Screening Some Extracellular Enzymes of Wild Mushrooms from Pt. Ravishankar Shukla University Campus

Srishti Verma¹, Visheshta Valvi², Kamlesh Kumar Shukla^*

^{1, 2, *}School of Studies in Biotechnology, Pt.  Ravi Shankar Shukla University, Raipur (C.G.)

 *Corresponding author E-mail: kshukla26@yahoo.co.in

Abstract:

Wild mushrooms are well known to produce wide range of bioactive metabolites and different types of enzymes. In this study 5 wild mushroom samples were collected which belongs to different groups. Samples were isolated and observed the culture characteristics, during the growth of mycelia many biochemical changes are known to occur, as a result of which enzymes are secreted extracellularly to degrade the insoluble materials into the substrates. Primary screening of extracellular amylase and cellulose were carried out by plate culture method in the GYP media with soluble starch to test the amylase activity and for cellulase, CMC (Carboxymethyl cellulose) plate assay was used. All the mushroom cultures differ in context of extracellular enzymatic activity. The activity of amylase enzyme was substantially higher in all the mushroom cultures. In the screening of cellulase enzyme two cultures were observed as positive. Present study suggests the capacity of these wild mushrooms in the production of biotechnologically useful enzymes with great industrial importance.

Keywords: Amylase, Cellulase, Enzyme, Screening, Wild Mushroom

List of Abbreviations:

                   A-          :       After

                   AV        :       Average

                   B-          :       Before

                CMC        :       Carboxymethylcellulose

                DS            :       Dietary Supplement

                E.E           :       Extracellular Enzyme

                Gxm        :       Glucoonoxylomanan⁺1 Variant

                GYP         :       Glucose Yeast Peptone media

                HEPA      :       High Efficiency Particulate Air

                LAF         :       Laminar Air Flow

                MM         :       Medicinal Mushroom

                MW         :       MiliQ Water

                PDA        :       Potato Dextrose Agar

                pH           :       potential of  Hydrogen

                psi          :       Pounds per Square Inch

                SD           :        Standard Deviation

1.     Introduction

Mushrooms are good source of several enzymes with biotechnological and industrial applications. Enzymes are large protein molecules which catalyze all interrelated reactions in a living cell. The vital concept of enzymes was finally discredited by Buchner (1897). Enzymes that functions insidethe cells in which it was produced are ‘Intracellular Enzymes’. These are correspond to the organized ferments.Similarly which are produced by the cells and secreted to other part of the body are called ‘Extracellular Enzymes’. These enzymes correspond to the unorganized ferments.

Amylase belongs to a group of starch degrading enzymes with importance in the biotechnology industries. Specific enzymes classified within this group include α amylase, β-amylase, gluco-amylase (also known as amyloglucosidase), pullulanase and inso amylase. Amylases are, classified into two categories, endoamylases and exoamylases (Ghosh et al., 2019). Endoamylases catalyse hydrolysis in a random manner in the interior of the starch molecule. This action causes the formation of linear and branched oligosaccharides of various chain lengths. Exoamylases hydrolyse from the non-reducing end, successfully resulting in short end products. A large array of amylases, are involved in the complete breakdown of starch. Enzymatic degradation of starch yields glucose, maltose and other low molecular weight sugars (Gupta et al., 2012). Also, enzymatically - mediated isomerisation of glucose yields high-fructose syrups. Abundant supplies of starch may be obtained from seeds and tubers, such as corn, wheat, rice tapioca and potato. The widespread availability of starch from such inexpensive sources, coupled with largescale production of amylolytic enzymes, facilitates the production of syrups containing glucose, fructose or maltose, which are of considerable importance in the food and confectionery industry. Furthermore, they may be produced quite competitively when compared with the production of sucrose, which is obtained directly from traditional sources such as sugar-beet or sugarcane (Morita et al., 2018). Starch may be hydrolyzed by chemical or enzymatic means.

An enzyme that converts cellulose into glucose or disaccharides is known as cellulases. It is not a single enzyme, it is a group of enzyme which is mainly composed of: endoglucanase, exoglucanase, cellobiohydrolases and β-glucosidase. Cellulose is a homopolymer consisting of a linear chain of several hundred to many thousands of β- anhydroglucose units (β- 1,4 linked D-glucose units). Each of the β-anhydroglucose units consist of 3 hydroxyl group (OH), one primary (C6 position) and two secondary (C2 and C3 position). The intra and inter chain hydrogen bonding network makes cellulose a relatively stable polymer and gives the cellulose fibrils high axial stiffness. Amylase and cellulose both have broad application in textile, food, drinks, paper, detergents, and animal feed industries (James and Lee, 1997; Pandey et al., 2000). Additionally cellulases are used for the production of fruit juice, beer, wine and bioconversion of lignocellulosic biomass to ethanol fuel (Khaund and Joshi,2014).In the present work, enzymatic properties of collected wild mushrooms were investigated to evaluate their bioprospection potential.

