International Journal of Current Research and Review
ISSN: 2231-2196 (Print)ISSN: 0975-5241 (Online)
Bootstrap Slider

Indexed and Abstracted in: SCOPUS, Crossref, CAS Abstracts, Publons, CiteFactor, Open J-Gate, ROAD, Indian Citation Index (ICI), Indian Journals Index (IJINDEX), Internet Archive, IP Indexing, Google Scholar, Scientific Indexing Services, Index Copernicus, Science Central, Revistas Medicas Portuguesas, EBSCO, BOAI, SOROS, NEWJOUR, ResearchGATE, Ulrich's Periodicals Directory, DocStoc, PdfCast, getCITED, SkyDrive, Citebase, e-Print, WorldCat (World's largest network of library content and services), Electronic Journals Library by University Library of Regensburg, SciPeople.

Search Articles

Track manuscript

Readers around the world

Full Html

IJCRR - Vol 04 ISSUE 16, August, 2012

Pages: 146-151

Date of Publication: 28-Aug-2012


Print Article   Download XML  Download PDF

PROTEASE ACTIVITY OF PEPTOCOCCUS ACTIVUS

Author: Zinnat Shahina, Mohammad Towhid Hossain, Mohammad Abdul Hakim

Category: General Sciences

Abstract:Peptococcus activus are anaerobic, non motile, non spore forming gram positive cocci. The objective of this study was to investigate the availability of this bacteria and their ability to protease production. Eleven different bacterial colonies were isolated from shrimp and dry fish(shrimp).Of them, eight colonies(Z1-Z8) were isolated from shrimp and rest of the three isolated from dry fish (Z9-Z11).Among them peptococcus activus which was named Z10 before identification exhibited highest protease production at temperature 37ºC,medium pH 5.0 and 48 hours of incubation period. Maximum protease activity was achieved at 400C for 70 minutes and pH 5.0 along with 2% gelatin as substrate concentration during enzyme substrate reaction.

Keywords: Peptococcus activus , Protease, Extracellular protease, enzyme activity.

Full Text:

INTRODUCTION
Proteases represent the class of enzymes which play an important role as well as their commercial applications. Commercial proteases account for nearly 60% of the total industrial enzyme market. This proteolytic enzyme finds numerous applications in pharmaceutical, food and detergent industries [1]. Proteases of commercial importance are produced from microbial, animal and plant sources [2].Microorganisms are a great source of proteases owing to the limited space required for their cultivation and their ready susceptibility to genetic manipulation. For the production of enzymes for industrial use, isolation and characterization of new promising strain is a continuous process[3].As protease represents one of the major groups of industrial enzymes, it is worthwhile to screen microbes from new habitats for proteases with novel properties to meet the needs of rapidly growing industries[4]. The aim of this study was to isolate a bacterium capable of producing protease which was isolated from dry fish. The ability of the bacteria to grow and produce protease under different environmental conditions was studied. The microorganism was capable of producing large amount of protease when the condition varies. Effects of pH, temperature and substrate concentration of protease were evaluated.

