CHITINASE PRODUCTION BY ASPERGILLUS TERREUS FROM MARINE WASTES AND ITS EFFICACY IN ANTIFUNGAL ACTIVITY

The present investigation aims to isolate and identify chitinase producing mycoflora and the chitinase production by efficient Aspergillus terreus on different shell powder wastes substrates such as prawn, crab and molluscs at an extracellular and intracellular level and its efficacy in assessing the antifungal activity. Shell fish wastes disposed soil samples were collected and screened for chitinolytic fungi (hydrolyzing zone). The chitinase production on different substrates was assessed and the enzyme extracts were screened for antifungal activity by agar well and disc diffusion methods. Among the number of mycoflora isolated only 4 fungal strains Aspergillus terreus, A. fumigatus, A. flavus, and A. clavatus showed maximum hydrolyzing zones. A significantly highest hydrolyzing zone (clearance zone) of 20 mm (out of colony diameter of 65 mm) was obtained by Aspergillus terreus. Among the different substrates (prawn, crab and molluscs shell powders at 1% concentration), highest chitinase activity was recorded by Aspergillus terreus on prawn shell powder waste followed by crab shell powder waste at an intra and extra cellular level. The chitinase produced from prawn shell powder waste as substrates by A. terreus showed maximum zone of inhibition in both agar well (28.0 mm) and disc diffusion (24.0 mm) methods against the pathogenic fungi, A. flavus compared to positive control (Fluconazole). The chitinase produced from A. terreus might have synthesized some antifungal compounds which may be responsible for killing pathogenic fungi.


ISSN: 2320-5407
Int. J. Adv. Res. 8(09), 1399-1404 1400 production. The chitin degrading enzyme, chitinases (EC 3.2.1.14) are a glycosyle hydrolase group of enzymes that catalyse the hydrolysis of insoluble chitin into monomer and oligomer. The microbial extracellular chitinases play an important role in pharmaceutical industry due to their immense potential application like isolation of protoplast of fungi and yeast, preparation of SCP, control of pathogenic fungi, production of bio-pesticides and control of mosquito propagation (Halder et al. 2012). Different types of substrates which contain chitin have been tried for the production of chitinases, which include fungal cell walls, prawn, crab and shrimp shells (Tagawa and Okazaki 1991). Some pre-treated chitin is used as a substrate for microbial chitinases production (Wang and Chang, 1997). Molluscs which are widespread and abundant play an important role in the food chain and are important food sources for larger animals from fish to whales. Shrimp processing industry plays an important role in global economy. The global annual recovery of chitin from the processing of marine crustacean waste is estimated to be around 37,300 metric tons (Sheikh and Deshpande 1993). The shell fish industry is operative among all the costal countries and contribute hugely to to the food delicacies. During the processing of prawns, shrimps and lobsters mostly, the meat is taken, while the shell and head portions are discarded as wastes which results in huge amount of waste generated throughout the world. The main industrial source of chitin is shellfish waste from processing of crab and shrimp. Biological control, or the use of microorganisms or their secretion to prevent plant diseases offers an attractive alternative or supplement for the control of plant diseases without the negative impact of chemical control. Therefore, biological control tactics have become an important approach to facilitate sustainable agriculture (Wang 1999). Chitinases are generally related with the production of antifungal compounds and extracellular hydrolytic enzyme (chitinases and 1, 3-β glucanase). Chitinolytic enzymes are efficient in lysing the fungal cell wall. The microorganisms that produce these enzymes are capable of eradicating fungal diseases that are a problem for global agricultural production.

Isolation of Fungal Species
Prawn, crab and molluscs shell wastes disposed soil samples were collected from the disposal area in Cochin district, Kerala. The samples were serially diluted and plated on Potato Dextrose Agar medium (PDA) and incubated for seven days at 30°C. After incubation, the plates were observed for fungal growth and were subcultured and maintained on PDA slants at 4°C. The fungal isolates were identified based on their morphology, mycelia structure and spore formation (Barron 1968;Ellis 1976;Domsche and Gams 1972).

Screening of Soil Fungal Isolates for Chitinolytic Activity
The identified fungal strains were grown individually for screening for chitinolytic activity on colloidal chitin agar plates (Sherief et al. 1991a). The clearance zone formed around the colonies was determined by adding 0.1 percent congored solution and counter stained with 1M NaCl for 15-20 min. A clear zone of chitinase hydrolysis gave an indication of chitinase producing microorganisms. The diameter of the clear zone was measured to provide a quantitative comparison of chitinolytic activity.

Enzyme production
Erlenmeyer flasks containing 100ml of colloidal chitin liquid medium (Sherief et al. 1991b), was sterilized at 1 atm for 15 minutes. After cooling, one ml of Streptomycin sulphate (10,000 ppm) was added and incubated for 5, 7, 9 and 11 days at 30°C under static conditions. The clear filtrate obtained through Whatman No. 40 was used as a source of extracellular enzyme. A quantity of 5.0 g of the washed mycelia mat was macerated in five ml of sodium phosphate buffer of pH 7.0 in a pre-chilled mortar and pestle with a pinch of acid washed sand. The homogenate was centrifuged and the supernatant served as crude source of intracellular enzyme.

