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Table of Contents1. Introduction….

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21.1 Proteases and their uses:….

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. 21.2 Isolation from plant sources over microbial sources:..

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Aim and Objectives:….

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. 44. Materials and Methods…..

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………………………….. 44.1 Germination conditions……………………………………………………………… 44.2 Isolation and preparation of Protease Enzyme (crude extract)……… 44.3 Assay of Protease Enzyme…………………………………………………………. 45. Result…………………………………………………………………………… 56. Discussion……………………………………………………………………. 57. Future Perspective…………………………………………………………. 68. Summary……………………………………………………………………… 69. References……………………………………………………………………. 7 List ofTablesTable 5.1:Specific protease activity in sample——————————5     1. Introduction 1.1 Proteases and their uses:                Proteases orproteolytic enzymes are enzymes that are seen to have multiple biologicalapplications in plants and animals, such as germination, inflammatoryprocesses, complement activation and many more. They can be divided into twobroad subcategories, namely exotoxins and endotoxins.          For commercialpurposes, proteases are used in leather, food and textile industries. Of bothclasses of proteases, endopeptidases have much more significance in industrialuse. They have the following applications:·       Processing of chymosin in cheesepreparation ·       Soy sauce preparation ·       Meat tenderization·       As an alternative to chemicaldetergents in the leather industry 1.2 Isolation from plant sources over microbialsources:          For industrialpurposes, proteases are usually isolated from microbial sources. But of recent,plant sources have showed promise due to a variety of factors, such as theirbroad substrate specificity, allowing for more flexible substrate preparationsfor varied plant sources. They also have a more wide range of permissibletemperature, pH and other factors such as the presence of organic compounds.          Proteases areseen to be at a high level especially during and post germination in seeds.This is due to the fact that these enzymes play an important role in thevarious biochemical mechanisms involved in germination and also in the initialstages of development. Of plant seeds, legume seeds have higher levels ofglobulin and albumin storage proteins, which are used for the nourishment ofthe seedling. Due to the above factors, the following investigation ofcomparing the protease content in multiple samples has been performed. 2. Review of Literature·       2.1In 2013, Ranajit Kumar Shaha and Shyam Sundar Shaha studied and compared thegerminating conditions and protease activity in multiple leguminous samplesincluding black gram, green gram, etc. They further performed time coursestudies for the same and obtained an outline of when best to perform protease isolationfor maximum yield. ·       2.2In 2013, Anupama V., Marimuthu M. and others studied and performed the partialcharacterization of proteases from underutilized and common food legumes. Theirmethod of isolation was seen to be a very simple, inexpensive procedure for theisolation of the enzyme.  ·       2.3In 2016, Diego F. Coelho and Elias Basile Tambourgiperformed a study on the use of Azocasein as a substrate for protease activitydetermination. Their research provided a reliable procedure for analyzing thebiological activity of proteolytic enzymes. ·       2.4In 2014, M. Akhtaruzzaman and Tanjina Rahman presented thecharacterization of protease enzymes from seven leguminous seeds. Their studyshowed which seeds showed the highest quantity of the proteolytic enzyme.                                                                                    3. Aim and Objectives:3.1 AimTocompare and contrast the protease content in green gram (Vigna radiata) and Bengal gram (Cicerarietinum). 3.2 Objectives3.2.1 To prepare acrude enzyme from the sample obtained through straining and precipitationtechniques3.2.2 To estimate theprotease content in the samples using UV/Spectrophotometry3.2.3 To compare theprotease content in the samples and identify whichever has the higher amountpresent. 4. Materials and Methods4.1 Germination conditions          Thesamples chosen for this procedure (green gram and Bengal gram) were cleanedthoroughly. They were surface sterilized with 70% ethanol and repeatedly washedwith distilled water. The seeds were then allowed to germinate in roomtemperature (28-30°C) in cycles of 12 hours darkness and 12 hours light for 24hours. During this phase, they were wrapped in moistened cloth and kept in aclosed vessel during the dark phases. After 24 hours, they were used for the experiment. 