MATERIALS AND METHODS
Microorganisms
The study of protease activity by the bacterial isolate Peptococcus activus was carried out at the Department of Microbiology, University of Chittagong, starting from August 2006 to April 2007, which were isolated from dry fish(shrimp). Isolation and screening of the isolates For the isolation of proteolytic microorganisms enrichment technique was followed. Primary screening was done by three methods – Boiled egg albumin degradation, Skimmed milk casein hydrolysis and Gelatin hydrolysis. During secondary screening 3 different broth media such as (i) Peptone 2%-yeast extract 1%,dextrose 2% [5],(ii) Tryptone 1%-yeast extract 0.5%-dextrose 0.1% [6],(iii) Gelatin 1%- yeast extract-0.2%-glucose 1%-K2HPO4-0.3%- KH2PO4 0.1%-MgSO4.7H2O trace [7] broth were used. Measurement of enzyme activity Protease activity was done by the modified method of Hayashi et.al.,[8],as followed by Meyers and Ahearn [9].Enzyme solution (3ml) was incubated with 3ml of 1% casein as substrate in 3ml of citrate phosphate buffer at 350C in a water bath for 1 hour. The reaction was terminated by 20% TCA and was further incubated for 1 hour it was then filtered through Whatman no.40.One ml enzyme substrate mixture was taken into a test tube and to this 2ml of 20% Na2C03 was added, followed by 1 ml of Folin Ciocalteu Reagent was added and shaken well. After 30 minutes 6ml distilled water was added and absorbance of the solution was measured at 650 nm in a spectrophotometer. The amount of amino acid released in the reaction was calculated from a standard curve previously plotted from the known concentration of tyrosine. Enzyme activity was expressed in Unit, which was defined as the amount of enzyme that releases 1 ?g of tyrosine /ml of crude extract /hour. Optimization of culture conditions A. Effect of incubation time, temperature and medium pH To ascertain the effect of culture conditions the present study was carried out at different incubation time (24, 48, 72 and 96 hours), pH (such as 5.0, 6.0, 7.0, 8.0 and 8.5 respectively) and temperature (such as 30?2 0C, 37?2 0C, and 45?2 0C respectively).Their effect of temperature on biomass characteristic, biomass yield and protease production was recorded. B. Effect of carbon sources The production of extracellular proteases using different carbon source and the original nitrogen source (peptone) were studied in the liquid culture medium [6]. Five carbon sources (sucrose, dextrose, fructose, starch, and maltose) were added to the medium and their effect on the production of protease, extracellular protein and biomass yield were recorded. Factors affecting enzyme activity To determine the optimum condition for the crude enzyme activity several factors such as pH (ranging form 5.0 to 8.5), optimum temperature (35 to 45?2 0C), incubation time (50, 60, 70, 80 and 90 minutes ), substrate concentration (i.e. 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0%) substrate specificity (using Casein, Egg albumin, BSA and Gelatin) were observed for highest enzyme activity. RESULTS AND DISCUSSION Primary screening confirmed that the strain Z10 showed greater albumin degradation, casein and gelatin hydrolysis. After primary screening this bacteria allowed to grow in three liquid media and maximum enzyme activity was found in the trypton-yeast extract-dextrose broth. On the basis of morphological (including size, shape; arrangement of the cell; presence and absence of spores; acid fast reaction, gram reaction etc), cultural, physiological and biochemical properties the strain was characterized. Then compared with the standard description of “Bergey’s Manual of Determinative Bacteriology”, 8th ed. [10]. The bacterial isolate Z10 was found to belong to the genus Peptococcus and provisionally identified as Peptococcus activus (Fig: 1.a,b,c,d, e & f). Optimization of different cultural conditions Effects of incubation period The Bacterial isolate Z10 (Peptococcus activus) showed maximum protease production 48.61 U/ml) after 48 hrs of incubation period (Fig-2.a) but highest biomass (1.020 absorbance at 600nm) was recorded after 72 hrs of incubation time. The pH of the culture filtrate were ranged from 5.05 to 5.77 . Production of maximum protease within 48-hours by bacteria was reported by Das et al. [11]. Effects of temperature The influence of incubation temperature on protease production by bacteria was studied by several workers [12-13].Among the different incubation temperatures tested for protease production 37ºC was found to be optimum for this isolate. At this temperature highest protease activity was 52.5 U/m1 and the highest biomass yield was 1.072 absorbance at 600nm (Fig-2.b). Effects of pH In the present study, maximum protease production was achieved at medium pH 5.0 (Fig-2.c). Production of protease at acidic medium pH ranged form 4.5 to 6.5 by microbes was observed by North [14]. Effect of carbon source Maximum production of protease was achieved by the isolate when gelatin was used as a nitrogen source and glucose as a carbon source (Fig-2.d). Factors involved in enzyme activity Enzyme activity depends upon the pH, temperature, incubation time, substrate concentration and many others factors. So it is necessary to find out different limiting factor for maximum activity of proteases.Thus the crude enzymes obtained were studied for the determination of different factors for maximum protease activity. Enzyme-substrate reaction incubation time The culture filtrate of bacterial isolate Z10 exhibited highest protease activity when enzyme substrate reaction mixtures were incubated at 40 ºC for 70 minutes (Fig-3) Substrate concentration The crude enzyme extract of isolate Z10 (Peptococcus activus) were allowed to react with different substrate concentrations(0.5- 3.0%) and maximum activity was recorded with 2.0% substrate concentration (fig-4). Substrate specificity Different substrate such as casein, gelatin, bovine serum albumin (BSA) and egg albumin were used to observe the efficiency of enzyme activity on different substrate and gelatin gave the highest value of activity (77.8 U/ml) with the crude of bacterial isolate Z10 (Peptococcus activus) (Fig-5). Temperature and pH Temperature and pH are also most important limiting factors, which markedly influenced enzyme activity. Maximum protease activity of crude enzyme extract of bacterial isolate Z10 was recorded at temperature 40ºC and pH 5.0 (Fig:6).similar temperature was also observed by Lee [15].

