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com

Production and characterization of fungal extracellular β-mannanase

Felicia C. Adesina*, Omolola A. Oluboyede, Oludare S. Agunbiade, Bolanle O.

Aderibigbe, Olatunji H. Kolade, Ekundayo M. Oluwole.
Department of Microbiology, Lead City University, Ibadan, Nigeria
*adesinafelicia@yahoo.com

ABSTRACT
Fungal isolates obtained from degrading wood were screened for their β-mannanase
production on plate agar containing locust bean gum as main carbon source. Two isolates
with relatively large zones of hydrolysis of mannan on the plate agar were identified and used
to produce β-mannanase in submerged fermentation and assayed at two day interval for six
days in stationary and agitated flasks. The amount of protein produced by these isolates into
the liquid medium was also monitored every other day for six days in differently incubated
flasks. β-mannanase produced by the isolates was afterwards characterized. All the isolates
screened showed zones of clearance on plate agar. Isolates with relatively large zones of
clearance among the screened isolates were identified as Aspergillus niger and Trichoderma
spp. Highest mannanase activity recorded for A. niger (0.427U/ml) and Trichoderma
spp(0.473U/ml) was on the 4th day of incubation in agitated flasks. Highest total protein
released by A. niger and Trichoderma spp. into the medium were 1.296mg/ml and
1.158mg/ml respectively on days 4 and 6 in agitated flasks. A.niger and Trichoderma spp.
had their highest specific β-mannanase activity of 0.466U/ml in agitated flask and 1.066U/ml
in stationary flask on the 6th and 4th days of incubation respectively. β- mannanase by both
isolates showed highest activity at pH 5 while peak activity of β- mannanase by A. niger was
at 30oC and Trichoderma spp. was at 30oC and 50oC.
Keywords: β - mannanase, Trichderma spp., Aspergilus niger, Screeening. Degrading wood
{Citation: Felicia C. Adesina, Omolola A. Oluboyede, Oludare S. Agunbiade, Bolanle O.
Aderibigbe, Olatunji H. Kolade, Ekundayo M. Oluwole. Production and characterization of
fungal extracellular β-mannanase. American Journal of Research Communication, 2013:}
www.usa-journals.com, ISSN: 2325-4076.

INTRODUCTION
β -d-Mannopyranosyl residues are widely distributed in nature occurring abundantly as
building blocks of several plant structural and storage polysaccharides (Buckeridge and dos
Santos, 2000). β -1,4-linked d-mannopyranosyl residues also form the main chain of
galactomannans occurring in various plant They are abundant particularly as building blocks
of several plant structural and storage polysaccharides. Together with d-glucopyranosyl
residues, d-mannopyranosyl residues form a β -1,4-linked backbone of galactoglucomannans
which is a type of hemicellulose in wood, (Timell, 1967; de Vries and Viser, 2001). Fungi
like Aspergillus tamarii (Civas et al., 1984), A. niger (Ademark et al., 1998; Siti Norita et al.,
2010), Sporotrichum cellulophilum (Araujo & Ward, 1991), Scopulariosis candida (Mudau
and Setati, 2008), Aspergillus fumigatus (Puchart et al., 2004) and Trichoderma reesei
(Arisan Atac et al., 1993) have been targeted for β-mannanases isolation. β-endomannanases
commonly produced by Aspergilli are responsible for degradation of mannan component of

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plant polysacchrides. β - mannanases hydrolyze the backbone of galactoglucomannans,

producing mannooligosaccharides. The ability of A. niger to grow in palm kernel cake waste
containing mannan-based polysaccharides, for manannase production has been reported
(Abd-Aziz et al., 2008). The biotechnological potential of mannan-hydrolysing enzymes, in
particular the mannanases, has been demonstrated within various industries. Industrially
useful mannanase have recently attracted attention due to their role in the pulp and paper
industry to remove the hemicelluloses from pulps (Gubitz et al.,1997) and in pulp bleaching
processes. This positive role has minimized the use of environmentally harmful bleaching
chemicals in the pulp and paper industry (Lahtinen et al., 1995; Cuevas et al., 1996).
Mannanases have potential application in animal feed production (Wu et al., 2005; Lee et al.,
2005; Sae-Lee, 2007) and laundry detergents (Schafer et al., 2002). Bioconversion of
agriculture waste containing mannan-based polysaccharides into valuable products such as
animal feeds also required microorganisms capable of producing mannan degrading enzymes.
Mannanases are also used for the extraction of vegetable oils from leguminous seeds and the
clarification of fruit-juices in the food industry (Christgau et al., 1994). They are useful in
reducing the viscosity of extracts during manufacture of instant coffee, chocolate and cacao
liquor (Belitz & Grosch, 1987; Francoise et al., 1996) to lower the cost for subsequent
evaporation and drying (Wong & Saddler, 1993). Mannanases are potentially used in the
pharmaceutical industry for the production of physiologically interesting oligosaccharides
(Christgau et al., 1994). The objective of this study was to screen 15 fungal isolates obtained
from degrading wood for the production of β-mannanase, induce the most potent fungal
isolates to produce the enzyme in submerged fermentation flasks and characterize the enzyme
biochemically.

