Dermatophytes are mycelial and keratinophylic fungi of the mold group, originally saprobial, but have adapted themselves to animal and human parasitism through evolution. Microsporum gypseum is a geophilic dermatophyte, frequently transmitted by fungal spores to man-soil contact in rural areas. It grows relatively rapidly and matures in 6 to 10 days. The texture of the colony is powdery to granular and the color is beige to cinnamon brown. From the reverse, it is yellow to brownish red. It produces septate hyphae, macroconidia and microconidia. Macroconidia are abundant, fusiform and symmetrical in shape with rounded ends. The walls of macroconidia are thin and rough and they contain 3-6 cells. Microconidia are moderately numerous in number, club-shaped and located along the hyphae. Microsporum usually produces a single inflammatory skin or scalp lesion. Invaded hairs show an ectothrix infection but do not fluoresce under Wood's ultra-violet light. It had been reported as a etiological agent of dermatophytosis in AIDS patients (Giudice et al., 1997). Several enzymes produced by dermatophytes, particularly keratinases, are considered to play a role in the virulence of the fungus.
Proteolytic enzymes are largely used in the industry for biotechnological applications
involving the hydrolysis of protein substrates. Proteases constitute an important
fraction of the global enzyme sales and a relevant part of this market is accounted
by bacterial proteases (Rao et al., 1998). Bacterial keratinases (E.C.
126.96.36.199) are of particular interest because of their action on insoluble keratin
substrates and generally on a broad range of protein substrates (Lin et al.,
1995). These enzymes have been studied for de-hairing processes in the leather
industry (Raju et al., 1996). Cultivation conditions are essential in
successful production of an enzyme and optimization of parameters such as pH,
temperature and media composition is important in developing the cultivation
process. Despite all the work that has been done on production of proteolytic
enzymes, relatively little information is available on keratinases (Wang and
Shih, 1999). Keratinolytic fungi are able to attack and destroy keratin, whereas
keratinophilic fungi accompany those utilizing only non-keratinous components
of keratinous substrata or the products of keratin decomposition. Vanbreuseghems
hair baiting method has been mainly used for examination of keratinolytic and
keratinophilic fungi in sewage sludge and other organic waste and waste-contaminated
The objective of this study was to isolation, screening, purification and determination of the enzymatic activity of extracellular keratinase from dermatophyte Microsporum gypseum.
MATERIALS AND METHODS
Collection of Samples
Fifty protein rich soil samples with sandy texture and grey to black in
colour were collected from the different part of Bangalore, India during January
2006. The research was carried out at Integrated Biotechnology Extension (IBX)
Lab, Bangalore, India
Isolation, Screening, Selection and Identification of Keratinases Producing
Bacteria (Vanbreuseghems Hair Baiting Method)
The isolation of keratinases producing fungi was carried out using hair
bait technique. Half filled sterile petriplates with soil samples were taken.
Short (2-3 cm)strands of steriliged defatted human hair was spreaded over surface
of the soil. Fifteen milliliter of sterile water was added to the soil to facilitate
germination of fungal spores. Antibiotic streptromycin (40 mg L-1)
was added to prevent bacterial growth. These complete set was incubated at room
temperature in a dark for 4 weeks. Inoculum from hairs with fungus growth were
taken and placed on plate of Sabourauds dextrose agar and incubated at
25°C for 7 days. After performing the microscopy, biochemicals, bromocresol
purple test, hair perforation test the organism was confirmed as Microsporum
Mass Production and Extraction of Enzyme
Mass production of enzyme was done by submerged fermentation, Microsporum
gypseum was inoculated into the medium containing human hair (2.6 g L-1)
and incubated at 25°C for 5 days and then in shaker for 7 days. At the end
of the growth period, the mycelium and residual hair were removed from the culture
fluid by filtration. The filtrate was taken out and precipitated with ammonium
Purification of the Enzyme
The purification was carried out by dialysis and ion-exchange chromatography.
Estimation of Total Protein by Lowrys Method
Different dilutions of Bovine Serum Albumin (BSA) solutions were prepared
by mixing stock BSA solution and water in the test tube. The final volume in
each of the test tubes is 1 mL. The BSA range is 0 to 200 μg mL-1.
Five milliliter of alkaline copper sulphate reagent (analytical reagent) was
added and mixed well. This solution was incubated at room temperature for 10
min. Then 0.5 mL of reagent Folin Ciocalteau solution (reagent solutions) was
added to each tube and incubated for 30 min in dark. The absorbance was taken
in the spectrophotometer at 660 nm using blank. The absorbance against protein
concentration was plotted to get a standard calibration curve. The absorbance
of purified and unpurified sample was taken and the concentration of the unknown
sample was calculated.
