【作者】 梁达奉；

【导师】 余林；

【作者基本信息】 广东工业大学 ， 应用化学， 2011， 博士

【摘要】 葡聚糖(Glucan)是以葡萄糖为单体的聚合物的总称。制糖工艺过程出现的葡聚糖主要为α-葡聚糖,又称右旋糖酐(dextran),是以α-D-葡萄糖为单体、以1-6键连接的直链为主链(约占95%以上)形成的高分子聚合多糖,其分子式为(C6Hio05)n。从蔗汁到精制产品,所有制糖工艺过程自始至终存在葡聚糖所引起的不良的影响。葡聚糖的产生和存在使蔗糖损失,明显增大糖液的粘度,降低糖液的过滤性,影响煮糖、结晶与分蜜,增加制炼成本,并且严重影响糖产品的质量。本论文针对制糖过程存在的α-葡聚糖的降解、检测的难题,构建并筛选了高效表达α-葡聚糖酶的基因工程菌株,并进行发酵试验研究；对发酵所产α-葡聚糖酶在甘蔗制糖过程α-葡聚糖的分解清除和医药领域微分子右旋糖酐的生产中进行相关的应用研究；成功制备了α.葡聚糖特异性单克隆抗体并研制出α-葡聚糖检测试剂盒。主要实验研究工作如下：1、α-葡聚糖酶基因工程菌的构建与筛选通过基因工程方法,构建pPICZ a A-DEX重组质粒,将源自青霉的α-葡聚糖酶基因整合进入毕赤酵母基因组,实现了外源α-葡聚糖酶融合蛋白的表达。所构建的X-33/pPICZaA-DEX毕赤酵母工程菌株,以甲醇为诱导物,其外源基因通过醇氧化酶AOX启动子的调控,得以高效转录表达。在国际上首次构建以三磷酸甘油醛脱氢酶GAP启动子作为外源基因启动子的pGAPZ a A-DEX重组质粒,进而构建KM71H/pGAPZ a A-DEX毕赤酵母工程菌株。该菌株在表达外源蛋白时,外源基因通过GAP启动子的调控,在以甘油或葡萄糖等为碳源的培养基上无需诱导物的存在就可以表达,产酶量和菌体密度具有较强的趋势一致性。2、α-葡聚糖酶基因工程菌的发酵对X-33/pPICZaA-DEX毕赤酵母工程菌株产酶培养基进行优化,探讨了温度、溶氧、初始pH、接种量、诱导阶段甲醇浓度和补料间隔时间等对产酶的影响。5L发酵罐流加培养甲醇诱导型工程菌株产酶,在优化的发酵工艺参数条件下,α-葡聚糖酶的活力最高可达1048 U/mL,国内领先,并通过了广东省科技厅组织的技术鉴定；5L发酵罐流加培养KM71 H/pGAPZaA-DEX产酶,在优化的发酵工艺参数条件下,α-葡聚糖酶的活力最高可达398 U/mL。以上两种不同类型的工程菌各有特点,探索完善其相关的发酵工艺,可为大规模工业生产α-葡聚糖酶打下基础。3、α-葡聚糖特异性单克隆抗体的制备及其检测试剂盒的研制以Dextran T-40-BSA交联物免疫小鼠,用常规方法进行细胞融合,间接ELISA方法筛选阳性克隆,同时采用有限稀释法进行杂交瘤细胞的亚克隆和培养,成功建立了7株杂交瘤细胞株,其效价均大于106；经纯化的抗体其重链分子量为50000,轻链分子量为23000；与BSA、OVA,蔗糖,葡萄糖等无任何交叉反应,证明抗体的特异性。运用获得的单克隆抗体建立α-葡聚糖定量检测免疫比浊法,在国内首次成功研制α-葡聚糖定量检测试剂盒,于2010年7月通过国家质检总局验收并获高度评价,国际先进,解决了α-葡聚糖(右旋糖酐)快速、准确、便捷检测的难题。α-葡聚糖定量检测免疫比浊法有望成为国家标准方法乃至国际统一分析法。4、α-葡聚糖酶的酶学性质及其稳定性研究研究了α-葡聚糖酶的酶学性质,表明α-葡聚糖酶的最适反应温度为45-50℃,最适反应pH=4.5-5.0；酶的储存稳定性较好,pH=4.5-5.0时葡聚糖酶较稳定,4℃保存,半衰期约为35天。采用葡萄糖作保护剂研究了其对α-葡聚糖酶的稳定性影响,表明葡萄糖可以极大地减少酶活的损失；以蔗糖作为保护剂,在室温下酶活基本没有损失。5、α-葡聚糖酶在制糖过程中的应用研究在实验室研究中,以降粘率、除色率、葡聚糖去除率等指标为判断依据综合评价α-葡聚糖酶对蔗汁澄清的效果,通过实验确定α-葡聚糖酶的各种使用条件。2009/2010榨季在广东省台山市海宴华侨农场糖厂进行模拟生产工艺试验,在不同清净工艺中对蔗汁的澄清效果试验表明,α-葡聚糖酶对甘蔗制糖过程蔗汁的清净有较大的帮助,对提高澄清汁的纯度尤为明显；α-葡聚糖酶处理低纯度蔗汁后其葡聚糖含量和色值都有较大的下降,表明葡聚糖酶对低纯度蔗汁的效果相当明显。6、α-葡聚糖酶在合成微分子右旋糖酐中的应用研究创新性地提出肠膜明串珠菌发酵蔗糖产微分子右旋糖酐新工艺,在发酵过程中应用α-葡聚糖酶进行催化水解协同作用,通过控制α-葡聚糖酶的添加量和添加时间控制右旋糖酐的分子量,一步法直接发酵生产微分子右旋糖酐。培养基中13%的蔗糖在24小时内转化率达90%以上,在发酵16小时时添加0.8-1.2 U/mLα-葡聚糖酶,可一步合成分子量为3000-8000Da的右旋糖酐；用超滤膜截留发酵液,可分离提纯得到符合《中华人民共和国药典》要求的右旋糖酐。新工艺解决了传统工艺中右旋糖酐分子量无法控制、酸水解废水污染大以及分离纯化有机溶剂用量大等问题。更多还原

