【作者】 王贺；

【导师】 杨瑞金；

【作者基本信息】 江南大学 ， 食品科学， 2013， 博士

【摘要】 乳果糖是一种功能性低聚糖，已广泛应用于医药、食品和动物饲料领域。目前，商业化上主要采用化学异构化法生产乳果糖。但由于化学异构化法存在碱性催化剂的分离、反应副产物多、高能耗等问题，近年来一些学者开展了酶法转化乳糖合成乳果糖的研究。本实验室在前期研究中发现利用固定化β-半乳糖苷酶和固定化葡萄糖异构酶组成的双酶系统在环己烷-缓冲液两相体系中合成的乳果糖产量达到151g/L，乳糖转化为乳果糖的转化率为18.8%。本研究试图利用芽孢表面展示系统克服固定化酶的弱点如酶源的生产和纯化以及固定化过程中酶活的损失等，以达到基因工程手段固定化乳果糖制备用酶的目的，推动酶法制备乳果糖技术的发展。芽孢表面展示技术是一种采用基因工程手段的定向固定化方法，它已应用于抗体的制备和活性疫苗的生产等领域。与抗体和疫苗的生产相比，利用芽孢表面展示技术构建生物催化剂的研究则相对滞后。因此，本论文拟将乳果糖制备用酶展示在枯草芽孢杆菌芽孢的表面，并以此芽孢表面展示系统转化乳糖研究其制备乳果糖的能力。具体地，本论文获得的研究结果阐述如下：首先，以增强型绿色荧光蛋白为靶蛋白，研究芽孢crust层蛋白CotX、CotY和CotZ作为分子载体的潜力。荧光强度分析结果发现，由上述三种crust层蛋白转化得到的重组芽孢与比野生型芽孢相比，它们的荧光强度显著增强，表明CotX、CotY和CotZ可以将增强型绿色荧光蛋白展示在芽孢的表面。为此，选取了一种四聚体蛋白β-半乳糖苷酶作为第二个靶蛋白，验证上述三种crust层蛋白作为分子载体展示β-半乳糖苷酶活力的能力，展示的β-半乳糖苷酶活力分别为0.39、0.12和0.25U/mg芽孢（干重），即展示效率为CotX>CotZ>CotY。以CotX为分子载体和β-半乳糖苷酶为靶蛋白系统研究了β-半乳糖苷酶在芽孢表面的存在情况。Western blot、荧光显微镜和流式细胞仪实验证实了CotX可以将β-半乳糖苷酶展示在芽孢的表面。芽孢表面展示β-半乳糖苷酶活力的测定结果表明β-半乳糖苷酶与CotX的融合蛋白可以在枯草芽孢杆菌芽孢的表面进行活性表达。芽孢表面展示β-半乳糖苷酶的最适pH为6.0，比游离的重组β-半乳糖苷酶的最适pH向酸性方向偏移了0.5单位；它的最适温度为75℃，较游离的重组β-半乳糖苷酶的最适温度提高了5℃。进一步研究了整合型和游离型表达方式对β-半乳糖苷酶在芽孢表面展示效果的影响，两种表达方式得到的活力分别为0.42和1.34U/mg芽孢（干重），表明游离型表达方式更有利于β-半乳糖苷酶在芽孢表面进行展示。利用CotX为分子载体构建了葡萄糖异构酶的芽孢表面展示系统。Western blot可以检测到芽孢衣壳中融合蛋白CotX-葡萄糖异构酶的存在，流式细胞仪分析也显示重组芽孢表面的荧光强度明显增强，但却检测不到葡萄糖异构酶活力。以CotX为分子载体构建的芽孢表面展示系统可以在芽孢表面表达葡萄糖异构酶蛋白，但展示的酶蛋白没有活性的原因有待进一步探究。开展了以β-半乳糖苷酶芽孢表面展示系统作为固定化基质吸附葡萄糖异构酶构建以乳糖为原料制备乳果糖的固定化双酶系统的研究。在优化的吸附条件下即pH4.0、芽孢浓度1×10¹²/mL和葡萄糖异构酶添加酶量为2.0mg，得到的固定化双酶系统中β-半乳糖苷酶和葡萄糖异构酶的活力分别为14.2U/mL和41.2U/mL。该双酶系统在优化的反应条件下即乳糖浓度为300g/L、65℃和pH7.5时，乳果糖产量达到37.5g/L，乳糖转化率为12.5%。最后，利用CotX为分子载体构建了纤维二糖差向异构酶的芽孢表面展示系统，并开展了该展示系统作为催化剂转化乳糖合成乳果糖的研究。结果表明，在乳糖浓度为700g/L、80℃和pH8.0条件下反应2h，乳果糖产量为247.2g/L，乳糖转化率达到35.3%，远高于实验室前期研究中构建的β-半乳糖苷酶—葡萄糖异构酶固定化双酶系统和本研究构建的以β-半乳糖苷酶芽孢表面展示系统作为固定化基质吸附葡萄糖异构酶组成的双酶系统。更多还原

