Sanjay Telang

Government Science and Commerce College, Benazir, Bhopal, India.

DOI : https://dx.doi.org/10.13005/bpj/430

Abstract

Proteases have been isolated from A. niger isolates originally isolated from common carp (Cyprinus carpio). The three isolates showed slight difference in their productivity of the enzyme. The characteristics of the enzyme from three isolates however was same i.e. alkaline in nature. The alkaline proteases are industrially very important.

Keywords

Fungal pathogen; Cyprinus carpio; Proteases Activity

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Copy the following to cite this article: Telang S. Aspergillus niger from Common Carp (Cyprinus carpio) with Proteases Activity. Biomed Pharmacol J 2013;6(2)

Copy the following to cite this URL: Telang S. Aspergillus niger from Common Carp (Cyprinus carpio) with Proteases Activity. Biomed Pharmacol J 2013;6(2). Available from: http://biomedpharmajournal.org/?p=2768

Introduction

Proteases constitute one of the most important groups of industrial  enzymes  and  have  applications  in  different industries  for  example  in  detergent,  food,  feed,  pharmaceutical, leather, silk and for recovery of silver from used X-ray  films  (Anisworth,  1994;  Fujiwara,  1993).  This  enzyme  accounts  for  30%  of  the  total world  enzyme  production  (Horikoshi,  1996).  A variety of microorganisms such as bacteria,  fungi,  yeasts  and  actinomycetes  are  known  to produce  this  enzyme (Reese et al., 1950; Taguchi et al., 1983;  Kim  et  al.,  1993;  Manjeet  et  al.,  1998).

The objective of this work was to isolate protease from the fungus Aspergillus niger originally isolated from the skin of common carp.

Materials and Methods

Fungal strain and maintenance

The fungal strains A. niger BEN1, A. niger BEN3 and A. niger BEN7 were used to isolate protease. The strains were maintained in refrigerator at 4^oC on PDA slant.

Liquid culture

Minimal synthetic medium supplemented with casein (1% w/v) was used to raise liquid culture in flask of size 25 ml. A 100 ml medium was inoculated with about 10⁷ spores of the fungus and the medium was incubated at 37^oC on a shaking incubator.

Preparation of enzyme source

After three, four and five days of growth, the broth was centrifuged at 10000 rpm and the supernatant was used as source of enzyme.

Assay of enzyme

The  protease  activity  was  assayed  by  casein  digestion  method (Kunitz,  1947).  The  reaction  mixture  contained  suitably  diluted enzyme  and  casein  in  0.1M  sodium  carbonate  buffer  pH  10.  The reaction  mixture  was  incubated  at  40°C  for  10  min.  The  reaction was terminated by the  addition  of 3 ml of 10% trichloroacetic acid. The  terminated  reaction  mixture  was  incubated  at  room  temperature  for  30  min.  The  precipitate  formed  was  filtered  through  Whatman No. 1 filter paper. The absorbance of the filtrate was measured at  280  nm.  Tyrosine  was  used  as  standard.  One  unit  of  protease activity  is  defined  as  the  amount  of  enzyme  which  liberates  one micromoles  of  tyrosine  per  minute  per  gram  dry  substrate  under experimental  conditions.  Protein  was  estimated  by  the  method  of Lowry et al. (1951).

Effect of pH

The pH optimum of the enzyme was determined by varying the pH of the reaction mixtures using the following buffers (100 mM): sodium acetate (pH 3.0-5.5), sodium phosphate (pH 6.0-7.0) and Tris-HCl (pH 7.5-8.0).

The protease activity at different period of incubation is given in table 1.

Table 1: Protease activity shown by the fungal isolates in different period of growth.

Fungal isolates	Proteases activity (unit/ml) in the broth of different period of growth
	Third day	Fourth Day	Fifth day	Sixth day
A. niger BEN1	2	3	4	4
A. niger BEN3	2	4	5	5
A. niger BEN7	2	3	4	4

 

The enzyme activity in the broth was found to increase from third day and became constant with effect from fifth day. The activity was found to be a bit better (5 unit/ml) in case of the strain A. niger BEN3.

The effect of pH on enzyme activity is shown by the figure 1. The enzyme showed maximum activity at alkaline pH, although residual activity continued to be seen upto a pH of 3.

Figure 1: Effect of pH on the proease activity from three isolates. Click here to View figure

 

The alkaline protease is very important industrially. Earlier alkaline protease has been reported from fungi and bacteria (Veloorvalappil et al., 2013).

Conclusions

niger isolated originally from carps produced proteases under in vitro condition.

Of the three strains BEN3 produced more unit of enzymes under parallel conditions All the three isolates produced alkaline proteases.

References

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