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.
SNo. Characteristics
|
Mushroom samples
|
|
M2
|
M3
|
M4
|
M5
|
1
|
Color
|
White
|
White
|
White
|
White
|
White
|
2
|
Form
|
Irregular
|
Circular
|
Filamentous
|
Filamentous
|
Rhizoid
|
3
|
Margin
|
Filiform
|
Entire
|
Filiform
|
Filiform
|
Serrate
|
4
|
Elevation
|
Raised
|
Flat
|
Raised
|
Flat
|
Raised
|
5
|
Opacity
|
Opaque
|
Opaque
|
Opaque
|
Opaque
|
Translucent
|
Fig.3:
Colony of different mushroom samples on PDA; R- reverse phase, F-front phase.
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.
SNo
|
AC. No.
|
Zone
measurement (cm)
|
Activity
|
Result
|
Before Activity
|
After activity
|
1
|
M1
|
4.13±0.50
|
4.5±0.20
|
0.36±0.20
|
Positive
|
2
|
M2
|
2.63±0.35
|
3.36±0.37
|
0.73±0.11
|
Positive
|
3
|
M3
|
2.0±0.26
|
3.53±0.25
|
1.53±0.49
|
Positive
|
4
|
M4
|
1.46±0.05
|
2.63±0.15
|
1.16±0.15
|
Positive
|
5
|
M5
|
1.66±0.15
|
2.23±0.30
|
0.56±0.15
|
Positive
|
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.
SNo
|
AC. No.
|
Zone
measurement (cm)
|
Activity
|
Result
|
Before Activity
|
After activity
|
1
|
M1
|
1.6±0.10
|
1.36±1.01
|
0.43±0.05
|
Positive
|
2
|
M2
|
2.13±0.20
|
2.13±0.20
|
-
|
Negative
|
3
|
M3
|
1.06±0.11
|
1.63±0.15
|
0.56±0.05
|
Positive
|
4
|
M4
|
1.20±0.20
|
1.40±0.17
|
0.20±0.10
|
Positive
|
5
|
M5
|
0.76±0.11
|
0.76±0.11
|
-
|
Negative
|
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.
S.No.
|
Mushroom samples
|
Total Activity
|
Amylase
|
Cellulase
|
1
|
M1
|
(+)
|
(+)
|
2
|
M2
|
(+ +)
|
(-)
|
3
|
M3
|
(+
+ +)
|
(+
+)
|
4
|
M4
|
(+ + +)
|
(+)
|
5.
|
M5
|
(+
+)
|
(-)
|
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.
References
Chaman
S, Sharma G, Shalini, Reshi AK (2013) Study of antimicrobial properties of Catharanthus roseus by agar well
diffusion method. International of
Pharmaceutical and Applied Sciences, 3:
65-68.
Claessen, D., Rozen, D. E., Kuipers, O. P.,
Søgaard-Andersen, L., & Van Wezel, G. P. (2014). Bacterial solutions to
multicellularity: a tale of biofilms, filaments and fruiting bodies. Nature
Reviews Microbiology, 12(2): 115-124.
Debnath, G., Das, P., & Saha, A. K. (2020). Screening and
characterization of extracellular cellulase enzyme produced by wild edible
mushroom Pleurotus giganteus. Indian Journal of Natural Products and
Resources (IJNPR)[Formerly Natural Product Radiance (NPR)], 10(3):
195-199.
Fen, L., Xuwei, Z., Nanyi, L., Puyu, Z., Shuang, Z, Xue, Z.,&
Haiping, L. (2014). Screening of lignocellulose-degrading superior mushroom
strains and determination of their CMCase and laccase activity. The
Scientific World Journal.
Ghosh, A., Chatterjee, B., & Das, A. (1991).
Purification and characterization of glucoamylase of Aspergillus terreus NA‐170
mutant. Journal of Applied Bacteriology, 71(2): 162-169.
Goud, M. J. P., Suryam, A., Lakshmipathi, V., & Charya, M. S.
(2009). Extracellular hydrolytic enzyme profiles of certain South Indian
basidiomycetes. African Journal of Biotechnology, 8(3): 354-360.
Gupta
R., Gigras P., Mohapatra H., Goswami V.K. and Chauhan B. (2012). Microbial α-amylase: a b α-amylase
biotechnological perpective. Journal of
Process Biochemistry: 20: 1-18.
Gupta,
R., Q.K. Beg and P. Lorenz. (2012). Bacterial alkaline proteases: molecular
approaches and industrial applications. App. Microbiol. Biotechnol. 59: 15–32.
James, J. A., & Lee, B. H. (1997). Glucoamylases: microbial sources,
industrial applications and molecular biology—a review. Journal of Food
Biochemistry, 21(6): 1-52.
Khaund, P., & Joshi, S. R. (2014). Enzymatic profiling of wild
edible mushrooms consumed by the ethnic tribes of India. Journal of the
Korean Society for Applied Biological Chemistry, 57(2): 263-271.
Krupodorova, T., Ivanova, T., & Barshteyn, V. (2021). Screening of
extracellular enzymatic activity of macrofungi. Journal of Microbiology,
Biotechnology and Food Sciences, 315-318.
Morita,
H., Matsunaga, M., Mizuno, K., & Fujio, Y. (1998). A comparison of raw
starch-digesting glucoamylase production in liquid and solid cultures of
Rhizopus strains. The Journal of General and Applied Microbiology, 44(3):
211-216.
Natrajan, K.C. et
al. (2005). Biodiversity of Agarics from Nilgiri Biosphere Reserve, Western
Ghats, India. Current Science. 12: 1890-1892.
Pandey A, Nigam P, Socol CR, Socol VT, Singh D, and Mohan R (2000).
Advances in microbial amylases - review.
Biotechnol Appl Biochem31:
135–52.
Robert
de Oliveira Gusmão1, Lucas Santos Solidade1, Lício Fábio Almeida Andrade
Ferreira1, Fábia Giovana do Val de Assis2 , Alisson Rodrigues da Cruz1 and
Patrícia Lopes Leal (2018). Filamentous
fungi producing enzymes under fermentation in cassava liquid waste. Acta Scientiarum. Biological Sciences, 40: e41512.
Stamets, P., &
Chilton, J. S. (1983). The mushroom cultivator. First Washington.