A Stable Serine Protease, Wrightin, from the Latex of the Plant Wrightia tinctoria (Roxb.) R. Br.: Purification and Biochemical Properties

Abstract

Today proteases have become an integral part of the food and feed industry, and plant latex could be a potential source of novel proteases with unique substrate specificities and biochemical properties. A new protease named "wrightin" is purified from the latex of the plant Wrightia tinctoria (Family Apocynaceae) by cation-exchange chromatography. The enzyme is a monomer having a molecular mass of 57.9 kDa (MALDI-TOF), an isoelectric point of 6.0, and an extinction coefficient (epsilon1%280) of 36.4. Optimum activity is achieved at a pH of 7.5-10 and a temperature of 70 degrees C. Wrightin hydrolyzes denatured natural substrates such as casein, azoalbumin, and hemoglobin with high specific activity; for example, the Km value is 50 microM for casein as substrate. Wrightin showed weak amidolytic activity toward L-Ala-Ala-p-nitroanilide but completely failed to hydrolyze N-alpha-benzoyl- DL-arginine-p-nitroanilide (BAPNA), a preferred substrate for trypsin-like enzymes. Complete inhibition of enzyme activity by serine protease inhibitors such as PMSF and DFP indicates that the enzyme belongs to the serine protease class. The enzyme was not inhibited by SBTI and resists autodigestion. Wrightin is remarkably thermostable, retaining complete activity at 70 degrees C after 60 min of incubation and 74% of activity after 30 min of incubation at 80 degrees. Besides, the enzyme is very stable over a broad range of pH from 5.0 to 11.5 and remains active in the presence of various denaturants, surfactants, organic solvents, and metal ions. Thus, wrightin might be a potential candidate for various applications in the food and biotechnological industries, especially in operations requiring high temperatures.

Induced Responses to Herbivory and Jasmonate in Three Milkweed Species

“…Various types are found in the latex of plants that belong to a diversity of phylogenetic clades. For example, cysteine proteases are reported in latex of families such as Caricaceae, Moraceae, and Apocynaceae (Light et al 1964;Sgarbieri et al 1964;Arribére et al 1998), and serine protease in Moraceae, Euphorbiaceae, Apocynaceae, Convolvulaceae (Arima et al 2000;Tomar et al 2008). Direct evidence for the involvement of cysteine proteases in plant resistance against herbivores came from experiments in which the toxicity of wild fig (Ficus virgata) against two generalist herbivores disappeared when E-64, a specific cysteine protease inhibitor, was painted on leaf surfaces (Konno et al 2004).…”

Leaf structure, microanalysis and characterization of the latex protein profile of Pachystroma longifolium (Nees) I.M. Jonhst. (Euphorbiaceae) in a seasonally dry Atlantic Forest

“…and diff erent protein classes (Agrawal & Konno 2009). Various latexes are known to contain glycosidases (Giordani & Lafon 1993), proteases (Kimmel & Smith 1954;Sgarbieri et al 1964;Lynn & Clevette-Radford 1987b;Arribére et al 1998;Arima et al 2000;Tomar et al 2008), acid phosphatases (Lynn & Clevette-Radford 1987a), amylases (Lynn & Clevette-Radford 1987a), chitinases (Jekel et al 1991), hevein (Van Parijs et al 1991), proteinase inhibitors (Archer 1983;Lin & Lu 1994), b-1,3-glucanase (Chye & Cheung 1995) and various other enzymatic activities (Lynn & Clevette-Radford 1987b;Agrawal & Konno 2009).…”

Wrightia tinctoria R. Br.-a review on its ethnobotany, pharmacognosy and pharmacological profile

“…Similarly, a new protease named "Wrightia" purified from the latex of the plant W. tinctoria is thought to be a potential candidate for various applications in food and biotechnological industries [72]. The "777 oil", a coded drug of Siddha system of medicine, has been derived from the leaves of W. tinctoria by insulation with coconut oil as a base.…”