Study On Stability And Stabilizer Of Rhizopus Oryzae Lipase

Lipases,a group of hydrolytic enzymes,have been widely used in the food,biodiesel,washing industry,biopharmaceutical and other fields.Among them,Rhizopus oryzae lipase?ROL?is a typical one and has been commercialized,which is often used to efficiently prepare structured triacylglycerol,biodiesel and solution of chiral compounds.But like most enzymes,ROL also exhibits thermal instability.In order to broaden its application scope by increasing its thermal stability,the fermented crude enzyme was used to investigate the stabilizers and the mechanism of inactivation in high temperatures.The main research and results are summarized as follows.1.The crude fermented ROL was incubated in a water-bath at 30-80?for some time to examine inactivation procedure.The relative activity of ROL remained more than 95%after being kept for 3 h in the water-bath at 30-50?.The activity loss was insignificant.However,when the temperature rose to 60?,the relative activity of the enzyme only retained 29.50%indicating that the enzyme was inactivated to a large extent.When the temperature further increased to 70?and 80?,the remnant relative activity of the enzyme was respectively 21.76%and 9.26%after being inactivated for only 3 minutes.The result showed that most amount of enzyme had been inactivated in such high temperature.Furthermore,an investigation was conducted into the mechanism of thermal inactivation at higher temperature.The change of relative activity was consistent with that of relative protein concentration in the supernatant.And the enzyme was found to fit two-step inactivation model,meaning there was a partial inactive state between active state and inactive state.2.After a series of isolation and purification of ROL fermented in our laboratory,the high purity enzyme was obtained,of which the purification fold and the recovery rate were 1.62 and 10.83%,respectively.And then,the enzymes including the crude lipase and the purified one were inactivated in water-bath with different temperatures.The stability of the crude enzyme was found to be higher than that of the purified one.The circular dichroism?CD?,endogenous fluorescence spectrum and exogenous fluorescence spectrum were used to detect the strucrural change of the purified enzyme under different temperatures.With the increase of temperature,the negative groove characterizing its secondary structure disappeared gradually,indicating that the secondary structure of the enzyme was destroyed gradually.Moreover,the increase of endogenous fluorescence intensity indicates that the aromatic amino acid residues originally embedded in the enzyme molecule were gradually exposed with the opening of the structure.The increase in intensity of the exogenous fluorescent light demonstrated that the hydrophobic groups were also gradually exposed.All the results proved that the second and third structures of ROL were gradually destroyed with the increase of temperature during the procedure of heat inactivation.3.Through screening of various factors and the single factorial optimization experiment,glucose,sorbitol,NaCl,MgCl₂ and polyoxyethylene pyrrolidone-30?PVP-30?were determined as raw materials for ROL heat stabilizer.Then,via significant factor screening experiments,the significant factors were determined,they were glucose,sorbitol,and sodium chloride,and then the optimal concentration was approximated by the steepest climbing test.Finally,the optimized mixing ratio was obtained by the response surface methodology:when the glucose concentration was 35.940%?m/v?,the sorbitol concentration was 34.816%?m/v?,and the sodium chloride concentration was 2.504mol/L,the relative enzyme activity was predicted to be 97.715%,which well coincides with the practical value.