【作者】 马晓静；

【导师】 宋欣；

【作者基本信息】 山东大学 ， 微生物学， 2012， 博士

【摘要】 表面活性剂是一类最重要的精细化工产品,广泛应用于家庭、工业和农业。几乎每一种产品和人们生活的各个方面都离不开表面活性剂。随着人们对环境问题认识的加深,从源头上减少污染和治理污染的同时不产生二次污染已成为人们的共识,在这方面生物表面活性剂是未来表面活性剂的发展趋势。由于槐糖脂具有高产量、生物可降解性,低毒性及良好的环境兼容性等优点,可以应用于石油、环境保护、化妆品、洗涤剂、药学领域及纳米科技等工业领域,越来越引起人们的关注。因此槐糖脂被认为是一种最有发展和应用潜力的、可能替代化学表面活性剂的可再生的生物表面活性剂,有着巨大的社会效应、经济效益和生态效益。本文的主要研究内容及结果如下：1.槐糖脂合成的氮源代谢调控研究国内外槐糖脂合成的代谢调控的相关研究主要集中在碳源对产槐糖脂菌株——Candida bombicola的影响,氮源对槐糖脂合成代谢的影响的相关研究则相对较少。本实验室前期筛选得到一株槐糖脂高产菌株——拟威克酵母(Wickerhamiella domercqiae var. sophorolipid CGMCC1576),本文则研究了氮源对W. domercqiae合成槐糖脂的影响。选取了十种氮源用于槐糖脂的发酵生产,结果发现有机氮源促进菌体生长,促进内酯型槐糖脂的合成：无机氮源抑制内酯型槐糖脂的合成,显著降低内酯型槐糖脂占总槐糖脂的比例。培养基中无机氮源NH4+的浓度越高,发酵液pH值越低,内酯型槐糖脂的产量越低,内酯型槐糖脂在总槐糖脂中所占的比例越低。发酵液的pH值和pH调节剂的种类均影响W. domercqiae生长及合成槐糖脂。pH调节剂C6H5O7Na3或NaOH可以提高发酵液的pH值以及增加菌体生物量和内酯型槐糖脂的产量;pH调节剂CaCO3可以提高发酵液的pH值却使内酯型槐糖脂的产量显著降低。以上结果表明,无机氮源通过改变发酵液的pH值,但不仅仅通过改变pH值影响槐糖脂的合成。利用TLC、IR及HPLC等色谱技术,比较了最具有代表性的有机氮源——酵母粉和无机氮源——硫酸铵两种条件下所得到的槐糖脂粗品的结构和组成的差异,并使用制备型HPLC、MS及GC-MS对硫酸铵条件下所得到的槐糖脂粗品中的主要组分进行了纯化、制备和结构鉴定。2.利用新一代Solexa测序技术分析研究W. domercqiae在不同氮源条件下的基因表达差异目前没有槐糖脂产生菌基因表达调控的相关研究,因此在全基因组水平研究氮源对W. domercqiae在转录水平的影响,有助于筛选差异表达基因,深入了解槐糖脂合成通路相关基因的表达调控机理。首次利用基于新一代Solexa的数字化基因表达谱测序技术构建了W.domercqiae在酵母粉、硫酸铵两种氮源培养条件下的两个表达谱数据库。酵母粉条件下总共得到了6,011,524条测序标签,其中包括64,329种不同的标签；硫酸铵条件下得到了72,375种5,886,784条测序标签。两个表达谱数据库的比较结果显示,表达倍数变化大于1的表达标签共有4,291种,其中,在酵母粉条件下上调(Ratio (YE/NS)>1)的表达标签有2,020种,下调(Ratio (YE/NS)<1)的表达标签2,271种。以FDR<0.001且倍数差异在2倍以上作为显著差异表达的标准,共得到显著差异基因709种,其中硫酸铵条件下显著上调的表达标签392种,显著下调的表达标签317种。将表达标签与W. domercqiae的全基因组数据进行比对,结果显示,96%以上的表达标签能够比对到来源于W. domercqiae的参考基因上。数据库YE和NS中分别有70.73%和67.73%的表达标签比对到参考基因上。分析比较比对后的表达谱数据库中的显著差异基因发现,在酵母粉条件下,参与槐糖脂合成途径的11个相关基因(如葡萄糖基转移酶、乙酰基转移酶及酯酶或脂肪酶等)显著上调；而在硫酸铵条件下,仅有2个参与槐糖脂合成途径的酯酶或脂肪酶基因显著上调。由于没有发现催化内酯化反应的内酯化酶基因的存在,而酯酶或脂肪酶具有催化酯化反应的能力,因此我们在全基因组范围内对编码酯酶或脂肪酶的基因进行了筛选,共得到了27种有差异的编码酯酶或脂肪酶的基因,为后续利用基因工程手段构建基因工程菌提供了有价值的参考。3.不同结构槐糖脂的表面活性和生物活性比较研究槐糖脂是酵母菌分泌的,由多种槐糖脂分子组成的混合物。不同种类和结构的槐糖脂分子具有不同的理化活性和生物活性,可以应用于不同领域。