Production and Partial Purification of Cellulase by Aspergillus niger and A. fumigatus Fermented in Coir waste and Sawdust
G Immanuel, C Bhagavath, P Iyappa Raj, P Esakkiraj, A Palavesam
a. fumigatus, a. niger, coir waste, dns, fpa, sawdust
G Immanuel, C Bhagavath, P Iyappa Raj, P Esakkiraj, A Palavesam. Production and Partial Purification of Cellulase by Aspergillus niger and A. fumigatus Fermented in Coir waste and Sawdust. The Internet Journal of Microbiology. 2006 Volume 3 Number 1.
It was the goal to investigate the cellulase enzyme production ability of fungal strains such as
) and pH 6 (0.052 IU ml
) was recorded by
Cellulose is commonly degraded by an enzyme called cellulase. This enzyme is produced by several microorganisms, commonly by bacteria and fungi (Bahkali, 1996; Magnelli and Forchiassin, 1999; Shin et al., 2000; Immanuel et al., 2006). Although a large number of microorganisms are capable of degrading cellulose, only a few of these produce significant quantities of cell free enzymes capable of completely hydrolysing crystalline cellulose
Fungal genera like
Several studies were carried out to produce cellulolytic enzymes from biowaste degradation process by many microorganisms including fungi such as
Some features of natural cllulosic materials are known to inhabit their degradation / bioconversion (Solomon et al., 1990 and 1999). These are degree of crystalinity, lignification and the capillary structure of cellulose to celluylolytic enzymes and other hydrolytic agents (Fan et al., 1987). However, many physical, chemical and microbial pre-treatment methods for enhancing bioconversion of cellulosic materials have been reported (Kumakura, 1997; Wu and Lee, 1997; Kanosh et al., 1999; Solomon et al., 1999).
Since the production of cellulase enzyme is a major process and economically viable, much work has been done on the production of cellulase from lignocellulosics and major attention has been given to use baggase as substrate (Kanosh et al., 1999; Solomon et al., 1999). The bioconversion of various complex cellulosic waste materials such as baggase (Kanosh et al., 1999), corncob (Ojumu et al., 2003); saw dust (Solomon et al., 1999) have been reported. Likewise coir fibres are major biowaste discarded along with coir retting effluent to estuarine environment. Yet literature related to coir fibre as a carbohydrate source and cellulolytic activity by microorganisms involved in coir retting process is not studied properly. Hence, the present study was carried out to determine the cellulolytic enzyme activity of fungi,
Materials and Methods
Isolation of cellulolytic fungi
The soil samples were aseptically collected from the coir retting ground of Rajakkamangalam retting zone area, Kanyakumari District, Tamil Nadu, India. The collected samples were sprinkled over the Czapek- Dox agar plates and were incubated for a period of 5 days at 30°C. The growth of fungal colonies was observed after incubation period. The individual colonies were isolated and restreaked on the same agar. Then the cultures were observed under the microscope to identify the specific species based on the following identification and characteristic features described by Sundarraj (2003). Based on the culture and morphological characteristics, two common fungal strains such as
Cellulase activity determination
The cellulase activity was determined by streaking the identified fungal cultures individually on the carboxy methyl cellulase agar plates and was incubated at 30°C. After 5 days of growth, the zone was identified around the culture by treating the plate with Congo red and NaOH.
Collection of Biowaste - substrates
Saw dust and coir dust are bio wastes and or abundantly available substrates. Both these substrates were aseptically collected from the respective industries.
Pre-treatment of substrates
The raw substrates were sun dried individually to reduce the moisture content to make them more susceptible for crushing. The crushed substrates were then sieved individually to get powder form. Then the substrates were soaked individually in 1% sodium hydroxide solution (NaOH) in the ratio 1: 10 (substrate: solution) for two hours at room temperature. After which, they were washed for free of chemicals and autoclaved at 121°C for one hour. The treated substrates were then filtered and washed with distilled water until the wash water become neutral (Gharpuray et al., 1983; Solomon et al., 1999).
The isolated cultures of
To 100 ml of the optimised culture medium (Table 1), a piece of mycelia from respective species was inoculated under controlled conditions. Then it was kept in a shaker (200 rpm) at 35°C for a day (Solomon et al., 1999). Simultaneously, separate media were prepared for coir as well as saw dust substrates.
Determination of reducing sugars and cellulase activity
The total amount of reducing sugars in 1.0 ml supernatant was determined by modified Dinitro salicylic method (DNS) of Miller (1972) and also Cellulase activity was determined by filter paper activity (FPA) method of Stephen et al. (2003). Filter paper activity is a combined assay for Endoglucanase (Cx) and Exoglucanase (Ci) cellulase.
