Abstract: The highest total activity of cellulases by Dactylella aquatica and Cylindrocarpon heteronemum was obtained after 9 days incubation at pH 11-14 and 20-25°C, respectively. The enzyme was produced in the presence of cellulose as a sole carbon source. CaCl2 at level 1000 ppm was the most convent for cellulyltic activity of D. aquatica where as growth at this concentration was nearly 1/3 that of control. Although CaCl2 was effective in decreasing cellulyltic activity of C. heteronemum. Growth of D. aquatica and C. heteronemum inhibited by the presence of CaSO4. Polygalacturonase (PG), Pectin Lyase (PL) and pectinmethylestrase (PME) were investigated qualitatively in most active of aquatic fungi.
INTRODUCTION
There is a group of deuteromycotina which are known by the conidial state in aquatic habitats, some of these species are also can found in terrestrial habitats. These fungi are referred to as Hyphomycetes. Aquatic Hyphomycetes colonize wood as well as leaves in streams (Kane, 1978; Sanders and Andersen, 1979; Willoughby and Archer, 1973; Khattab, 1991; Shearer and Webster, 1991; Yuen et al., 1998). Maximum leaching of sugars from twigs is within 3 weeks of submersion; but some leaching continues for up to 12 weeks (Willoughby and Archer, 1973). To exploit plant cell wall polymers, aquatic hyphomycetes possess enzymes such as pectinases; hemicellulases and cellulases which show ability to degrade lignin (Dwt et al., 1992; Schearer, 1992; Rodrigues and Graca, 1997). Aquatic hyphomycetes were investigated for ligninolytic activity (Barlocher and Kendrick, 1974; Sanders and Andersen, 1979; Jones, 1981, 1985; Fisher et al., 1983; Dwt et al., 1992; Abdel-Raheem, 1992).
The cellulolyltic activity of leaves during different stages of decomposition and at two season of contrasting temperature was examined in an attempt to correlate the patterns of enzymatic activity with the associated microbial community (Sinsabaugh et al., 1981; Hodgkiss and Leung, 1986). The mechanism of enzymatic hydrolysis of cellulose by filter paper cellulose (C1) and carboxymethyl cellulose (Cx) involved a two step process with an initial activation by non-hydrolytic enzyme followed by the hydrolytic carboxymethyl cellulase. Existence of the non-hydrolytic enzyme has not been confirmed. The isolation of filter paper cellulase and carboxymethyl cellulase enzymes was attempted by workers and later these were tentatively identified as exo-cellobiohydrolase and endo-1, 4-β-D-glucanase. Both of these enzymes exhibit a distinctive mode of action toward cellulosic substrate. The present concept of crystalline or native cellulose hydrolysis involves three major enzymes as cellobiohydrolase, endoglucanase and β-glucosidase (β-D-glycoside glucohydrolase). The biological decomposition of pectin is an important degradation process. In nature, pectin is degraded by enzymatic systems produced by a wide variety of saprophytic and phytopathogenic microorganisms, including bacteria and fungi (Rombouts and Pilnik, 1980). Pectinases are group of enzymes that hydrolyze pectin by different mechanisms (Fogarty and Kelly, 1982) and are divided into: pectin methylestrase (PME), polygalacturonase (PG) and pectin and pectatelyase (PNI and PL). For each class of enzymes there may be hydrolyze and lyases that clear randomly the appropriate polymers. Polygalacturonases have been isolated from the culture media of various mesophilic fungi such as Aspergillus japonicus (Tshii and Yokotsuka, 1972) Sclerotinia sclrotiorum (Kenon and Waksman, 1990) and Colletotrichum lindemuthianum (English et al., 1972; Kenon and Waksman, 1990). The enzymes are secreted by the microorganisms and some of them macerate some plants effectively (Scott-Craig et al., 1990; Miyairi et al., 1985) obtained a polygalacturonases from the culture medium of Stereum purpureum and showed that the enzyme caused silver leaf symptoms on apple trees. Recent studies of polygalacturonases have indicated that this enzyme is involved in the signaling systems of plant that regulated defensive and development at processes (Ryan and Farmer, 1991; Fischer and Bennett, 1991; Ferrari et al., 2003; Sicilia et al., 2005; Nighojkar et al., 2006).
