Studies On The Zymologic Characteristics Of Phospholipase D In Preparation Of Phosphatidylglycerol

This paper focused on the catalytic performance and secondary structure of phospholipase D in various anhydrous organic solvents with corresponding determination methodologies developed. The structure-activity relationship of phospholipase D was also discussed. The main contents and results of this study are as follows:The effect of log P (octanol-water partition coefficient) of organic solvents upon the catalytic activity of phospholipase D was reviewed. An extreme log P value will restrict phospholipase D's activity. The most appropriate solvent for phospholipase D is diethyl ether, followed by ethyl acetate, chloroform and methylene chloride. The phospholipase D from peanuts has specificity on the sources of substrates phospholipids, i.e. lecithin from soybean is more effectively catalyzed to transphosphatidylation than that from egg. The study on the noticeable factors shows the favorable conditions: phospholipase D is 2% of weight of substrates, whose concentration is 10mg/ml; the weight of ion-exchange resin is 100mg. In addition, the pretreatments for phospholipase D can enhance its catalytic activity to different extent. However, Ca2+ plays a critical role for the activity of phospholipase D. And the requirement of the concentration of Ca2+ is very strict, i.e. Ca2+ of concentration over the appreciate value will severely restrict the activity of phospholipase D. The optimum parameters of pretreatments are: the concentration of Ca2+ is 80mM, the KCl is 98% (actually, the higher the better) of weight of lyophilized powder at pH 5.58.The secondary structures of phospholipase D under different conditions were also rigorously measured. The results show that lyophilization process obviously changed the secondary structures of phospholipase D by decreasing the contents ofα-helix and increasingβ-sheet. The lyophilized powders with the presence of high proportions of salt can partially maintain the secondary structures of phospholipase D in hydrous solvents, while the pH statements, which are reflected by theβ-structure maintenance, can also be reserved after the process of lyophilization and suspending in organic solvents. The secondary structures of phospholipase D suspending in organic solvents are mostly as same as that of lyophilized powders. Nevertheless, the secondary structures of phospholipase D in the powders with pretreatments are affected in different extent by the solvents, among which, benzene, toluene, n-hexane and acetonitrile are more effective than chloroform, methylene chloride, diethyl ether and ethyl acetate.The rigorous and credible determination methodologies were developed to quantitively measure the PG contents in samples by HPLC and the secondary structures of phospholipase D by FTIR.The relationship between the secondary structures and catalytic activities was investigated with the methods of statistics, which helped to set up the multiple linear regression model of this relationship: y = 63.368 + 1.491x1– 2.213x2– 5.207x5 and the main factors regression model: y = 48.9593 + 0.0249x1– 0.0455x2– 0.0061x3– 0.4601x5 where y is the concentration of PG and x1, x2, x3 and x4 (which have been removed) are respectively the contents ofα-helix,β-sheet,β-turn and irregular structures, and x5 is log P of organic solvents.