Dinitrosalicyclic method (DNS)
It is an alternative to Nelson-Somogyi method. It is simple sensitive and adoptable during handling of a large number of samples at a time. The culture filtrate was collected from the fermentation media by centrifugation. 1 ml of culture filtrate was taken in a test tube and it was equalised with 2ml of distilled water. To the prepared culture filtrate, 3 ml of DNS reagent was added. The contents in the test tubes were heated in a boiling water bath for 5 min. After heating, the contents were allowed to cool at room temperature. At the time of cooling, 7 ml of freshly prepared 40% sodium potassium tartarate solution was added. After cooling, the samples were read at 510 nm in a U.V. spectrophotometer (Techomp-2500). The amount of reducing sugar was determined using a standard graph.
Filter paper assay method (FPA)
It is a combined assay for endo and exo β-1, 4 glucanase. The substrate used was Whatman No. 1 filter paper. 2 ml of crystalline cellulose solution was taken in a test tube [filter paper – (50 mg) and dissolved in 0.2 M sodium acetate buffer (pH 5.5)]. To this tube, 0.5 ml of the culture filtrate was added (enzyme solution). The mixture was incubated at 35°C for one hour and the reaction was terminated by adding 2 ml of DNS reagent. Then it was heated in a boiling water bath for 5 min and then 1 ml of potassium sodium tartarate (40%) was added to the warm tubes. The tubes were allowed to cool and the absorbance was read at 540 nm in a U.V. spectrophotometer. The enzyme production was expressed as the mg glucose released per minute per mg of protein.
Effect of pH on the production of cellulase
The optimised media were prepared using the individual substrates and the pH was set at different level such as 5, 6, 7, 8 and 9 respectively by adding 1% NaOH and concentrated HCl. Then the media were autoclaved. Later they were inoculated with a piece of mycelia and were placed in a shaker (150 rpm) at 30°C for 5 days. Simultaneously, for both the organisms and both the substrates, assay was carried out separately.
Effect of temperature on the production of cellulase
The optimised media were prepared individually by using the substrates and autoclaved. Later it was inoculated with a piece of mycelia and was set at different temperatures 20, 30, 40 and 50°C respectively. The effect of temperature on the production of cellulolytic enzyme was determined by growing the organisms at the above temperatures. Simultaneously for both the organisms and both the substrates, separate assay was carried out. The enzyme solution obtained from these two (pH and temperature) experiments was individually optimised based on Dinitro-salicyclic acid and filter paper activity methods as described earlier.
Partial purification of cellulose
Enzyme source preparation
The fungal organisms
Alcohol precipitation of cellulase
100 ml of crude enzyme (it is the culture filtrate of the respective organisms grown individually) from each source (both organisms and substrates) were taken individually and along with that 500 ml each of ethyl alcohol was added. They were allowed for precipitation for an hour and then centrifuged at 5000 rpm for 10 min. The precipitated enzymes from each source were refrigerated (4°C) until further analysis.
The precipitate collected from each source was dissolved individually in 30 ml of sodium acetate buffer (0.2 M) at pH 5.5 and were dialyzed against the same buffer overnight at 4°C.
Analysis of enzyme fractions by Poly Acrylamide Gel Electrophoresis(PAGE)
The dialysed enzyme samples from each source was analysed through PAGE using specific standard molecular marker. The gel obtained was photographed and scanned using a gel documentation system (SYNGENE, UK). Then the molecular weight of individual enzyme fractions was determined by referring the molecular weight of the marker.
The results obtained in the present study were subjected to SD and One-Way ANOVA described by Zar (1974).
