【作者】 韩瑞枝；

【导师】 堵国成；

【作者基本信息】 江南大学 ， 发酵工程， 2013， 博士

【摘要】 2-氧-D-吡喃型葡萄糖基-L-抗坏血酸（2-O-D-glucopyranosyl-L-ascorbic acid，AA-2G），是一种L-抗坏血酸（L-ascorbic acid，L-AA;俗称Vitamin C）的衍生物。它克服了L-AA在水溶液中（特别是存在光、热和金属离子等因素）容易被氧化的缺点，增强了其在水溶液中的稳定性。目前，AA-2G的生产方法主要是生物合成法，其中常用的催化剂为环糊精葡萄糖基转移酶（Cyclodextrin glycosyltransferase，CGTase）。在CGTase催化合成AA-2G的过程中，由于α-和β-环糊精具有较高的转化率，常被用作糖基供体。然而，由于α-环糊精价格昂贵，β-环糊精很难溶于水，所以从生产成本考虑，两者均不适合大规模生产AA-2G。寻求便宜易溶的底物取代α-和β-环糊精作为糖基供体生产AA-2G具有重要意义。本论文以来源于Paenibacillus macerans JFB05-01的CGTase为研究对象，通过不同方法对其改造，提高其利用麦芽糊精及可溶性淀粉等便宜易溶的底物生产AA-2G的转化率。主要结果如下：1.以P. macerans JFB05-01基因组为模板，PCR扩增获得不含自身信号肽的cgt基因，插入质粒pET-20b(+)中构建pET-20b(+)/cgt重组质粒，并导入E. coli BL21(DE3)中表达。经硫酸铵沉淀及镍柱纯化后，获得较纯的CGTase，SDS-PAGE验证其分子量约为75kDa。2.以pET-20b(+)/cgt为模板，对47位点的赖氨酸定点饱和突变，通过比较突变体利用麦芽糊精合成AA-2G的转化效率，筛选出了4种阳性突变体：K47V、K47F、K47L和K47W。纯化后对它们酶学性质的研究发现：它们利用麦芽糊精合成AA-2G时，最适反应温度为36℃，最适反应pH为5.5，最适底物配比为麦芽糊精/L-AA=1/1。K47V、K47F、K47L和K47W分别在反应8h、12h、24h和12h时AA-2G产量最高。K47L合成AA-2G最高产量为1.97g L-1，比原始CGTase提高了64%。动力学研究发现，与原始CGTase相比，四种突变酶针对麦芽糊精的米氏常数Km分别降低了25%、19%、23%和14%，说明它们对麦芽糊精亲和力增强。而Kcat/Km均提高说明催化效率提高。同时比较了它们不同催化反应活力，发现突变酶环化活力降低而歧化活力增强。最后通过同源模拟CGTase的三维晶体结构对实验结果做了推测性解释。3.通过对+2亚位点的3个位点氨基酸（Y260、Y195和Q265）定点饱和突变，并选取各自最优突变体进行组合突变，最终获得7种阳性突变酶：Y195S、Y260R、Q265K、Y260R/Q265K、Y260R/Y195S、Q265K/Y195S和Y260R/Q265K/Y195S。突变酶Y260R/Q265K/Y195S以麦芽糊精作为糖基供体生产AA-2G的产量为1.88g L-1，比原始CGTase提高了60%。酶学性质研究发现，突变酶Y260R/Q265K和Y260R/Q265K/Y195S的最适反应pH为6.5。突变酶Y260R和Q265K最适反应pH为5.0，Y195S和Y260R/Y195S最适pH为5.5。突变酶Q265K/Y195S最适pH为7.0。它们的最适反应温度均为36℃。动力学研究表明，七种突变酶针对麦芽糊精的米氏常数Km降低，说明它们对麦芽糊精亲和力增强。而Kcat/Km均提高说明催化效率提高。不同催化反应活力比较，发现突变酶环化活力降低，而水解活力和歧化活力增强。最后通过同源模拟CGTase的三维晶体结构对实验结果做了推测性解释。4.通过对CGTase中-6亚位点的4个位点氨基酸（Y167、G179、G180和N193）迭代饱和突变，最终获得四种阳性突变酶：Y167S、Y167S/G179K、Y167S/G179K/G193R、Y167S/G179K/G193R/G180R。纯化后酶学性质研究发现，突变酶最适温度从原始型的36℃变为28℃；最适pH较原始型也发生不同程度的变化。在最佳反应条件下，纯化后的Y167S/G179K/G193R/G180R利用麦芽糊精转化合成AA-2G的最高产量为2.12g L-1，比原始CGTase提高了84%。分别对突变酶和原始酶的环化，水解和歧化活力测定发现突变酶环化活力基本丧失，水解和歧化活力增强。此外，动力学研究表明突变株对麦芽糊精的底物结合能力较原始型有所提高。最后通过同源模拟CGTase的三维晶体结构对实验结果做了推测性解释。5.将来自于碱性Alkalimonas amylolytica淀粉酶的CBM与来自于P. macerans CGTase融合，构建了两种嵌合酶：CGT-CBMAmy和CGT△E-CBMAmy并纯化。纯化后酶学性质研究表明：以可溶性淀粉为糖基供体生产AA-2G时，嵌合酶最适温度从原始型的36℃变为28℃；最适pH则原始型CGTase相同为pH6.5。AA-2G产量分别是原始型CGTase的5.94和3.94倍。分别对嵌合酶和原始酶的环化，水解和歧化活力测定发现嵌合酶环化活力基本丧失，水解和歧化活力增强。此外，动力学研究表明嵌合酶对麦芽糊精的底物结合能力较原始酶有所提高。并通过同源模拟对实验结果做了可能性解释。6.通过将6种自组双亲短肽融合到来源于P. macerans CGTase的N-末端，构建了6种重组酶：SAP1-CGTase~SAP6-CGTase。纯化后进行酶学性质研究，发现6种重组酶的环化活力跟原始CGTase比较均有较大程度的下降，而SAP5-CGTase和SAP6-CGTase的歧化活力增加，并且以可溶性淀粉为糖基供体时，这两种重组酶合成AA-2G产量分别比原始CGTase提高了近1.33倍和2.36倍。动力学研究表明它们对可溶性淀粉底物结合能力增强。通过对原始CGTase和SAP6-CGTase三维结构的模拟及分析，为实验结果提供了一个推测性的解释依据。更多还原