MATERIALS AND METHODS

Sample sources
Decaying wood samples were collected into clean, properly labeled, polythene bags obtained
from different locations in Ibadan metropolis, Oyo state Nigeria where they are cast as waste.
The samples were taken to the laboratory for further work.
Preparation of Culture medium for fungal isolation
Potato dextrose agar medium was prepared in 500ml Erlenmeyer flask according to
manufacturer’s instruction and sterilized by autoclaving at 121oC (15 psi) for 15 minutes.
This medium provided a balance mixture of required nutrients that permitted the rapid growth
of the fungal isolates.
Isolation and screening of fungal isolates
Each degrading wood sample was macerated into powder and transferred into potato dextrose
agar plates. The plates were thereafter incubated at 25oC + 2 oC. Screening of the isolates for
mannanase activity was performed using a chemically defined medium with the following
composition in g/l: Mg SO4.7H2O-0.5g; Fe SO4.7H2O-0.05g; KH2PO4-1g; agar 10g; KCl-
0.5g;NaNO3-2g, Locust bean gum-0.5% (w/v) dissolved in 0.1M citrate buffer at pH5.6. The
medium was sterilized by autoclaving at 121°C and 15 psi for 15 minutes and afterwards left
to cool to 40oC and decanted to petridishes. Agar plates were inoculated with the fungal
isolates and incubated for 5 days at 25oC + 2 oC. Culture plates were afterwards flooded with
0.4% (w/v) Congo red solution and 0.5M NaCl solution and for 15 minutes and mannan
hydrolysis observed by appearance of clear zones around the fungal growth (Downie et al.,

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1994).. The relative activity was calculated as the percentage of the ratio of the diameter of
the clearing zone to the diameter of the fungi growth. Isolates with high relative mannanase
activities were selected for production of the enzyme. Selected isolates were maintained on
agar slants of a medium composed of the following in g/l: Mg SO4.7H2O- 0.5g ; Fe
SO4.7H2O- 0.05g; KH2PO4 -1g; agar 10g; KCl-0.5g;NaNO3-2g, Locust bean gum-0.5%(w/v)
dissolved in 0.1M citrate buffer with final pH adjusted to 5.6 and stored at 4oC until used
(Wiley et al., 2008).
Identification of selected Fungal isolates
The selected fungal isolates were identified using their cultural and morphological
characteristics, (Domsch et al.,1980) and (Kiffer Morrelet, 1994).
Mannanase Production
50 ml aliquots of a chemically defined medium with the following composition in g/l: Mg
SO4.7H2O- 0.12g ; Fe SO4.7H2O- 0.05g; KH2PO4 -0.25g; KCl-0.5g;NaNO3-0.25g, Locust
bean gum-0.5%(w/v), peptone water-3.75g 0.1M in citrate buffer( pH 5.6) were dispensed in
250ml Erlenmeyer flasks. The medium was sterilised at 121oC for 15 minutes and allowed to
cool afterwards. Each flask was inoculated with 2ml fungal spores suspension obtained from
7 day old slant cultures and incubated for 6 days at 28+2oC. Some of the incubated flasks
were subjected to agitation while some were incubated in stationary state. Each treatment was
carried out in triplicates and the results obtained throughout the work were the arithmetic
mean of three experiments + mean error. Samples from each flask were taken at 2 day
intervals for analysis.
Preparation of crude Enzyme
At the end of each incubation period, fungal cells were separated by contact method (Krishna
et al.,1996) and crude filtrates were centrifuged at 6000 rpm for 15 minutes in a cold
centrifuge to remove the cells. The supernatant was taken as the crude enzyme solution
(Naggar et al., 2006).
Determination of Protein Content
Protein content was determined by the method of Lowry et al., (1951). Standard curve was
plotted using Bovine serum albumin (Sigma, U.SA.).
Assay for mannanase activity
0.5 % (w/v) locust bean gum dissolved in 0.1 M citrate buffer at pH5.6 was used as the
substrate mixture. 1 ml of the substrate mixture was added to 1ml of the crude enzyme
solution and incubated in a water bath at 50oC for 15 minutes. Afterwards 1ml of
dinitrosalicylic acid (DNS) was added to 1 ml of each enzyme – substrate mixture and boiled
for 5 minutes. The absorbance of the mixture was thereafter measured at 540 nm in a
spectrophotometer ((752W UV-VIS Spectrophotometer). The amount of mannose released
was determined by the method of Miller, (1959). One unit of mannanase was defined as the
amount of mannanase that released 1 micro mole of mannose in one millimeter of the
reaction mixture under the assay conditions.

