Introduction

Thermophillic fungi mainly are responsible for  compost maturation (Miller 1996). Fungi have been isolated from sewage, more or less polluted soil and water by different workers (Cooke 1952, 1954, 1957, 1959; Backer Snaw 1955; Niebla and Aquero, 1982; Rebox et al. 1984). Fungi are important enzyme producers since their enzymes are produced externally (Borgestrom and Brackman 1984; Venkat Eshawarlu and Reddy, 1993; Hang and Woodams 1990; Ferreira Costa and Pralta, 1999). Lipase (E.C.3.1.1.3) occur extensively in nature and catalyses the hydrolysis of triacylglycerols in aqueous media. In this study, the synthesis of extracellular lipases by two lipolytic thermophilic species sporotrichum thermophile and Aspergillus fumigatus and the conditions which determine their activities were examined. Therefore the present investigation has been under taken for the thermophilic fungi isolated from different sites of Bareilly.

Materials and Methods

Samples collection

Samples were collected from the sites contaminated with lipids, oil and decaying organic matter from five different sites. Samples were collected as undecomposed, semidecomposed and totally decomposed.

Isolation of lipolytic molds

Selective isolation of fungi will be made using enriched (YPS’s) medium by using recent technique (Warcup 1950). PH is 7.0 and temperature is 45⁰C.

Identification of fungi

Identification of fungi is done with the help of camera lucida.

Extraction of Lipase

Lipase was extracted by  the method of Yamada and Machida (1962). Isolated fungus were grown on modified Czapek’s dox medium. Composition is oil cake 30 gm, MgSO4.7H₂O  0.5 gm, KCl 0.5 gm, NaNO₃ 2.0 gm , K₂HPO₄  1.0 gm, FeSO₄ 0.01 gm.

Lipase specific activity

Lipase specific activity was measured by universal titrimetric method using automatic titrator from Radiometer (Copenhagen Denmark) .

Following reagents were prepared for enzyme assay –

Coconut oil                 –          1% O/W emulsion as substrate

Calcium Chloride        –          0.1 M solution

Sodium Hydroxide     –          0.05 N solution

Phosphate Buffer        –­          0.1 M solution

Stock solution A         –          0.1 M K₂HPO₄

Stock solution B         –          0.1 M KH₂PO₄

Phenolphthalein indicator

Calculation

Lipase specific activity was calculated using the formula given by Peled. N, Krenz (1981).

U/ml = (V_S–V_b ) x 0.05 x 10³ x D x 18.5

18.5     –           Total assay media.

V_S        –           Titration value.

V_b        –           Blank  titration value.

0.05     –           Molar concentration of NaOH used for neutralization.

10³       –           factor for µ mole.

D         –           Dilution.

One unit of lipase specific activity was defined as the amount which liberated one µ mole of acid per minute at 45⁰C. (somkuti and Babel , 1967) .

Protein estimation

The protein contents of the filtrates were determined using the method of Lowry et al (1951) with crystalline bovine albumin as standard.

Effect of pH on lipase specific activity 

The effect of pH on lipase specific activity was determined by using 0.1 M phosphate buffer pH range from 4 to 8.5.

Results and Discussions

It has been found that lipase specific activity of different isolates vary at different pH. Aspergillus fumigatus showed maximum lipase specific activity 3.870±0.0026 U/mg protein at pH 6.0 on 5^th day (table 1), while Sporotrichum thermophile showed maximum lipase specific activity at 1.847±0.0254 U/mg protein at pH 7.0 on 5^th day (table 2). Aspergillus fumigatus showed lowest lipase specific activity 0.337±0.0015 U/mg protein at pH 8.0 on 3^rd day (table 1), while Sporotrichum thermophile showed lowest lipase specific activity at pH 0.445±0.254 U/mg  protein on 5^th day (table 2).

Optimum range of pH for lipase specific activity was 6-7.

Table 1: Effect of pH on the activity of lipase by Aspergillus fumigatus from city waste of Bareilly. 

Specific activity (U/mg Protein)

Table 2: Effect of pH on the activity of lipase by Sporotrichum thermophile from city waste of Bareilly.

Specific activity (U/mg Protein)

Conclusion

pH 6.0 was optimal for lipases of Aspergillus fumigatus while pH 7 was best for Sporotrichum thermophile. Similar peaks were recorded for thermophilic fungi by Somkuti and Babel (1968).

References

1. Cooke, W. B., Fungi in Polluted Water and Sewage Water, Isolation technique, Sewage Industrial Waste, 26, 661-674 (1954).
2. Cooke, W. B., Checklist of Fungi Isolated from Sewage and Polluted Water, Sydowia II Bein, 1, 146-175 (1957).
3. Cooke, W. B., Are Fungi Important in Sewage Treatment? Public Works Magazine,113-114 (1959).
4. Cooke, W. B., The Role of Fungi in Environmental Sanitation. Development in Industrial Microbiology, 3, 313-318 (1969).
5. Miller, G. L., Use of dinitrosalicyclic acid Reagent for Determination of Reducing Sugar, Chem., 31, 426-428 (1959).
6. Somkuti, G. A. and Babel, F. J. Lipase Activity of Mucor Pusillus. Microbial, 16, 617-619 (1968).