Determination of Activity of Keratinases by Folin Ciocalteau (FC) Method
The standard solution of amino acid, tyrosine was dispersed in a series
of test tubes in the range of 0 to 2 mL and the volume of each tube was made
up to 2 mL with water and 6 mL of alkaline copper sulphate reagent was added
and incubated at room temperature for 15 min. 0.5 mL of Folin Ciocalteau solution
was added and incubated at room temperature for 30 min. The optical density
was taken using 660 nm filter using spectrophotometer. Fifty milligram of substrate
(hair), 6 mL of 28 mM phosphate buffer, 1 mL of enzyme sample was incubated
at room temperature. 2.0 mL of 10% TCA was added to stop the reaction. Centrifuged
at 3000 rpm for 10 min. 1.0 mL of supernatant, 2.5 mL of 0.5 N NaOH and 0.5
mL of Folin Ciocalteau solution were added and incubated for 30 minutes at room
temperature. Amino acids released were estimated by taking optical density at
660 nm. the control was prepared without incubation. The absorbance against
tyrosine concentration was plotted to get a standard calibration curve. The
absorbance of purified and unpurified sample was taken and the concentration
of the unknown sample was calculated.
Determination of Keratinases Activity
The effect of pH, temperature, activator (MgCl2), inhibitor (ZnCl2)
was also studied.
| Micro molar of PNP Liberated/Molecular weight of PNP
||μmole mL-1 60 min at 37°C
| Enzyme activity/Protein concentration
||μmole mg-1 60 min at 37°C
Table 1 shows the protein concentration, enzyme activity and the specific activity of the 1 mL of sample at 37°C.
From the obtained result the total protein content of the purified enzyme was 1.33 fold. The enzyme activity was 2 fold and the specific activity was 1.49 fold.
The charecterisation studies were carried out with effect of pH, temperature, activator (MgCl2), inhibitor (ZnCl2) and tabulated in Table 2.
Maximum enzyme activity and enzyme yields were observed at temperature range
25-45°C and pH ranging from 7.0 to 9.0. Maximum enzyme activity was obtained
with more alkaline pH values (Table 2). Although the production
pattern of keratinase was similar for the different pH values tested, lower
activities were observed at pH 5.0 and 15°C.
|| Total protein concentration, enzyme activity and the specific
activity of keratinases at 37°C
||Characterization of keratinases
The study clearly indicates the presence of the enzyme keratinases in the dermatophyte Microsporum gypseum. One milliliter of the purified sample contain 80 μg of protein, 1.09 μ mole mL-1 60 min enzyme activity and 13.6 μ mole mg-1 60 min specific activity which was higher with respect to the unpurified one. The purified and unpurified state of the enzyme was judged by SDS/PAGE. Purified enzyme showed a single band of molecular weight of 33 kDa. Characterization studies showed optimum activity at alkaline pH and at 35°C. The enzyme kinetics increased with increased concentration of MgCl2 and decreasedwith increased concentration ofZnCl2.
Maximum biomass and keratinase activity were observed from pH 7.0 to 9.0, which agrees with those described for most feather-degrading Bacillus (Wang and Shih, 1999; Suntornsuk and Sutornsuk, 2003). For production of keratinase by B. licheniformis and a recombinant B. subtilis, uncontrolled pH operation was more favorable than the controlled pH operations (Wang and Shih, 1999). The same was observed for alkaline protease production by B. licheniformis (Çalik et al., 2002).
It is thought that the abundance of keratinous debris favors the growth of keratinolytic and keratinophilic fungi in sludge, Microsporum gypseum in particular. A keratinase was produced by Microsporum gypseum under different growth conditions. This enzyme has been shown to be useful for biotechnological purposes such as hydrolysis of poultry feathers (Riffel et al., 2003a) and de-hairing of bovine pelts (Riffel et al., 2003b).
Although the production of proteases in complex growth media often promotes exuberant growth and high enzyme yields (Johnvesly and Naik, 2001; Joo et al., 2002), their expensive cost makes them unsuitable for a large-scale production. It seems more adequate to use raw materials like some wastes from the food industry as a basis of the culture.
The recent finding that B. licheniformis PWD-1 keratinase cause enzymatic breakdown of prion protein PrPSc (Langeveld et al., 2003) leave open a novel relevant application for broad range of keratinase. In this study, the optimum conditions for keratinase synthesis by the Microsporum gypseum were determined, which is an essential step for the production of adequate amounts for application in research field and other industrial areas.