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Characterisation of Enzyme
The crude enzyme solution was used for this test; the temperature optima of the crude β-
mannanases were determined by incubating the enzymes with 0.5%(w/v) locust bean gum
substrate for 30 min at different temperatures ranges of 30oC, 35oC, 40oC, 45oC and 50oC at
pH 5.6, followed by determination of reducing sugars in the mixture. Optimum pH for the
enzyme was also determined by monitoring the amount of reducing sugar released at
different pH ranges of 3, 4, 5, 6, 7 and 8 for 30 minutes.

RESULTS
Fifteen fungal isolates were obtained, the mean relative mannanase activities of the isolates
on plate screening test are shown on Table 1. Based on their high mean relative mannanase
activities (Table 1), isolates 3e2 and 3i were selected and identified.Isolates 3e2 and 3i were
identified as Aspergillus niger and Trichoderma sp. based on their cultural and
morphological characteristics respectively (Domsch et al.,1980) and (Kiffer Morrelet , 2000).
Table 1 shows the relative mannanase activities of screened fungal isolates from degrading
wood material. Isolates 3i(61.67) had the highest relative mannanase activity among them
followed by isolate 3e2(36.70), this informs why they were chosen for the production of
mannanase in submerged fermentation.

Table 1: Mean relative β-mannanase activity of isolates

ISOLATES CODE Mean relative mannanase

activities(%) of isolates
3a 19.33+0.064d
2f 15.67+0.176gh
2e 18.67+0.176e
3i 61.67+0.145a
2a 12.67+0.176j
1g 19.13+0.231d
3e1 11.73+0.634k
3d 16.00+0.231g
2b 13.70+0.174h
2d 13.67+0.073h
1b 13.33+0.145i
1a 18.33+0.145e
3e2 36.70+0.291b
3g 23.70+0.136c
1c 17.33+0,088f
AF 22.67+0.12cd

Each value is the mean of triplicate results + mean error. Means with different superscript within the same row are
significantly different (p<0.05).

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Tables 2 shows the mannanase activities of each isolate on days 2, 4 and 6 of incubation in
stationary and agitated flasks respectively. Trichoderma spp. had its highest mannanase
activity in stationary flasks on the second day of incubation and the least on the sixth day
with no significant differences in activities on days 2 and 4 of incubation. However A niger
had the highest mannanase activity on the 6th day of incubation and least on the 2nd day, there
was significant difference in mannanase activity of this fungus on the days assays were
carried out. In agitated flasks, mannanase activity of Trichoderma spp. and A.niger were
highest on the 4th day if incubation and least on the second day for both fungi. There was
significance difference in the mannanase activities of A.niger in all the days assays were
carried out while for Trichoderma spp., there was no significant difference in activity on the
second and sixth days of incubation.
Total protein released into the medium by each of the fungal isolates are shown on Table 3,
protein was maximally produced into the medium in stationary flasks by Trichoderma spp.
and A.niger on the 6th day of incubation and least on the 4th and 2nd days respectively. In
agitated flasks, protein was released maximally by Trichoderma spp. on the 4th day and by A.
niger on the 6th day of incubation. Least amount of protein was released by Trichoderma spp.
into the medium in agitated flasks on the 6th day and by A. niger on the 2nd day of incubation.
There was significant difference in the amount of protein released by each fungus on the
different days assays were carried out.
Results of specific β - mannanase activity of the isolates were shown on Table 4.
Trichoderma spp. had its highest specific mannanase activity on day 4 of incubation while A.
niger had its highest on day 6 in stationary flasks. In agitated flasks, however Trichoderma
spp. had highest specific mannanase activity of 0.466U/mg on day 6 while 0.402U/mg was
recorded for A. niger on day 6 of incubation respectively. There was significant difference in
specific mannanase activities of each fungus throughout incubation.
Tables 5 and 6 show the result of characterisation of the β – mannanase produced by the
isolates. β – mannanase produced by Trichoderma spp. had optimum activity at two
temperatures (30oC and 50oC). Optimum activity for β –mannanase produced by A.niger was
at 30oC, although the activity was still stable at 35oC with a value that is not significantly
different from the value obtained at 30oC as shown on Table 5. There was also no significant
difference in the activity of this enzyme at 40oC and 45oC however the values dropped to
about half of the values obtained at 30oC and 35oC.

