Strain improvement of a new Keratinolytic Fungi

 

Venkatesh Kamath B¹, Hawa Hashim², Kamesh Kumar Keshri², Swati Tripathy³, Subrahmanyam. V. M¹

¹Department of Pharmaceutical Biotechnology, Manipal College of Pharmaceutical Sciences,

Manipal Academy of Higher Education. Manipal – 576104.

²Final Year BPharm Students, Manipal College of Pharmaceutical Sciences,

Manipal Academy of Higher Education. Manipal – 576104.

³PG Student, Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education,

Manipal-576104.

 

ABSTRACT:

Keratinase is a protease enzyme. It degrades the keratin protein present in the hair, skin, nails etc. It can be applied in various fields of therapeutics such as in the treatment of onychomycosis, hair dandruff and recently in skin cancer as well. It can also be applied in various textile and leather industries. The SPF1 fungi isolated from the poultry farm around Manipal, India was found to produce the inducible keratinase enzyme. The strain was exposed to UV radiation to induce the UV mutation and the yield was found to have improved from 147.52µg/ml to 288.00µg/ml. The SPF1 strain was also plated in Sabouraud Dextrose Agar and 8 different colonies were selected to compare their keratinolytic activities.

 

 

 

1. INTRODUCTION:

Keratin is a type of heteropolymeric protein which is insoluble¹, fibrous in nature and provides structural integrity to the epithelial cells. Keratinase is a type of protease enzyme which has the ability of hydrolyzing the peptide bonds specifically in keratin into shorter products. Many microorganisms belonging to bacteria, fungi², actinomycetes etc. have been reported to have the ability of producing keratinase in the presence of keratin substrate. Keratinase has found its applications in the treatment of psoriasis, acne, onychomycosis, hair dandruff and a certain types of skin cancer. It can also be used in the manufacture of leather garments³, glues⁴, films, coatings etc. It can also be used to develop organic fertilizers and animal feed and in the treatment of waste water. The objective of this research was to improve the yield of the keratinase enzyme produced by the new isolated SPF1 fungi using UV mutation.

 

The SPFI fungus which was isolated from a poultry farm around Manipal, Udupi district was found to have keratinolytic activity which was observed using equivalent amount of tyrosine produced when the organism was introduced to the basal salt media containing 1% feather waste. The tyrosine content was estimated using Lowry’s method. The SPF1 fungi was then plated on Sabouraud Dextrose Agar and various colonies having different extent of keratinolytic activity were observed. The strain was also exposed to UV radiation for various time periods and the yield of the keratinase enzyme produced was seen to have improved.

 

2. METHODOLOGY:

i.      Microorganism and maintenance of culture:

The microorganism used was a fungus (SPF1 strain) which was previously isolated from a poultry farm around Manipal, Udupi District. The microorganism was grown on Sabouraud dextrose agar slants at 27^oC for 4-5 days.

 

ii.    Standardization of tyrosine:

a.     Preparation of tyrosine stock solution:

Tyrosine stock solution was prepared which had a concentration of 1000µg/ml (1mg/ml).

This was done by accurate weighing 100mg of L-tyrosine powder and which was transferred to a 100ml volumetric flask, mixed and dissolved in distilled water and the volume was made up to the mark with distilled water. From the above stock solution, different concentrations of working stock solutions were prepared and diluted to the final volume (10ml) using distilled water

 

b.     Preparation of Lowry’s reagents:

Reagent A:

1g of sodium carbonate was weighed, transferred to a 100ml beaker and dissolved in 50ml distilled water.

0.2g of sodium hydroxide was weighed, transferred to another 100ml beaker and dissolved in 50ml distilled water.

Both the solutions were mixed.

 

Reagent B:

0.156g of copper sulphate was weighed, transferred to a 100ml beaker and dissolved in 10ml of distilled water.

To this, crystals of sodium potassium tartrate weighing 0.237g were added.

100ml of the reagent A and 2ml of the reagent B were mixed in a 250ml beaker and was labelled as Lowry’s reagent.