2.     Material and Method

2.1.  Collection of wild mushrooms

Fruiting body of wild mushrooms were collected from Pt. Ravishankar Shukla University campus, during rainy season 2020. Note all the morphological characters and photographs were taken. Samples were safely kept in labelled polybags (Natrajan et al., 2005).

2.2.  Isolation of collected samples

Mushrooms are fragile in nature so immediate processing is required. Isolation was done by tissue culture technique (Stamets and Chilton, 1983). Fresh tissue picked from stipe and transferred in the center of freshly prepared culture media (PDA) plates. All plates were sealed and incubate at 26±2°C for 5-7 days. Observe everyday growth of the mycelium, till it fully grow.Cultures were used as mother culture for screening of extracellular enzymatic activity. Stock cultures were maintained on PDA slants at 4°C.

2.3.  Preparation of culture media

Potato Dextrose Agar (PDA) (Chaman et al., 2013) was prepared for isolation and growth of mushroom culture.The medium composition used was as follows:

For 1000ml-

Potato – 250g

Dextrose – 20g

Agar – 15g

Distilled Water – 1000ml/1L

pH – 5.6

All the media constituents, except agar were dissolved in 500ml of distilled water and pH of it was adjusted to 5.6. Thereafter, agar was added slowly to it and heated for complete solubilization. Now, this media was sterilized using autoclave at 121֯C for 45 min. at 15 psi. Thereafter, media was poured on to petri plates under the sterile conditions (inside the LAF). Plates were allowed to solidify.

2.4.  Enzymatic screening of mushroom samples:

2.4.1.    Test for amylase

Primary screening was carried out by plate culture method.Glucose Yeast Peptone (GYP) medium with 0.2% soluble starch was used.Composition of Media was as follows:

The GYP media (1000ml) considered of:

Peptone – 10g

Yeast – 5g

Dextrose – 20g

Agar – 15g

Starch – 2%

pH – 7.0

For Lugol’s Iodine Solution (100ml):

Potassium Iodide- 10g

Iodine- 5g

GYP media was autoclaved and poured in petri plates. All plates were prepared at least one day before inoculation to avoid contamination. Culture inoculation was performed in biosafety cabinet.  Prepared the LAF, Sterile the working area (HEPA-filter, laminar flow hood) for the inoculation where the glasswares and other things (inoculating loop, spirit lamp, parafilm, marker, glovesand bag sealer) were sterilized properly in it. Before inoculation sterilization of the cabinet is necessary, so it was done by giving UV light treatment for 15mins and then wipe the cabinet surface with cotton dipped into 70% ethanol and air flow was on. At the time of fungi inoculation air flow is to be stopped so that spores don’t spread inside the cabinet. After that small amount of growing mycelium from the mother petri-dish were picked up with the help of sterilized inoculating needle and transferred to a fresh petri-dish containing GYP media (pH-6.0)with2% soluble starch as sole carbon source (Claessen et al., 2014). Seal the petri-dishes with parafilm, handle them with care, then placed the petri plates in the incubator at 26±2 ֯C and check everyday growth of the mycelium, till the proper growth observed. After incubation, the plates were flooded by 3% Lugol’s iodine solution and were incubated at room temperature for 5 to 10 minutes. Formation of a clear zone surrounding the colony was considered as a positive result for amylase production. Negative result was set on violet color plate.

2.4.2.    Test for cellulase

For cellulase, CMC (Carboxymethyl cellulose) plate assay was used:

The GYP media (1000ml) considered of:

Peptone – 10g

Yeast – 5g

Dextrose – 20g

Agar – 15g

CMC – 0.5%

pH – 7.0

For congo red solution (100ml):

Congo red- 0.2%

NaCl – 1M

GYP media is prepared autoclaved and poured 10-15ml each petri-plate in a biosafety cabinet, left for solidification. Rest for atleast one day to avoid contamination. After that small amount of growing mycelium from the mother petri-dish were picked up with the help of sterilized inoculating needle and transferred to a fresh petri-dish containing GYP media (pH-6.0) with 0.5% CMC as sole carbon source (Claessen et al., 2014). The Petri plates were incubated at 26±2 ֯C and check everyday growth of the mycelium, till the proper growth observed. After incubation, the plates were stained by 0.2% congo red and rest the plates for 15minutes, then rinse the plates with 1M of NaCl solution.Transparent zone was observed around the colony for positive result. Negative result was set on red color plate.

3.     Result and Discussion

3.1.  Collected wild mushrooms

 Fig. 1: Collected wild mushroom belong to different groups; M1, M3, M5 gilled; M2 polypore; M4 puffball.

