Abstract: The purpose of this study was to characterization of xylanase producing Fusarium solani isolate and optimization of cultural conditions for xylanase enzyme production. Screening of Fusarium solani isolate was based on the diameter of the clear zone formation in oat spelt xylan agar plates, Fusarium solani isolate F7 was selected and optimized for xylanase enzyme production using cheaper substrate like wheat straw, rice straw, rice bran and wood husk. Maximum enzyme activity was observed in wheat straw (78.32 U mL-1). Optimum pH and temperature for xylanase activity were found to be 5.5 and 30° at 3% substrate concentration. In purification step, 75% ammonium sulphate saturation was found to be suitable giving maximum xylanase activity. Purified xylanase yielded single band with a molecular weight of 89 kDa. The use of wheat straw as a major carbon source is particularly valuable because oat spelt xylan is very expensive, The Fusarium solani F7 isolate proved to be a promising microorganism for xylanase production.
INTRODUCTION
Xylans are the major hemicelluloses in wood from angiosperms, where they account for 15-30% of the total dry weight. In the gymnosperms however, xylans contribute only 7-12% of the total dry weight. The structure of xylans is complex and their complete biodegradation requires the concerted action of xylanases enzymes. Xylans are heterogeneous polysaccharides with a backbone consisting of beta-1, 4 linked D-xylosyl residues. Endo-beta-1, 4 xylanases are the main enzymes responsible for cleavage of the linkages within the xylan backbone (Amani et al., 2007). To date xylanase has gained increasing attention because of its various biotechnological applications and have great potential for industrial applications mainly for the bioconversion of lignocelluloses to sugar, ethanol and other useful substances (Viikari et al., 1994). They are extensively used in pre-treatment of forage crops and other lignocellulosic biomass, added to swine and poultry cereal-based diets to improve nutrient utilization, flour modification for bakery products and saccharification of agricultural, industrial and municipal wastes (Sá-Pereira et al., 2002). Moreover, it is reported that xylanases have been widely used for clarifying fruit juices and wine (Hang and Woodams, 1997), food processing industries and in the production of several valuable products like xylitol and ethanol (Salles et al., 2005) and improving the nutritional properties of agricultural silage and grain feed (Kuhad and Sing, 1993). To reach commercial feasibility, enzyme production must be increased by introducing a more potent strain and by optimizing culture conditions. Xylanases are produced by numerous microorganisms among which the fungi are the most potent producers (Pham et al., 1997). Haltrich et al. (1996) gave an overview of fungal xylanases and showed that the enzyme can be produced by a number of microorganisms including bacteria, yeasts and filamentous fungi such as Trichoderma, Aspergillus, Penicillium Fusarium, Chaetomium, Humicola, Talaromyces and many others. Fusarium oxysporum and other Fusarium sp. has been shown to be a promising organism for enhanced production of xylanases (Chirstakopoullos et al., 1999). Extracellular enzymes are considered important from the industrial viewpoint as they ease the extraction procedure.The use of purified xylan as a substrate to induce xylanase synthesis increases the cost of enzyme production. Therefore, for commercial applications, there have been attempts to develop a bioprocess to produce xylanase in high quantities from simple and inexpensive substrates. Wheat straw, wheat bran and corncob has been shown to be an efficient substrate in the production of xylanase and the enzyme production is related to the type and concentrations of nutrients and growth conditions (Alam et al., 1994; Hoq and Deckwer, 1995; Haltrich et al., 1996; Gawande and Kamat, 1999; Kang et al., 2004; Sonia et al., 2005). Filamentous fungi are interesting procedures of this enzyme from an industrial point of view due to extracellular release of xylanases, higher yield compared to bacteria and yeast and production of several auxiliary enzymes that are necessary for debranching of the substituted xylanase (Haltrich et al., 1996).
Thus, since there is less reports on efficient xylanase production by the fungus Fusarium solani, therefore the objective of this study to consolidate information on optimization process for xylanase production.
MATERIALS AND METHODS
Isolation of Fusarium solani Isolate
The soil sample for the isolation of Fusarium solani were collected
from root zone of different nearby areas of Faizabad region, U.P., India (Lat.
26° 47’ N; Long. 82° 12’ E). The Fusarium solani culture were isolated
from soil samples by serial dilution method of Clark et al.
(1988), 1 g soil sample was dissolved in 100 mL sterilized distilled water.
The soil suspension was diluted up to 10+3 to 10-3 and
0.5 mL of diluted suspension was used. The study was carried out at Microbiology
Laboratory, Dr. R.M.L. Awadh University, Faizabad-224001, UP, India during 2007-2008.
Identification of Fusarium solani Isolate
Identification was based on cell and colony morphology characteristics as
per method described by Booth (1971). The young colonies
of Fusarium solani were aseptically picked up and transferred to PDA
slants and incubated at 28± 2 ° for 4-5 days for maximum growth.
