Site Directed Mutagenesis Study Of Phospholipase A₁and Its Effects On Enzymatic Properties

Phospholipase A1(PLA1) is a kind of enzyme which can specifically catalyze ester bondof phospholipids and have the lipase activity at the same time. PLA1has both importantphysiological functions and highly application values. Besides, lysophospholipid, thehydrolysate of PLA1, is also an excellent surfactant. Based on the method of rational design,by analyzing the crystal structure of Thermomyces lanuginosus (TLL), locus R103, R106andE109locate catalytic center around were selected to be mutated to study the effects of keyresidues on enzymatic properties, which will provide the theory basis for the research onPLA1. The main contents as following:1. The expression and enzymatic properties analysis of PLA1wild-typeThe expression vector of wild-type was constructed and linearized by restriction enzymeBlnI, then transformed into P. pastoris X-33which were grown and expression in YPD liquidmedium. Subsequently, the methods of ammonium sulfate precipitation, dialysis andanion-exchange chromatography were performed to purified protein. By analyzing theenzymatic properties of PLA1, we found that the optimum temperature of catalyzingphospholipids was55℃，and the optimum pH was5.0. The specific activity to catalyzephospholipids was1541.240U/g. While for the lipase activity, the optimum temperature was45℃, optimum pH was7.0，and its specific activity was1541.240U/g. For the reactionkinetics parameters, the Kmvalue of catalyzing phospholipids was0.051M, and the Kcat/Kmvalue was920.278s-1*M-1. The Kmvalue of catalyzing triglyceride was0.024M, and theKcat/Kmvalue was1498.435s-1*M-1.2. Study on the enzymatic properties of R103mutantsAfter analyzing the enzymatic properties of R103mutants, we found that R103M andR103K changed the optimum pH of phospholipase activity from5.0to6.0, and R103Achanged the optimum pH of lipase activity from7.0to5.0. Besides, the mutation changed theoptimum temperature of PLA1at the same time. The optimum temperature of phospholipaseactivity of R103K had no changes, and the other mutants were decreased by5to10degrees.While for the lipase activity, an increase was observed on R013K. However, R103M、R103Kand R103A mutations improved the activity of PLA1. The Kmvalues of R103M、R103K、 mutants decreased substantially; R103K mutant increased the Kcat/Kmvalues from920.278s-1*M-1to1855.9s-1*M-1; the Kmvalues of R103M、E103K mutants catalyzing estersdecreased substantially; The Kcat/Kmvalues of R103K mutant catalyzing esters increased from920.278s-1*M-1to2015.7s-1*M-1.3. Study on the enzymatic properties of R106mutantsR106mutations changed the optimum pH of lipase activity from7.0to6.0, and theoptimum pH of catalyzing phospholipids did not change. The optimum temperature of R106mutants catalyzing phospholipids decreased to50℃, and R106K increased optimumtemperature of its lipase activity. The Kmvalues of R106A and R106K mutants improvedsubstantially compared with that of wild type and decreased the Kcat/Kmat the same time,while R106M was just the opposite. The Kmvalues of R106M and R106K mutants catalyzingesters decreased substantially and improved the Kcat/Kmvalues from1498.435s-1*M-1to2769.9s-1*M-1, while R106A was just the opposite.4. Study on the enzymatic properties of E109mutantsE109L mutations changed the optimum pH of phospholipase activity from5.0to6.0, andmutation also changed the optimum temperature of PLA1; E109L mutation changed theoptimum pH of lipase activity from7.0to6.0and kept the optimum temperature at45℃.E109Q changed the optimum pH of lipase activity from7.0to5.0，and changed the optimumtemperature from55℃to50℃, and the optimum pH did not change. E109Q improved lipaseactivity of PLA1, and the lipase activity of E109L did not change too much. The Kmvalues ofE109L mutants decreased substantially, and Kcat/Kmincreased compared with that of wild type.E109Q improved the Kmvalues of PLA1, and decreased Kcat/Kmvalues compared with that ofwild type.5. The CD analysis of PLA1wild type and its mutantsThe second structures (α-helix, β-sheet etc.) of mutants did not change significantlycompared with PLA1wild-type by the mean of circular dichroism spectrum indicating thatmutations did not affect second structures significantly.6. Study on the oil degumming and its application of PLA1This paper mainly studied the catalytic properties of PLA1and its mutants in soybean oildegummed system. Results are as follows: R103A、R106M and E109Q have good results on degumming as well as R106A and R106K. While for R103M、R103K and E109L mutants, thedegumming effects were not observed.