Useful Extracellular Enzymatic Activity of Mycelial Culture of Some Edible Mushrooms of Odisha

Biodegradation involves microbes for the solublization of insoluble macromolecules like keratin, cellulose, collagen, lignin, chitin and casein which depends on the secretion of extracellular enzymes in their substrates Abbas et al. [1], Bockle et al. [2], Friedrich et al. [3], Allpress et al. [4] this kind of activities shown by the microbes helps in the mineral balance in nature. In the same way term “Biodeterioration” is mainly pronounced for the undesirable activity of microbes on useful substances, which causes immense economic losses. Overall in all the conditions, microorganisms produces extracellular enzymes, which helps for hydrolyzing the complex organic substances into simpler forms that can be utilized and assimilated by them or released in surrounding free environment Rao et al. [5], Kumar and Takagi [6] Kirk et al. [7] Nehra [8], Amoozegara et al. [9]. The production of useful enzymes by plant and animal sources such as proteases, cellulase, Xylanase and lipase are not sufficient to meet the current industrial demands which has drawn upon the interest of researchers towards microbial enzymes, since they have been known to possess almost all features desired for useful biotechnological as well as other industrial applications Forgatty and Kelly, [10] Singh [11] Beg et al. [12], Ellaiah et al. [13] Nascimento et al. [14], Gouda et al.[ 15]. Ectomycorrhizal fungi show varying phosphatase activity between species, resulting in different efficiency of phosphate solublization of host plant Ho and Zak [16]. Moreover factors like edaphic components, pH and mineral constituents can modify the conformation of enzymes and affect their activities Eivazi and Tabatabai [17].


Introduction
Biodegradation involves microbes for the solublization of insoluble macromolecules like keratin, cellulose, collagen, lignin, chitin and casein which depends on the secretion of extracellular enzymes in their substrates Abbas et al. [1], Bockle et al. [2], Friedrich et al. [3], Allpress et al. [4] this kind of activities shown by the microbes helps in the mineral balance in nature. In the same way term "Biodeterioration" is mainly pronounced for the undesirable activity of microbes on useful substances, which causes immense economic losses. Overall in all the conditions, microorganisms produces extracellular enzymes, which helps for hydrolyzing the complex organic substances into simpler forms that can be utilized and assimilated by them or released in surrounding free environment Rao et al. [5], Kumar and Takagi [6] Kirk et al. [7] Nehra [8], Amoozegara et al. [9]. The production of useful enzymes by plant and animal sources such as proteases, cellulase, Xylanase and lipase are not sufficient to meet the current industrial demands which has drawn upon the interest of researchers towards microbial enzymes, since they have been known to possess almost all features desired for useful biotechnological as well as other industrial applications Forgatty and Kelly, [10] Singh [11] Beg et al. [12], Ellaiah et al. [13] Nascimento et al. [14], Gouda et al. [ 15]. Ectomycorrhizal fungi show varying phosphatase activity between species, resulting in different efficiency of phosphate solublization of host plant Ho and Zak [16]. Moreover factors like edaphic components, pH and mineral constituents can modify the conformation of enzymes and affect their activities Eivazi and Tabatabai [17].

S. no Species Amylase Protease Lipase Xylanase Cellulase L-Asparaginase
Organic acid production IAA Phosphate solublizing

Collection and identification
Mushrooms species of R. lepida, R. brevipes, R. nigricans, M. procera, V. volvaceae, L. tuberregium and C. indica were collected from tropical moist deciduous and semi ever green forest of Odisha (India). For identification, macroscopic and microscopic examination of pileus, stipe, veil, ring, volva, lamellae and gills etc. were taken into consideration according to Largent [33].

Preparation of master culture plate
Wild edible mushrooms were collected from the different forest divisions of Odisha after gathering the information regarding the edibility of these mushrooms. They were brought in the laboratory and surface sterilization was done by using 0.1% HgCl 2 solution, subsequently washing was done with the sterilized distilled water and tissue was transferred to the malt extract agar medium aseptically and incubated at 28 °C to get mushroom mycelium. Subsequent inoculations were done in order to get pure culture.

Amylase activity
Starch agar media was used in order to screen the starch hydrolysis activity, inoculation of the respective mushroom mycelium was done and plates were incubated at 28°C. After the appreciable amount of the growth of mycelium, 1% iodine solution was added to the plates. Clear zone was observed for the organisms showing positive results.

Cellulase activity
Sodium salt of carboxymethylcellulose (0.5%) was used as the substrate for this test. After the mycelial colonization, the plates were flooded with 0.2% Congo red solution, after the incubation period of 15 minutes plates were washed with 1M NaCl solution and clear zone was observed for the cellulase producing species.

Lipase activity
Spirit blue agar media with tween-20 was used for the screening of lipase activity. After the requisite amount of growth of mushroom mycelium a clear precipitate was observed for the lipase producers.