Screening for Xylanolytic Activities
The fungal isolates formed were subcultured to purity and examined
for xylanolytic activities. Screening for xylanolytic activities was performed
on Malt Extract Agar (MEA) containing 0.1% (w/v) of xylan from oat spelt.
Positive xylanolytic isolates were detected based on the clear zones of
hydrolysis on the xylan. The potential isolates were subcultured and maintained
on MEA slants. The slants were stored at 4 ° prior to use.
Xylanase Production Medium
Erlenmeyer flasks (250 mL) containing 10 g of wheat straw were added with
the Mandels and Sternburg’s basal medium (Mandels and Sternburg,
1976) just to wet the wheat straw. The Mandel’s medium was prepared with
the following composition (g L-1) 10.0 g; urea, 0.3; peptone, 0.75;
yeast extract, 0.25; (NH4)2SO4, 1.4; KH2PO4,
2.0; CaCl2, 0.3; MgSO4.7H2O, 0.3 and trace
elements (mg L-1): FeSO4.7H2O, 5; MnSO4.
4H2O, 1.6; ZnSO4.7H2O, 1.4 and CoCl2.6H2O,
20.0 and Tween 80 0.1% (v/v) (pH 5.0). The flasks were inoculated with 2 mL
of spore suspension prepared from a week old PDA slants of the culture grown
at 30°. Sterile distilled water containing 0.1% (v/v) Tween 80 was used to
prepare fungal spore suspension. Inoculated flasks were incubated at 30 ° under
static conditions for 10 days.
Inoculum and Fermentation
A inoculum size of conidia (Each mL of cells suspension contained
2.0x106 cells) was transferred from a stock culture in 250
mL flask containing 50 mL of growth medium. The flasks were incubated
for 72 h at 28± 2 ° on a rotatory shaker at 150 rpm.
Preparation of Enzyme
The enzyme from each flask was extracted using 50 mL of 0.05M citrate
buffer (pH 5.3) and filtered through a wet muslin cloth by squeezing.
The extract was centrifuged at 5000 rpm for 20 min. The clear supernatant
was partially purified by ammonium sulphate fractionation at 4 ° to
achieve 75% saturation and dialysed using the same buffer for 24 h with
three intermittent changes.
Xylanase Assay
The supernatant was used as a source for enzyme sample. Xylanase activity
was measured with the optimized method described by Bailey
et al. (1992), using 1% oat spelt xylan, (Himedia, Mumbai, India)
as the substrate. The solution of xylan and the enzyme at appropriate dilution
were incubated at 55 ° for 7 min and the reducing sugars were determined by
the dinitrosalicylic acid method described by Miller (1959),
with xylose as standard. The released xylose was measured spectrophotometrically
at 540 nm. One unit of xylanase was defined as the amount of enzyme (protein)
in milligram required for hydrolysis of starch to produce a millimolar of reducing
sugar in 1 h under above assay conditions. The specific activity was defined
as number of units per gram protein. All experiments were repeated thrice.
SDS-PAGE of Xylanase Enzyme
The cell free supernatant of crude enzyme was essayed on sodium dodecyl
sulphate-poly acrylamide gel electrophoresis for protein profiling to
confirm the molecular weight of xylanase enzyme.
Optimization of Process Parameters
The optimization of composition of medium and cultural conditions
was carried out based on stepwise modification of the governing parameters
for xylanase production. The effect of various substrates, consisting
of rice straw, wheat straw, rice bran and wood husk was examined by adding
10 g of each substrate in a 250 mL Erlenmeyer flask with 10 mL of sterile
distilled water, which was added to moisture the substrates. Cultivation
was carried out at ambient temperature (28± 2°) for 7 days.
Changing the pH 3 to 8 in the production medium the effect of pH was observed.
The effect of cultivation temperature on the enzyme production was examined
at different temperatures starting from 25 to 50° with 5° intervals.
The effects of incubation period were evaluated by 24 h interval by checking
the enzyme activity. The optimization was carried out by adding standardized
concentration wheat straw using supplemented carbon sources like glucose,
sucrose, galactose, starch, dextrin, xylose, sorbitol, corboxymethyl cellulose,
fructose and nitrogen sources like were peptone, urea, yeast extract,
sodium nitrate, ammonium sulphate, ammonium nitrate and beef extract,
were also tried.
