Immobilized tyrosinase enzyme has been used to convert L-tyrosine to L-DOPA.
• Tyrosinase obtained commercially from mushrooms has been immobilized on various supports to produce L-DOPA from tyrosine, using L-ascorbate as the reducing agent .
i. A total of 252 mg DOPA was produced in 7 hours (Seetharam and Saville, 2002).
ii. A production rate of 53.97 mg L-1 h-1 was achieved for an optimal total incubation period of 24 h by Carvalho et al. (2000) (1.3 g of L-DOPA).
iii. Up to 73 mg L-1 of L-DOPA was produced by immobilizing tyrosinase on to activated agar particles (Norouzian et al., 2007).
However, mushroom tyrosinase is expensive, since it is an intracellular enzyme that is produced in small amounts.
o Microbial synthesis of L-DOPA using the conversion of L-tyrosine by tyrosinase is eco-friendly and has attracted much attention in recent years as a replacement for L-DOPA from plant sources and chemical synthesis.
• Microbial biomass suspended in buffer has been used to convert L-tyrosine to L-DOPA.

Process Yield Reference
Mycelium of the fungus Aspergillus oryzae was suspended in buffer containing tyrosine in the presence of illite Nearly 1.7 mg mL-1 was produced in 60 minutes. Ali and Ikram-ul-Haq, 2006
Biomass of a bacterial strain belonging to of the yeast Yarrowia lipolytica corresponding to 2.5 mg mL-1 biomass was suspended in a buffer of pH 3.5, containing tyrosine and 2.0 mg mL-1 diatomite Up to 3.48 mg ml-1 was produced Ali et al., 2007
A submerged culture of the fungus Acremonium rutilum on potato-dextrose medium containing 5 mg ml-1 tyrosine, a temperature of 25°C, pH 5.0 and an inoculum size of 2.5 ml and an incubation time of 70 – 120 h was used Up to 0.89 mg ml-1 of L-DOPA was produced Krishnaveni et al., 2009
Biomass of a bacterial strain belonging to Bacillus, corresponding to 1 mg ml-1 was suspended in a buffer of pH 8.0 at 40°C for 60 minutes, together with CuSO4 and L-ascorbic acid and activated charcoal Up to 0.497 mg ml-1 L-DOPA was produced from 0.5 mg tyrosine Surwase and Jadhav, 2011
Biomass of a bacterial strain belonging to Brevundimonas sp. SGJ, corresponding to 2 g L-1 was suspended in a buffer of pH 8.0, , 2 g L-1 L-tyrosine, 0.04 g L-1 CuSO4, 0.02 g L-1 ascorbic acid and 0.5 g L-1 carrageenan at a temperature of 40°C 3.81 g L-1 of L-DOPA was produced from 4 g L-1 tyrosine after 18 h Surwase et al., 2012

o Recombinant organisms for production of L-DOPA have been attempted.
• Since tyrosine is an expensive substrate and separation of tyrosine from L-DOPA is difficult, Foor et al. (1993) developed a method by which the gene encoding inducing high levels of tyrosine phenol-lyase enzyme from Erwinia herbicola was expressed at high levels in Escherichia coli.
o L-DOPA was synthesized in yields of up to 105 mM L-DOPA (20.7 g L-1) from catechol, pyruvate and ammonia by induced cells.
• US Patent 5837504 (1998) describes an invention that uses a recombinant Escherichia coli harboring genes for tyrosinase. The enzyme converts L-tyrosine to L-DOPA. Biomass of the recombinant bacterium was suspended in a mixture containing 5 % glycerol and incubated for 50 hours with periodic additions of L-tyrosine to a total of 75 mM.
o A total of 48 mM or 9.2 g of L-DOPA accumulated after 50 h of incubation.
• A hyper-L-DOPA producing strain of Erwinia herbicola, carrying a mutant transcriptional regulator, an activator of the tyrosne phenol-lyase (Tpl) was constructed by Koyanagi et al. (2009).

