【作者】 吴建；

【导师】 魏东芝；

【作者基本信息】 华东理工大学 ， 生物化工， 2011， 博士

【摘要】 氧化葡萄糖酸杆菌(Gluconobacter oxydans)是一种在生物工业中广泛应用的微生物,它含有多种膜结合的脱氢酶,表现非常独特的、其它微生物无法比拟的不完全氧化特性,可立体选择性和区域选择性地氧化醇、糖等羟基化合物生成相应的醛,酮或酸。利用氧化葡萄糖酸杆菌中的膜结合甘油脱氢酶可直接将甘油区域选择性脱氢生成二羟基丙酮,而利用膜结合的乙醇脱氢酶可化学选择性氧化苯甲醇生成苯甲醛。本论文以二羟基丙酮和苯甲醛的合成为目标,针对性地研究转化过程中的关键影响参数,探讨酶或细胞活性的影响机制,提出相应的解决策略,实现二羟基丙酮合成过程的规模放大,建立相对高效的苯甲醛生物法合成工艺。第一部分,基于对依赖PPQ的膜结合甘油脱氢酶的认知进行了氧化葡萄糖酸杆菌转化甘油合成二羟基丙酮的过程研究及规模放大。(1)建立了一种简便、快速、同时检测发酵液中甘油和二羟基丙酮的气相色谱方法。利用N-甲基咪唑作为催化剂,可在室温下快速完成对发酵液中甘油和二羟基丙酮的乙酰化,乙酰化的产物用气相色谱检测,内标法定量。方法验证表明,甘油和二羟基丙酮在1-100酬的浓度范围内线性良好,检测限分别为0.013和0.025g/l。方法准确度,重现性好,样品回收率大于95%, RSD小于1%。(2)筛选了具有高甘油脱氢酶活性的菌株,进而比较了发酵法与静息细胞转化法对生产二羟基丙酮的影响,结果显示,静息细胞在菌体量,二羟基丙酮产率以及保留活细胞数等三个方面分别提高了43.2,18.1和27.6%。(3)在摇瓶中初步确定了氧化葡萄糖酸杆菌静息细胞转化甘油的适宜条件：反应温度30℃、pH 6.0、转速200rpm、细胞浓度5g/l(干重)、甘油浓度46g/l,摇瓶中二羟基丙酮的产率不超过30g/l。在此基础之上,在3.7L发酵罐进行转化试验,通过溶氧和pH的有效控制,5.0-8.0g/l的静息细胞可在8-16h内转化100-120g/l甘油,转化率大于98%,可获得平均13.75g/lh最大时空产率。高浓度二羟基丙酮对反应有强烈抑制,其使反应趋于停止的临界浓度在140g/l左右。溶氧是影响二羟丙酮产率的重要限制因素,可在保持最大通气量的情况下,依据反应液中溶氧和pH的变化进行搅拌转速的调控,以满足反应过程溶氧需求。(4)对于高浓度二羟基丙酮降低细胞催化活性的原因,流式细胞仪检测和红外光谱的分析结果初步证明：二羟基丙酮对氧化葡萄糖酸杆菌静息细胞生理状态没有显著影响,而二羟基丙酮对膜结合甘油脱氢酶的不可逆抑制(失活)才是降低细胞活性的主要原因。(5)分别在300L和30L发酵罐上成功实现了菌体培养和静息细胞转化的规模放大。随着反应器的逐级放大,菌体生长和甘油转化活性都有不同程度地提高。在300L发酵罐上可获得8.5g/l菌体,分别比摇瓶、30L罐提高了164.5%和11.6%；在30L发酵罐上转化初速度比3.7L发酵罐上提高了17.4%。30L发酵罐上进行了9批菌体重复利用试验,转化率均大于98%,累计转化甘油850g/l,是目前报道的最高水平。第二部分,基于对依赖PQQ的膜结合乙醇脱氢酶的认知完成了氧化葡萄糖酸杆菌静息细胞不完全氧化苯甲醇合成苯甲醛的过程研究。(1)确定了氧化葡萄糖酸杆菌中负责氧化苯甲醇合成苯甲醛的酶为膜结合的乙醇脱氢酶(ADH),其作用机制是有序机制,O2是先导底物。膜上只有一个乙醛脱氢酶(ALDH),可将生成的苯甲醛进一步氧化成苯甲酸。(2)针对由醛脱氢酶引起的苯甲醛过氧化问题,利用缺失膜结合乙醛脱氢酶基因的氧化葡萄糖酸杆菌,在单水相体系中可使苯甲醛的选择性达到67.3%,而原始菌只有2.8%。在两相体系中利用其可进一步改善苯甲醛选择性和转化速率,1h内可完全转化5g/l的苯甲醇,苯甲醛选择性可达100%,该结果优于目前报道的微生物合成苯甲醛的最高水平。(3)针对有机溶剂对静息细胞的毒性影响,利用海藻酸钠包埋固定化细胞进行苯甲醇的转化反应。利用响应面法优化了固定化细胞中的三个重要因素的水平,结果表明：海藻酸钠浓度、细胞浓度和固定化颗粒粒径分别为2.55%(w/v)、49.26g/l和2mm时,固定化细胞可获得最好的活性和稳定性。在两相体系中,固定化细胞的稳定性明显高于游离细胞,在10批反应后还可保留游离细胞活性的53.2%,而游离细胞仅剩15.7%。(4)利用对苯醌作为电子受体在厌氧条件下进行氧化葡萄糖酸杆菌催化苯甲醇合成苯甲醛的研究,可完全阻断苯甲醛到苯甲酸的过氧化,在水溶液中实现苯甲醛的高选择性合成。使用人工电子受体的厌氧反应体系,为水溶性羟基醛的生物法合成提供了新的思路。更多还原