CONCLUSION
The inability of the plant and animal proteases to meet current world demands has led to an increased interest in microbial proteases. Proteases from microbial sources are preferred to the enzymes from plant and animal sources since they possess almost all the characteristics desired for their biotechnological applications. Despite the extensive research on several aspects of proteases, there is a paucity of knowledge about the roles that govern the diverse specificity of these enzymes. Deciphering these secrets would enable us to exploit proteases for their application in biotechnology. Further investigation on this bacterial (peptococcus activus) species can possibly reveal it’s potentiality as a source of protease for any biotechnological approach.

ACKNOWLEDGEMENTS
Authors acknowledge the immense help received from the scholars whose articles are cited and included in references of their manuscript. The authors are also grateful to authors/editors/ publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed.

References:

1. Moon, S. H. and S.J. parulekar. 1991. A parametric study of protease production in batch and fed-batch cultures of Bacillus firmus. Biotechnol. Bioengg., 37: 467-483.

2. Patel, P.R., 1985.Enzymes iaolation and purification. In: Biotechnology: Applications and Research. Technomic Publishing Co. Inc., USA, pp: 534-564.

3. Kumar., A. Sachdev., A. Balasubramanyam., SD. Saxena., AK. And Lata, 2002. Optimization of conditions for production of Neutral and Alkaline protease from species of Bacillus and Pseudomonas. Ind. J. Microbiol.42:233-236.

4. Kumar, D., Savitri, N. Thakur, R. Verma and T. C. Bhalla, 2008.Microbial proteases and application as laundry detergent additives. Res. J. Microbial., 3: 661-672.

5. Amar, J.S., and Halvorson, H.O. 1975. Proteinase activities of Saccharomyces cerevisiae during sporulation. Journal of Bacteriol. 124(2): 863-869.

6. Matta,H.,V.Punj and S.S.Kanwar,1997.An immuno-dot assay for detection of thermostable protease from Pseudomonus sp.AFT-36 of dairy origin.Appl.Microbiol.,25:300-302.

7. Shalinisen and Satyanarayana, T. 1993. Optimization of alkaline protease production by thermophilic Bacillus licheniformis S-40. Indi J. Microbiol. 33(1): 43-47.

8. 8.Hayashi,L.D.,D.Fukushima and K.Mogi,1967.Agr.Biol.chem.., Tokyo,31:1237-1241.Cited from Physiology and Parasitism.Edt.By G.P.Agarwal and K.S.Bilgrami.Today and tomorrow’s printers and publishers,New Delhi,India,pp:194.

9. Meyers,S.P. and D.G.Ahearn, 1977. Mycologia,69:649-651. Cited from Physiology and Parasitism.Edt.By G.P.Agarwal and K.S.Bilgrami.Today and tomorrow’s printers and publishers,New Delhi,India,pp:195.

10. Buchanan,R.E. and Gibbons,N.E.(8th eds),1974.The William and Wilkons Co.Baltimore.

11. Das,F.,Hossain,MD.T.and Anwar,M.N.2005.Protease production by Bacillus Sphericus.TheChittagong univ.j.Sci,29(1):71-76.

12. Al-Saleh, A.A. and Zahran, A.S. 1997. Protease production by Pseudomonas fleorescens CM12 isolated from raw camel milk. Egypt. J. Dairy Sci. 25 (2): 327-336.

