A Novel Serine Protease Cryptolepain from Cryptolepis buchanani:  Purification and Biochemical Characterization

Abstract

A novel protease is purified to homogeneity from the latex of a medicinally important plant Cryptolepis buchanani of family Apocynaceae (formerly Asclepiadaceae). The enzyme named cryptolepain has a molecular mass of 50.5 kDa. The isoelectric point and extinction coefficient (epsilon280nm1%) are 6.0 and 26.4, respectively. Cryptolepain contains 15 tryptophans, 41 tyrosines, and eight cysteine residues forming four disulfide bridges. The detectable carbohydrate moiety in the enzyme was found to be 6-7%. Cryptolepain hydrolyzes denatured natural substrates like casein, azocasein, and azoalbumin with high specific activity. The protease is exclusively inhibited by serine protease inhibitors phenylmethansulfonyl fluoride and diisopropyl fluorophosphate. Hydrolysis of azoalbumin by the cryptolepain is optimal in the pH range of 8-10 and temperatures of 65-75 degrees C. The enzyme shows high stability against pH (2.5-11.5), temperature (up to 80 degrees C), and chemical denaturants. The Km value of the enzyme was found to be 10 microM with azocasein as the substrate. The N-terminal sequence of cryptolepain is unique and shows only little homology to other known serine proteases, which makes this enzyme an ideal candidate for our ongoing biochemical and structure-function investigations of proteases. Easy availability of the latex and simple purification procedures make the enzyme a good system for exploring the biophysical chemistry of serine proteases as well as applications in the food industry.

In-vitro Evaluation of Antifungal Activity of Ganoderma lucidum Against the Biofilm Producing Candida species

“…Absorbance was read in a spectrophotometer at 440 nm. 31,32,33 The phospholipase enzyme activity was determined by mixing the supernatant of the biofilm solution with phosphatidylcholine substrate for 1 hr at 37°C in 5% CO 2 and reading the absorbance in a spectrophotometer at 630 nm. 34 …”

Purification and characterization of a novel serine protease compositain from compositae (Scorzonera hispanica L.)

“…So it indicates that is an excellent enzyme for the food, leather, pharmaceutical and detergent industries. Most plant serine proteases show optimum activity in the temperature range of 30-60°C [28].…”

“…The serine protease from the latex of Cryptolepain contained 6-7 % carbohydrate [2]. Euphorbiaceae family also have carbohydrate in their molecular architecture.…”

“…They have multiple applications, for example, in detergent formulations, textile, food and pharmaceutical industries. Therefore, the industrial demand of proteolytic enzymes, with appropriate speciWcity and stability to pH, temperature and chemical agents, continues to motivate the search for new sources [2].…”

A kinetically stable plant subtilase with unique peptide mass fingerprints and dimerization properties

“…Such an oligomerization state is unprecedented for a plant serine protease to the best of our knowledge [4]. Recently characterized serine proteases from plant latex like cryptolepain and carnein have similar molecular weights but show single peaks on mass spectrometric analysis, indicating that they are monomeric proteins [25].…”