| Phosphatidylserine?PS?,as the main acidic phospholipid in the brain,has the effects of improving cognitive ability and anti-depression,and can be used in medicine,food and other fields.In recent years,people began to pay attention to the preparation of PS by enzymatic transformation because of the less content in nature and the difficulty of extraction,that is,soybean lecithin and L-serine were used as substrates,and phospholipase D?EC 3.1.4.4;PLD?was used as the catalyst to synthesize PS through transphosphatidylation.Although some achievements have been made,there are still some deficiencies:?1?The low level of PLD secreted by wild-type species is far below the industrial production standard;?2?Generally,transphosphatidylation is carried out in a biphasic system consisting of a water-immiscible organic solvent phase.In this study,Bacillus subtilis WB600was used as host to study the heterologous expression of PLD,and the formation conditions of PS in pure water phase were optimized.The main results are as follows:?1?Heterologous expression of PLD and optimization of signal peptide.The PLD gene from Streptomyces racemochromogenes was synthesized according to the codon-optimized for B.subtilis.Seven signal peptides originating from Bacillus subtilis were selected according to the number of charged N-terminal and hydrophobicity of H-terminal,and were fused into the N-terminus of mature PLD.The fusion fragments were cloned into the expression plasmid pSTOP and transfected into B.subtilis WB600,and the secretion efficiency of PLD under different signal peptide was compared.According to our analysis of enzyme activity,PLD was successfully expressed in B.subtilis WB600,with the fusion protein containing the signal peptide amyE having the highest extracellular PLD activity(11.3 U×mL-1),and in a PLD expression level 5.27-fold higher than when the endogenous signal peptide is used,and the fusion protein containing ywbN having the lowest extracellular activity(3.2 U×mL-1).?2?Optimization of expression plasmids.Three recombinant expression plasmids pSTOP-PLD-amyE-his,pMA0911-PLD-amyE-his and pP43-PLD-amyE-his were constructed.The recombinant bacteria containing these three plasmids were named STT1,ST2 and ST3,respectively.The results showed that the extracellular enzyme activities of ST2 and ST3 were higher than those of STT1.The highest enzyme activity of ST2 strain was 19.1 U×mL-1,which was about 69.03%higher than that of the control strain.Western Blot analysis of the supernatants of three strains showed that there was a single band at about 53 kDa,which was consistent with the reported band size.?3?RBS and spacer sequence optimization.RBS Calculator v2.0 software was used to optimize the RBS and spacer regions of the recombinant plasmid pMA0911-PLD-amyE-his.The RBS sequences with translation intensities 3,6,9,or 12 times higher than that of the original RBS sequence were selected to obtain the recombinant strains RS1,RS2,RS3 and RS4,respectively.The results showed that the highest extracellular enzyme activity of RS1 strain was 24.2 U×mL-1,which was 26.7%higher than that of control,while the extracellular enzyme activity of RS4 strain was 11.8%lower than that of control.We found that the extracellular secretion of PLD increased first and then decreased with the increase of RBS strength,indicating that the translation intensity of RBS was not positively correlated with PLD secretion.?4?Fermentation tank amplification experiment and analysis of PLD characterization.Enzym-atic activity was increased by about 5 times to 116.2 U×mL-1 in 15 L fermentor.The genetic stability test of RS1 strain showed that the retention rate of the recombinant plasmids was still 100%after 5generations.The extracellular supernatant of RS1 strain was purified and the enzymatic properties of PLD were determined.The results showed that the size of PLD protein band was about 53 kDa,which was consistent with the reported band size.The optimum reaction temperature was 45?,the optimum reaction pH was 5.5,and the enzyme could be preserved for a long time at 4?.?5?The PS production process in pure aqueous phase was optimized to avoid the use of organic phase.The optimum reaction conditions were:PC60:L-serine 1:10(g×g-1),5 mL enzyme 500 U,reaction temperature 45?,reaction pH 5.5,6 h reaction time,10%calcium chloride.After 6 hours,with PC60 as the substrate,the conversion of PS reached 96.7%,the remaining PC was 2.3%,and the by-product PA was only 0.2%.The efficient conversion of substrates to products is realized. |
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