【作者】 袁文杰；

【导师】 白凤武；

【作者基本信息】 大连理工大学 ， 生物化工， 2009， 博士

【摘要】 本文在驯化选育一株马克斯克鲁维酵母K.marxianus YX01的基础上,进行了同步糖化发酵菊粉和菊芋粗原料生产乙醇的研究工作。通过正交实验法对该菌株产菊粉酶培养基组成及培养条件进行了研究,确定培养基组成为（g/L）:菊粉40,酵母粉4,蛋白胨4,尿素1;在初始pH值5.0,温度30℃,转速150 rpm/min条件下培养菊粉酶活性达到57 U/mL。以菊粉为底物,该酶的最适水解温度为55℃,在60℃以下稳定性好;最适pH值为5.0,在pH4.6-5.2范围内酶稳定性好;该酶属于外切菊粉酶,8%的乙醇对其酶活力基本没有影响。K.marxianus YX01的种子液的培养时间为24 h,培养基初始pH最适为4.6,最适发酵温度为35℃;在通气量为50 ml/min和100 ml/min时菌体生长速度加快,发酵时间缩短,但不通气条件下糖醇转化率明显提高;在菊粉浓度235 g/L时,发酵终点乙醇浓度达到92.2 g/L,乙醇对糖的得率为0.436,为理论值的85.5%。不同浓度海水灌溉的菊芋都是乙醇发酵的良好底物,尤其以25%海水灌溉的为最好。粉碎粒度为40-80目范围内,对发酵没有明显影响。浓度分别为200、225和250g/L的25%海水灌溉的菊芋干粉乙醇发酵结果表明,底物浓度越高,发酵液黏度越大,混和程度越差。在底物浓度为200 g/L时,乙醇得率为0.439。进行批式补料发酵,将菊芋干粉浓度提高到280 g/L,发酵终点乙醇浓度达到83.75 g/L,为理论转化率的84.56%。采用有机酸色谱柱Aminex HPX-87H,以0.01 mol/L硫酸水溶液为流动相,流速为0.5ml/min,利用紫外和示差折光检测器为双通道检测手段,同时对K.marxianus YX01菊芋发酵液中的葡萄糖、丙酮酸、果糖、琥珀酸、乳酸、甘油、乙酸、乙醇进行了定量分析,发现主要的副产物为乳酸、甘油、乙酸,构建了乙醇的代谢网络。利用兼并引物,通过降落PCR法克隆了K.marxianus YX01的乳酸脱氢酶基因（LDH）片段,通过插入G418抗性基因构建基因破坏载体,利用同源重组对LDH基因进行了基因破坏。在生长条件下,破坏子T₄乳酸脱氢酶活力在120 h时比出发菌株降低了25%;在发酵条件下发酵终点时乳酸脱氢酶降低46%;乙醇、甘油、乙酸和乳酸的终浓度都出现不同程度的降低,但琥珀酸的浓度出现一定程度的提高。与出发菌株相比,破坏子的生长受到一定抑制,菊粉酶活力下降20-30%。破坏子T₄单位质量细胞的乙醇产量反而提高,由对照菌株的20.35 g/L/g（dcw）提高到T₄的22.64 g/L/g（dcw）。构建了菊粉酶基因的单拷贝整合表达载体,并转化K.marxianus YX01,获得整合子K/INU1。K/INU1的菊粉酶活力比出发菌株提高近一倍,发酵时间从对照的84h缩短到72h。以粗菊芋粉为底物进行的补料批式发酵发现整合子乙醇生成速度快于出发菌株。更多还原

