1. Introduction1.1Proteases and their uses: Proteases or proteolytic enzymes areenzymes that are seen to have multiple biological applications in plants andanimals, such as germination, inflammatory processes, complement activation andmany more. They can be divided into two broad subcategories, namely exotoxins and endotoxins. For commercial purposes, proteasesare used in leather, food and textile industries.
Of both classes of proteases,endopeptidases have much more significance in industrial use. They have thefollowing applications:· Processingof chymosin in cheese preparation · Soysauce preparation · Meattenderization· Asan alternative to chemical detergents in the leather industry 1.2Isolation from plant sources over microbial sources: For industrial purposes, proteases are usually isolatedfrom microbial sources. But of recent, plant sources have showed promise due toa variety of factors, such as their broad substrate specificity, allowing formore flexible substrate preparations for varied plant sources. They also have amore wide range of permissible temperature, pH and other factors such as thepresence of organic compounds.
Proteases are seen to be at a highlevel especially during and post germination in seeds. This is due to the factthat these enzymes play an important role in the various biochemical mechanismsinvolved in germination and also in the initial stages of development. Of plantseeds, legume seeds have higher levels of globulin and albumin storageproteins, which are used for the nourishment of the seedling. Due to the abovefactors, the following investigation of comparing the protease content inmultiple samples has been performed. 2. Reviewof Literature2.
1 In 2013, Ranajit Kumar Shaha and Shyam Sundar Shahastudied and compared the germinating conditions and protease activity inmultiple leguminous samples including black gram, green gram, etc. They furtherperformed time course studies for the same and obtained an outline of when bestto perform protease iisolation for maximum yield.2.2 In 2013, Anupama V.
, Marimuthu M. and others studiedand performed the partial characterization of proteases from underutilized andcommon food legumes. Their method of isolation was seen to be a very simple,inexpensive procedure for the isolation of the enzyme.
2.3 In 2016, Diego F. Coelho and EliasBasile Tambourgi performed a study on the use of Azocasein as a substrate forprotease activity determination. Their research provided a reliable procedurefor analyzing the biological activity of proteolytic enzymes.2.
4 In 2014, M. Akhtaruzzaman and TanjinaRahman presented the characterization of protease enzymes from seven leguminousseeds. Their study showed which seeds showed the highest quantity of theproteolytic enzyme. 3. Aim and Objectives:3.1 AimTo compare and contrast theprotease content in green gram (Vigna radiata)and Bengal gram (Cicer arietinum). 3.2Objectives3.
2.1 To preparea crude enzyme from the sample obtained through straining and precipitationtechniques3.2.2 To estimatethe protease content in the samples using UV/Spectrophotometry3.
2.3 To comparethe protease content in the samples and identify whichever has the higheramount present. 4.
Materials and Methods4.1 Germinationconditions The samples chosen for thisprocedure (green gram and Bengal gram) were cleaned thoroughly. They weresurface sterilized with 70% ethanol and repeatedly washed with distilled water.The seeds were then allowed to germinate in room temperature (28-30°C) incycles of 12 hours darkness and 12 hours light for 24 hours. During this phase,they were wrapped in moistened cloth and kept in a closed vessel during thedark phases.
After 24 hours, they were used for the experiment. 4.2Isolation and preparation of Protease Enzyme (crude extract) 50g of each of thesamples were weighed and homogenized with pre-chilled acetone. The mixture wasground finely in a chilled mortar and pestle. The obtained homogenate weretransferred to individual beakers and stored at 4°C. Thehomogenates were treated with an equal volume of chilled 10mM Tris-HCl bufferat pH 8, containing 2M NaCl for three hours. Afterwards, the extract mixtureswere filtered through Whatmann’s filter paper and the filtrates werecentrifuged at 10000rpm for 10 minutes at 4°C.
The obtained supernatant was thecrude extract. It was collected and stored at 4°C for further treatment. 4.3 Assayof Protease Enzyme The assay was made using Azocasein, achromomeric substrate in the following manner.
0.25ml of 1% Azocasein, preparedin 20mM sodium acetate buffer, was mixed with 0.15ml of the enzyme extract. Themixture was allowed to react for 60 minutes in 37°C.
The reaction was stoppedby adding 1.2ml of 10% Trichloro Acetic Acid (TCA). For preparation of thecontrol, the substrate was treated with 1.2ml of 10% TCA before the addition ofthe extract. The contents were incubated for 15 minutes in room temperaturebefore centrifugation at 3000rpm for 5 minutes.
1.2ml of the supernatant wastransferred to a separate tube and the absorbance was read at 440nm.