Study On The High-level Expression And Thermal Stability Of Pseudomonas Aeruginosa Lipoxygenase

Lipoxygenase(LOX),EC 1.13.11.12,is a non-heme iron-containing dioxide,capable of catalyzing the stereotactic peroxidation of fatty acids with the presence of at least one cis,cis-1,4-pentadiene system.LOX has a huge application prospects in the food processing industry,chemical industry,pharmaceutical industry and paper industry.The current commercial LOX products are extracted from soybeans.However,due to the presence of multiple isozymes in the soybean,the LOX product batch obtained by the extraction method.As a result,this kind of LOX has poor stability and is not conducive to the control of the final product quality and large-scale application.In the early work of the laboratory,the extracellular expression of P.aeruginosa BBE LOX in Escherichia coli Rosetta(DE3)was successfully achieved(N6).In this study,the above strain was used as the starting strain.Co-expression of antioxidant enzymes,atmospheric temperature(ARTP)mutagenesis and the optimization of induction conditions,which were conducted on the starting strain,intend to improve the production of LOX in the recombinant strain.At the same time,the thermal stability of LOX was studied.The thermal stability of LOX was improved by adding Transaminase(TGase).The main findings are as follows:1.Co-expression of antioxidant enzymes to improve LOX expressionThe superoxide dismutase genes(sodB and sodM)from P.aeruginosa BBE and catalase gene(katE)from E.coli were cloned into expression plasmid pRSFDuet-1,yielding the plasmids pRSF-sodB,p RSF-sodM and p RSF-katE.The recombinant plasmids were transformed into N6(a LOX expressing strain generated by E.coli)to obtain strains N6-B,N6-M and N6-K,respectively.Under the induction with 1 mmol·m L-1 IPTG at 20 oC,the total LOX activities of N6-B,N6-M and N6-K reached 21.6,28.1 and 7.1 U·m L-1,respectively.The yield of LOX in N6-B and N6-M was increased by 83% and 138% in contrast to N6(11.8 U·m L-1),respectively.It was also found that intracellular LOX production was nearly stable at 27.2 U·m L-1,which was 3.1 times that of LOX in N6 cells when the strains were induced for 32 h.2.Atmospheric temperature(ARTP)mutagenesis enhances LOX expressionN6-M was utilized as the original strain.Based on the lethal rate,the mutagenesis time was defined as 30 s.Screening the positive mutants using high-through put method,a mutant called E7 with LOX activity increased by 14.7% was finally achieved.Nevertheless,in terms of genetic stability,LOX activity of E7 can only be stable for 4 generations.After the fourth generation,the LOX yield reduced to the level of the starting strain.As a result,the high production characteristic of E7 cannot be inherited stably.3.Optimization of induction processIPTG concentration and cell concentration(OD600)were investigated by orthogonal experiments.Then the induction temperature of recombinant strain,N6-M,was optimized by single factor experiment.The result showed that the LOX yield was 29.2 U·m L-1,increased by 7.4% compared with the control strain.The optimized induction test was as follow: OD600=2.5,IPTG=2 mmol·mL-1,and inducing temperature=16 oC.3.Adding TGase to cross-link LOX to enhance the thermal stabilityThe thermal stability of the purified recombinant LOX was studied.The effect of TGase on the thermal stability of LOX under different conditions was investigated at 50 oC.The results showed that TGase could improve the thermal stability of LOX.By adding 0.13 U·mL-1 TGase and keep 20 oC for 30 min,the half-life(t1/2)of LOX was enhanced to 7.51 min,with an increase of 52.6% compared with the blank control(half-life 4.92 min).