IJRET: International Journal of Research in Engineering and Technology eISSN: 2319-1163 | pISSN: 2321-7308 _______________________________________________________________________________________ Volume: 03 Issue: 06 | Jun-2014, Available @ http://www.ijret.org 88 ISOLATION, PARTIAL PURIFICATION AND CHARACTERIZATION OF ALKALINE SERINE PROTEASE FROM SEEDS OF CUCUMIS MELO VAR AGRESTIS B. Gayatri Devi 1 , K.P.J.HemaLatha 2 1 Research Scholar, Department of Biochemistry, Andhra University, Andhra Pradesh, India 2 Professor, Department of Biochemistry, Andhra University, Andhra Pradesh, India Abstract The present study shows the isolation, partial purification and characterization of alkaline protease from the seeds of Cucumis melo var agrestis, by a four step purification process. Its molecular weight was estimated to be 54KDa. Enzyme showed maximum activity at p H 9.0 and optimum temperature at40 o Cwith casein as substrate. The enzyme exhibited homogeneity as attested by a single protein band on both native PAGE and SDS PAGE. It is a monomeric enzyme and nonglycoprotein in nature. The k m value of enzyme for casein as determined by double reciprocal plot was 2.5 mg/ml. It was strongly inhibited by PMSF but not by EDTA. The results indicate that the alkaline protease is a serine protease, similar to cucumisin from sarcocarp of melon fruit. Keywords: Cucumis melo var agrestis, purification, characterization, monomeric, homogeneity, serine protease, cucumisin. --------------------------------------------------------------------***------------------------------------------------------------------ 1. INTRODUCTION Proteases are group of enzymes which catalyse the cleavage of peptide bonds. They are also generally referred to proteolytic enzymes or proteinases. They are widely distributed in plants animals and microorganisms. Many proteases have been isolated from latex , fruits and seeds (Boller,1986). They have importance in both commercial and physiological fields. Nearly half of commercially available proteases are frequently used in food processing, tenderization of meat, brewing, cheese elaboration, bread manufacturing, leather and textile industry (Kaneda et. al.; 1997). Serine proteinases are class of proteases which come under endopeptidases, which have been purified from a number of plants in the past 30years (Antao & Malcata 2005). In general these enzymes are active at 40ºC and above, however, optimum pH vary greatly, from acidic to neutral and alkaline environments and show broad substrate specificity for proteins. Most serine proteases are inhibited by a class of specific inhibitors, such as DFP (diisopropyl- fluorophosphate) and PMSF (phenylmethylsulfonylfluoride). Alkaline proteases are ubiquitous in nature, found in animals, plants and microorganisms. Proteases which are active above p H 7 are called as Alkaline proteases. Alkaline proteases are involved in the mobilization of seed storage proteins in Dolichous lab lab during germination (Padmakar et al., 2005).Proteases are involved in the processing of pre proteins and maintenance of intracellular protease levels in seeds of horse gram (Jinka et al., 2009). In barley, hordolisin , a subtilisn like serine protease is not involved in degradation of hordein storage protein of barley (Nina Trep et al., 2000). Another protease SEP-1 a subtilisin like serine endopeitidase was isolated from germinated seeds of Hordeum vulgare L. cv. Morex. It was reported to be not involved in storage degradation (Debora Fontanini and Berne L. Jones, 2002).The proteases isolated from germinating finger millet were stable at pH 5 ( Vidyavathi et al., 1983). The most popular plant protease Cucumisins are a family of subtilisin like endopeptidases found in Cucurbitaceae (Kaneda and Tominaga, 1975; Arima et al ., 2000a; Uchikoba et al., 1995). Kaneda and Tominaga (1975) purified cucumisin for the first time from the sarcocarp of melons (Cucumis melo). Several cucumisin-like enzymes have also been isolated from other members of Cucurbitaceae family and from rice, maize, wheat and barley (Antao and Malcata 2005). Most of these enzymes show molecular mass in the range 50-80 kDa and exhibit broad specificity, preferring hydrophobic amino acid residues in the PI position (Arima et al., 2000b). Mature cucumisin (54 kDa) shows optimum pH in the range of 8-10 and is stable at 60ºC. Cucumisin is not affected by trypsin inhibitor from soybean (SBTI), Ovomucoid, cysteine proteinase inhibitors or EDTA, but is strongly inhibited by chloromethyl ketone derivatives of peptide substrates, PMSF and DFP ( Kaneda and