The present study was carried out to investigate aquatic hyphomycetes fungi occurring on colonizing wood as well as leaves in the river Nile posses enzymes such as cellulases and pectinases and show an ability to degrade the cell wall polymers subastances The effect of some factors such as temperature, pH, incubation period, carbon source and heavy metals were carried out on the cellulolytic activity.
MATERIALS AND METHODS
Fungal Isolate
D. aquatica and C. heteronemum were isolated from the River
Nile (Elquanater Elkhayria) by El- Shafy (2000).
Bioassay Medium for Detection of Pectinase Activity
The procedure used here was that used by Ammar et al. (1994)
Pectic Enzyme Assays
The methods used for enzyme assay were these described by Duncan (1969).
Screening for Cellulolytic Activity
The basal media were supplemented with different cellulosic sources, carboxymethyl
cellulose or filter paper Whatman No. 1 while were proved to be best sources
for the production of cellulase.
According to (Bland and Douglas, 1977) the basic medium used for cellulase production composed of the following ingredients (g L-1). Urea, 0.3; KH2PO4; 2(NH4)2SO4, 1.4; MgSO4.7H2O, 0.3; cellulose, 5; peptone, 1; FeSO4.7H2O, 1; MnSO4.7H2O, 1.6; ZnSO4.7H2O, 1.7; COCl2, 2 and distilled water 1 L.
Detection of Cellulase Activity in Liquid Media
This method was measured as described by Mandels et al. (1976a).
Factors Affecting Cellulase Activity
In this experiment two isolates of Dactylella aquatica and Cylindrocarpon
heteronemum which were the highest cellulase producers among the other isolated
from aquatic fungi tested were chosen (Table 1). The cellulolytic
activities of chosen fungi were investigated under different environmental factors.
Factors such as pH, temperature incubation period, carbon source and heavy metals
were considered.
| Table 1: | Cellulolytic activities of isolated fungi on solid medium |
Effect Hydrogen Ion Concentration
The effect of pH on cellulase enzymes production by Dactylella aquatica
and Cylindrocarpon heteronemum were determined at pH range from 3, 5,
7, 9, 11 and 14 using 0.1 N NaOH or 0.1 HCl.
Effect of Temperature
The effect of temperature on enzyme production was tested at 20, 25, 30,
35, 40, 45 and 50°C.
Effect of Incubation Period
Cellulase activity was determined for different incubation period, at zero
time 3, 5, 7, 9, 12 and 15 days.
Effect of Carbon Source on Cellulolytic Activity
The basal medium was contained 50 mL and weighting different carbon source
as glucose, sucrose, starch, autoclaved, inoculated with 1 mL of spore suspension
and incubate at optimum temperature.
Effect of Heavy metals
Prepare the basal medium and weight different molarity of heavy metals as
calcium, copper, manganese, magnesium and cobalt (mg L-1) and adjust
optimum pH thin distributed the medium in a conical flasks, each containing
50 mL and autoclave as usual. Inoculated each flask with 1 mL spore suspension
of Dactylella aquatica and Cylindrocarpon heteronemum then incubate
at optimum temperature for optimum incubation period and filter the mycelia
keep the filtrate cool until use for the assay.
Cellulolytic Activity Assay
Cellulase was measured in terms of filter paper cellulase activity (Cl),
carboxymethyl cellulose (Cx) by Mandel et al. (1976a).
Determination of Filter Paper Cellulase (Cl)
To 50 mg filter paper (Watman No. 1), 1 mL sodium acetate buffer pH 4 and
0.5 mL enzyme filtrate were added. The mixture was incubated at 50°C for
30 min (Sternberg, 1976; Nisizawa et al., 1978). The liberated reducing
sugars were measured as glucose using 1 mL of 3,5 dinitrosalcylic acid reagent.
Determination of Carboxymethyl Cellulose (Cx)
The method was based on that described by Su (1976).
Determination of β-Glucosidase (β-G)
To 0.5 mL of filtrate enzyme culture 0.5 mL of l% salicine was added and
incubated for 30 min at 50°C the liberated reducing sugars were measured
as glucose using 3,5 dinitrosalicylic acid reagent.