【Abstract】 Gluconobacter oxydans is known for its incomplete oxidation of a wide range of carbohydrates and alcohols in a bioprocess. The corresponding oxidative products are secreted almost completely into the medium. In this paper, the processes have researched for production of dihydroxyacetone (DHA) and benzaldehyde by the resting cell of G. oxydans. The DHA was be used widely in the cosmetic as sunless tanning tangent, and the benzaldehyde is the most important aromatic aldehyde in the industry.In the first section, the oxidation of glycerol to DHA by G. oxydans DSM 2003 based on the membrane-bound glycerol dehydrogenase was performed.A gas chromatographic method that accurately measured glycerol and DHA from fermentation broth is described. The method incorporates a sample derivatization reaction using N-methylimidazole as catalyst in the presence of acetic anhydride. The resulting derivatives were separated on a DB-5 capillary column and flame ionization detector. The present method exhibited a good linearity at the concentration range from 1 to 100 g/1 with regression coefficients of 0.9997 and 0.9998 for DHA and glycerol, respectively. The limits of detection were 0.025 and 0.013g/l. The recoveries of DHA and glycerol for fermentation sample were in the range of 95.37-98.76% with RSD below 1%.A comparative experiment has done for production of DHA between fermentation and resting cell bioconversion from the obtained dry cell weight (DCW), productivity and the cell physiological state. The results showed the conversions have the advantage than fermentation in the all aspects by resting cell.In the shake flask reaction, the productivity of DHA was no more than 30g/l under the optimization condition. To enhance DHA production, the process of bioconversion was improved in 3.7L bioreactor based on the optimized parameters in shaking flasks, by the optimization,100-120g/l of glycerol was converted completely to DHA by 5-8g/l DCW in 8-16h. The inhibition concentration of DHA was founded beyond 100g/l, and the critical concentration of DHA of reaction ceased was about 140g/l.The scale up process was operated in the 300L fermentor for cell culture and 30L fermentor for bioconversion of glycerol. It could be obtained the 8.5g/l DCW in the 300L fermentor, compared to the shaking flask and 30L seeds fermentor, the 164.5and 13.1%DCW could be improvement. In the 30L bioreactor for resting cell conversion, the initial velocity reached 15.2g/l, compared to the 3.7L, the 17.4% of improvement has been obtained. The nine batch bioconversion was completed in the 30L bioreactor, the conversion rate reached about 98% and the total concentration of glycerol converted was 850g/l.The resting cell physiological state was studied by flow cytometry, the results showed the influence of DHA on the cell physiological was smaller than catalytic activity of glycerol dehydrogenase In the second section, bioconversion of benzyl alcohol to benzaldehyde by G. oxydans M5 based on the membrane-bound alcoholdehydrogenasewas studied.The selective oxidation of primary alcohols to the corresponding aldehydes is one of the most important transformations in fundamental and industrial research owing to the carbonylic group as a versatile building block.The mutant strain defective in membrane-bound alcohol dehydrogenase (ADH) gene (GOX1067-1068) and the complementary strain were constructed and the results of oxidation of benzyl alcohol showed that the ADH was the key enzyme responsible for oxidation of benzyl alcohol to benzaldehyde in G. oxydans M5. The mechanism of oxidation benzyl alcohol by ADH is sequential ordered Bi Bi, and O2 is the leading substrate.The mutant strain with disruption of membrane-bound aldehyde dehydrogenase (ALDH) gene (GOX0585-0587) was be used for production of benzaldehyde. The gene strain (67.3%) showed obvious improvement for the selective toward benzaldehyde than the wild strain (2.8%), the 5g/l benzyl alcohol was be converted completely to benzaldehyde in the aqueous/isoctane biphasic system within 1h, the selectively toward benzaldehyde reached 100%.To avoid the organic solvents toxicity to resting cell, the immobilized cell was applied in the biphasic system for production of benzaldehyde. The response surface methodology was used for optimization the factors of alginate concentration, the cell load, diameters, the best results were 2.55% (w/v),49.26mg/ml,2mm, respectively. By the immobilized, the tolerance of cells has marked improvement. By ten recycles, the activity of immobilized cell has still retained 53.2%, but the free cell has only 15.7%.The oxidation benzyl alcohol was performed by resting cell of G. oxydans M5 under the anaerobic conditions using the p- benzoquinone accepted hydrogen. The results indicated the selectivity for benzaldehyde was reached 100% in the aqueous.更多还原