Collection was done from the campus of Pt. Ravishankar Shukla University, Raipur, different collection sites are shown in fig 2. Collected fruiting body of wild mushrooms are shown in the fig1. Different mushroom groups (including: gilled, polypore and puffball) were selected for enzymatic screening. Samples were coded as M1, M2, M3, M4 and M5.

Fig 2: Collection sites in the campus of Pt. Ravishankar Shukla University, Raipur.

3.2.  Culture characterization of collected samples

Mycelia cultures of mushroom were grown after 4-8 days of incubation at 27±2֯ C. These mycelia cultures were used as mother culture for the rest of the experiments. Cultures were regularly observed to check their growth pattern. Different isolated culture plates with having supportive media are shown in Fig.3 and culture characterization were depicted in Table 1.

Table 1: Culture characteristics of the mushroom samples.

3.3.  Qualitative screening of enzymes

3.3.1.    Amylase

When the optimum growth observed in GYP media plates,all the 5 different mushroom samples were screened for the production of extracellular amylase enzymes.Zone measurement of amylase activity has been presented in table 2. Qualitative test is based on visibility a clear inhibition zone was observed for positive result which shown in fig. 4

Fig.4: The illustration of amylase activityzone formation (left plate showing positive result and right plate showing negative result).

 Table 2: Zone measurement of amylase activity.

Note: The activity assays of all the enzymes were conducted in triplicate, with the preparation of each reaction control.As per Robert et al., (2018), zones with the diameter above (< 0.1cm) were considered positive for enzymatic activity, cm= centimeters.

Fig.5: Amylase activity of different mushroom samples.

All the samples investigated showed positive result for the production of amylase activity.The fig. 5 shows various activity zones formed by different mushroom colonies.

3.3.2.    Cellulase

When the optimum growth observed in GYP media plates containing carboxymethyl cellulase, all the 5 different mushroom samples were screened for the production of cellulose enzyme. Zone measurement of cellulase activity has been presented in table no. 3. Qualitative test is based on visibility a clear inhibition zone was formed in positive result and no zone formation indicated the negative result which are shown in fig 6.

Fig.6: Qualitative Screening Test for Cellulase

Table 3: Zone Measurement of cellulase activity.

Note: The activity assays of all the enzymes were conducted in triplicate, with the preparation of each reaction control. As per Robert et al., (2018), zones with the diameter above (< 0.1cm) were considered positive for enzymatic activity, cm= centimeters.

Fig. 7: Cellulase activity of different mushroom samples.

All the samples were investigated for the production of cellulase activity, three samples shown positive result but two were negative. Fig 6 shows different activity zones of mushroom colonies.

3.4.  Estimation of total enzyme activity

Based on the average activity of both the enzymes total activity was recorded and present in table 5.

Table 4: Total activity of enzymes.

Note: Robert et al., (2018) described that, zones with the diameter above (<0.1cm) were considered as (+) present, (+ +) moderate, (+ + +) strong activity, no activity were indicated as (-) absence of enzymatic activity (Each value represents the SD of three replicated cultures).

Fig. 8: Zone measurement of both the enzymes.

3.4.1.    Detection of total amylase activity

As the total enzymatic activity of amylase the results were depicted in the Table 5. All tested mushroom samples exhibited a positive result in this experiment, with slight different intensities between the samples. As compared to the average activity, the highest and lowest activities were observed in M3 with 1.53 cm activity and on the other hand M1 with 0.36cm activity.The result of this work was similar to the findings of Krupodorova et al., (2021); Debnath et al.,(2020); Goud et al.,(2009).

3.4.2.    Detection of total cellulase activity

As the total enzymatic activity of amylase the results were depicted in the Table 5. Out of the 5 tested samples 3 were positive and 2 were negative for cellulose test. As compared to the average activity, the highest and lowest activities were observed in M3 with 0.56cm activity and on the other hand M4 with 0.2cm activity. This study was closely related to the findings of Krupodorova et al., (2021); Debnath et al.,(2020); Goud et al.,(2009).

4.     Conclusion

The study of mushroom enzymatic activity is one of the important stages of understanding their physiological and biochemical features, and the revealing of macrofungi from different ecophysiological and taxonomical group. According to the present study it may be concluded that wild mushrooms are great source of extracellular amylase and cellulose having good activity at carboxymethyl cellulose (CMC) substrate. This can be considered the most interesting because of its enzyme amount and their good visualization. One of the main results of the study was obtaining for the first time data about the ability of some fungi to produce one or the other extracellular enzymes. This screening experiment helped us to select for future quantitative enzymatic determination of some perspective mushrooms. Preliminary screening of these mushroom species can provide a base work for the researchers to explore up to the purification of elucidation level.  In this work studied, the enzyme activities of different mushroom samples showed different reactions with individual growth rates. The availability of enzymes from mushrooms also remains a best viable option which can be used as a source for industrial amylase and cellulose with different applications such as production of detergents, paper, coffee, pulp, ethanol, textile, and many pharmaceutical industries.

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