4.2 Isolation and preparation of Protease Enzyme(crude extract)          50gof each of the samples were weighed and homogenized with pre-chilled acetone.The mixture was ground finely in a chilled mortar and pestle. The obtainedhomogenate were transferred to individual beakers and stored at 4°C.           The homogenates were treated with anequal volume of chilled 10mM Tris-HCl buffer at pH 8, containing 2M NaCl forthree hours. Afterwards, the extract mixtures were filtered through Whatmann’sfilter paper and the filtrates were centrifuged at 10000rpm for 10 minutes at4°C. The obtained supernatant was the crude extract. It was collected andstored at 4°C for further treatment. 4.3 Assay of Protease Enzyme          The assay was madeusing Azocasein, a chromomeric substrate in the following manner. 0.25ml of 1% Azocasein,prepared in 20mM sodium acetate buffer, was mixed with 0.15ml of the enzymeextract. The mixture was allowed to react for 60 minutes in 37°C. The reactionwas stopped by adding 1.2ml of 10% Trichloro Acetic Acid (TCA). For preparationof the control, the substrate was treated with 1.2ml of 10% TCA before theaddition of the extract. The contents were incubated for 15 minutes in roomtemperature before centrifugation at 3000rpm for 5 minutes.           1.2ml of thesupernatant was transferred to a separate tube and the absorbance was read at440nm. Calculations were performed using the following formula: Protease Activity= Protease activity is denoted in units (U) 5. Result          Table 5.1: Specific protease activity insample Common Name Botanical Name Specific protease activity (U/mg) at different pH Bengal Gram Cicer arietinum 0.008786 Green Gram Vigna radiata 0.003862                                                                                                                 6. Discussion          Protease activity was seen to be greater in Bengalgram when compared to green gram. It would thus seem to be a far more viableoption for protease isolation if don’t from plant sources for industrialpurposes. It must also be noted that a variety of factors are capable ofinfluencing protease activity, such as pH, temperature and other organicsolvents present in the reaction mixture. All reactions were primarily carriedout in 4°C, the pH found to be 6-7. Studies have shown that the protease ismost likely to be present in the vacuoles of the seed cells, and thus a moreacidic environment for the isolation might yield higher quantities of protease.           Currently, the leguminous sourcesmentioned are grossly underused, used primarily for nutrition in humansfollowed by animals. A viable method for isolating the protease and itseventual purification would allow for a far cheaper and reliable alternative tomicrobial sources. 7. Future Perspective          The isolation and purification of proteasefrom the source providing highest yield/mg of the enzyme could be studied.Furthermore, a cost comparison could be made with microbially obtained proteaseand that obtained from plant sources, whichever the more economical takenforward. 8. Summary          Protease is an enzyme that is seen to play arole in inflammation, germination, complement fixation and others in plants andanimals. It has multiple industrial and commercial applications in the food andleather industries. Protease is currently mostly obtained from microbialcultures as of now, but there are always risks associated such as contamination,expenses and many more. Now, isolation of protease is gathering good responsesfrom the biological community due to multiple advantages over the microbialroute.          Multiple leguminous seed samples weregerminated and the crude enzyme was isolated. Once isolated, the enzyme wastreated with an Azocasein substrate, the obtained mixture analyzed under theUV/Spectrophotometer for the protease concentration at 440nm.          It was seen that Bengal gram (Cicerarietinum) had much protease activity in comparisonto green gram (Vigna radiata), andthus the former is a much more viable candidate for the isolation of protease. 9. References·        AkhtaruzzamanM. et al (2012), Isolation andCharacterization protease enzyme from leguminous seeds, Agricultural ScienceResearch Journals, Vol. 2(8), pp. 434-440.·        RanajitKumar Shaha et al (2013), Isolationand Characterization of Cysteine protease from leguminous cotyledons, Journalof Advanced Laboratory Research in Biology, Volume 4, Issue 2, pp. 63-73·        AnupamaV. et al (2013), Optimization,Isolation and Partial Characterization of Proteases from underutilized andcommon food legumes, International Research Journal of Pharmacy, Volume 4(7),pp. 99-106·        DiegoCoelho F. et al (2016), AzocaseinSubstrate for Determination of Proteolytic Activity: Reexamining a TraditionalMethod Using Bromelain Samples, Hindawi Publishing Corporation, BioMed ResearchInternational, Volume 4, Article ID 8409183, 6 pages.·        Rawlings ND, Barrett AJ, Bateman A. (2011). Asparagine peptide lyases: a seventh catalytictype of proteolytic enzymes Journal of Biological Chemistry. 286 (44): 38321–8

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