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View Full Text-PDF Int.J.Curr.Microbiol.App.Sci (2016) 5(3): 345-353 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 5 Number 3(2016) pp. 345-353 Journal homepage: http://www.ijcmas.com Original Research Article http://dx.doi.org/10.20546/ijcmas.2016.503.041 Screening of Tyrosinase Producing Soil Actinomycetes from Shirala Region of Maharashtra, India Gare Sandip Subhash1*, D.D. Karad2 and S.W. Kulkarni3 1Department of Microbiology, Vishwasrao Naik Art’s, Commerce and Baba Naik Science Mahavidyalaya, Shirala 415408, Dist-Sangli M.S, India 2Departments of Microbiology and Research Center, Shri Shivaji Mahavidyalaya, Barshi-413 411, Dist-Solapur, MS, India 3Research Department of Microbiology, Shriman Bhausaheb Zadbuke Mahavidyalaya, Barshi 413 401, Dist-Solapur M.S, India *Corresponding author ABSTRACT K eywo rd s A total seven soil samples were collected from the Shirala region Dist-Sangli. The total 70 Actinomycete isolates were obtained from soil by performing serial Actinomyce tes, dilution technique and using Glycerol asparagine agar supplemented with Genus, Cycloheximide (100µg/ml). Actinomycete isolates were primarily identified to Streptomyces , genus level on the basis of their microscopic and cultural characteristics. Tyrosinase, Biochemical and other characters were used to identify isolates to species level. A Screening . total 70 Actinomycete isolates were identified by using modified DOS based MICRO-IS software such as PIBWin. Out of 70 Actinomycete isolates total Article Info 82.85% isolates were belonged to genus Streptomyces, 10% isolates were belonged Accepted: to genus Streptoverticillum, 2.8571% isolates were belonged to Nocardia, 2.8571% 15 February 2016 isolates were belonged to genus Micromonospora and rest of the 1.4285% isolate Available Online: were belonged to Actinomadura. The primary screening of 70 isolates for 10, March 2016 tyrosinase production were carried out and isolates were showed positive tyrosinase activity. Introduction The word actinomycete was taken from the unicellular organisms, belonging to order Greek. It has made from two different words actinomycetales highlighted with substrate and aerial mycelial growth (Lechevalier and such as ''atkis'' (a ray) and ''mykes'' (fungus), possess characteristics of both bacteria and Lechevalie, 1981). Actinomycetes reproduce fungi. Actinomycetes bear almost either by fission or by means of special distinctive features than bacteria and fungi, spores or conidia. They possess high (G+C) so they fixed into separate class. content in their DNA which is above 55 mol Actinomycetes are aerobic, spore forming, percent. The evidences of 16s ribosomal Gram positive bacteria grouped as branching cataloguing and DNA: rRNA pairing studies 345 Int.J.Curr.Microbiol.App.Sci (2016) 5(3): 345-353 suggested that phylogenetic relationship (including diagnostic applications, research among the group of actinomycetes and biotechnology) accounted for a further (Goodfellow and Williams, 1983; Korn- £700 million. Estimates of future demand Wendisch and Kutzner , 1992). The domain are in the range of £4000-5000 million bacteria has been currently proposed with 18 between 2015 and 2016, growing at a rate of major lineages out of them actinomycetes 6–7% annually. The developing economies represent one of the largest taxonomic unit. of the Asia Pacific Region, the Middle East (Ventura, et al., 2007) In soil actinomycetes and Africa are now seen to be emerging as from thread like filaments and which are the fastest growing markets for industrial most abundant organisms. The characteristic enzymes. earthy smell of freshly turned healthy soil is due to hypae produced at the time of Tyrosinase (EC 1.14.18.1) is a copper- growth.(Sprusansky, et al., 2005) The containing enzyme that have both actinomycetes are found in various habitats monophenolase or cresolase and diphenolase in nature (George, et al, 2012) and most or catecholase activity in which prominent group of microbes distributed monophenolase or cresolase activity of an throughout the natural ecosystems around enzyme tyrosinase expressed by catalyzing the world. (Srinivasan, et al., 1971) They are the o-hydroxylation of monophenols to the initially represented as soil inhabitants. corresponding catechols and diphenolase or (Kuster, 1968) catecholase catalysing the oxidation of diphenols or catechols to the corresponding Microbial secondary metabolites have great o-quinones. The enzyme can be found in a interest all over the world. Around 23,000 wide variety of organisms, ranging from bioactive secondary metabolites are reported prokaryotes to mammals. Tyrosinase can throughout the world in which 10,000 are take part in biosynthesis of melanin in which produced only by actinomycetes, hence