Mannanase produced by Trichoderma spp. had optimum activity at pH 5 while its activity
was least at pH 8. The activity of this enzyme started to drop pH 5 until it was least at pH8,
the activity of this enzyme at pH 7 was not significantly different from its activity at pH 8 as
shown on Table 6. In this work, A.niger produced a mannanase with optimum activity of
0.435U/ml also at pH 5 while its activity was lowest at pH 7.

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Table 2: β- Mannanase activities of Isolates on Different Incubation Periods in

stationary and agitated flasks

Mannanase activities (U/ml) of isolates on Different Incubation Period (Days)

(Stationary flasks) (Agitated flasks)
ISOLATES Day2S Day 4S Day 6S Day 2A Day 4A Day 6A
Trichoderma 0.263+0.002a 0.259+0.002a 0.066+0.001b 0.074+0.002b 0.473+0.003a 0.081+0.002b
sp.
A. niger 0.075+0.001c 0.143+0.002b 0.325+0.002a 0.054+0;002c 0.427+0.002a 0.172+0.002b
Each value is the mean of triplicate results + standard mean error. KEY: 2S, 4S, 6S= Cultures in Stationary flasks. 2A, 4A,
6A= Cultures in agitated flasks. Means with different superscript within the same row are significantly different (p<0.05).

Table 3: Total Protein released by isolates into fermentation medium in stationary and
agitated flasks

ISOLATES Protein content(mg/ml) on Different Incubation Period (Days)

(Stationary flasks) (Agitated flasks)
Day2S Day 4S Day 6S Day 2A Day 4A Day 6A
Trichoderma 0.694+0.002b 0.244+0.002c 0.946+0.002a 0.707+0.003b 1.158+0.002a 0.392+0.001c
sp.
A. niger 0.269+0.002c 0.871+0.002b 0.945+0.002a 0.882+0.002c 1.067+0.002b 1.296+0.002a
Each value is the mean of triplicate results + standard mean error. Means with different superscript within the same row are
significantly different (p<0.05). KEY: 2S, 4S, 6S= Cultures in Stationary flasks. 2A, 4A, 6A= Cultures in agitated flasks

Table 4: Specific β - Mannanase Activity of Isolates on different Days of Incubation in

stationary and agitated flasks

ISOLATES Specific mannanase activity of isolates (U/mg) on Different Incubation Days

(Stationary flasks) (Agitated flasks)
Day2S Day 4S Day 6S Day 2A Day 4A Day 6A
Trichoderma 0.373+0.001 1.066+0.002 0.070+0.002 0.112+0.001 0.411+0.002 0.466+0.001a
b a c c b

sp.
Aspergillus 0.276+0.002b 0.165+0.002c 0.345+0.002a 0.063+0.002c 0.402+0.004a 0.134+0.002b
niger
Each value is the mean of triplicate results + standard mean error. Means with different superscript within the same row are
significantly different (p<0.05). KEY: 2S, 4S, 6S= Cultures in Stationary flasks. 2A, 4A, 6A= Cultures in agitated flasks

Table 5: Optimum temperature of β - Mannanase produced by isolates

Isolates Mannanase activities(U/ml) of isolates at Different Temperatures(oC)

30 35 40 45 50
Trichoderma sp. 0.411+0.007a 0.358+0.001b 0.134+0.009c 0.072+0.010d 0.398+0.003a
A.niger 0.573+0.027a 0.522+0.020a 0.279+0.009b 0.278+0.009b 0.162+0.006c
Each value is the mean of triplicate results + mean error. Means with different superscript within the same row are
significantly different (p<0.05).