 

c.     Folin’s reagent:

Folin and ciocalteaou’s reagent was diluted using distilled water in a ratio of 1:1

 

d.     Standard plot preparation:

1ml each from the working stock solutions was taken in different test tubes and to each 4.5ml of Lowry’s reagent was added and kept for incubation at room temperature (27°C) for 10 minutes and then 0.5ml of the previously diluted Folin and ciocalteaou’s reagent was added and incubated at 27°C for 30 minutes.

 

A blank was prepared in a similar manner using 1ml of distilled water instead of the working stock solution.

 

After incubation, the absorbance was measured using the colorimeter at 660nm and the graph of absorbance against concentration was plotted.

 

iii.  Keratinolytic activity of the fungal (SPF1) isolates:

2.5ml of the fungal suspension in distilled water was made from the previously preserved SDA slants and inoculated in 75ml of the Basal salt media which contained 1% feather waste. The inoculated flask containing the media and the fungal suspension was incubated for 9 days at room temperature in an orbital shaker at 130rpm. After 9 days, 2ml was withdrawn from the flask and centrifuged for 20 minutes at 4000 rpm. 1ml of the supernatant was tested for keratinolytic activity using Lowry’s method. This was done in duplicate.

 

iv.   UV mutation:

The SPF1 strain from the previously preserved slants was taken and a 15ml suspension in sterile water was made and kept in a petri dish of 80mm in diameter and exposed to UV rays for 6,8 and 10 minutes at a distance of 25cm from the UV lamp. The suspension was then diluted in sterile water as shown in table.1ml of each of the diluted samples was plated on SDA and incubated at 27°C for 96 hours after which the colonies were transferred to the SDA slants, incubated for 5 days at room temperature and tested for the keratinolytic activity by Lowry’s method

 

v.     Morphology of the SPF1:

The fungal suspension was diluted to 10^-6 using sterile phosphate buffer pH 7.2 and plated on SDA and incubated for 5 days to observe the morphology of the colonies.

 

Also, the microorganism was stained using lactophenol cotton blue in a glass slide and observed under the microscope using high power.

 

3. RESULTS AND DISCUSSION:

 

Figure 1: Standard tyrosine plot

 

Table 1: Keratinolytic activity of the SPF1 fungus (wild strain)

 

 

 

 

 

Table 2: UV mutation

 

Figure 2: Morphology of SPF1 fungi plated on Sabouraud Dextrose Agar

 

The yield of keratinase produced by SPF1 fungi was found to have improved after exposing it to UV radiation. There is no much work done on the keratinolytic ability of this particular fungal strain earlier. Chemical method of mutation would have been used, but considering the time constraint, UV mutation was more feasible.

 

4. CONCLUSION:

The keratinolytic activity of the SPF1 fungi can be applied in the therapeutic fields such as the treatment of onychomycosis, psoriasis and other skin diseases such as dandruff. It is also useful in the industries and in the agricultural field. The wild strain SPF1 was found to produce tyrosine having concentration of 147.52µg/ml on the 9^th day. UV mutation was carried out and the colony that was exposed to UV for 6 minutes and was diluted 5 times gave the highest yield of 288.00µg/ml.

 

5. REFERENCES:

1.     Itisha Singh, R. K. S. Kushwaha. Keratinases and microbial degradation of Keratin. Adv. Appl. Sci. Res. 2015; 6(2):74-82.

2.     Beti Vidmar and Maša Vodovnik. Microbial Keratinases: Enzymes with Promising Biotechnological Applications. Food Technol Biotechnol. 2018; 56(3): 312–328.

3.     Gupta R, Ramnani P. Microbial keratinases and their prospective applications: an overview. Appl Microbiol Biotechnol. 2006;70(1):21-33.

4.     Veslava Matikevicience, Danute Masiliunience, Saulius Grigiskis. Degradation of keratin containing wastes by Bacteria with keratinolytic activity, Environment Technology Resources, 2009; 01:284-289.

 

Received on 23.01.2020            Modified on 19.03.2020

Accepted on 06.05.2020         © RJPT All right reserved