Enzyme production at varying pH
Cellulase enzyme production – DNS Method
Coir waste as substrate
The cellulose enzyme production by
Saw dust as substrate
Cellulase enzyme production – FPA method
Coir waste as substrate
The cellulase enzyme production by FPA method determined that
Saw dust as substrate
The fungal strain,
Enzyme production at varying temperature
Cellulase enzyme production – DNS method
Coir waste as substrate
The results revealed that the enzyme production by
Sawdust as substrate
The enzyme production by
Cellulase enzyme production – FPA method
Coir waste as substrate
At 20°C, the enzyme production by
Sawdust as substrate
The fungal strain
Electrophoresis studies (PAGE Analysis)
Fig. (5) represents the protein profile of cellulase produced by
Lane M: Marker; Lane A:
The protein fractions have been assigned numbers in order of the increasing mobility towards the anode. In case of
On comparing the enzyme production by both strains grown at two different substrate supplemented medium, coir waste showed maximum enzyme production by both organisms than that of saw dust. Comparison between the fungal strains showed
Major impediments to exploit the commercial potential of cellulases are the yield stability and cost of cellulase production. Therefore, research should also aim at exploiting the commercial potential of existing and new cellulase in nature (Coral et al., 2002). Agricultural residues such as corn stove, wheat straw, rice straw, baggase etc. were used in cellulase production (Rao et al., 1983; Chalal, 1996). Although, the raw materials are cheaper, pre-treatment is generally required to improve the utilizability of lignocellulosic materials and the cost is considerable (Liming Xia and Peilin Cen., 1999). In view of the above facts, in the present study, the natural waste materials such as coir waste as well as saw dust waste have effectively utilized as major carbon source for the production of cellulase enzyme by fungal strains.
A capacity to degrade cellulose is a character distributed among a wide variety of aerobic, facultative aerobic, anaerobic bacteria and fungi. The characters are restricted to a few species among several major taxa (Gooday, 1979). The important cellulolytic fungus like
In the present study, two fungal strains such as
The carbon sources induce production of cellulase, but amount produced is variable. This is because of the influence of substrate (carbon source) on the growth of cellulolytic organisms (Mandels and Reese, 1985; Zhu et al., 1988; Lakshmikant and Mathur, 1990). In the present study, two different substrates such as saw dust and coir wastes were used as major carbon source. Apart from this, some environmental factors are also influenced the growth of organisms as well as maximum production of enzymes will be at certain optimum temperature, pH, salt concentration etc. (Immanuel et al., 2006).
In the present study, the effect on environmental factors such as pH and temperature against
In the present study, the ability of cellulase degrading fungi,
In the filter paper assay method by using coir waste as substrate, high level of enzyme production was obtained at pH 7 (0.340 IU ml-1 by
Regarding the temperature influence on production of enzymes, the DNS method by using coir waste as substrate showed that high level of production was obtained at 40°C. i.e., 0.292 and 0.258 IU ml-1 by
For estimation of optimum temperature of enzyme, the enzyme activity was determined by carrying out the assay at several temperatures between 30 and 90°C. The optimum temperature was observed around 40°C (Coral et al., 2002). Here in the present study, it is evident that 40°C is optimum for the production of cellulase enzyme when coir waste used as major carbon source. While in case of saw dust used as carbon source, the temperature ranged from 40 – 50°C for optimum production of cellulase enzyme.
In case of pH, it was observed that the enzyme activity has a broad pH range between 3.0 and 9.0 (Coral et al., 2002). Here in the present study, it was found that pH 5 – 7 is optimum in case of coir waste as substrate, but pH 6 is optimum when sawdust used as substrate for enzyme production by
Thus it was seen that there were no arrangements regarding enzyme data results. Such different results may appear because of differences within the same genus. In addition, no comparative investigations have been published on the enzymes from these organisms but the difference appears to be small as difference in morphology between the species (Aunstrup et al., 1979). But pH requirement was determined at pH 5.0 in fungal species
A cellulytic enzyme from
In the present study with SDS-polyacrylamide gel electrophoresis two bands showing cellulolytic production were detected in each lane from the crude enzyme. The molecular weight was 32 kDa and 21 kDa in case of
Likewise coir and sawdust are suitable lignocellulosic bio wastes for the production of cellulase enzyme. From the present study, it could understand that sawdust is most suitable substrate for cellulase production when compared to that of baggase or corncob (Ojumu et al., 2003) as it gives highest yield of enzyme. The coir waste is most abundant lignocellulosic waste obtained in Kanyakumari district, Tamil Nadu, India, can be effectively used for cellulase production and it is concluded that it has high ability to produce cellulase enzyme.
Cellulase enzyme production accounts for 40% of cost in bioethanol synthesis. To reduce cost of production, the lignocellulosic substrates are used instead of synthetic cellulase due to their reasonable cost, high enzyme production capacity etc. The reduction in cost paves an economically easy way production of ethanol. It is an important issue to deal with the residue both the comprehensive utilization of lignocellulosic resources and for the prevention of environmental pollution.
G. Immanuel School of Industrial Fisheries Cochin University of Science and Technology Foreshore Road Ernakulam, Cochin-16 Kerala, India E.mail: firstname.lastname@example.org (or) email@example.com