Study On The Expression And Purification Of MGLP、Enzymatic Characterization And Its Application

Monoglyceride lipase is a kind of enzyme with a special substrate specificity, whichshows hydrolytic activity towards monoglycerides, but not diglycerides and triglycerides.Because of this substrate specificity, it can be used for the functional oils production. Untilnow only limited information on monoglycerides lipases is available. This paper studied onmonoglyceride lipase (MGLP) from a cell-free extract of the moderately thermophilic Bacillussp.H-257, and we described optimum expression conditions in prokaryotes system,purification, enzymatic characterization and catalytic application of the monoglyceride lipase.The results are as follows:1. The best conditions of expression and purification of MGLP: The final concentrationof IPTG is0.1mM, the optimal induced temperature and time combination was30℃-8h.CBD purification systems was employed for the purification of MGLP, and the CBD tag wasremoved by3C protein to obtain high purity MGLP, in order to prepare for researching onenzymatic characterization.2. Enzymatic characterization of MGLP: The optimum reaction temperature was60℃,MGLP was found to be stable even after3h incubation at40℃,50℃,60℃, respectively,and lose partial activity when the temperature increased to70℃; The optimum pH was9,and was relatively stable in the alkaline solution, and the maximum activity was observedfrom pH8to pH9; Enzyme activity remained nearly90%after incubation in differentconcentrations of organic solvents for1h, which indicated that MGLP was well tolerated withorganic solvents, and the impact is greater with the increasing concentration; Enzyme activitywas slightly inhibited by the presence of non-ionic surfactant (TritonX-100, Tween20,Tween80), and the inhibition hardly changed with the higher concentration. while an-ionicsurfactant (SDS) had strong inhibitory effect on enzymatic activity, and impact was greaterwith the increasing concentration; Enzymatic activity of MGLP did not change afterincubation with Ca2+for1h, and the other several heavy metal ions had a certain inhibitoryeffect on enzymatic activity. In addition, according to the relevant references, the MGLPshowed highest activity towards C8among substrates examined in the hydrolysis system, so choose octanoic acid and glycerol as the substrates for system optimization, results showedthat the optimum conditions: The optimum temperature was50℃, the ratio of water to thesubstrate was1%, enzyme loading was150U/g, molar ratio of glycerol/caprylic acid was5:1.In this system, MGLP showed specific selectivity towards different carbon chain saturatedfatty acids, but the activity of catalytic esterification was low. With the growth of the carbonchain, the trend of esterification rate was declining. These results indicated that MGLPshowed higher selectivity towards short carbon-chain fatty acids.3. Study on the removal of monoglyceride from esterification products, and the optimumconditions of MGLP hydrolysis of caprylic monoglyceride were explored. The optimumhydrolysis system: The ratio of water to the substrate was90%, enzyme loading was500U/g,and the optimum reaction temperature was50℃. In this system, The hydrolysis reactionproceeded for24h and approximately reached equilibrium after72h of reaction, and thecontent of monoglyceride in the reaction mixture decreased from the initial34.59%to5%.