【作者】 赵玉萍；

【导师】 徐岩；

【作者基本信息】 江南大学 ， 发酵工程， 2014， 博士

【摘要】 γ-癸内酯（γ-decalacton，GDL），又名丙位癸内酯，化学名称为γ-正己基丁内酯，天然存在于水果及发酵产品中。GDL因具有诱人的桃香味和低香气阈值，在食用香料及制药、化学合成等领域得到了广泛应用，人们对该产品的需求量日益增大，然而在自然界中尚未发现此香料的大量存在，而化学法生产的却是消旋体。用生物法生产的GDL具有天然等同的特点，目前国际市场也没有天然旋光性GDL产品供应，因此，用生物法生产GDL具有广阔的前景。本论文筛选获得了产GDL的耶鲁维亚解脂酵母（Yarrowia lipolytica），以不断提高GDL产量为目标进行了较为系统、完整的研究。采用基因组改组方法提高GDL产量，并对发酵条件进行优化，建立了菌体生长动力学模型和GDL生产动力学模型。为减少底物和产物对细胞的毒性提高GDL产量，以有机复合无机材料制备固定化细胞，提高了细胞的通透性和机械强度，延长了使用寿命。为进一步增加胞内外的物质交换，促进蓖麻油底物进入细胞及GDL产物的泌出，建立了生物转化的离子液体系。通过对影响GDL产量和ACO同工酶酶活因素分析，发现吡啶环具有较好的促进或刺激作用。对照了传统溶剂和超临界二氧化碳萃取法对GDL的提取效果。具体内容有以下几个方面：（1）根据GDL在碱性条件下与盐酸羟胺反应生成中间产物异羟肟酸，异羟肟酸可与三价铁反应生成有色络合物的显色反应原理，建立了准确性和重复性均很好的GDL分光光度检测法，检测范围为：0.125-8.14g L-1。基于该检测方法，建立了96孔微培养板高通量筛选方法，用96孔微培养板培养菌体，再对发酵液进行显色反应，用酶标仪检测吸光度，最终从4327个样本中，获得了215株产GDL菌株，其中产量最高为179mg L-1，经26S rRNA测序结果，该菌株确定为Yarrowia lipolytica。（2）以紫外诱变获得的正向突变株作为基因组改组的出发菌库，获得了原生质体制备和融合的最佳条件，采用递推式融合法，经过三轮融合，获得了产量最高且遗传稳定性较好的菌株G3-3.21，产量为3.75g L-1，是出发菌株的6.58倍。通过对正向突变株ACO酶活和GDL产量的比对分析，发现GDL的高产不仅要求ACO同工酶对长链底物降解能力较强，还需对中链和短链底物的降解偏弱，才有利于GDL的累积且能够在一定时间内维持此最高产量，不会在短时间内未来得及内环化又被进一步β-氧化了。（3）为提高GDL产量，采用田口设计法对8个因素进行优化，确定主要影响因素为麸皮、复合氮源和初始pH。然后采用响应面分析法对主要影响因素进一步优化，获得最优发酵条件，GDL产量达到4.767g·L-1。麦麸中烟酸的吡啶环对于GDL的生产可能有较好的促进作用。对Z703和G3-3.21的菌体生长动力学和产物形成动力学进行研究，其菌体生长模型分别为dXdd=0.309(1Xt15.2)X和XXdt=0.438(116.7)X，GDL生成动力学模型分别是dPdPdt=0.00105X和dt=0.00784X。（4）为减少底物和产物对细胞的毒性，采用固定化细胞转化技术，为提高通透性和机械强度，采用有机海藻酸钠复合无机凹凸棒石粘土固定化细胞，GDL产量达6.15g L-1，比游离细胞高1.32倍，且可重复使用3次。构建了生物相容性较好的离子液转化体系，增加了胞内外的物质交换，GDL产量达7.85g·L-1。与相同的阴离子相比，当阳离子为吡啶离子时，GDL产量和ACO酶活普遍高于咪唑离子；当阴离子为溴离子和硝酸离子时，GDL产量和ACO酶活均低于阴离子为四氟磷酸离子和六氟磷酸离子。（5）研究了游离细胞的破壁条件，对照未破壁发酵液，GDL产量提高14.06%。通过酸化和加热实验，证明本研究中所采用的耶氏酵母本身就具有内酯化能力，不需在萃取前，通过加热酸化将前体物4-羟基-癸酸人工内酯化成GDL。研究了固定化细胞离子液体系生物转化GDL的提取工艺，采用乙酸丁酯萃取法，萃取率最高达86.2%；采用超临界二氧化碳的最佳萃取率为91.7%。更多还原