RESULTS AND DISCUSSION
The application of xylanases are widely spread in paper and pulp industries
as well as in food and pharmaceutical industries. In present investigation,
purification of xylanase was carried out. The molecular weight of the single
protein band was calculated to be about 89 kDa (Fig. 1). Microbial
xylanases are single subunit proteins with molecular masses within a the range
of 8-145 kDa (Kulkarni et al., 1999). In a earlier
study, a purified xylanase enzyme preparation also showed a single protein band
on sodium dodecyl sulphate-poly acrylamide gel electrophoresis and the molecular
weight of this enzyme was found to be 24 kDa (Sardar et
al., 2000).
| Fig. 1: | Purification of xylanase from Fusarium solani
F7 |
Selection and Identification of Isolate Fusarium solani
Based on the screening programme, a total of 16 isolates were capable
of exhibiting xylanolytic activities on MEA-xylan agar with the diameter of
the clear zones ranging from 34 to 48 mm. However, 5 isolates consisting of
F2, F5, F7, F11, F13 and F15 were selected for further confirmation using the
MEA-xylan agar plates. The isolates F7 demonstrated reproducible zones of hydrolysis
of 48 mm diameter and this isolate was selected as the potential producer of
xylanase. Isolate F7 was identified based on the structural morphologies as
observed under the light and scanning electron microscopes. It was observed
that the isolate possessed distinct macro and mico conidia formation. Macro-conidia
were of 3-5 celled slightly curved at the pointed ends, typically canoe shaped
whereas micro-conidia 1-celled, ovoid. Based on these characteristics, isolate
Fusarium solani isolates were identified to be Fusarium solani (Booth,
1971) and isolate F7 was used for further investigation for optimization
of xylanase producing property.
Effect of Incubation Period
The xylanase activity was determined after every 24 h of incubation in order
to determine the optimum incubation period for maximum production of xylanase.
The enzyme production however, started after 24 h of inoculation and showed
maximum production (56.31 U mL-1) on 6th day of incubation period
at 30° (Fig. 2). In some fungi, high xylanase production
has been shown to be linked strictly to cellulose production due to time course
or incubation period (Haltrich et al., 1996; Chirstakopoullos
et al., 1999; Kang et al., 2004).
Effect of Temperature
Temperature is one of the important parameters that determine the success
of optimization system. Therefore, the effect of temperature on xylanase production
by Fusarium solani F7 was examined at various temperature ranges 20 to
50° for 48 h and the results obtained are shown in Fig. 3.
The production of xylanase was maximum at the temperature near to ambient temperature
(30°) with an activity of 58.8 U mL-1 and the growth about 2.2 mg
mL-1. A lower activity in the range of 6.42-22.11 U mL-1
and growth of about 1.10-1.42 mg mL-1 substrate were obtained with
cultivation temperatures lower or above the ambient temperature. At 20°, the
activity of xylanase was 6.42 U mL-1. The results obtained indicated
that the enzyme production corresponded closely to the growth of the fungus.
The optimum temperature for xylanase production is similar to the optimum temperature
for the growth of the fungus. This observation was in agreement with those reported
by Chirstakopoullos et al. (1999) and Biswas
et al. (1988, 1990), who showed that the highest
xylanase activities were obtained at temperatures that were optimum for the
growth of the fungi. Fusarium solani F7, the ambient temperature which
was the optimum temperature for xylanase production was similar to the temperature
of the natural habitat of the fungus where it was initially isolated.
| Fig. 2: | Effect of incubation period on xylanase production |
| Fig. 3: | Effect of temperature on production of xylanase and
fungal biomass |
Effect of pH
The initial pH of medium was adjusted to variable pH range by adding the
0.1 N HCl. Enzyme purified was tested in the pH range (pH 3 to 8). The production
of xylanase was found to be the best at pH 5.5 Below and above this pH production
of xylanase was significantly lower (Fig. 4). The results
showed that xylanase production by Fusarium solani F7 was much dependent
on pH and the optimum initial pH was between pH = 5.0 and 5.5 (Fig.
4). However, when the pH was increased or decreased to values other than
5.0 and 5.5, the production of xylanase gradually decreased. This might be due
to the fact that alkaline pH has inhibitory effect on the growth of Fusarium
solani F7 (an acidophilic fungi) and enzyme production. xylanases from different
organisms show an optimum pH within a range of 4.0-7.0. However, certain xylanases
from Aspergillus kawachii, Penicillium herque and some other fungi
including Fusarium oxysporum exhibit an optimum pH more on the acidic
side (pH 2.0-6.0) (Funaguma et al., 1991; Ito
et al., 1992; Kulkarni et al., 1999).
Endoxylanase 1 and 2 from Aspergillus awamori show an optimum pH at 5.5-6.0
and 5.0, respectively (Kormelink et al., 1992).
| Fig. 4: | Effect of pH on enzyme activity |
| Fig. 5: | Production of xylanase influenced by substrates and
their concentration |
Effect of Substrate and Their Concentration
Standard xylanase activity (52.81 U mL-1) was obtained
in the medium containing 1% wheat straw after 144 h of incubation in comparison
with the, Rice Straw, Rice bran and wood husk (Fig. 5).
