{"id":2768,"date":"2015-04-28T07:15:25","date_gmt":"2015-04-28T07:15:25","guid":{"rendered":"http:\/\/biomedpharmajournal.org\/?p=2768"},"modified":"2020-04-26T07:36:55","modified_gmt":"2020-04-26T07:36:55","slug":"aspergillus-niger-from-common-carp-cyprinus-carpio-with-proteases-activity","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol6no2\/aspergillus-niger-from-common-carp-cyprinus-carpio-with-proteases-activity\/","title":{"rendered":"Aspergillus niger from Common Carp (Cyprinus carpio) with Proteases Activity"},"content":{"rendered":"

Introduction<\/strong><\/p>\n

Proteases constitute one of the most important groups of industrial\u00a0 enzymes\u00a0 and\u00a0 have\u00a0 applications\u00a0 in\u00a0 different industries\u00a0 for\u00a0 example\u00a0 in\u00a0 detergent,\u00a0 food,\u00a0 feed,\u00a0 pharmaceutical, leather, silk and for recovery of silver from used X-ray\u00a0 films\u00a0 (Anisworth,\u00a0 1994;\u00a0 Fujiwara,\u00a0 1993).\u00a0 This\u00a0 enzyme\u00a0 accounts\u00a0 for\u00a0 30%\u00a0 of\u00a0 the\u00a0 total world\u00a0 enzyme\u00a0 production\u00a0 (Horikoshi,\u00a0 1996).\u00a0 A variety of microorganisms such as bacteria,\u00a0 fungi,\u00a0 yeasts\u00a0 and\u00a0 actinomycetes\u00a0 are\u00a0 known\u00a0 to produce\u00a0 this\u00a0 enzyme (Reese et al<\/em>., 1950; Taguchi et al<\/em>., 1983;\u00a0 Kim\u00a0 et\u00a0 al<\/em>.,\u00a0 1993;\u00a0 Manjeet\u00a0 et\u00a0 al<\/em>.,\u00a0 1998).<\/p>\n

The objective of this work was to isolate protease from the fungus Aspergillus niger<\/em> originally isolated from the skin of common carp.<\/p>\n

Materials and Methods<\/strong><\/p>\n

Fungal strain and maintenance<\/strong><\/p>\n

The fungal strains A. niger<\/em> BEN1, A. niger<\/em> BEN3 and A. niger<\/em> BEN7 were used to isolate protease. The strains were maintained in refrigerator at 4o<\/sup>C on PDA slant.<\/p>\n

Liquid culture<\/strong><\/p>\n

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<\/sup> spores of the fungus and the medium was incubated at 37o<\/sup>C on a shaking incubator.<\/p>\n

Preparation of enzyme source<\/strong><\/p>\n

After three, four and five days of growth, the broth was centrifuged at 10000 rpm and the supernatant was used as source of enzyme.<\/p>\n

Assay of enzyme<\/strong><\/p>\n

The\u00a0 protease\u00a0 activity\u00a0 was\u00a0 assayed\u00a0 by\u00a0 casein\u00a0 digestion\u00a0 method (Kunitz,\u00a0 1947).\u00a0 The\u00a0 reaction\u00a0 mixture\u00a0 contained\u00a0 suitably\u00a0 diluted enzyme\u00a0 and\u00a0 casein\u00a0 in\u00a0 0.1M\u00a0 sodium\u00a0 carbonate\u00a0 buffer\u00a0 pH\u00a0 10.\u00a0 The reaction\u00a0 mixture\u00a0 was\u00a0 incubated\u00a0 at\u00a0 40\u00b0C\u00a0 for\u00a0 10\u00a0 min.\u00a0 The\u00a0 reaction was terminated by the\u00a0 addition\u00a0 of 3 ml of 10% trichloroacetic acid. The\u00a0 terminated\u00a0 reaction\u00a0 mixture\u00a0 was\u00a0 incubated\u00a0 at\u00a0 room\u00a0 temperature\u00a0 for\u00a0 30\u00a0 min.\u00a0 The\u00a0 precipitate\u00a0 formed\u00a0 was\u00a0 filtered\u00a0 through\u00a0 Whatman No. 1 filter paper. The absorbance of the filtrate was measured at\u00a0 280\u00a0 nm.\u00a0 Tyrosine\u00a0 was\u00a0 used \u00a0as\u00a0 standard.\u00a0 One\u00a0 unit\u00a0 of\u00a0 protease activity\u00a0 is\u00a0 defined\u00a0 as\u00a0 the\u00a0 amount\u00a0 of\u00a0 enzyme\u00a0 which\u00a0 liberates\u00a0 one micromoles\u00a0 of\u00a0 tyrosine\u00a0 per\u00a0 minute\u00a0 per\u00a0 gram\u00a0 dry\u00a0 substrate\u00a0 under experimental\u00a0 conditions.\u00a0 Protein\u00a0 was\u00a0 estimated\u00a0 by\u00a0 the\u00a0 method\u00a0 of Lowry et al. (1951).<\/p>\n

Effect of pH<\/strong><\/p>\n

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).<\/p>\n

The protease activity at different period of incubation is given in table 1.<\/p>\n