monophenolase or cresolase activity of an contributing 45% of all bioactive microbial enzyme tyrosinase can catalyses substrate metabolites discovered and among tyrosine into 3,4-dihydroxyphenylalanin or actinomycetes, near about 7,600 compounds DOPA (o-diphenol) by ortho-hydroxylation are produced by Streptomyces species. reaction and an enzyme diphenolase or (Berdy, 2005) Their metabolic potential catecholase activity of an enzyme tyrosinase offers a strong area of research. can catalyse substrate 3,4- Accordingly, the role of actinomycetes in dihydroxyphenylalanin or DOPA into biotechnology and medicine is well known dopaquinone (o-quinone) by oxidation and these industries are always looking for reaction followed by o-quinone turn to novelty bioactive compounds. melanin pigments through series of enzymatic and nonenzymatic reactions. The value of the world enzyme market has (Robb, 1984; Whitaker, 1995) Along with increased steadily from £110 million in 1960 tyrosine and L-DOPA numerous other to £200 million in 1970, £270 million in substrates such as phenols and diphenols are 1980, £1 000 million in 1990 and over £2 converted to corresponding diphenols and 000 million in 2010. Food and beverage quinones respectively by an enzyme enzymes represented the largest sector of the tyrosinase. Basically, the monophenol industrial enzymes market in 2010, with a hydroxylase and diphenoloxidase activities value of £750 million, and the market for of tyrosinase are used in environmental enzymes for technical applications technology for the detoxification of phenol- containing waste waters and contaminated 346 Int.J.Curr.Microbiol.App.Sci (2016) 5(3): 345-353 soils as a construction of a biosensor for the so far studied to some extent throughout the detection of phenolic compound. (Dos world by scientists. Geographically Shirala Santos, et al., 2013) In pharmaceutical region is situated at heavy rain fall. No one industries tyrosinase has been used for the had reported actinomycetes from this region production of o-diphenols such as L-DOPA which can produce tyrosinase. This study and dopamine for the treatment of focuses on screening of actinomycetes Parkinson’s disease and also have been which have ability to produce an enzyme reported as a marker in melanoma patients tyrosinase. (Gradilone, et al., 2010) and as a target for the activation of prodrugs (Jawaid, et al., Materials and Methods 2009) in food industries for modification of food proteins via crosslinking affects. Materials (Monogioudi, et al., 2011) Synthetic melanin is also used for protection against 1. Soil sample- seven soil samples were radiation (UV, X-ray, and gamma ray), collected from the villages around Shirala cation exchangers, drug carriers, region antioxidants, antiviral agents, or immunogen. There is considerable Dist.-Sangli, M.S. India and used in this information representing the great potential study for isolation of actinomycetes. of this enzyme for food, medicine, and agricultural industries as well as for 2. Glycerol asparagine broth, Glycerol analytical and environmental purposes. asparagine agar and Cycloheximide. (Claus and Filip, 1988) 3. Skimmed milk agar, Tyrosine agar, While most of the studies on actinobacteria Peptone yeast extract iron agar, Tyrosine have focused on antibiotic production, only broth and Chloroform. few reports have focused on their enzymatic potential. Streptomyces genus were Methods extensively studied because of their capacity to produce antibiotics and enzymes of Isolation of Actinomycetes industrial importance (Bahrim and Negoita, 2007) Bacterial tyrosinase with new features The soil samples were collected from the like high-temperature stability, (Kong, et al., villages around Shirala region Dist.-Sangli, 2000) or a broader substrate spectrum M.S. India and enrichment of soil samples (Wang, et al., 2000) open further areas of were carried out in Glycerol asparagine applications. The tyrosinase from broth supplemented with Cycloheximide Streptomyces species are non modified (100μg/ml). A 10-fold serial dilution of the monomeric proteins with relatively low sample was prepared up to 10-6 and 0.1ml molecular mass. These enzymes are secreted aliquots of each dilution was inoculated into to the surrounding medium, where they are Glycerol asparagine agar (L asparagine- involved in extracellular melanin 0.1g, K2HPO4-0.1g, glycerol- 1%, trace salt production. solution- 0.1ml, agar- 2.5g, distilled water- 100ml pH-7.4). To avoid the growth of An enzyme tyrosinase has remarkable fungal contaminant medium were properties and many applications in different supplemented with Cycloheximide sectors as mentioned above. Different (100μg/ml). Plates were incubated at room aspects of an enzyme tyrosinase have been 347
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