In order to determine the best amount of wheat straw for xylanase production,
different concentrations (1-6% by mass per volume) were tested.
The results showed that the highest yield of xylanase was 78.32 U mL-1 with 3% wheat straw (Fig. 5). Increasing the concentration for more than 3% resulted in a significant decrease in xylanase activity. This might be attributed to the fact that high concentration of substrate led to the increase in medium viscosity, which influenced the mixture medium components and oxygen transfer. Similar results were obtained by other researchers by using high concentrations of lignocellulosic materials as substrates for enzyme production (Cao et al., 2008). Wheat straw and wheat bran has been known for being ideally suitable for xylanase production in T. aurantiacus and Penicillium citrinum cultures (Kalogeris et al., 1998; Nair et al., 2008).
Effect of Supplemented Carbon Source
The production of primary metabolites by microorganisms is highly influenced
by their growth, which is determined by the availability of the nutrients in
the substrates. Therefore, it is expected that the improvement of the nutritional
value of wheat straw by the supplementation of carbon will also improve the
growth of Fusarium solani F7 and subsequently the enzyme production.
Flask containing production media supplemented with carbon sources (glucose,
sucrose, starch, carboxymethyl cellulose, fructose, sorbitol, xylose, galactose
and dextrin) were tested for the influence of carbon sources at 1% concentration.
Figure 6 shows the supplementation of sugars, which may act
either as carbon sources or inducers. Starch, Sucrose and dextrin, fructose
and sorbitol and galactose were moderate source carbon sources for xylanase
production. Carboxymethyl cellulose was poor producer of xylanase (Fig.
6). As shown in the Fig. 6, the addition of xylose resulted
in an increment of 42% in xylanase production from other supplemented carbon
sources. These findings confirmed that the size of carbon source was an important
factor in xylanase production (Kalogeris et al., 1998).
| Fig. 6: | Production of xylanase influenced by t carbon sources
with 3% wheat straw as substrate |
Xylose has been described as an effective inducer and carbon source for xylanase production in several microorganisms for xylanase production including A. pullulans (Priem et al., 1991), Fusarium oxysporum (Chirstakopoullos et al., 1999) and T. lanuginosus (Purkarthofer et al., 1993). Xylan is costly for large-scale production of xylanases, lignocellulosic materials can be used as cost-effective substrates for xylanase production (Haltrich et al., 1996; Beg et al., 2000). Various lignocellulosic materials and microbial cultures have been used successfully in solid-state fermentation for xylanase production (Topakas et al., 2003; Sonia et al., 2005). The significant difference in xylanase titers, when wheat straw was used as the carbon source may be attributed to its hemicellulose nature and favorable degradability, the presence of some nutrients in the carbon source (Sonia et al., 2005).
Effect of Supplemented Nitrogen Source
Effect of different nitrogen sources viz. peptone, urea, yeast extract,
sodium nitrate, ammonium sulphate, ammonium nitrate and beef extract on the
production of xylanase was studied, it was observed that ammonium sulphate,
yeast extract and beef extract caused poor enzyme production. Peptone and sodium
nitrate supported maximum production of enzyme whereas ammonium nitrate and
urea produced considerable amount of xylanase. The optimum concentration of
peptone was 0.3% (Fig. 7). The results obtained also did not
exhibit any significant difference between the organic or inorganic nitrogen
sources on the production of xylanase by Fusarium solani F7. These results
are in agreement with those reported in the literature where fungi were found
to produce higher xylanase activities on organic nitrogen sources (Purkarthofer
et al., 1993; Lemos et al., 2001).
The mineral salt solution, distilled water, organic nitrogen source solution
and mineral salt solution+ organic nitrogen source solution used as MA supported
xylanase production and extracellular protein synthesis to varying degrees.
The effects of several MAs have been reported on xylanases production in SSF
(Grajek, 1986, 1987; Alam
et al., 1994; Gutierrez-Correa and Tengerdy, 1998;
Chirstakopoullos et al., 1999).
| Fig. 7: | Effect of different nitrogen sources at 0.3% concentrations
|
CONCLUSION
The results obtained from the present study indicates that significant improvement of xylanase production by Fusarium solani F7 isolate could be obtained by selective use of nutrients and growth conditions. Since oat spelt xylan is an expensive substrate for commercial scale xylanase production, the possibility of using wheat straw for xylanase production was investigated. Wheat straw (3% by mass per volume) could be used as a less expensive substrate for efficient xylanase production (78.32 U mL-1). This observation is interesting due to the low cost of these carbon sources.
ACKNOWLEDGMENTS
We are thankful to Head, Department of Microbiology, Dr. R.M.L. Awadh University for research support to complete this study. Dr. L.P. Yadava, Central Institute for Sub-Tropical Horticulture, Lucknow, UP, India for providing the research guidance.