13. Sharmin, S., Hossain, M.T. and Anwar,M.N.2005. Isolation and characterization of a protease producing Bacteria Bacillus amovivorus and optimization of some factors of culture conditions for protease production, Journal of Biological Sciences 5(3):358-362.

14. Lee W., Young-Jecho, Gyu-Mok S. and Cheong Choi. 1992. Characteristic and action pattern of alkaline protease produced from Bacillus sp. CW-1121. Korean Biochem J. 24(6): 537-542.



Research Incentive Schemes

Awards, Research and Publication incentive Schemes by IJCRR

Best Article Award: 

One article from every issue is selected for the ‘Best Article Award’. Authors of selected ‘Best Article’ are rewarded with a certificate. IJCRR Editorial Board members select one ‘Best Article’ from the published issue based on originality, novelty, social usefulness of the work. The corresponding author of selected ‘Best Article Award’ is communicated and information of award is displayed on IJCRR’s website. Drop a mail to editor@ijcrr.com for more details.

Women Researcher Award:

This award is instituted to encourage women researchers to publish her work in IJCRR. Women researcher, who intends to publish her research work in IJCRR as the first author is eligible to apply for this award. Editorial Board members decide on the selection of women researchers based on the originality, novelty, and social contribution of the research work. The corresponding author of the selected manuscript is communicated and information is displayed on IJCRR’s website. Under this award selected women, the author is eligible for publication incentives. Drop a mail to editor@ijcrr.com for more details.

Emerging Researcher Award:

‘Emerging Researcher Award’ is instituted to encourage student researchers to publish their work in IJCRR. Student researchers, who intend to publish their research or review work in IJCRR as the first author are eligible to apply for this award. Editorial Board members decide on the selection of student researchers for the said award based on originality, novelty, and social applicability of the research work. Under this award selected student researcher is eligible for publication incentives. Drop a mail to editor@ijcrr.com for more details.


Best Article Award

A Study by Juna Byun et al. entitled "Study on Difference in Coronavirus-19 Related Anxiety between Face-to-face and Non-face-to-face Classes among University Students in South Korea" is awarded Best Article for Vol 12 issue 16
A Study by Sudha Ramachandra & Vinay Chavan entitled "Enhanced-Hybrid-Age Layered Population Structure (E-Hybrid-ALPS): A Genetic Algorithm with Adaptive Crossover for Molecular Docking Studies of Drug Discovery Process" is awarded Best article for Vol 12 issue 15
A Study by Varsha M. Shindhe et al. entitled "A Study on Effect of Smokeless Tobacco on Pulmonary Function Tests in Class IV Workers of USM-KLE (Universiti Sains Malaysia-Karnataka Lingayat Education Society) International Medical Programme, Belagavi" is awarded Best article of Vol 12 issue 14, July 2020
A study by Amruta Choudhary et al. entitled "Family Planning Knowledge, Attitude and Practice Among Women of Reproductive Age from Rural Area of Central India" is awarded Best Article for special issue "Modern Therapeutics Applications"
A study by Raunak Das entitled "Study of Cardiovascular Dysfunctions in Interstitial Lung Diseas epatients by Correlating the Levels of Serum NT PRO BNP and Microalbuminuria (Biomarkers of Cardiovascular Dysfunction) with Echocardiographic, Bronchoscopic and HighResolution Computed Tomography Findings of These ILD Patients" is awarded Best Article of Vol 12 issue 13 
A Study by Kannamani Ramasamy et al. entitled "COVID-19 Situation at Chennai City – Forecasting for the Better Pandemic Management" is awarded best article for  Vol 12 issue 12
A Study by Muhammet Lutfi SELCUK and Fatma COLAKOGLU entitled "Distinction of Gray and White Matter for Some Histological Staining Methods in New Zealand Rabbit's Brain" is awarded best article for  Vol 12 issue 11
A Study by Anamul Haq et al. entitled "Etiology of Abnormal Uterine Bleeding in Adolescents – Emphasis Upon Polycystic Ovarian Syndrome" is awarded best article for  Vol 12 issue 10
A Study by Arpita M. et al entitled "Estimation of Reference Interval of Serum Progesterone During Three Trimesters of Normal Pregnancy in a Tertiary Care Hospital of Kolkata" is awarded best article for  Vol 12 issue 09
A Study by Ilona Gracie De Souza & Pavan Kumar G. entitled "Effect of Releasing Myofascial Chain in Patients with Patellofemoral Pain Syndrome - A Randomized Clinical Trial" is awarded best article for  Vol 12 issue 08
A Study by Virendra Atam et. al. entitled "Clinical Profile and Short - Term Mortality Predictors in Acute Stroke with Emphasis on Stress Hyperglycemia and THRIVE Score : An Observational Study" is awarded best article for  Vol 12 issue 07
A Study by K. Krupashree et. al. entitled "Protective Effects of Picrorhizakurroa Against Fumonisin B1 Induced Hepatotoxicity in Mice" is awarded best article for issue Vol 10 issue 20
A study by Mithun K.P. et al "Larvicidal Activity of Crude Solanum Nigrum Leaf and Berries Extract Against Dengue Vector-Aedesaegypti" is awarded Best Article for Vol 10 issue 14 of IJCRR
A study by Asha Menon "Women in Child Care and Early Education: Truly Nontraditional Work" is awarded Best Article for Vol 10 issue 13
A study by Deep J. M. "Prevalence of Molar-Incisor Hypomineralization in 7-13 Years Old Children of Biratnagar, Nepal: A Cross Sectional Study" is awarded Best Article for Vol 10 issue 11 of IJCRR
A review by Chitra et al to analyse relation between Obesity and Type 2 diabetes is awarded 'Best Article' for Vol 10 issue 10 by IJCRR. 
A study by Karanpreet et al "Pregnancy Induced Hypertension: A Study on Its Multisystem Involvement" is given Best Paper Award for Vol 10 issue 09
Late to bed everyday? You may die early, get depression
Egg a day tied to lower risk of heart disease
88 Percent Of Delhi Population Has Vitamin D Deficiency: ASSOCHAM Report

