【作者】 王星星；

【导师】 汝少国；

【作者基本信息】 中国海洋大学 ， 生态学， 2013， 博士

【摘要】 本文利用表面展示质粒pINA1317-YICPW110将来源于假单胞菌(Pseudomonas sp.)WBC-3的甲基对硫磷水解酶(methyl parathion hydrolase, MPH)展示在解脂耶罗威亚酵母(Yarrowia lipolytica)Po1h细胞表面，研究了重组工程菌的催化特性和降解性能。该工程菌安全无害、不携带抗性基因、目的基因表达无需诱导且可稳定遗传，而且避免了采用非表面展示重组工程菌进行甲基对硫磷降解时需要破碎细胞、酶纯化时活性降低、酶与底物接触不充分等问题；为进一步提高全细胞酶的重复使用性和连续操作的稳定性，通过将绿色木霉(TrichodermaViride)内切葡聚糖酶IV(EG IV)的纤维素结合域(cellulose binding domain, CBD)与甲基对硫磷水解酶的融合蛋白展示在Y. lipolytica Po1h细胞表面，构建了可特异吸附于纤维素基质上的全细胞甲基对硫磷水解酶，采用固定化全细胞酶对甲基对硫磷进行降解研究，解决了甲基对硫磷水解酶采用常规固定化方法出现的活力下降、操作繁琐、费用昂贵等问题。本文构建了一种操作简便、稳定高效、安全无害的固定化全细胞甲基对硫磷水解酶，开辟了国内甲基对硫磷降解的新思路和新手段，为甲基对硫磷降解提供了理论依据和技术支撑。主要研究结果如下：(1)利用表面展示质粒pINA1317-YlCWP110将Pseudomonas sp.WBC-3的甲基对硫磷水解酶在Y. lipolytica Po1h上进行了表面展示，通过免疫荧光检测和蛋白酶敏感性分析确定甲基对硫磷水解酶已成功展示于Y. lipolytica Po1h细胞表面。筛选得到甲基对硫磷水解酶酶活力最高的转化子Z51，其甲基对硫磷水解酶比酶活力为36.5±0.5U/mg cells(450.6±7.3U/mL cell suspension)，酶活力优于已有的多种重组表达的甲基对硫磷水解酶活力。(2)表面展示甲基对硫磷水解酶的最适作用pH为9.5，在pH4.0-11.5之间较稳定；最适作用温度为40℃，在40℃以下稳定性较好。Co2+、Mn2+、 Ni2+、Cu2+对表面展示甲基对硫磷水解酶活性有激活作用，Ag+、Li+、Ba2+、Hg2+对展示酶活力有抑制作用，K+、Zn2+、Ca2+、Mg2+、Fe2+、Fe3+、Na+对展示酶活力没有明显的影响。在10.0mL反应体中的最佳水解条件为100mg/L甲基对硫磷、2.6×107cells/ml表面展示甲基对硫磷水解酶的酵母细胞、反应时间30min，甲基对硫磷水解率可达90.8%。全细胞酶终浓度为2×107cells/ml、甲基对硫磷浓度为20.0mg/L的不同水体40℃、180rpm振荡反应40min，甲基对硫磷的降解率依次为淡水(pH9.5)98.7%、淡水(pH6.8)97.0%、海水(pH9.5)96.5%和海水(pH8.2)94.4%，全细胞酶重复使用4次后，仍可保持70%以上的活力。全细胞酶在4℃放置30天，仍能保持94%的活性。(3)采用重叠延伸PCR技术，构建绿色木霉EG IV的CBD和Pseudomonassp.WBC-3MPH的融合基因片段，利用表面展示质粒pINA1317-YlCWP110将MPH-CBD融合蛋白在Y. lipolytica Po1h酵母上进行了表面展示，通过免疫荧光检测和蛋白酶敏感性分析确定MPH-CBD融合蛋白已成功展示于Y. lipolytica Po1h细胞表面。筛选得到甲基对硫磷水解酶酶活力最高的转化子M3，其甲基对硫磷水解酶比酶活力为33.1±1.1U/mg cells(381.0±9.2U/mL cell suspension)，甲基对硫磷水解酶的活性基本未受到CBD的影响。(4)表面展示MPH-CBD融合蛋白的转化子M3细胞吸附纤维素基质的最适pH为8.0，最适温度为4℃，在4℃-40℃之间具有良好的吸附性。M3细胞通过表面展示的MPH-CBD融合蛋白对纤维素基质具有较高的吸附能力，且细胞与纤维素基质的的结合非常牢固。将M3细胞固定于微晶纤维素上，制成固定化全细胞酶。填充有固定化全细胞酶的柱式生物反应器于流速为120ml/h、pH为9.5、温度为室温25℃时，对26.0mg/L甲基对硫磷的降解率在10min时即可达到80%以上。在随后的60min内，甲基对硫磷的降解率一直保持在80%左右。反应器在30天内对26.0mg/L的甲基对硫磷可保持约77%的降解率。更多还原

【Abstract】 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.更多还原