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Phospholipase A1 (PLA1,EC3.1.1.32) is an enzyme that hydrolyzes phospholipids and produces 2-acyllysophospholipids and fatty acids.It is widely applied in many fields such as vegetable oil degumming,bread making,dairy processing and egg yolk industry.For reasons of quickly and high efficiently improved the catalytic capability and optimized the catalytic property,directed evolution plays an important role in the modification of enzymes.In this study,error-prone PCR and DNA Shuffling technology was used to build the mutation library of phospholipase A1 gene( plaB) from Serratia marcescens PL-06 which was screened by our laboratory.And an convenient screening method has been established based on the size of hydrolysis halo in the screening plate used phospholipids as carbon source.After one round of error-prone PCR and two rounds of DNA shuffling,the mutant M2 was screened from the mutation library.The enzyme activity of M2 reached 23.7U/mL,which improved 48.13% compared to the parent enzyme,furthermore M2 displayed better thermal and the low pH stability.Protein sequence analysis showed that there were six amino acid had been changed,and the mutation sites 166 (Gly-Ala),210 (Gly-Ala),187 (Gly-Met) and 269 (Ile-Ala) were near from the predicted enzyme active center.The mutations at the sites 166,210,and 269 increased the rigidity of the catalytic center and this change might be responsible for the enhancement of the thermal stability of the mutant enzyme.By the comparison the three-dimensional structure,the amino acid change of 166(Gly-Ala) caused the absence of an α-helix in the enzyme structure and this change might be related to the increase in enzyme activity.