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Table 6: Optimum pH of β -Mannanase produced by isolates

Isolates Mannanase activities(U/ml) of isolates at Different pH

3 4 5 6 7 8
Trichoderma 0.288+0.003b 0.230+0.009c 0.418+0.007a 0.113+0.00e 0.191+0.003d 0.104+0.003e
sp.
A.niger 0.245+0.001c 0.228+0.003d 0.435+0.001a 0.339+0.001b 0.112+0.001e 0.154+0.001d
Each value is the mean of triplicate results + mean error. Means with different superscript within the same row are
significantly different (p<0.05).

DISCUSSION
Growing interest in potential application of beta 1, 4 - mannanase in various industries has
triggered increasing research towards biochemical characterisation of these enzymes.
Consequently beta mannanases of fungal and bacterial sources have been characterized
(Ferreira and Filho, 2004).
Two of the isolates obtained from the decaying wood samples, namely Trichoderma spp. and
Aspergillus niger had relatively high mannanase activity. This is in correlation with other
researchers who have used Aspergillus niger and Trichoderma sp. in the production of
various enzymatic and non-enzymatic bioproteins such as mannanase, glucanase and
cellulases (Pentilla et al., 1998; Viikari et al., 1998; Fadel 2001; Immanuel et al., 2007).
Hemicellulloses are one of the most abundant polymers in nature and one of the very
important enzyme for the digestion of this component is β-mannanase enzyme. It hydrolyzes
mannan yielding mannotriose and mannobiose (Stalbrand et al., 1993). The screening result
showed that Trichoderma sp. showed the highest mannanase activity followed by A.niger
cultures.  The results showed that A. niger and Trichoderma spp. obtained in this work can
produce β-mannanase enzyme when cultured in medium containing locust bean gum as sole
carbon source. Extracellular proteins with significant mannanase activity were obtained from
the cultures( Tables 2,3 and 4). Most species of  Trichoderma and A. niger are notable
producers of extracellular enzymes including important plant cell-wall hydrolyzing enzymes
such as mannanases (de Vries and Visser, 2001). The protein levels and mannanase activities
of the crude enzyme preparations however differed significantly.

The lowest protein released by Trichoderma spp. in agitated flasks was on the 6th day of
incubation while for stationary was on the fourth day. This is the basis for the high specific
mannanase activity obtained for this isolate on these days (Table 4). However for A.niger, the
specific mannanase activity of this isolate was highest on the 6th day and 4th day for stationary
and agitated flasks respectively which happens not to be the days lowest protein levels were
recorded for this isolate (Table 4).

The cultures in agitated flasks were observed to have higher mannanase activity and protein
content than those in static flasks. This may be attributed to the oxygen limitation that could
be a serious problem in the shaken culture due to the highly non-Newtonian medium caused
by the filamentous growth of each of the isolates (Grobwindhager et al., 1999).

The β - mannanase produced by Trichoderma spp. in this study has maximum activity at 30
°C and 50 °C as shown in Table 5. These peaks may indicate that this isolate secreted two or
more distinct β - mannanase into their environment. Viikari et al. (1993) reported that

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mannanases were usually secreted into the culture fluid as multiple enzyme form. Gübitz et
al. (1996) also reported two mannanases from Sclerotium rolfsii which had optimum
temperature between 72 °C and 74 °C. The optimum temperature obtained for mannanase by
A. niger falls within the range for mesophilic fungi. The activity of β - mannanase by
Trichoderma spp. and A. niger was highest at pH 5 (Table 6) and remain more active to
around neutral pH region. The β -mannanases reported so far exhibit acidic to neutral pH
optima just like this isolates and temperature optima between 40oC and 70oC (Stalbrand et al.,
1993; Puchart et al., 2004), The pH optimum (pH 5) obtained for these β - mannanases was
similar to those of fungal β -mannanases that usually lie between pH 3 – 5.5 (Stalbrand et al.,
1993; Christgau et al., 1994; Ademark et al., 1998; Sachslehner and Haltrich, 1999; Siti
Norita et al., 2010). However, it was lower than those reported for bacterial β - mannanases
which have pH optima close to neutral pH (Viikari et al., 1993). Recently, a β - mannanase
produced by Scopulariopsis candida was reported to be most active at pH 6 (Mudau and
Setati, 2008).

CONCLUSSION
Degrading wood and wood shavings are abundantly available as waste in our environment.
This work shows that easily grown fungal isolates – Trichoderma spp and A. niger from
degrading wood could be used to produce mannanases which are of great industrial
importance. The obtained mannanases of the fungal isolates had optimum temperatures and
pH that lied within the range previously reported for fungal mannanases.

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