Table 1: Protease activity shown by the fungal isolates in different period of growth.<\/strong><\/p>\n\n\n\n\n\n\n\n
Fungal isolates<\/strong><\/td>\nProteases activity (unit\/ml) in the broth of different period of growth<\/strong><\/td>\n<\/tr>\n
Third day<\/strong><\/td>\nFourth Day<\/strong><\/td>\nFifth day<\/strong><\/td>\nSixth day<\/strong><\/td>\n<\/tr>\n
A. niger <\/em>BEN1<\/strong><\/td>\n2<\/td>\n3<\/td>\n4<\/td>\n4<\/td>\n<\/tr>\n
A. niger <\/em>BEN3<\/strong><\/td>\n2<\/td>\n4<\/td>\n5<\/td>\n5<\/td>\n<\/tr>\n
A. niger <\/em>BEN7<\/strong><\/td>\n2<\/td>\n3<\/td>\n4<\/td>\n4<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

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<\/em> BEN3.<\/p>\n

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.<\/p>\n\n\n\n
\u00a0\"Figure<\/td>\n\n

Figure 1: Effect of pH on the proease activity from three isolates<\/strong>.<\/p>\n

\n

Click here to View figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

The alkaline protease is very important industrially. Earlier alkaline protease has been reported from fungi and bacteria (Veloorvalappil et al<\/em>., 2013).<\/p>\n

Conclusions<\/strong><\/p>\n

niger isolated originally from carps produced proteases under in vitro<\/em> condition.<\/p>\n

Of the three strains BEN3 produced more unit of enzymes under parallel conditions All the three isolates produced alkaline proteases.<\/p>\n

References<\/strong><\/p>\n

    \n
  1. Anisworth, J.\u00a0 Soap\u00a0 and\u00a0 detergents.\u00a0 Chem.\u00a0 Eng.\u00a0 News<\/em>., 72: 34-59 (1994)..<\/li>\n
  2. Fujiwara, N. Production of\u00a0 thermophilic\u00a0 alkaline\u00a0 protease\u00a0 from Bacillus<\/em> B18. J. Biotechnol., <\/em>30:245-256 (1993).<\/li>\n
  3. Horikoshi, Alkalophils\u00a0 from\u00a0 an\u00a0 industrial\u00a0 point\u00a0 of\u00a0 view.\u00a0 FEMS Microbiol. Rev., <\/em>18: 259-270 (1996).<\/li>\n
  4. Reese, E.T., Sin, R.G.H. and Levinson, H.S. The biological degradation of cellulose\u00a0 derivatives\u00a0 and\u00a0 its\u00a0 relationship\u00a0 to\u00a0 the\u00a0 mechanism\u00a0 of\u00a0 cellu-lose hydrolysis. J. Bacteriol.<\/em>, 59: 480 \u2013 485 (1950).<\/li>\n
  5. Taguchi, H., Hamoki, M., Matsuzava, H. and Ohta, J. Heat stable extra-cellular proteolytic enzyme produced by Thermus caldophilous<\/em> strain GK24 an extremely thermophilic bacterium. Biochem.<\/em>, 93: 7 \u2013 13 (1983).<\/li>\n
  6. Kim, ,\u00a0 Dhillon, J.,\u00a0 Chaudhary, S. and\u00a0 Singh,\u00a0 R. Properties\u00a0 of\u00a0 alkaline protease isolated from Nocardiopsis dassonvillei<\/em>. Korean Biochem. J.<\/em>, 26: 81- 85 (1993).<\/li>\n
  7. Manjeet, K., Dhillon, S., Chaudhary, S. and Singh, R. production purification and\u00a0 characterization\u00a0 of\u00a0 a\u00a0 thermostable\u00a0 alkaline\u00a0 protease from Bacillus polymyxa<\/em>. Indian J. Microbiol.<\/em>, 38: 63 \u2013 67 (1998).<\/li>\n
  8. Kunitz, M. Crystalline soyabean\u00a0 trypsin\u00a0 inhibitor\u00a0 \u00a0 General\u00a0 pro-perties. \u00a0\u00a0J. Gen. Physiol.<\/em>, 30: 291\u2013310 (1947).<\/li>\n
  9. Lowry, O.H., Rosebrough, N.J., Farr and Randall, R.J. Protein measurement with Folin phenol reagent. Biol. Chem.<\/em>, 193: 265 \u2013 275. (1951).<\/li>\n
  10. Veloorvalappil, N. J., Robinson, B. S., Selvanesan, P., Sasidharan, S., Kizhakkepawothail, N. U., Sreedharan, S., Prakasan, P., Moolakkariyil,S.J. and Sailas, B. Versatility of microbial proteases. Advances in Enzyme Research.<\/em>, 1: 39-51 (2013).<\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"

    Introduction Proteases constitute one of the most important groups of […]<\/p>\n","protected":false},"author":2,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[17],"tags":[],"class_list":["post-2768","post","type-post","status-publish","format-standard","hentry","category-vol6no2"],"_links":{"self":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts\/2768","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/users\/2"}],"replies":[{"embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/comments?post=2768"}],"version-history":[{"count":5,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts\/2768\/revisions"}],"predecessor-version":[{"id":33247,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts\/2768\/revisions\/33247"}],"wp:attachment":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/media?parent=2768"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/categories?post=2768"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/tags?post=2768"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}