【作者】 李影；

【导师】 陈晟；

【作者基本信息】 江南大学 ， 发酵工程， 2019， 硕士

【摘要】 D-甘露糖是一种六碳糖,主要用作食品营养补充剂,同时还具有预防尿路感染、抗炎症等生理作用,目前已广泛应用于医药、食品、饲料工业和生命科学等领域。最新研究结果表明,甘露糖能影响肿瘤的生长。目前工业上主要使用生物酶法制备D-甘露糖。与植物提取法和化学法相比,酶法制备D-甘露糖具有生产成本低、转化率高、反应过程简单、反应条件温和等优势。D-甘露糖异构酶（D-mannose isomerase,简称D-MIase,EC 5.3.1.7）可催化D-果糖和D-甘露糖之间的可逆异构化反应,是D-甘露糖生产的关键酶。本研究首先将来源于Pseudomonas geniculata的AGE family epimerase/isomerase（AGEase）在大肠杆菌（Escherichia coli BL21（DE3））中表达,通过进行酶学性质分析将其定性为D-MIase。通过定点突变技术获得了热稳定性提高的重组P.geniculata D-MIase突变体,并对以D-果糖为底物生产D-甘露糖的工艺进行优化。通过3-L罐发酵优化实现了重组P.geniculata D-MIase的高效表达。为了探索该重组D-MIase表达量较高的原因,进一步进行了蛋白质解折叠/重折叠的研究。主要研究结果如下:（1）通过PCR从P.geniculata中获得了AGEase的基因age,构建重组质粒pET-24a-age,并转化至E.coli BL21（DE3）中。重组E.coli BL21（DE3）/pET-24a-age摇瓶发酵24 h后,胞内酶活达284790 U?mL^-1,蛋白质表达量为5.3 mg?mL^-1。蛋白电泳显示在43 kDa处出现目的条带,表明来源于P.geniculata的AGEase在E.coli BL21（DE3）中成功表达。（2）通过硫酸铵盐析沉淀和GE凝胶柱Superdex 200（10/300 GL）对重组P.geniculata AGEase进行了纯化,并考察其酶学性质。结果表明,重组P.geniculata AGEase的最适温度为60℃,最适反应pH为7.5,在pH 7.5时,其50℃和60℃的半衰期分别为4 h和0.5 h。重组P.geniculata AGEase对D-甘露糖的K_m、k_cat及k_cat/K_m分别为48.74±8.4 mM、84062±725 s^-1、1724.7±86 s^-1·mM^-1,比活为53208 U·mg^-1。通过研究该重组P.geniculata AGEase的底物特异性、酶反应动力学和三维结构活性中心分析,可把该酶归类为AGE酶家族中的YihS酶类的D-MIase。（3）通过定点突变技术对重组P.geniculata D-MIase进行改造,获得了突变体C126A和E402W,相比野生型50℃半衰期均提高了50%。在此基础上对上述两位点进行叠加突变,获得突变体C126A/E402W,其50℃半衰期比野生型提高了75%。利用该突变体C126A/E402W催化D-果糖制备D-甘露糖,并对该工艺进行优化。以D-果糖为底物利用重组P.geniculata D-MIase制备D-甘露糖的最优条件为:在pH 7.5,50℃,加酶量为450 U·mL^-1,底物D-果糖浓度为20%时,酶转化率最高为39.3%,此时D-甘露糖产量为78 g·L^-1;以30%D-果糖为底物时,D-甘露糖的产量达到最大值104.4 g·L^-1,此时酶转化率为34.8%。（4）探究了重组菌E.coli BL21（DE3）/pET-24a-age在3-L罐中发酵诱导温度对菌体生长及产酶的影响。结果表明:当诱导温度为25℃时,重组P.geniculata D-MIase酶活最高达2535197 U?mL^-1,约为摇瓶水平的9倍;蛋白表达量为50 mg?mL^-1,约为摇瓶水平的9.4倍。为了探究该重组菌株E.coli BL21（DE3）/pET-24a-age产重组P.geniculata D-MIase表达量较高的原因,进一步进行了蛋白质解折叠/重折叠的研究。研究发现,蛋白质重折叠速率较慢有助于其正确折叠,可溶性表达量更高;蛋白质重折叠速率过快可能导致其在表达过程中错误折叠并形成大量包涵体,可溶性表达量低。更多还原

