And Discussion3.1. Purification from the CYP26 Storage & Stability protease from Red Pitaya. A single
And Discussion3.1. Purification of the Protease from Red Pitaya. A single protein using the protease activity was purified from the red pitaya peel by ammonium sulphate precipitation, cation exchange chromatography on a SP-Sepharose column, and gel filtration chromatography on Sephacryl S-200. Table 1 summarizes the study of purification of the protease from pitaya peel. The extracted enzyme was precipitated with ammonium sulphate and, according to the outcomes, 600 saturation produced the highest purification by a factor of 9.four using a yield of 83.2 amongst the other ammonium sulphate concentrations. The concentrated fraction was then loaded onto the cation exchange chromatography column (SP-Sepharose). The enzyme was ACAT2 custom synthesis eluted in the column having a salt concentration of 1.five M NaCl. The enzyme activity and proteins had been located in one peak right after elution (Figure 1(a)). The protease from red pitaya peel was purified by a factor of much more thanBioMed Research InternationalTable 1: Purification step in the thermoalkaline protease from Hylocereus polyrhizus peel.Purification actions Crude extract Ammonium sulphate precipitation Cation exchange chromatography Gel filtration chromatographyTotal protein (mg) 44.two 3.9 0.three 0.Total activity (U) 557.two 462.four 412.eight 397.Particular activity (Umg) 12.6 118.four 1312.9 2787.Purification fold 1 9.four 104.two 221.Yield ( ) 100 83.2 74.1 71.Fold purification calculated with respect towards the precise activity on the crude extract.Absorbance protein at 280 nm30 40 50 Fraction number160 140 120 one hundred 80 60 40 2030 40 50 Fraction number400 350 300 250 200 150 100 50100 80 60 40 20Serine protease Protein 280 nmSerine protease (UmL) NaCl concentration (molarity) Protein 280 nm(a) (b)Figure 1: Cation exchange and gel filtration chromatography plots. (a) shows the cation exchange chromatography on SP-Sepharose (when the column was equilibrated with Tris-HCL at pH 8.0). The protein of interest eluted inside 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 within the identical buffer.104.2 having a 74.1 yield, with its certain activity equal to 1312.9 Umg proteins (Table 1). The active fractions of cation exchange chromatography were separated by Sephacryl S-200 gel filtration chromatography (Figure 1(b)). After this step, protease was purified by a aspect of 221.two having a recovery of 71.3 as well as a certain activity of 2787.1 Umg proteins, respectively (Table 1). The gel filtration chromatography approach and ion exchange chromatography employed within this study have also been employed effectively for the protease purified from latex of Euphorbia milii from sweet potato roots [17, 18]. 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 (352) have been collected and concentrated. The purified protease was homogenous because it gave a single protein bond on SDS-PAGE. The molecular weight in the protease by SDS-PAGE was around 26.7 kDa (Figure two). The molecular weight obtained by Sephadex G-200 and DEAESephadex column chromatography was also around 26.7 kDa (Figure two). It could be observed that the enzymatic activity was eluted in one peak, which coincided with all the peak of protein. Fractions of this peak (469) have been collected and concentrated. The purified protease was homogenous as it gave a single protein band on SDS-PAGE. Molecular weight in the protease.
