Purification and kinetic study of protease enzyme from kesinai (streblus asper) leave

Abstract

A study was carried out to purify protease enzyme from kesinai leaves extract. The protease enzyme was extracted by using phosphate buffer at pH 7.5. The crude enzyme extract was further with the purification technique by using heat-treatment aqueous two phase system with ratio (w/w) of 2:4.5:1.6:1.5:0.4 crude enzymes, distilled water, PEG, MgSO4 and NaCl accordingly. The separation achieved after centrifugation and was proceed to freeze dry. The observation on enzyme extracted was done and found to have change in color as it was purified. The absorbance of the enzyme was measured by using SHIMADZU spectrophotometer to calculate the enzyme activity and protein concentration. The enzyme activity was increase gradually after being purified and freeze dry with 6.17 and 6.96 U/mL accordingly. The protein concentration for the enzyme decrease dramatically after being purify and freeze dry with 526 and 411 mg/mL. The recovery of protease enzyme was enhanced after freeze dry with purification fold 19.03 and percent yield of 31.1 %. The study was further by investigating kinetic study of sample before and after freeze dry. The VMax and KM value for sample before freeze dry were 0.0324 s-1 and 0.1243 M while for sample after freeze dry were 0.0309 s-1 and 0.0371 M accordingly. The results show that the protease enzyme after freeze drying had a higher affinity for the substrate and higher proteolytic activity than the protease before freeze dry.