【作者】 于大为；

【导师】 冯雁；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 我们将来源于深海古菌Aeropyrum pernix K1的嗜热脂肪酶APE1547基因在E. coli BL21 (DE3)中克隆并表达。该重组嗜热脂肪酶是重要的工业酶制剂品种之一,可以催化解脂、酯交换、酯合成等反应,广泛应用于油脂加工、食品、医药、日化等工业。本文对该工程菌的发酵条件进行了系统的研究。考察了大肠杆菌在摇瓶中表达该酶的基本条件包括温度、pH值、DO值、质粒稳定性、诱导剂和诱导时机等;利用生物统计学方法优化了以玉米浆为发酵基质的大肠杆菌培养基组成,达到了国产蛋白胨,酵母粉培养基的水平。我们使用玉米浆培养基在发酵罐中对该工程菌进行高密度发酵,建立了指数流加葡萄糖实现高密度发酵和高浓度乳糖诱导表达嗜热脂肪酶的发酵生产工艺,并用1吨发酵罐进行了中试实验;根据嗜热脂肪酶耐高温的特点,在产物后处理过程中创造性地提出了加热破碎法,在大肠杆菌破碎的同时,嗜热脂肪酶被纯化,经过冷冻干燥制备出不同纯度的嗜热脂肪酶制剂样品。在本研究中我们应用生物统计学方法确立了以玉米浆为主的发酵基质;确立了一个从小规模到中试规模的发酵生产重组嗜热脂肪酶的方法;建立了适合于嗜热脂肪酶生产的后处理工艺。我们系统地优化了整个发酵过程,为下一步的大规模工业生产搭建了新的平台。更多还原

【Abstract】 As biological catalyst, enzyme has the advantages of high efficient abilityof catalyzing and specificity of substrate. Most of enzymes come frommesophilic bacteria, they have many advantages but these enzymes meet withlimitations at the extreme reactive condition. Thus, we need to develop a newsource of enzymes to adapt to the reaction at the extreme condition. With thesuccessful separation of the heat-resistant DNA Polymerase from Thermusaquaticus, PCR accomplished a historical leap from adding the enzymes eachcircle to adding the enzyme to the whole circles only once, which broke a newchapter for the application of thermophilic bacteria and thermophilic enzymes.Thermophilic enzymes and hyperthermophilic enzymes not only have the sameadvantage of high efficient ability of catalyzing as mesophilic enzymes but alsocan keep high stability at high temperature and strongly resist the organicsolvents and denaturants. So they have important latent applicable values inmany areas. Lipase is the enzyme that catalyzes triglyceride to glycerol and fattyacid. It has already widely applied to the industry of eradicator, grease chemistry,food, organic synthesis, paper making, biological exterior active agent andmedication for its several important characters: substrate specificity, stereoselectivity, position selectivity and the ability to catalyze the reaction oninterface between the aqueous solution and the non-aqueous solution.In the flask experiments, it was confirmed that the recombinanthyperthermophilic lipase got the best production at 37 ℃, pH7.8 , 2%inoculation and round speed 180 rpm. When the OD600 reached 0.8, theinduction was performed. After that the culture continued for another 12 hoursto get the best production. The plasmid stability is usually sustained by theampicillin pressure. But as ampicillin is too expensive for large scale culture ofrecombinant E.coli, we substitute penicillin sodium for ampicillin to sustain theplasmid stability. The optimal amount of the penicillin sodium concentration is6μg/ml.Response surface methodology is an empirical statistical modelingtechnique employed for multiple regression analysis using quantitative dataobtained from properly designed experiments to solve multivariable equationssimultaneously. Recombinant hyperthermophilic lipase was produced from theagricultural waste product corn steep liquor. The effect of corn steep liquor,mineral salt and trace metals on hyperthermophilic eslipase production wasinvestigated by means of a five-level three-factor central composite rotatabledesign. The final composition of the medium is as follows: corn steep liquor(24.3g/L), mineral salt solution (16.5ml/L) and trace elements solution(12.7ml/L). The predicted response was 261.60U/ml and the actual response was251.39U/ml, which proves the validity of the culculation.In this study, a series of bioreactor cultivations with similar growth phaseswas performed to develop the subsequent induction phase with the aim ofachieving high levels of hyperthermophilic lipase expression using lactose as theinducer molecule. 10L corn steep liquor medium with the 10g/L glucose wasused for the batch fermentation. The transition from the batch to the fed-batchphase is indicated experimentally by the rapid raise in DO concentration, whichcorresponds to the depletion of the glucose added at the beginning of the batchphase. During the fed-batch phase, the 500g/L glucose was added and thespecific growth rate was kept at 0.15h-1. When the OD600 gets 100, the inductionwas performed with 1.4g per gram of dry cell weight of lactose. Thehyperthermophilic lipase activity got 1100U/ml. After that, we carry out thefermentation of the mutation bacteria E. coli APE1547R526V using theoptimization of culture conditions and induction strategy on 1 ton fermentor.The hyperthermophilic lipase activity got over 6300U/ml.The membrane structure of E. coli became permeable by heat treatment,which results in the hyperthermophilic lipase release. At the same time ofdisruption, the hyperthermophilic lipase was purified because most of theintracellular motley proteins would be denatured and precipitated by heattreatment. Furthermore, the effect of thermolysis and chemical method wascompared. A series of hyperthermophilic lipase samples with different puritieswere obtained.In this study, we found a suitable way to produce the recombinanthyperthermophilic lipase from small scale to pilot scale and the fermentationprocess was optimized. It provides a base to the industrial scale production ofhyperthermophilic lipase.更多还原