List of Awardees

Awardees of COVID-19 Research

Woman Researcher Award

A Study by Neha Garg et al. entitled "Optimization of the Response to nCOVID-19 Pandemic in Pregnant Women – An Urgent Appeal in Indian Scenario" published in Vol 12 issue 09

A Study by Sana Parveen and Shraddha Jain entitled "Pathophysiologic Enigma of COVID-19 Pandemic with Clinical Correlates" published in Vol 12 issue 13

A Study by Rashmi Jain et al. entitled "Current Consensus Review Article on Drugs and Biologics against nCOVID-19 – A Systematic Review" published in Vol 12 issue 09

Emerging Researcher Award

A Study by Madhan Jeyaraman et al. entitled "Vitamin-D: An Immune Shield Against nCOVID-19" published in Vol 12 issue 09


RSS feed

Indexed and Abstracted in


Antiplagiarism Policy: IJCRR strongly condemn and discourage practice of plagiarism. All received manuscripts have to pass through "Plagiarism Detection Software" test before forwarding for peer review. We consider "Plagiarism is a crime"

IJCRR Code of Conduct: We at IJCRR voluntarily adopt policies on Code of Conduct, and Code of Ethics given by OASPA and COPE. To know about IJCRRs Code of Conduct, Code of Ethics, Artical Retraction policy, Digital Preservation Policy, and Journals Licence policy click here

Disclaimer: International Journal of Current Research and Review (JICRR) provides platform for researchers to publish and discuss their original research and review work. IJCRR can not be held responsible for views, opinions and written statements of researchers published in this journal.



Company name

International Journal of Current Research and Review (JICRR) provides platform for researchers to publish and discuss their original research and review work. IJCRR can not be held responsible for views, opinions and written statements of researchers published in this journal

Contact

148, IMSR Building, Ayurvedic Layout,
        Near NIT Complex, Sakkardara,
        Nagpur-24, Maharashtra State, India

editor@ijcrr.com

editor.ijcrr@gmail.com


Copyright © 2020 IJCRR. Specialized online journals by ubijournal .Website by Ubitech solutions