Determination of Reducing Sugar in the Crud Enzyme Filtration
To 0.5 mL enzyme filtrate, 1 mL of 1% 3, 5 dinitrosalicylic acid reagent
was added in a test tube, then heated for 3 min in boiling water bath, cooled
in ice bath. Absorbency reading was taken at 540 nm using speckol spectrophotometer
pure glucose was prepared as reference for constructing the calibration curve
(Willis and Scott, 1988).
Enzyme Precipitation Using Ammonium Sulphate
Grow the organisms on the optimum growth; then filter the culture and transfer
the filtrate into fresh tube or tubes. According to the table of ammonium sulfate
apply the different concentration as, Zero, 10, 20, 30, 40, 50% as follow for
each concentration. Calculate the needed grams of ammonium sulfate according
to the used volume of filtrate. Add the salt gradually and shake gently, after
complete dissolving of the salt, leave the tube to stand for 30 min at 15000
rpm at 4°C. Transfer the supernatant into fresh tube and keep the ppt (as
No. 1). The supernatant is Sl. To the supernatant add more grams of ammonium
sulfate according to the next wanted concentration dissolve gently and leave
to stand at room temperature for 30 min. Then centrifuge for 20 min at 15000
rpm. at 4°C after centrifugation, transfer the supernatant into fresh and
keep as ppt No. 2. The supernatant is S2. Carry on all the wanted concentration
following the same steps as before. All the obtained ppts. were dissolved in
equal volumes of the used buffer. See the activity of the enzyme in the ppts.
Solution and in the different supematants (S1, S2, S3,......).
Statistical Analysis
Analysis of variances were performed with Program Minitab Throughout the
result were calculated. All replicates were repeated at lest 6 times.
RESULTS
The ability of fungal isolates to hydrolyze cellulose and pectin are shown in (Table 1). A strong cellulalyltic ability were shown by Cylindrocarpon heteronemum, Dactylella aquatica. However, the ability of these isolates to produce pectinolytic enzymes were variable. The most active isolates were Rhizopus sp., C. heteronemum, F. chlamidosporium, F. merisopedium and C. candidum. The remaining species had limited cellulalyltic and pectinolytic ability.
Effect of Temperature on Cellulase Activity
The maximum dry biomass production in Dactylella aquatica was obtained
at 25°C. Biomass markedly decreased by the rise in temperature (Table
2).
| Table 2: | Effect of different temperatures on biomass (dry weight g L-1) and cellulolytic activity (μ mL-1) of Dactylella aquatica and Cylindrocarpon heteronemum |
| C1: Filter paper cellulase, Cx: Carboxymethyl cellulase, βG: β-glucosidase: salicinase, TA: Total Activity, PC: Protein Concentration, SA: Specific Activity (μ mg-1 protein), LSD: Least Significant Difference | |
On the other hand, filter paper cellulase (Ci) and β-glucosidase showed insignificant differences among the studied range of temperature (15-50°C). However, carboxymethyl cellulase (Cx) activity increased significantly and reached maximum activity (277.8 μ L-1) at 25°C, the rise in the incubation temperature to 50°C resulted in a significant decrease in the activity and reached about 43% that recorded at 25°C. In general, 25°C was the most convenient temperature for the production of total cellulases (575 μ L-1) in Dactylella aquatica. Higher temperature decreased total activity significantly. The highest specific activity (18.9 μ mg-1) has been observed at temperature 50°C which produced the least total activity (356.7 μ L-1).
In case of Cylindrocarpon heteronemum, the response of cellulases to the various studied temperatures was not very clear, in case of filter paper cellulase (Ci), activity was increased significantly in the temperature range (20-30°C), then started to decline and reached its minimal value at 50°C. On the other hand, carboxymethyl cellulase (Cx) showed its highest activity at temperature range (15-20°C), then the activity decreased to reach its lowest value at 50°C. B-glucosidase activity showed insignificant differences in the range of tested temperature. Maximum growth represented by dry biomass (2.9 g L-1) obtained when the cultures incubated at 20°C. The highest total activity was also obtained at 20°C (438.4 μ L-1).
