Purification and biochemical characterization of phytocystatin fromBrassica alba

Abstract

Phytocystatins belong to the family of cysteine proteinases inhibitors. They are ubiquitously found in plants and carry out various significant physiological functions. These plant derived inhibitors are gaining wide consideration as potential candidate in engineering transgenic crops and in drug designing. Hence it is crucial to identify these inhibitors from various plant sources. In the present study a phytocystatin has been isolated and purified by a simple two-step procedure using ammonium sulfate saturation and gel filtration chromatography on Sephacryl S-100HR from Brassica alba seeds (yellow mustard seeds).The protein was purified to homogeneity with 60.3% yield and 180-fold of purification. The molecular mass of the mustard seed cystatin was estimated to be nearly 26,000 Da by sodium dodecyl sulfate polyacrylamide gel electrophoresis as well as by gel filtration chromatography. The stokes radius and diffusion coefficient of the mustard cystatin were found to be 23A° and 9.4 × 10(-7) cm(2) s(-1) respectively. The isolated phytocystatin was found to be stable in the pH range of 6-8 and is thermostable up to 60 °C. Kinetic analysis revealed that the phytocystatin exhibited non-competitive type of inhibition and inhibited papain more efficiently (K(i) = 3 × 10(-7) M) than ficin (K(i) = 6.6 × 10(-7) M) and bromelain (K(i) = 7.7 × 10(-7) M respectively). CD spectral analysis shows that it possesses 17.11% alpha helical content.

Physico-chemical and in-silico analysis of a phytocystatin purified fromBrassica junceacultivar RoAgro 5444

“…The α-helical content of the inhibitor calculated using the BestSel online program is 18.8% while the β sheet and β turn conformations are 21% and 15% respectively. Reports have shown comparable secondary structural contents for phytocystatins from pineapple (Irene et al, 2012) , yellow mustard (Ahmed et al 2016), Phaseolus mungo (Sharma et al 2006). The calculated secondary structure for mustard cystatin is similar to animal D r a f t cystatins like goat pancreatic (17%) and human placental (21%) cystatins previously described in our lab (Priyadarshini and Bano 2010;Rashid et al 2006).…”

“…Most of the purified phytocystatins are stable to extremes of temperature and pH (Pernas et al 1998). The ability of the purified B.juncea seed cystatin to remain stable over a wide pH range of 3-9 and temperature range of 30-60ºC holds it in good stead as compared to other Brassica phytocystatins like yellow mustard (B.alba) cystatin (Ahmed et al, 2016) and chinese cabbage (B.rapa) cystatin (Hong et al, 2012) that show stability in the pH ranges of 6-8 and up to pH 7 respectively. The storage stability of the Indian mustard cystatin shows that it is viable up to 4 months when stored at -20ºC and 45 days when stored at 4ºC.…”

Insight into the biochemical characterization of phytocystatin from Glycine max and its interaction with Cd+2 and Ni+2

“…The protein migrated as a single band under both reducing and nonreducing conditions suggesting the monomeric nature of the SBPC. A similar result was also reported in yellow mustard seeds . The purified SBPC was found to be devoid of both carbohydrate moieties as well as sulfhydryl groups which are in accordance with other inhibitors reported earlier in plant cystatin like garlic phytocystatin .…”

“…In the present study, a phytocystatin from soybean has been purified with better yield and fold purification by slightly modifying the method of reported earlier . Various biophysical techniques were used to further characterize the purified phytocystatin and to explain their interaction with papain.…”

A Comprehensive Review on Kiwifruit Allergy: Pathogenesis, Diagnosis, Management, and Potential Modification of Allergens Through Processing

“…Act d 3 is a 40 kDa protein attached to complex glycans (cross-reactive carbohydrate determinants; Palacin et al, 2008). Act d 4 is a 12 to 16 kDa phytocystatin and is considered a cysteine proteinase inhibitor (Ahmed, Shamsi, & Bano, 2016;Bublin et al, 2010).…”