【Abstract】 Dextran, a kind of bacterial polysaccharides, has been recognized as a serious problem in sugar processing over 100 years. Dextran (C6H10O5)n is synthesized by microorganisms such as Streptococcus, Lactobacillus and Leuconostoc mesenteroides. The glucose monomers are predominantly linked by a(1,6) bonds in their major chains with a variable percentage of a(1,3) and occasional a(1,2) or a(1,4) branched linkages. The presence of dextran is associated with the processing problems from sugar cane juice to refined products. The dextran in sugar factories leads to a falsely high polarization, increased viscosity, lower evaporation rates, elongated crystals and increase of sugar loss to molasses.The aim of this work was to determine and eliminate the dextran level during sugar production. A monoclonal antibody against dextran was developed and an assaying kit base on the antibody for the dextran detecting was established. Then engineering strains of Pichia pastoris highly expressing an extracellular a-dextranase were constructed and screened. After fermentation in the optimal conditions, the activity of a-dextranase can reach 1048U/mL. The application of the recombinant dextranase was identified in the removal of dextran from sugar products and also in the production of lower molecular dextran in the medical and pharmacologic field.The main work is listed below:1. Construction and screening of engineered strains with recombinant plasmid encoding dextranaseThe a-dextranase gene from Penicillium minioluteum was cloned into vector pPICZa A and was expressed in Pichia pastoris under the control of AOX promoter. The engineered strain with recombinant plasmid X-33/pPICZaA-DEX was induced to express a-dextranase by methanol. We also constructed a recombinant plasmid of KM71H/pGAPZa A-DEX, with GAP promoter. The P. pastoris containing this plasmid expressed a-dextranase under the control of GAP promoter, without inductors in the glycerol or glucose medium. It was found that the yield of the a-dextranase is relative to the density of the density of engineered strains.2. Optimization of fermentation conditions for the expression of a-dextranaseThe fermentation conditions were optimized for P. pastoris with X-33/pPICZaA-DEX. Impact of temperature, dissolved oxygen, initial pH, inoculation size, methanol level in the induction step and interval time of feed supplement on the production of a-dextranase was analyzed. The dextranase activity from P. pastoris with X-33/pPICZaA-DEX culture and KM71H/pGAPZa A-DEX was determined to be 1048U/mL and 398 U/mL, respectively.3. Development of anti-dextran monoclonal antibody and establishment of assaying kit for dextranDextran was conjugated with BSA and then injected into Balb/c mice for the immunization. Cells of spleens and SP2/0 were fused by the hybridoma technology and the growing hybridomas were screened by indirect ELISA for their ability to secret high titer antibody against dextran. After cloning and subcloning by the Limiting Dilution method,7 hybridomas were selected, all of which exhibit titers higher than 1×106 in the culture supernatant. After purification of the culture supernatant, the antibody was analyzed to be specific against dextran, without cross-reacting with BSA, OVA, sucrose and glucose. Based on the newly-generated antibody, an immunonephelometric kit for quantitative detection dextran was developed. The kit passed the inspection and acceptance of the State Quality Inspection Administration of China in July,2010. The kit was proved be a rapid, correct and convenient tool for detecting dextran in raw material, by-products and end-products of sugar. This kit also can be applied to quantify dextran in medical and pharmacological field. The immunonephelometric assay is also respected to be a national standard detecting method, even a unitive analytical approach across the world. 4. Characterization of the a-dextranaseDextranase precipitated from the fermentation broth using ammonium sulfate was analyzed. Results showed that the optimal temperature for dextranase was 47-50℃and the optimal pH was 4.5-5.0. The stability of the a-dextranase was tested and results showed better stability in pH of 4-5. Half time of the dextranase was approximately 35 days when stored at 4℃.5. Application of a-dextranase in the sugar-making industryThe evaluating parameters for the clarification efficiency of a-dextranase on the sugar juice were viscosity reducing rate, color removal rate and dextran removal rate. Sugar juice showed better purity and color when treated with a-dextranase during various clarification process. Content of dextran and value of color of sugar cane juice in low purity decreased significantly after treated with a-dextranase. This indicated that a-dextranase was remarkably efficient on sugar cane juice in low purity.6. Application of dextranase in the production of low molecular mass dextranHere we also put forward the original technology of producing low molecular mass of dextran from fermentation of sucrose using Leuconostoc mesenteroides. Dextranase was added to hydrolyze the large polysaccharides into smaller ones. The molecular weight of dextran can be controlled by adjusting the a-dextranase adding level and adding time. Dextran with molecular weight of 3000-8000Da can be synthesized in one step when 0.8-1.2U/mL a-dextranase were added into the culture which had been fermented for 16h. The dextran was separated from the broth by ultra-filtration membrane and the obtained dextran meets the criterion of dextran in "Pharmacopoeia of the People’s Republic of China". The new technology solved the problems of uncontrolled molecular mass of dextran, severe pollution of wastewater, and large amount of organic solvent used when purifying.更多还原