Cation exchange chromatography on SP-Sepharose (when the column was equilibrated with Tris-HCL at pH eight.0). The protein of interest eluted within the unbound samples. (b) The nonretained fraction from SP-Sepharose 200 was loaded to gel filtration chromatography on Sephacryl S-200. Column was eluted with linear salt gradient in the identical buffer.104.2 using a 74.1 yield, with its particular activity equal to 1312.9 U/mg proteins (Table 1). The active fractions of cation exchange chromatography have been separated by Sephacryl S-200 gel filtration chromatography (Figure 1(b)). Immediately after this step, protease was purified by a issue of 221.2 using a recovery of 71.three along with a precise activity of 2787.1 U/mg proteins, respectively (Table 1). The gel filtration chromatography technique and ion exchange chromatography used within this study have also been employed effectively for the protease purified from latex of Euphorbia milii from sweet potato roots [17, 18]. It can be observed that the enzymatic activity was eluted in 1 peak, which coincided using the peak of protein.Tegafur Fractions of this peak (352) have been collected and concentrated.Levosimendan The purified protease was homogenous because it gave a single protein bond on SDS-PAGE.PMID:23746961 The molecular weight of the protease by SDS-PAGE was around 26.7 kDa (Figure 2). The molecular weight obtained by Sephadex G-200 and DEAESephadex column chromatography was also around 26.7 kDa (Figure two). It may be observed that the enzymatic activity was eluted in a single peak, which coincided using the peak of protein. Fractions of this peak (469) have been collected and concentrated. The purified protease was homogenous since it gave a single protein band on SDS-PAGE. Molecular weight of your protease by SDS-PAGE was approximately 26.7 kDa (Figure two). The molecular weight obtained by SPSepharose and Sephacryl S-200 column chromatography was also approximately 26.7 kDa (Figure 2).M 55.six 42.7 34.6 27.0 20.0 14.3 six.Purified proteaseFigure two: SDS-PAGE of your purified protease. M: regular protein markers; lane 1: crude enzyme; lane two: ammonium sulphateprecipitated enzyme; lane 3: purified enzyme on SP-Sepharose (cation exchange); lane 4: purified enzyme on Sephacryl S-200 (gel filtration).3.2. Optimum Temperature and Thermal Stability in the Purified Protease. The purified protease from red pitaya peel was active and steady throughout a wide temperature variety (20 C to 75 C). The temperature for the maximum protease activity was 70 C. At both 80 and 90 C, the protease was really active, with virtually 60 and 35 activity, respectively. As a result, theNaCl concentration (molarity)100 90 80 70 60 50 40 30 20 10500 450 400 350 300 250 200 150 one hundred 50Serine protease (U/mL)Serine protease (U/mL)Absorbance at 280 nmBioMed Study International120 Relative activity ( ) one hundred 80 60 40 20 0 0 20 40 60 80 Temperature ( C)(a)120 Residual activity ( ) one hundred 80 60 40 20 0 -20 0 20 40 60 80 Temperature ( C)(b)120 Residual activity ( ) Relative activity ( ) one hundred 80 60 40 20 0 0 2(c)120 one hundred 80 60 40 20 0 0 2(d)six pH6 pHFigure 3: The optimum temperature (a), thermal stability (b), optimum pH (c), and pH stability (d) of purified thermoalkaline protease have been investigated.final results reveal that the optimum temperature for the enzyme is 70 C (Figure three(a)). Analysis with the thermal stability on the protease showed that the enzyme retained a lot more than 90 of its activity in the selection of 20 to 80 C, however the enzyme activity was significantly ( 0.05) decreased at temperature above 80 C. The residual.