Chitinase assay
Chitinase activity was determined by the production of N-acetyl-D-glucosamine using DNS method. The reaction mixture consisted of 1ml of 0.2 M sodium phosphate buffer (pH 7.0) containing 10 percent of colloidal chitin and 1ml enzyme solution. The mixture was kept in water bath at 50°C for 1 hr. The reaction was terminated by adding 1 ml of 1 percent NaOH and boiled for 5 minutes at 100°C and centrifuged for 10 minutes. 1 ml of the supernatant was mixed with 1ml DNS solution and boiled at 100°C for 10 minutes. Finally, the content was cooled and 5ml of distilled water was added to the mixture. The absorbance was read at 535 nm and the protein content was determined by the method of Lowry et al.1951.

Effect of different substrates on enzyme production
The isolated fungal strain was incubated in the production medium containing 0.50g of substrates like prawn shell, crab shell and molluscs shell powder . The enzyme activity for each trial was estimated at 5 th , 7 th , 9 th and 11 th day of incubation in triplicates.

Screening of the extracts for antifungal activity
Test fungal strains of Aspergillus niger, A. flavus, A. fumigatus kept in the fungal culture bank of Department of Botany, Avinashilingam University, Coimbatore were taken and maintained as subculture in slants. Antifungal studies were carried out by agar well and disc diffusion methods.

Agar well diffusion method (Smania et al. 1995)
Each fungal suspension of test microorganisms were spread separately over the surface of PDA plates with cotton swap and five wells each of 6mm diameters were made on the agar. The wells were filled with 20μl each of enzyme (obtained from different substrates) along with negative control (phosphate buffer) and positive control (fluconazole) at the centre. The plates were incubated at 30°C for 76 hours. The zone of inhibition was calculated by measuring the diameter of clear zone formed around the well.

Disc diffusion method (Bauer et al. 1966)
Plates were prepared by pouring 20 ml of sterile PDA medium into the sterile petriplates and were inoculated with test organisms. Sterile paper disc (Whatman No.1, 3.6 mm diameters) impregnated with 20μl quantity each enzyme, phosphate buffer (as negative control) and positive control (fluconazole) and were air dried and placed on the PDA plates. The plates were incubated at 30°C for 76 hours. The zone of inhibition was calculated by measuring the diameter of clear zone formed around the disc.  (Table 1)   Values are mean of three triplicates Umg -1 = 1μ mol of N-acetyl-D-glucosamine released min -1 mg -1 Protein.

Assessing Chitinase Production on Different Substrates (Table 2)
Among the different substrates (prawn, crab and molluscs shell powder wastes), chitinase activity by Aspergillus terreus showed an enhancement from 2.36 Umg -1 to 4.89 Umg -1 enzyme protein up to 7 th day of incubation and it gradually decreased to 1.75 Umg -1 enzyme protein in prawn shell powder waste as substrate compared to control with an enhancement from 0.51 Umg -1 to 0.72 Umg -1 enzyme protein and it declined to 0.25 Umg -1 enzyme protein on the 11 th day of incubation. The least enhanced activity was observed in molluscs shell powder waste from 1.34 Umg -1 to 3.49 Umg -1 enzyme protein and it decreased to 0.92 Umg -1 than the control (from 0.47 Umg -1 to 0.77 Umg -1 enzyme protein and declined to 0.38 Umg -1 enzyme protein) on the 11 th day of incubation at an intracellular level. At an extracellular level, the chitinase activity by Aspergillus terreus was increased from 1.17 Umg -1 to 5.29 Umg -1 enzyme proteins up to 7 th day of incubation and it gradually declined to 1.36 Umg -1 enzyme protien on the 11 th day of incubation than the control from 0.42Umg -1 to 0.81 Umg -1 enzyme proteins in prawn shell powder waste as substrate. The minimal activity observed was in molluscs shell powder waste from1.45Umg -1 to 3.37 Umg -1 enzyme protein up to 7 th day of incubation and it decreased to 0.73Umg -1 enzyme protein than the control (from 0.55 Umg -1 to 0.70 Umg -1 enzyme protein and decreased to 0.38 Umg -1 enzyme protein) on the 11 th day of incubation. The present result is on par with the report of (Rattanakit et al. 2002) who found that the chitinase production by Aspergillus sp. S1-13 was increased to 58% -65% when, shrimp powder was used as a substrate.

Screening of the enzyme extracts for antifungal activity (Table 3) Agar well diffusion method
It was observed from the results of the agar well diffusion method that among the enzyme produced from different substrates by Aspergillus terreus, highest zone of inhibition of 28.0 mm was observed against phytopathogenic fungi, Aspergillus flavus from the chitinase produced from the prawn powder waste as a substrate. This was higher than that observed (26.0 mm) in positive control (fluconazole). The minimum zone of inhibition observed was 10.0 mm in case of chitinase produced by molluscs powder waste against the phytopathogenic fungus A.niger.

Conclusion:-
Thus, it can be deduced from the present investigation, that the candidate Aspergillus terreus, a chitinolytic fungus can be effectively harnessed for its maximum production by using cost effective, cheap substrates (shell fish wastes). The chitinase produced from A. terreus might have synthesized some antifungal compounds which may be responsible for killing pathogenic fungi. From the sea food waste, commercially important enzyme chitinase could be produced and it not only solves the environmental problem of disposal of waste in an ecofriendly manner but also promotes the production of value added products of economic benefits at a cheaper rate.