Cloning,Expression And Biochemical Characterization Of Phospholipase D From Aspergillus Niger

Phospholipase D(PLD,EC 3.1.4.4),belonging to phospholipases superfamily,mainly catalyzes the hydrolysis of phosphatidylcholine into signaling molecule phosphatidic acid.In farmaceutical industry,the base transfer reaction(transphosphatidylation)of PLD can be used to modify phospholipids to prepare single and naturally rare phospholipids.However,with recent progress in practical applications,which necessitates the need to develop PLDs with special properties,such as high enzyme activity and unique biochemical characteristics.In the present study,the two genes putatively encoding phospholipase Ds from Aspergillus niger were cloned,expressed and biochemically characterized.The main results obtained are as follows:Two OFRs(pldA and pldB)putatively encoding phospholipase Ds from A.niger were identified by by bioinformatics tools.These two genes were 2499 and 3645 bp in size,and encoded 832-amino-acid and 1214-amino-acid proteins,respectively.Phylogenetic tree construction and multiple sequence alignment showed that proteins PldA and PldB shared high similarity with PLDs from A.nidulans FGSC 26 and A.kawachii IFO 4308,respectively,with the similarities of 81.35%and 95.60%.Besides,they contained the two highly conservative HKD motifs(HxKxxxxDxxxxxxGS(G))and fungal PLD-specific motif YIENQFF,thus belonging to fungal PLD family.Total RNA was extracted from A.niger F0510 and cDNA synthesized by reverse transcription,which was used as a template for the amplification and expression of individual genes in Pichia pastoris.The two recombinant strains,GS-pldA and GS-pldB,were finally constructed.In the shaing flask experiments,the activities of the recombinant enzymes PldA and PldB were 0.59 and 0.43U/mL.These two enzymes were then purified to elctrophoretic homogeneity by ammonium sulfate precipitation,desalting and gel chromatography,with the purification fold and yiled of 22.2 and 25.1%,and 47.9 and 31.7%,respectively.The optimum temperature and pH of PldA were determined to be 30? and 7.0,It was stable at 30-50? and pH 7.0-9.0.The optimum temperature and pH of P1dB were determined to be 40? and 6.0,respectively.It was stable at 30-40? and pH 5.0-7.0.Their activities were significantly enhanced by Ca2+,Mn2+,Ba2+ and Tris,but were completely inhibited by acetonitrile.Other metal ions and chemicals exerted different inhibitory effects on their activities.Besides,pldA and pldB can be kept at 4? for more than 30 days.Two recombinant phospholipase Ds can hydrolyze the substrate lecithin to produce phosphatidate.