Phospholipids have been widely used in food, medicine, cosmetics, and other fields because of their unique chemical structure and healthcare functions. Phospholipase D (PLD) is a key biocatalyst for the biotransformation of phospholipids. Here, an autodisplay expression system was constructed for rapid screening of mutants, and PLD variants were recombined using DNA shuffling technology and three beneficial mutations were obtained. The results of enzymatic performance and sequence information comparison indicated that C-terminal amino acids exerted a greater impact on the correct folding of PLDs, and N-terminal amino acids are more important for catalytic reaction. The best-performing recombinant enzyme in transphosphatidylation reactions was Recom-34, with a phosphatidylserine content accounting for 80.3% of total phospholipids and a 3.24-fold increased conversion rate compared to the parent enzyme. This study demonstrates great significance for screening ideal biocatalysts, facilitating soluble expression of inclusion body proteins, and identifying key amino acids.