Effect of pH on Cellulase Activity
Cellulolytic activity of Cylindrocarpon heteronemum and Dactylella
aquatica was investigated under a wide range of pH (3-14). Although growth
was markedly inhibited under conditions of high acidity or alkalinity, cellulases
were not affected in the same manner observed for biomass production. pH 7 was
the best for maximum production of biomass in case of Dactylella aquatica
(2.1 g L-1) while pH 5 produced maximum biomass in case of Cylindrocarpon
heteronemum (2.5 g L-1).
The results of (Table 3) showed clearly that β-glucosidase activity was insignificantly changed under all tested pH values (3-14). In case of Dactylella aquatica slight increase in C1 activity was observed at pH 5, while Cx activity significantly increased at pH 7. In general total cellulases activity significantly increased at pH 7 with activity (417.8 μ L-1) compared to (370 μ L-1) at pH 3 and (374.4 μ L-1) at pH 14.
In case of Cylindrocarpon heteronemum, the most obvious observation was the significant increase in C1, which reached (161.1 μ L-1) at pH 5. Total activity was also maximum (418.9 μ L-1) at pH 5. Variable values of specific activity were obtained under different pH conditions, pH 11 and 14 showed the highest values in both fungi.
| Table 3: | Effect of different pH values on biomass (dry weight g L-1) and cellulolytic activity (μ mL-1) of Dactylella aquatica and Cylindrocarpon heteronemum |
| C1: Filter paper cellulase, Cx: Carboxymethyl cellulase, βG: β-glucosidase: Salicinase, TA: Total Activity, PC: Protein Concentration, SA: Specific Activity (μ mg-1 protein), LSD: Least Significant Difference | |
| Table 4: | Effect of incubation periods on biomass (dry weight g L-1) and cellulolytic activity (μ mL-1) of Dactylella aquatica and Cylindrocarpon heteronemum |
| C1: Filter paper cellulase, Cx: Carboxymethyl cellulase, βG: β-glucosidase: Salicinase, TA: Total Activity, PC: Protein Concentration, SA: Specific Activity (μ mg-1 protein), LSD: Least Significant Difference | |
Effect of Incubation Period
Table 4 shows the effect of incubation period on cellulases
as well as biomass production and specific activity. It is obvious that growth
of Dactylella aquatica reached its maximal growth after 12 days of incubation
(4.5 g L-1), while maximum growth (4.8 g L-1) was observed
after 9 days of incubation in case of C. heteronemum. The results showed
clearly that cellulases, especially C1 and β-glucosidase significantly
increased by the increase in incubation period and reached their maximum activity
(200 and 168.4 μ L-1, respectively) in 12 days of incubation
in case of Dactylella aquatica. Total activity showed the same trend
and produced maximum value (515 μ L-1) after the same period
of incubation.
In case of Cylindrocarpon heteronemum, the most observed increase was in case of C1, which reached its maximum activity (174.8 μ L-1) in 9 days of incubation. Total activity was at its highest level (448.6 μ L-1) after the same period of incubation. It is clear that cellulolytic activity in both fungi was related to fungal growth.
Effect of Different Carbon Sources
Four carbon sources, representing mono, di- and polysaccharides were examined
for their ability to induce cellulolytic activity in D. aquatica and
C. heteronemum (Table 5). The most effective carbon
source was cellulose, which resulted in total activity as much as 5 times that
of starch and 10 times as that in the presence of sucrose. β-glucosidase
activity represented about 40% that of the total activity. Although growth was
highest in the presence of glucose yet cellulases were not detectable in the
filtrate.
