Construction Of A Genetic Yarrowia Lipolytica Surface-displaying Methyl Parathion Hydrolase And Study On The Immobilization Of The Whole Cell Enzyme

In this study，the methyl parathion hydrolase (MPH) gene of Pseudomonas sp.WBC-3was cloned into the multiple cloning site of the surface display vectorpINA1317-YlCWP110and displayed on the surface of Yarrowia lipolytica Po1h cells,and the catalytic characteristic and degradation property of the recombinantengineered yeast were investigated. This engineered yeast was safe and didn’t carryantibiotic resistance genes, and its expression of recombinant MPH gene which wasgenetically stable was free from special induction. And it will avoid cell lysis, activitydecrease during the process of enzyme purification and obstacle of enzyme andsubstrates contact when using non-surface-display recombinant engineered strains formethyl parathion (MP) degradation. In order to further improve the repeatability andcontinuous operation stability of whole cell enzyme, the MPH-CBD fusion protein ofcellulose binding domain (CBD) of endo-glucanase IV from Trichoderma Viride andMPH from Pseudomonas sp. WBC-3was displayed on the surface of Yarrowialipolytica Po1h cells to construct the whole-cell methyl parathion hydrolase whichspecifically bounded to cellulose supports, by which the inferiority of activitydecrease, complicate operation and high cost of traditional MPH immobilizationmethod is solved. In this study, a kind of convenient, stable, effective and safeimmobilized whole-cell methyl parathion hydyolase was constructed and provided anew perspective and method of methyl parathion degradation, and it may serve as thetheoretical basis and technological support for degradation of methyl parathion. Themain research achievements of this study were as follows:(1) The methyl parathion hydrolase of Pseudomonas sp. WBC-3was displayed on the cells of Yarrowia lipolytica Po1h using surface display vectorpINA1317-YlCWP110, the authenticity of the display of methyl parathion hydrolaseon the cell surface of Y.lipolytica Po1h has been confirmed by immunofluorescenceand proteinase accessibility assay. Transformant Z51was selected with the highestmethyl parathion hydrolase activity of36.5±0.5U/mg cells(450.6±7.3U/mL cellsuspension), which preceded that of many present recombinant methyl parathionhydrolases.(2) The displayed methyl parathion hydrolase had the optimal pH of9.5and theoptimal temperature of40℃respectively and was stable in the pH range of4.0–11.5and up to40℃. The displayed methyl parathion hydrolase was stimulated by Co2+、Mn2+、Ni2+and Cu2+, and was inhibited by Ag+、Li+、Ba2+、Hg2+, however it was notaffected by K+、Zn2+、Ca2+、Mg2+、Fe2+、Fe3+、Na+. The optimal hydrolysis conditionin10.0mL of the reaction mixture was2.6×107cells/ml of yeast cell concentration,100mg/L of the substrate concentration and30min of reaction time, under which90.8%of methyl parathion was hydrolyzed. Different kind of water containing20.0mg/L ofmethyl parathion and2×107cells/m of the yeast cells displaying MPH was incubatedat40℃by shaking at180rpm for40min,98.7,97.0,96.5and94.4%of methylparathion in tap water (pH9.5), tap water (pH6.8), seawater (pH9.5) and naturalseawater (pH8.2) were hydrolyzed, respectively. Over70.0%of the activitymaintained after reused for four times and almost94%of whole cell MPH activityretained over a period of30days.(3) By employing overlap extension PCR, the fusion gene segment of CBD ofendo-cellulase IV from Trichoderma Viride and MPH from Pseudomonas sp. WBC-3was constructed and the MPH-CBD fusion protein was displayed on the cells ofYarrowia lipolytica Po1h with pINA1317-YlCWP110. The authenticity of the displayof MPH-CBD fusion protein on the cell surface of Y.lipolytica Po1h has beenconfirmed by immunofluorescence and proteinase accessibility assay. TransformantM3was selected with the highest methyl parathion hydrolase activity of33.1±1.1U/mg cells (381.0±9.2U/mL cell suspension), which indicated that themethyl parathion hydrolase activity was not significantly affected by CBD. (4) The optimal conditions for M3cells displayed the MPH-CBD fusion proteinbinding to cellulose substrate was at pH8.0and40℃respectively and M3cells alsoshowed high binding affinity to cellulose substrate at the temperature range of4℃-40℃. M3cells exhibited a high ability for attaching to cellulose substrate throughMPH-CBD fusion protein, and this bond was extremely tight. M3cellsimmobilization was performed by using microcrystalline cellulose as cellulose matrix.The bioreactor column packed with immobilized whole cell enzyme could hydrolyzeover80%of26.0mg/L methyl parathion in the10th minute under the condition of25oC, pH9.5and the120ml/h of flow rate, and the degradation ratio was maintained atabout80%in the subsequent60minutes.77%degradation ratio was retained against26.0mg/L methyl parathion over a period of30days.