Literature:
Ali, S. and Ikram-ul-Haq. 2006. Innovative effect of Illite on improved microbiological conversion of L-tyrosine to 3,4 dihydroxy phenyl L-alanine (L-DOPA) by Aspergills oryzae ME2 under acidic reaction conditions. Current Microbiology 53: 351-357.
Ali, S., J.L. Schultz and Ikram-ul-Haq. 2007. High performance microbiological transformation of L-tyrosine to L-dopa by Yarrowia lipolytica NRRL-143. BMC Biotechnology 7: 50-57.
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.
Davie, C. 2008. A review of Parkinson’s disease. British Medical Bulletin 86: 109-127.
Foor, F., N. Morin andK.A. Bostian. 1993. Production of L-dihydroxyphenylalanine in Escherichia coli with the tyrosine phenol-lyase gene cloned from Erwinia herbicola. Applied and Environmental Microbiology 59: 3070-3075.
Garcı´a-Borro´ n, J.C. and Solano, F. (2002) Molecular anatomy of tyrosinase and its related proteins: beyond the histidine bound metal catalytic center. Pigment Cell Res 15, 162–173.
Koyanagai, T., Katayama, T., Suzuki, H., Nakazawab, H., Yokozei, K., and Kumagai, H. 2005. Effective production of 3,4-dihydroxyphenylal L-alanine (L-DOPA) with Erwinia herbicola cells carrying a mutant transcriptional regulator TyrR. Journal of Bioechnology 115: 303-306.
Koyanagai T., T. Katayama, H. Suzuki, A. Onishi, K. Yokozeki and H. Kumagai. 2009. Hyperproduction of 3,4-dihydroxyphenyl-L-alanine (L-DOPA) using Erwinia herbicola cells carrying a mutant transcriptional regulator TyrR. Bioscience Biotechnology and Biochemistry 73: 1221-1223.
Krishnaveni, R., V. Rathod, M.S. Thakur and Y.F. Neelgund. 2009. Transformation of L-tyrosine to L-DOPA by a novel fungus, Acremonium rutilum, under submerged fermentation. Current Microbiology 58: 122-128.
Norouzian D., e al. 2007. Immobilization of mushroom tyrosinase by different methods in order to transform L-tyrosine to L-3,4 dihydroxyphenylalanine (L-dopa). Biotechnology 6: 436-439.
Rani N., B. Joy and T.E. 2007. Cell suspension cultures of Portulaca grandiflora as potent catalysts for biotransformation of L-tyrosine into L-DOPA, an anti-Parkinson’s drug.
Seetharam G and BA Saville. 2002. L-DPA production from tyrosinase immobilized on zeolite. 2002. Enzyme and Microbial Technology 31: 747-753.
Surwase S.N. and J.P. Jadhav. 2011. Bioconversion of L-tyrosine to L-DOPA by a novel bacterium Bacillus sp. JPJ. Amino Acids 41: 495-506.


Myko Tech’s process to produce DOPA and advantages

The highlight of Myko Tech’s technology are as follows.

• L-DOPA is presently expensive, because the enzymes involved in its synthesis, such as the tyrosinase, are intracellular and difficult to produce in large amounts. Myko Tech uses a patented technology wherein high levels of extracellular tyrosinase enzyme produced by a fungus are used, making the process inexpensive.
• High levels of L-DOPA are produced, more than what is presently produced from other technologies. A minimum of1 g of DOPA can be produced in vitro by 1 litre of enzyme.
• The cost is expected to be much cheaper than the presently available L-DOPA.
• The enzyme may be immobilized to yield higher levels of L-DOPA per liter enzyme used.

Problem this idea/invention addresses:
Myko Tech has developed a process to produce L-DOPA, the drug of choice for Parkinson’s disease, in a highly economical manner. Parkinson’s disease is a neurodegernerative disorder that particularly affects the aged. In Western Europe, nearly 4 % of those above 80 years are affected by the disease. A major feature of Parkinson’s disease is the reduced levels of dopamine, an important signaling molecule in the nervous system. In human beings, dopamine is the byproduct of L-DOPA. L-DOPA is the precursor of neutransmitters dopamine, norepinephrine (nonadrenaline), and epinephrine (adrenaline) collectively known as catecholamines. The world market for L-DOPA is about 250 tons per year and the total market volume is about $101 billion per year (Koyanagari et al., 2005). Various brand names containing L-DOPA, such as Parcopa, Sinemet, Atamet, Stalevo, Tidomet, Madopar, Prolopa are used to treat idiopathic Parkinson's disease, postencephalitic parkinsonism, and symptomatic parkinsonism, since 1967. L-DOPA is presently produced by enzymatic or chemical synthesis and is highly expensive. Myko Tech’s process using extracellular tyrosinase enzyme from a microorganism is a highly economical process.

Auxiliary products or services for sale:
This technology is linked to the technology on production melanin, also from Myko Tech. All negotiations will be for both the technologies together.

Sale or license. Negotiable.

Technology for sale or license: Patented microbial strain, technical knowhow, patent rights.

Cost: Negotiable.
Upfront payment: US$ 150,000.
Staggered over the next two years: US$ 150,000; or 5% royalty over profit in the next 10 years.
Total: US$ 300,000.

Company
Myko Tech Private Limited,
313, Vainguinnim Valley,
Dona Paula, Goa – 403 004.

Contact person: Dr Seshagiri Raghukumar
E-mail: s_raghukumar@mykotech.com
Phone Number: +91(0832)256070
Mobile: +919890450007

Asking price: [CONTACT SELLER]
Available for consultation? Yes

Invention #11872
Date posted: 2013-09-09

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