【Abstract】 2-O-D-glucopyranosyl-L-ascorbic acid (AA-2G), one of L-ascoribc acid (L-AA)derivatives, is prepared by transferring a glycosyl residue from α-1,4-glucan to the C2position of L-AA. AA-2G overcomes the extreme instability of L-AA in aqueous solutionespecially under particular oxidative conditions such as heat, light and metal ions, improvesthe stability in aqueous solution. Nowadays biological transformation is the main approachesfor AA-2G synthesis and cyclodextrin glycosyltransferase (CGTase) is generally consideredto be the most effective biological catalyst. Usually, α-and β-cyclodextrin are used asglycosyl donors for AA-2G production with CGTase as the catalyst because of their hightransformation efficiencies. However, neither is suitable for large-scale production of AA-2Gowing to the high cost of α-cyclodextrin and low solubility of β-cyclodextrin in aqueoussolution. To find a cheap and easily soluble substrate replacing α-and β-cyclodextrin forAA-2G industrial production is the important future development direction.In this study, molecular modification was performed on the CGTase from Paenibacillusmacerans to improve its specificity for maltodextrin and soluble starch, both of which wereregarded as cheap and easily soluble glycosyl donor for AA-2G synthesis. The main researchcontents are listed as follows:1. We obtained the cgt gene encoding α-CGTase from P. macerans JFB05-01by polymerasechain reaction (PCR) with the genome of P. macerans JFB05-01as the template. Then thecgt gene was cloned into the pET-20b(+) victor, and the expression plasmidpET-20b(+)/cgt was constructed and transformed into the host E. coli BL21(DE3). Afterexpression and purification, we got the purified CGTase which was approximately75kDa.2. Site-saturation engineering of Lys47in CGTase was performed with pET-20b(+)/cgt asthe template. When using maltodextrin as glycosyl donor, four mutants K47F, K47L,K47V and K47W showed higher AA-2G yield as compared with that produced by thewild-type CGTase. The enzymatic characteristics suggested that their optimal temperature,pH and substrate ratio were36oC, pH5.5and maltodextrin/L-AA=1/1, respectively. Attheir optimal conditions, K47V，K47F，K47L and K47W got their highest AA-2G yieldsat8,12,24and12h, respectively. The highest AA-2G titer produced by K47L was1.97g L-1, which increased64%than the wild type CGTase (WT). The reaction kineticsanalysis showed that the Km(maltodextrin) of the four mutants were lower than the WTCGTase while the Kcat/Kmof them were higher than the WT. It was also found thatcompared with the WT, the four mutants had relatively lower cyclization activities andhigher disproportionation activities. Finally, the possible reasons were further explored bystructure modeling of the mutant CGTases.3. Combination site-saturation engineering was performed on Tyr260, Tyr195and Asn265of the CGTase and seven positive mutants Y195S，Y260R，Q265K，Y260R/Q265K，Y260R/Y195S， Q265K/Y195S and Y260R/Q265K/Y195S were obtained. After purification, the highest AA-2G titer produced by Y260R/Q265K/Y195S withmaltodextrin as the glycosyl donor was1.88g L-1, which increased60%than WT. Theenzymatic characteristics suggested that their optimal temperature were36oC. And for