【Abstract】 Kluyveromyces marxianus is characterized by its thermal tolerant feature,and it is also fast growing and able to utilize broad range of substrates.Most importantly,it has the ability to produce inulinase,which enables direct ferementation of ethanol using inulin,the main carbohydrate in Jerusalem artichoke.In this study,a yeast strain of K.marxianus YX01 was selected for ethanol production using Jerusasalem artichoke.The extracellular inulinase production by K.marxianus YX01 was optimized and 57 U/mL inulinase was produced in shake flask culture,with the medium composition:inulin 40,yeast extract 4,peptone 4 and urea 1 g/L,under the culture conditions:pH 5.0,30℃and 150 rpm for 120 h.The optimal temperature and pH for the inulinase to hydrolysize inulin were 55℃and 5.0,but the enzyme was validated to be stable at the temperature as high as 60℃and the range of pH from 4.6 to 5.2.The activity ratio of the inulinase toward inulin and sucrose was 1.7 and 8%ethanol showed no significant effect on its activity.The growth and ethanol fermentation of K.marxianus YX01 were studied using inulin as substrate.The activity of inulinase,which attributes to the hydrolysis of inulin,was monitored in both shake flask culture and bioreactor.The optimum seed culture time is 24 h,and the pH of medium is 4.6.The optimum temperatures were 38℃for growth and inulinase production, and 35℃for ethanol fermentation.Aeration was not necessary for ethanol fermentation with the K.marxianus YX01 from inulin.Furthermore,the impact of aeration and substrate concentration was studied through batch fermentation in the 2.5 L bioreactor,and the results indicated that the average ethanol fermentation time was decreased at the aeration rates of 50 mL/min and 100 mL/min,but higher ethanol yield was achieved under non-aeration conditions with more substrate directed to ethanol production.The ethanol concentration of 92.2 g/L was obtained with the substrate containing 235 g/L inulin,and the ethanol yield was calculated to be 0.436,equivalent to 85.5%of its theoretical value.The fresh Jerusalem artichoke tubers grown in salina and irrigated with 25%and 50% seawater mixed with fresh water were further examined for ethanol fermentation with the K. marxianus YX01,and a higher ethanol yield was achieved for the Jerusalem artichoke tuber irrigated with 25%seawater.Furthermore,the dry meal of the Jerusalem artichoke tubers irrigated with 25%seawater was examined for ethanol fermentation at three solid concentrations of 200,225 and 250 g/L,and the highest ethanol yield of 0.439,or 86.1%of the theoretical value of 0.511,was achieved for the slurry with a solid concentration of 200 g/L.Finally,Jerusalem artichoke grown in salina and irrigated with seawater was fermented without sterilization treatment to further improve the economic competitiveness of the fermentation process,and 83.75 g/L ethanol was obtained with the substrate containing 280 g/L dry Jerusalem artichoke meal,with an ethanol yield of 0.432,indicating the Jerusalem artichoke could be an alternative feedstock for grain-based fuel ethanol production. An efficient high performance liquid chromatographic（HPLC） method for simultaneous determination of ethanol,sugar and organic acids in the K.marxianus YX01 culture broth was developed.Aminex HPX-87H organic acids analysis column was used and the mobile phage was 0.01 mol/L sulfic acid solution with the flow rate of 0.5 ml/min.The components of the culture broth containing glucose,fructose,ethanol,acetic acid,glycerol,lactic acid were detected with UV and RID dual detectors.The main by-products of the ethanol fermentation were determined to be lactic acid,glycerol and acetic acid.This method ensured rapid and accurate quantification of the metabolites of the K.marxianus YX01 in the ethanol fermentation from Jerusalem artichoke,and provided basis for the future metabolic flux analysis as well.Fragments of the lactic dehydrogenase gene（LDH） gene sequence from K.marxianus YX01 were cloned using degenerate primers and touch-down PCR.Kam gene conferring G418 resistance was inserted into the clone vector,and the LDH gene of the K.marxianus YX01was disrupted by homologous recombination.Cell growth and ethanol fermentation of the disruptant T₄ were investigated,and the results showed that the LDH activity of T₄ was 75%of that of the control under growth conditions and 54%of that of control in the fermentation process,and the concentrations of ethanol,glycerol,acetic acid and lactic acid in T4 were decreased,but the production of succinic acid production increased.The growth of T₄ was deficient comparing with the control,and the inulinase activity of T₄ was decreased 20-30%.However,due to its low biomass yield,the final specific ethanol productivity of T₄ was 11.25%higher that of the control strain（22.64 g/L/g（dcw） comparing to 20.35 g/L/g （dcw））.Improvement of the inulinase activity and stable expression of inulinase gene is beneficial to improve the ethanol productivity.To this end,inulinase gene INU with its native promoter was amplified from K.marxianus YX01 by PCR,and integrative vector with the HO flanking sequences was constructed.The integration vector was linerized by Not I and then transformed into K.marxianus YX01.The inulinase activity and ethanol fermentation performance of the transformant K/INU1 were investigated.The inulinase activity in K/INU1 doubled that of the control,and the fermentation time was shortened from 84 h in the control strain to 72 h in K/INU1,with similar final ethanol titer in the two strains.Using raw Jerusalem artichoke flour,fed batch culture was performed,and it was revealed that the fermentation rate of K/INU1 is faster than that of the wild strain,with similar final ethanol titer.Multicopy integration of INU will be an efficient way to further improve the ethanol fermentation performance of K.marxianus YX01.更多还原