【Abstract】 Lactulose is a typical prebiotic disaccharide. Owing to its perceived health benefits,lactulose has been widely used in the fields of medical, food and animal feed. Commerciallyavailable lactulose is produced today mainly through the chemical isomerization of lactoseunder alkaline media. However, requirement of removal of catalysts and byproducts, andenergy requirement are the major drawbacks of such chemical process. In the last decade,enzymatic bioconversion of lactose into lactulose has been extensively studied to overcomethe above-mentioned problems. A study from our laboratory demonstrated that lactulose canbe successfully synthesized using immobilized β-galactosidase and immobilized glucoseisomerase dual-enzymatic system in organic-aqueous two-phase media. Under optimalconditions, the maximum lactulose concentration was approximately151g/L with aconversion yield of18.8%. Spore surface display technology, likely oriented immobilizationtechnology, enables the presentation of antigens or proteins of biological interest on thesurface of B. subtilis spores and has been successfully used for the production of vaccine andantibody. However, few studies have investigated the use of spore surface display system forthe development of whole-cell biocatalysts. In the case of immobilized lactulose-producingenzymes, very ofen, enzyme production was required and therefore the cost of the processwas increased. In considering both the disadvantages of the immobilized lactulose-producingenzymes and the excellent features of spore-surface display system, the primary aim of thepresent study was to display lactulose-producing enzymes on the spore surface. Theutilization of spore surface display system not only simplifies the procedure and also willfacilitate the lactulose production. Accordingly, an investigation of the display oflactulose-producing enzymes on the spore surface was carried out and the obtainedrecombinant spores were employed as whole cell biocatalyst for the lactulose production. Abriefly description of the obtained experimental results is given in the following sections.Firstly, three crust proteins CotX, CotY and CotZ were used as potential fusion partnersto target enhanced green fluorescence protein, encoded by the gene egfp, on the B. subtilisspores. Based on the results of the flourescence intensity experiment, the recombinant sporesof three engineered strains showed increased flouresence intensities than that of the wild typeB. subtilis168spores. To further confirm the possibility of CotX, CotY and CotZ acting asfusion partners, a tetrameric β-galactosidase was used as the second model protein. TheCotX-fused spore-displayed β-galactosidase exhibited an enzyme activity of0.39U/mg spores(dry weight), while the enzyme activities of CotY and CotZ-based recombinant protein were0.12and0.25U/mg spores. Data from the spore-displayed β-galactosidase assay showed thatthe display efficiency of β-galactosidase on the spore was in the order CotX>CotZ>CotY.An investigation into the utilization of CotX as a carrier for the display ofβ-galactosidase at the pore surface was performed. A combination of western blot,immunofluoresence microscope and flow cytometry was used to demonstrate that theCotX-linked fusion protein was successfully expressed on the B. subtilis spores. Further, theresulting spore-displayed β-galactosidase was able to hydrolyze ONPG, indicating that β-galactosidase was actively expressed on the spores. Next, the biochemical properties ofspore-displayed β-galactosidase were characterized in details. The optimum pH ofspore-displayed β-galactosidase was found to be6.0, decreased by a0.5pH unit as comparedto the recombinant enzyme. The spore-displayed β-galactosidase displayed its highest activityat75℃, a slightly higher than the purified recombinant enzyme (70℃). In additoin, the effectof the integrated and non-integrated expression models on the display efficiency ofβ-galactosidase on the spores was studied. It was found that the activities of spore-displayedβ-galactosidases obtained from the two different expression ways were determined to be0.42and1.34U/mg spores, respectively, suggesting that the use of non-integrated model is a betterchoice for the expression of β-galactosidase on the spores.To widen the application fields of spore-surface display systems, another CotX-basedspore display system that allows the expression of a thermostable glucose isomerase under thecontrol of the native CotX promoter PcotXwas constructed. Western blot was used to detect thepresence of the CotX-linked glucose isomerase fusion protein. Flow cyrometry showed that asignificant increase in flourescence intensity was observed for the recombinat spores ascompared to the wild type B. subtilis168spores. However, the recombinant spores exhibitedundetectalbe activity. These results evidenced that the CotX-based glucose isomerase fusionprotein was displayed, but not actively expressed on the spores. Further study is needed toreveal how the fusion protein folds. Besides this, the recombinant B. subtilis sporesexpressing β-galactosidase were used as a novel matrix for the immobilization of glucoseisomerase. The effcets of the pH value of buffer, the concentration of spore suspension andthe amount of the added enzyme on the adsorption efficiency of recombinant glucoseisomerase were optimized. Under optimal conditions, i.e., pH4.0sodium phosphate-citratebuffer,1×1012/mL spores concentration and2.0mg added enzyme amount, the activities ofspore-displayed β-galactosidase and adsorpted glucose isomerase was14.2and41.2U/mL.The obtained recombinant spores co-displaying β-galactosidase and glucose isomerase werefurther employed as a bi-enzyme system to enzymatically convert lactose into lactulose.Under optimal condition, i.e.,300g/L initial lactose concentration,65℃and pH7.5, thelactulose yield reached37.5g/L with a lactose conversion rate of12.5%.Finally, the CotX was used as a carrier to construct a spore-surface display system inwhich a new type lactulose-producing enzyme cellobiose2-epimerase was expressed on thespores. The resulting spore-displayed cellobiose2-epimerase was employed as a whole cellbiocatalyst to produce lactulose from lactose. Under optimal condition, i.e.,700g/L initiallactose concentration,80℃and pH8.0, the maximum lactulose yield was247.2g/L with alactose conversion rate of35.3%. The lactulose yield obtained in this study was much higherthan those of previous resulsts obtained from the dual-enzyme system in which consisted ofimmobilized β-galactosidase and immobilized glucose isomerase, and obtained from therecombinant spores co-diplaying β-galactosidase and glucose isomerase.更多还原