theoptimal pH: Y260R/Q265K and Y260R/Q265K/Y195S were pH6.5, Y260R and Q265Kwere pH5.0, Y195S and Y260R/Y195S were pH5.5, Q265K/Y195S was pH7.0. Thereaction kinetics analysis showed that the affinity and catalytic efficiency of the sevenmutants towards maltodextrin were higher than the WT. It was also found that comparedwith the WT, the four mutants had relatively lower cyclization activities and higherdisproportionation activities. Finally, the possible reasons were further explored bystructure modeling of the mutant CGTases.4. Iterative saturation mutagenesis (ISM) was performed on-6subsite of the CGTase toimprove the maltodextrin specificity for AA-2G synthesis. Four positive mutants Y167S,Y167S/G179K， Y167S/G179K/G193R and Y167S/G179K/G193R/G180R wereobtained and purified. The enzymatic characteristics showed that the optimal temperatureof the mutants was28oC (different from WT), and the optimal pH were also differentfrom WT. The highest AA-2G titer produced by Y167S/G179K/G193R/G180R withmaltodextrin as the glycosyl donor was2.12g L-1, which increased84%than WT. Thereaction kinetics analysis showed that the affinity ability towards maltodextrin of the fourmutants was higher than WT. It was also found that compared with the WT, the fourmutants had relatively lower cyclization activities and higher disproportionation activities.Finally, the possible reasons were further explored by structure modeling of the mutantCGTases.5. Two chimeric enzymes CGT-CBMAmyand CGT△E-CBMAmywere obtained by fusing acarbohydrate-binding module (CBM) from Alkalimonas amylolytica α-amylase (CBMAmy)to cyclodextrin glycosyltransferase (CGTase) from P. macerans. The enzymaticcharacteristics showed that when using soluble starch as the glycosyl donor for AA-2Gsynthesis, the optimal temperature and pH of the two chimeric enzymes were28oC andpH6.5. The highest AA-2G titers of CGT-CBMAmyand CGT△E-CBMAmywere3.94-and5.94-fold of yield from WT. The reaction kinetics analysis showed that the affinity abilityof the two chimeric enzymes towards soluble starch improved. Compared to WT, the twofusion enzymes had relatively high hydrolysis and disproportionation activities. Finally,the possible reasons were further explored by structure modeling of the mutant CGTases.6. Fuse six self-assembling amphipathic peptides (SAPs) to the N-terminal of CGTase toimprove AA-2G synthesis with soluble starch as the glycosyl donor. The enzymaticcharacteristics showed that all fusion enzymes have lower cyclization activities than WT.Compared to WT, SAP5-CGTase and SAP6-CGTase increased the disproportionationactivities, and the AA-2G titers produced by them increased about1.33-and2.36-fold.The reaction kinetics analysis showed that the affinity ability of the two fusion enzymestowards soluble starch improved compared to WT. Finally, the possible reasons werefurther explored by structure modeling of the mutant CGTases.更多还原