Protease activity
Gelatin agar media was used for the assessment of extracellular protease activity. After the required amount of the mycelial growth, plates were flooded with the reagent containing 15% HgCl 2 and 20% HCl. A visible zone was observed for the protease producing organisms.

Xylanase activity
Medium containing xylan was used for the screening of Xylanase activity in mushroom species. After the appreciable growth of mushroom mycelia in the plate it was flooded with 0.1% Congo red, incubated for 30 minutes and washed with 1M NaCl solution. Plates were observed for the formation of clear zone for the positive organism.

L-Asparaginase activity
For testing L-Asparaginase activity, medium containing 1% L-asparagine was used where it served as substrate, after the mycelial growth in the plate it was flooded with Nessler's reagent. Plates showing pink coloration after the addition were recorded as extracellular L-asparaginase producer.

Organic acid production
Modified Sperber's medium was used for the screening of the mushroom mycelium for the production of organic acid where bromocresol green was used as a indicator, plates after the inoculation of the respective mushroom mycelium were incubated at 28 °C in triplicates, the acid production in the medium was determined by the yellow zone formed around the colonies.

IAA production
The mushroom mycelium were inoculated in triplicates in a specific medium i.e JNF medium and pH 5.5 was maintained, the plates were incubated at 28 °C for 10 days. After observing proper growth in plates Salkowski' S reagent was added, pink

Results and Discussion
Amylase is the widely distributed fungal enzyme used for the hydrolysis of starch, reports suggests that these enzymes are used for the fruiting body formation mainly in the ectomycorrhizal mushrooms Terashita et al. [35], Hur et al. [36], Hur et al. [37]. In the present studies only M. procera and R. brevipes showed a very little amylase activity where as no activity was found in rest of the mushroom species. Reports from Sławińska and Kalbarczyk [38] shows that cellulase activity in some Pleurotus species is more than amylase activity hence similar findings was recorded in the present studies in case of R. brevipes, R. nigricans, R. lepida and C. indica. A good cellulase activity was found in the R. nigricans while moderate activity was recorded in R. brevipes.
Fungal lipases are produced extracellularly and widely used in the food industry (Sharma et al., 2001). However, very less findings regarding lipolytic activity from the edible fungi has been reported such as Agaricus bisporus Wang et al. [39], Lentinus edodes Zhu et al. [40] and Antrodia cinnamomea Lin et al. [41] and Shu et al. [42] in the present studies lipolytic activity was seen in R. brevipes and L. tuberregium whereas no activity was found rest of the species. Protease is served as an industrially as well as pharmaceutically important enzyme which is distributed among the various sources Rao et al. [43] Choi and Shin [44], Choi and Sa [45] some mushroom species also produce protease enzymes Lee et al. [46], Park et al. [47], Kim et al. [48]. A very weak proteolytic activity was observed in R. brevipes whereas no such activity was seen in other studied mushroom mycelia.
In general it is considered that most of the microbial L-asparaginase is intracellular in nature except some, which is secreted outside the cell Savitri et al. [49], Narayana et al. [50]. However no records are available regarding some species which is presently studied such as R. brevipes, R. nigricans and R. lepida regarding the exogenous production of L-asparaginase. Present findings suggest that maximum L-asparaginase activity was recorded in R. nigricans and R. brevipes where as moderate production of the enzyme was recorded in M. procera and R. lepida.
Many researches demonstrate that IAA can be synthesized by utilizing tryptophan by plants and bacteria. Only few reports are there regarding phytohormonic potentialities of mushrooms, some reports from Tsivileva et al. [51] and Bose et al. [52] suggests that even mushrooms are also capable of synthesizing IAA if tryptophan is used as a substrate. In the present work good activity was shown by the L. tuberregium, moderate activity by R. nigricans and C. indica where as poor activity was recorded in R. lepida and R. brevipes.
Phosphate solublization by some ectomycorrhizal mushrooms are important aspect to be studied, reports suggest that ectomycorrhizal mushrooms solublises insoluble phosphate and helps plant for the P intake Arumanayagam and Arunmani [53]. R. nigricans showed the best phosphate solublization while in all the mushroom species studied no activity was found. Wild edible mushrooms are reported for the organic acid production Valentano et al. [54]. In the present screening, extracellular production of organic acid was seen only in R. nigricans where other species showed no such activity [55].
The availability of enzymes from mushroom species also remains a best viable option which needs to be further explored. Production of enzymes by the agro waste can used for production of the enzymes by the mushroom mycelium which may be the alternative path for the reuse of agro wastes, since in many cases agro wastes serves as a substrates for the mushroom cultivation. Presently screened enzymes can meet the industrial needs if the optimization of these enzymes will be done by amendment of nutritional and other factors. Preliminary screening of these mushroom species can provide a base work for the researchers to explore upto the purification and elucidation level.