Expression And Directed Evolution Of Phospholipase C In Bacillus Subtilis

The phospholipid, which has unfavorable effects on the flavor, odor, appearance, and shelf life of the oil, is the major component of the stable colloidal suspensions. To improve the quality of the oil product, phospholipids need to be removed in the deguming process. There are a number of limitations in the traditional duguming method, such as high-energy-consumption and high-pollution. Therefore, more and more researchers turned to enzymatic deguming. Phospholipase C (PLC, E.C.3.1.4.3) is a kind of enzyme which specifically hydrolyzes phospholipid into phosphate monoester and diglyceride. When used in the deguming process, as one of the hydrolysis products, the hydrophilic phosphate monoester can be easily removed by a small amount of water and the other product diglyceride can increase the retention of the crude oil. Considering the environmental and economic factors, phospholipase C has a promising prospect in enzyme deguming. But the application is heldback by the high production price, which is the key issue at the moment.This work aimed to bring down the PLC production cost by heterologous expression and enzyme engineering. Taking the complexity of phospholipids in colloidal suspensions and the safety issue in food industry into consideration, we chose PLCBc from Bacillus cereus (the sequence number in PDB:2FGN_A) as the study object. PLCBc has a relatively broad substrate spectrum and is the only phospholipase of bacterial source that is untoxic to mammals.Because the overexpression of PLC might inhibit cell growth, we chose a constitutive promoter HpaⅡ which is not strictly a promoter and modified the plasmid for optimal expression. As the next step, cloning and extracellular expression of PLCBc in the GRAS (generally recognized as safe) organism Bacillus subtilis was accomplished by fusion it with the signal peptide from plasmid pP43NMK. This is for the first time that PLC was successfully expressed in B. subtilis and the activity was detected with mixed phospholipids as the real substrate, which turned out to be 56.14 U/mL fermentation broth.Since the hydrolysis of mixed phosphopids by PLC gives a range of different products, which leads to difficulty in detection, this work linked PLC with an alkaline phsophatase to further hydrolyze the phosphate monoester to release phosphate, thus a high-throughput screening method could be constructed for indirect determination of the PLC activity based on the concentration of phosphate. The alkaline phosphatase from Escherichia coli K-12, which specifically hydrolyzes the dephosphorylation of phosphate monoester but not degrades diphosphate or triphosphate linkages, was therefore chosen to construct a coupled reaction system together with the PLCBc to establish a high-throughput screening method in B.subtilis based on the real substrate phospholipids. After two rounds of directed evolution, three obviously positive mutants were obtained. For the mutant K51E, the mutation occured at the 8th site in the mature peptide, resulting in an increase of 23% in the specific activity as compared with the wild type. The subtle changes in protein conformation resulted from the amino acid change might lead to significant changes in catalytic performance. In the mutant T16I/S27T/K51E, mutations were found in the signal peptide, and the volume activity was improved by 20%, which was probably caused by the enhanced secretion capacity. The third mutant had anonsense mutation at the 57th amino acid (His:CAT/CAC) and the protein expression level was possibly elevated by this change contributing to higher translating effiency, which also led to an activity increase of 20%.In this work, the analysis of the mutants indicated that the amimo acid residues at a distance from the active cavity could also have an apparent influence on the catalytic activity of the enzyme, which makes a significant complementation to the previous conclusions regarding the functions of the important amino acids in the proximity of the activity center of PLCbc. Mutagenesis at sites far away from the active cavity is also a major advantage of directed evolution over rational design. Meanwhile, there is so far no report on directed evolution of PLC in B.subtilis. The high-throughput screening method established in this work could be applied in the future researches on PLC to optimize the enzyme characterization and promotes the application of enzyme deguming process employing PLC in food industry.