【Abstract】 Gamma-decalactone (GDL) is the lactone of4-hydroxydecanoic acid (4HDA), found inthe fruits and the fermentation products naturally. This compound exhibits an oily-peachyaroma and has been widely used in food additives, pharmaceutical and chemical synthesisindustries. People’s demands for the product are increasing, but it’s not found occuring widelyin natural, and it synthetised by chemical method is racemic molecule. GDL synthetised bybiological method has the characteristics of natural equivalent, and the optical activity GDLproduct has not supplied in the current international market. Therefore, the biological methodhas broad prospects.In this paper, the strain of Yarrowia lipolytica was screened for GDL production. Asystematic and complete research was carried on in order to increase GDL production. Firstly,the genome shuffling method was employed to improve the yield of GDL. And the cultureconditions were optimized and the models for cell growth kinetics and GDL productiondynamics were established. Then an organic-inorganic composite material was prepared andused for immobilizing cells. The composite material could not only improve the mechanicalstrength and permeability of the cells but also prolong their service life. The bioconversion ofionic liquid system was established to increase the material exchange between the inside andoutside of cells. The analysis results of influence factors on the GDL production and ACOisozyme activity indicated that pyridine ring showed a preferable function of promotion andstimulation. Finally the GDL extraction efficiency of the supercritical carbon dioxide methodwas compared with the traditional solvent method. The details are as follows:(1) Under alkaline conditions, GDL reacts with hydrochloric acid hydroxylamine to formhydroxamic acid, which can reaction with ferric iron and further generate a colored complex.The spectrophotometric assay of GDL was established with good accuracy andreproducibility with a detection range from0.125to8.14g L-1. In order to improve thescreening efficiency of GDL producing strains, a high-throughput screening method wasperformed on the96-well microculture plates. Cells were cultured on96-well microplates.And GDL in the fermented broth was determined by using this newly rapidspectrophotometric quantitative determination method. As a result of screening,215GDL-producing strains were selected from4327samples. The highest production of thesestrains was179mg L-1. This strain was identified to be Yarrowia lipolytica according to thecell morphology, culture characteristics and the sequence of26S rRNA.(2) Ultraviolet radiation was performed to establish the positive mutant library. Theoptimal conditions for protoplast formation and fusion were obtained. By using recursivefusion method and after three rounds of fusion, a mutant strain, named G3-3.21, was obtainedand showed the best genetic stability and highest GDL yield with3.75g L-1, which was6.58times higher than that of parental strain. The results of comparative analysis on the mutantstrains indicated that GDL production required the Acyl-CoA oxidase (ACO) isozymes havinghigher ability of degrading the long chain substrates and lower ability of degrading the middleor short chain substrates. This was in favor of GDL accumulating and maintaining the highest yield in a certain period and avoiding the consequent β-oxidization before its formationthrough intramolecular cyclization.(3) In order to improve GDL production, the eight variables were optimized by using theTaguchi designed method. The wheat bran, complex nitrogen source (yeast extract and(NH4)2HPO4) and initial pH were determined to be the major effect factors. The centralcomposite design in response surface methodology was used to further optimize these mainfactors. As a result, the optimal medium composition was determined, and GDL productionwas4.767g L-1. The analysis of wheat bran components showed that the nicotinic acid couldimprove the yield of GDL greatly due to the promoting effect of pyridine ring in the nicotinicacid. By analysis on the characteristics of the cell growth and GDL production, the models forcells growth kinetics and GDL production dynamics were established. The growth kineticsmodel for Z703and G3-3.21strains weredX0.309(XdXdt=115.2)Xandd=0.438(1X,t16.7)Xrespectively. The GDL production dynamics model for the Z703and G3-3.21strain weredPdt=0.00105Xand dPdt=0.00784X, respectively.(4) In order to reduce the toxicity of substrate and product for cells, Organic alginatesodium compouned by inorganic attapulgite clay was used for cell immobilization. GDLproduction of immobilized cells was up to6.15g L-1,1.32times higher than that of free cells.In addition, the cells could be used for3times. In addition, ionic liquid with better biologicalcompatibility was introduced in this study to increase the material exchange between theinside and outside of the cells. In this ionic liquid biotransformation system, GDL productionwas7.85g·L-1. Compared with the same anion, when the cation was pyridine, GDLproduction and ACO activity were generally higher than those of imidazole ion. when theanions were bromine ion and nitrate ion, GDL production and the ACO activity were lowerthan those of tetra-fluoride phosphate ion and hexa-fluoride phosphate ion.(5) The wall-breaking conditions of the free cells were studied, total GDL produciton(both inside and outside the cells) was5.11g L-1, which was increased by14.06%comparedwith the produciton outside the cells. Acidification and heating experiments showed that theyeast adopted in this study had lactonization ability itself and could catalyze4-hydroxyl-decanoic lactone into GDL without heating and acidification. The extractiontechnology of GDL of immobilized cell in ionic liquid biotransformation system wasresearched and extraction effects of the two methods were compared. The extraction rate was86.2%by the butyl acetate extraction method, while the extraction rate was91.7%bysupercritical CO2extraction method.更多还原