【Abstract】 D-mannose is a kind of six-carbon sugar,which is mainly used as a food supplement.It also has physiological functions such as preventing urinary tract infection and anti-inflammation.It has been widely used in medicine,food,feed industry and life sciences.At present,the industrial preparation of D-mannose is mainly carried out by biological enzymatic method.Compared with the plant extraction method and the chemical method,the enzymatic preparation of D-mannose has the advantages of low production cost,high conversion efficiency,simple reaction process and mild reaction conditions.D-mannose isomerase（D-MIase）catalyzes the reversible isomerization reaction between D-fructose and D-mannose and is a key enzyme in D-mannose production.In this study,AGE family epimerase/isomerase（AGEase）derived from P.geniculata was expressed in E.coli BL21（DE3）firstly and characterized as D-MIase by enzymatic properties analysis.Recombinant P.geniculata D-MIase mutant with improved thermal stability was obtained by site-directed mutagenesis,and the process of producing D-MIase using D-fructose as substrate was optimized.The high-efficiency expression of D-MIase was achieved by 3-L fermentor fermentation optimization.In order to explore the reason for the high expression level of recombinant D-MIase,further studies on protein unfolding/refolding were conducted.The main findings are as follows:（1）The gene age of AGEase was obtained from P.geniculata by PCR technique,and the recombinant plasmid pET-24a-age was constructed and transformed into E.coli BL21（DE3）.The recombinant strain E.coli BL21（DE3）/pET-24a-age was cultured for 24 h in shake flasks,the intracellular enzyme activity reached 284790 U·mL^-1,and the protein expression was 5.3mg·mL^-1.Protein electrophoresis showed a target band appeared at 43 kDa,indicating that AGEase derived from Pseudomonas geniculata was successfully expressed in E.coli BL21（DE3）.（2）The recombinant P.geniculata AGEase was purified by ammonium sulfate precipitation and Superdex 200（10/300 GL）gel column,and its enzymatic properties were investied.The results showed that the optimum temperature of recombinant P.geniculata AGEase is 60℃,and the optimum pH is 7.5.At pH 7.5,the half-lives at 50℃and 60℃are 4h and 0.5 h,respectively.The K_m,k_catat and k_cat/K_m of recombinant P.geniculata AGEase for D-mannose were 48.74±8.4 mM,84062±725 s^-1,1724.7±86 s^-1·mM^-1,respectively,and the specific activity was 53208 U·mg^-1.The recombinant enzyme can be classified into the D-MIase of the YihS enzyme in the AGE enzyme family by studying the substrate specificity,enzyme reaction kinetics and three-dimensional structure active center analysis of the recombinant P.geniculata AGEase.（3）The recombinant P.geniculata D-MIase was modified by site-directed mutagenesis to obtain mutants C126A and E402W,the 50℃half-life were 50%higher than the wild type.On the basis of this,the above two points were superimposed and mutagenized to obtain mutant C126A/E402W,and its half-life at 50℃was increased by 75%compared with wild type.D-mannose was prepared by catalyzing D-fructose using the mutant C126A/E402W,and the process was optimized.The optimal conditions for the preparation of D-mannose by using D-fructose as the substrate using recombinant P.geniculata D-MIase are:The amount of recombinant P.geniculata D-MIase is 450 U·mL^-1,pH 7.5,50℃,the substrate D-fructose concentration is 20%,at this time the enzyme conversion efficiency is up to 39.3%,and the D-mannose yield is 78 g·L^-1;when 30%D-fructose was used as the substrate,the yield of D-mannose reached a maximum of 104.4 g·L^-1,and the enzyme conversion efficiency was34.8%.（4）Explored the effect of fermentor induction temperature on the growth and enzyme production of recombinant E.coli BL21（DE3）/pET-24a-age in3 L tank.The results showed that when the induction temperature was 25℃,the enzyme activity of recombinant P.geniculata D-MIase was the highest,reached 2535197 U·mL^-1,which was about 9 times of the shake flask level.The protein expression of recombinant P.geniculata D-MIase was the highest,reaching 50 mg·mL^-1,which is about 9.4 times of the shake flask level.In order to investigate the reason for the high expression level of the recombinant E.coli BL21（DE3）/pET-24a-age,further studies on protein unfolding/refolding were conducted.The study found that the slow rate of protein refolding helps its correct folding,and the soluble expression is higher.The protein refolding rate is too fast,which may cause the protein to fold incorrectly during expression and form a large number of inclusion bodies with low expression.更多还原