| Table 5: | Effect of carbon sources on biomass (dry weight g L-1) and cellulolytic activity (μ mL-1) of Dactylella aquatica and Cylindrocarpon heteronemum |
| ND: Not Determined, C1: Filter paper cellulase, Cx: Carboxymethyl cellulase, βG: β-glucosidase: salicinase, TA: Total Activity, PC: Protein Concentration, SA: Specific Activity (μ mg-1 protein), LSD: Least Significant Difference | |
| Table 6: | Effect of different CaCl2 concentrations on biomass (dry weight g L-1) and cellulolytic activity (μ mL-1) of Dactylella aquatica and Cylindrocarpon heteronemum |
| ND: Not Determined, C1: Filter paper cellulase, Cx: Carboxymethyl cellulase, βG: β-glucosidase: salicinase, TA: Total Activity, PC: Protein Concentration, SA: Specific Activity (TA/PC) (u mg-1 protein), LSD: Least Significant Difference | |
The same trend has been observed in case of C. heteronemum. The presence of cellulose markedly induced cellulyltic activity, which produced (380.4 μ L-1) while in other carbon sources, totals activities were 150.2 and 148.2 μ L-1 for starch and sucrose, respectively. In both fungi Specific Activity (SA) was higher in case of cellulose than in the other used carbon sources.
Effect of Calcium Chloride
In Table 6 variable responses of tested fungi in the presence
of different concentrations of calcium chloride as well as under control conditions.
In case of D. aquatica, filter paper cellulase C1 activity significantly
decreased by the addition of CaCl2 to the growth medium. All tested
CaCl2 concentrations showed the same response with the exception
of the concentration (1000 ppm). Total activity, at this concentration was nearly
the same as that under control conditions without the addition of CaCl2.
All detected fractions of cellulases were more or less as the same levels of
controls.
Higher concentrations resulted in a marked decrease in activity, which was about 40%, that of the control when added at the level 1500 ppm. It seems that CaCl2 at level 1000 ppm was the most convenient for cellulolytic activity of D. aquatica, the growth at this concentration was nearly 1/3 that of control.
On the other hand, in the case of C. heteronemum growth was markedly inhibited by the addition of CaCl2 salt to the growth medium, the lowest used concentration (250 ppm) produced nearly 1/3 that of the dry biomass produced under control conditions (in absence of CaCl2). The developed growth under the stress of high concentration of Cad; (2000 ppm) was only 5% that of the control. Activities of filter paper cellulase C1 and carboxymethyl cellulase Cx were reversibly related to CaCl2 concentration, their activities decreased significantly with increase in CaCl2 concentration and reached minimal value when growth medium amended with (15 00 ppm) CaCl2.
| Table 7: | Effect of different CaSO4 concentrations on biomass (dry weight g L-1) and cellulolytic activity (u mL-1) of Dactylella aquatica and Cylindrocarpon heteronemum |
| ND: Not Determined, C1: Filter paper cellulase, Cx: Carboxymethyl cellulase, βG: β-glucosidase: salicinase, TA: Total Activity, PC: Protein Concentration, SA: Specific Activity (TA/PC), LSD: Least Significant Difference | |
| Table 8: | Pectenasic activites of isolated fungi on solid medium |
| Table 9: | Pectinolytic activity |
However, β-glucosidase activity was significantly higher at low concentrations (250 and 500 ppm) and started to decline by increasing concentration to reach the lowest value at (1500 ppm) CaCl2. In general, calcium chloride was effective in decreasing cellulolytic activity of C. heteronemum.
Effect of Calcium Sulphate
The results of (Table 7) showed the response of tested
fungi towards the addition of different concentrations of CaSO4 to
growth medium. The trend in this experiment is very clear.
Growth and biomass production markedly inhibited by the presence of CaSO4 in growth medium and such inhibition in activity increased by the increase in CaSO4 concentration and at the highest applied one (2000 ppm), growth completely ceased in D. aquatica and C.. heteronemum. Also total cellulolytic activity as well as all detected fractions of cellulases (C1, Cx and β-G) followed the same trend as in growth. Addition of CaSO4 to growth medium markedly decreased cellulases activity. The activity decreased with the activity of both fungi. However, Cx fraction in the case of D. aquatica showed significant increase in activity (178.9 μ L-1) compared to (148.4 μ L-1) of the control, then decreased at higher concentration (1000 ppm). In case of C. heteronemum the activity of Cx fraction showed slight decrease than control.
No activity has been detected when MgSO4 applied in higher concentration than (1000 ppm) in case of D. aquatica and (500 ppm) in case of C. heteronemum.
Pectinase Activity
Table 8 showed the ability of the isolates to produce
pectinolytic enzymes, which were variable as detected by the formation of a
clear zone around the fungal growth on solid medium. Infindepera adhaerens
had the least activity, while Rhizopus sp., C. heteronemum,
Fusarium chlamidosporium, F. merisopedium and C. candidum
were the highest pectinase producers.
