细胞表面展示有机磷水解酶的恶臭假单胞菌工程菌的构建及全细胞酶活性分析

Construction and Whole-cell Catalytic Activity Analysis of Engineered Pseudomonas putida with Cell-surface Displayed Organophosphorus Hydrolase

利用丁香假单胞菌(Pseudomonas syringae)冰核基因的N-末端(inpn)作为锚定单元(anchoring mo-tif),通过与来自黄杆菌属(Flavobacteriumsp.)的有机磷水解酶基因opd构建融合基因inpn-opd,并连接于假单胞菌表达载体pYMBP,然后导入恶臭假单胞菌(P.putida)野生型菌株AB92019,获得了能在其细胞表面展示有机磷水解酶并具有全细胞酶催化活性的重组工程菌MMBL-opd。SDS-PAGE结果表明,融合基因能表达产生80 ku的蛋白质。重组菌MMBL-opd在无抗性LB固体培养基上能稳定生长,所携带的外源质粒的稳定性达到100%;在添加100μmol/L Co2+培养基上28℃培养48 h,表面展示的有机磷水解酶具有最高全细胞酶活性,为0.036 U/mg。用蛋白酶K消化处理重组菌表面蛋白可使其全细胞酶活降低92%。重组菌在PBS缓冲液中于4℃条件下保存30 d,仍能保持93%的全细胞酶活。

In the present study,N-termini sequence（inpn） of ice-nucleating protein gene from Pseudomonas syringae,was employed as the anchoring motif to direct the cell-surface display of a heterologous organophosphorus hydrolase in P.putida wild-type strain AB92019.A chimera gene with inpn and organophosphorus hydrolase encoding gene（opd） from Xanthomonas sp.,was initially constructed and then introduced into AB92019 when mediated by a Pseudomonas expression vector pYMBP.Consequently,the resulting engineered strain BBML-opd was endowed a whole-cell catalytic activity of organophosporus hydrolase.SDS-PAGE profile confirmed that a fusion protein was expressed in MMBL-opd with the deduced 80 ku of protein,and OPH activity declined to 92% of total whole-cell activity by treating with proteinase K on the surface proteins.The impact on whole-cell catalytic activity by culture temperature and incubation time was also investigated.It showed that a maximum 0.036 U/mg cell dry weight of whole-cell catalytic activity could be obtained when culturing the strain 48 h under 28 ℃ and a Co^2＋ concentration of 100 mol/L was given.Moreover,by determining the successive growth of MMBL-opd in the absence of the antibiotic,it showed that the stability of the introduced plasmid is 100% when inoculating in succession for 7 times and culturing for 168 h;and when leaving MMBLopd in PBS buffer for 30 d under 4 ℃ condition,it still maintained 93% of the whole-cell catalytic activity.