Polygalactoronase (PG), Pectin Lyase (PL) and Pectin Estrase (PE) were investigated qualitatively in the most active isolates (Table 9). PE was not detected in the three tested isolates, while PG and PL were detected in C. heteronemum and C. candidum. PL was only detected in F. chlamidosporium.
DISCUSSION
The results of the present study showed that cellulolytic activity of Cylindrocarpon heteronemum and Dactylla aquatica respond variably to temperature and hydrogen ion concentration. Maximum activity was maintained at 20°C and pH 5 in case of C. heteronemum while 25°C pH 7 were optimum for the activity in case of D. aquatica. The results are in agreement with that of Mohamed (1998) which showed that pH 5 was the most suitable for maximum production of cellulases in case of Aspergillus fumigatus and Penicillin notatum. Also, Sayed (1989) reported that pH value between 4-5 was convenient for enzyme formation by A. flavus and the shift in pH level over this range resulted in a decrease in the activity. Hazaa (1985) showed that pH between 5-7 was optimum for cellulases activity in Micropolyspora thermogluca. In Rhizopus sp. optimum pH for cellulases was 5.5 as reported by Kassim (1983) and Taj Alden and Alkenany (1993). Considering incubation temperature variable results obtained by Mohamed (1998), optimum temperature for cellulolytic activity was 27.5 and 35°C for P. notatum and A. fumigatus, respectively.
Au et al. (1992) reported that tested aquatic Hyphomycetes showed higher cellulolytic activity in the winter than summer leaf litter. On the other hand, Parado and Forchiassin (1999) found that temperature between 50-55°C was the optimal temperature for cellulase system in Nectria catalinensis while Mandeep et al. (1994) recorded the optimum temperature (28°C) for the secretion of the 3 components of cellulase complex in vitro and pH value 6. The type of carbon source in the growth medium is very important factor in the synthesis of cellulases. Crystalline cellulase was the most effective carbon in inducing cellulases in the present study, while in case of glucose no cellulases were detected.
The present study results are in agreement with that of Coughlan and Ljungdahl (1988) and Gritzali and Brown (1979). They found that cellulase formation by T. reesei was inducable by cellulase repressed by monosaccharides such as glucose. The same findings were reported by Merivuori et al. (1984) in the same fungus. Also, Hazaa (1985) and Tsao and Chaiang (1983) reported that cellulase was a good inducer for cellulases by Micropolyspera thermogluca. On the other hand, Robert and Christian (1991) suggested that cellulase formation by T. reesei is not at all repressed by carbon sources and that formation of cellulase on lactose is most probably due to induction.
It has been shown that calcium is required for growth and development of certain Oomycetes (Giffm, 1966; Cameron and Lejohn, 1972). Also, it is widely implicated in several biochemical processes (O'Day, 1990; Jacson and Heath, 1989). So it can be accepted as an essential micronutrient of fungi. Also, magnesium requirements may be considered essential for enzyme activation and ATP metabolism, in general both required in very low concentrations. The present study showed clearly the inhibitory effect of the tested salts of calcium and magnesium on fungal growth and cellulalytic activity specially when applied in high concentrations. The inhibitory effect of high concentrations of calcium and magnesium salts may be attributed to the interference with the utilization of other essential microelements by fungal cells. Also, the shift on pH toward more acidity by the presence of sulphate and chloride ions may be considered as on important factor in decreasing the tested fungi (the recorded optimum pH was between 5-7).
Fungal pectinases are very important in maceration of plant leaves and other plant fragments and the present investigation showed the presence of pectinases in variable amounts in the isolates detected during the study. Pectinases and cellulases are very important in leaf maceration and degradation, so favorable conditions are required to degrade plant fragments and make them available as feed for water inhabitants.
In general, aquatic habitat of moderates conditions, i.e., temperature between 20-30°C, pH between 5.5-7.5, optimum. concentration of salts will maintain good conditions for growth and enzymatic activity and consequently water fungi will be able to degrade plant fragment and render them available for utilization by living organisms.