使用制备型HPLC制备得到了W. domercqiae利用油酸为第二碳源发酵所得的槐糖脂粗品中的6种主要组分,并用MS和GC-MS分析了这6种槐糖脂纯品的结构。结果发现他们的结构的区别在于槐糖部分乙酰化程度的不同(一个乙酰基或两个乙酰基),脂肪酸部分碳链长度(16碳或18碳)和不饱和度(两个双键或一个双键)的不同以及是否内酯化(酸型或内酯型)。研究了不同结构的槐糖脂纯品的理化性质和生物活性,发现槐糖脂分子的乙酰化程度、脂肪酸部分的碳链长度和不饱和度以及是否内酯化都会影响其表面活性、乳化能力、细胞毒性和生物可降解性。4.脱木素木糖渣制备槐糖脂合成底物的研究生产成本较高是槐糖脂目前还处于实验室研究阶段,尚未进行大规模的工业化生产和走向市场的主要原因。为了降低槐糖脂的生产成本,我们开展了以纤维素原料——脱木素木糖渣(DCCR)为底物进行槐糖脂生产所需亲水性碳源(脱木素木糖渣糖化液中所含的葡萄糖)、疏水性碳源(单细胞油)及氮源(单细胞蛋白)的制备的相关研究。考察了相同滤纸酶活添加量的3种商品纤维素酶(KDN酶液、JNK酶液和A10酶粉)在3种糖化缓冲体系(pH48的H2O、0.05M pH4.8HAc-NaAc缓冲液及0.2M pH4.8HAc-NaAc缓冲液)中酶解DCCR的效果,综合考虑后,确定KDN酶液为DCCR糖化的最优纤维索酶液。考察了产油酵母——Cryptococcus curvatus ATCC96219在不同脱木素木糖渣水解液(DCCRH)及脱毒后DCCRH中的生长和产油情况。发现C. curvatus在未经脱毒处理的pH48的H20和0.05M的HAc-NaAc DCCRH中可以正常生长,菌体干重分别达到了1786g/1和15.32g/l,油脂含量分别为27.44%和4021%。而在未经脱毒处理的0.2M HAc-NaAc DCCRH中不能生长。经脱毒处理后,C. curvatus在三种糖化液中均可以正常生长,菌体干重分别达到了10.57g/l,20.49g/l和1736g/l油脂含量也提高至22.13%,4674%和44.35%。用GC-MS对所得油脂的脂肪酸组成进行了比较分析,发现其脂肪酸组成差别不大,均与植物油类似,以棕榈酸、油酸和硬脂酸为主。通过正交实验设计和进一步优化确定了C. curvatus的最优发酵培养基配方,研究并比较了在最优配方下,C. curvatus在经不同脱毒处理后的DCCRH中的生长和产油情况,确定了含有5.0g/l酵母粉和20.0m1/l无机盐溶液的活性炭吸附后的0.05M HAC-NaAc DCCRH为最优的C. curvatus细胞生长和积累油脂的培养基,在此条件下,单细胞油产量和油脂系数分别达到了10.94g/l和55.41%。高压破碎发酵完成后所得C. curvatus菌体细胞,细胞破碎液中所含细胞碎片和油脂可以用作槐糖脂合成所需氮源和部分疏水性碳源。5.脱木素木糖渣生产槐糖脂的研究考察了不同糖化缓冲体系和脱毒处理方法对W. domercqiae生长和产槐糖脂的影响。发现当用KDN纤维素酶液在0.2M HAC-NaAc缓冲液中酶解DCCR所得水解液进行槐糖脂的发酵时,槐糖脂的产量最高；活性炭吸附(C)处理水解液可以进一步提高槐糖脂的产量,因此在后续实验中,选择使用上述DCCRH进行槐糖脂的合成的相关研究。以经活性炭吸附处理后的水解液为底物,优化了W. domercqiae利用DCCRH和油酸产槐糖脂的培养基配方,确定了糖化液培养基中最优酵母粉浓度和葡萄糖总浓度分别为1.5g/l和60.0g/l。在1.5L发酵罐中考察了补加葡萄糖、控制pH和补加菜籽油对W. domercqiae利用DCCRH和菜籽油产槐糖脂的影响,并对不同条件下所得槐糖脂的组成进行了HPLC分析。考察了W. domercqiae利用DCCR水解液和单细胞油以及利用DCCR水解液和细胞破碎液合成槐糖脂的能力。结果显示,W. domercqiae可以利用DCCRH和单细胞油合成槐糖脂,糖化液的脱毒处理可以提高内酯型、总槐糖脂的产量和内酯型槐糖脂占总槐糖脂的比例。在未处理和脱毒处理后的DCCRH培养基中,总槐糖脂的产量分别达到了3908g/l和42.06g/l。当使用C. curvatus细胞破碎液替代槐糖脂合成所需氮源和部分疏水性碳源时,在未处理和脱毒处理后的DCCRH培养基中,总槐糖脂的产量分别达到了37.19g/l和48.97g/l。因此我们认为DCCR是一种有潜力的、可用于槐糖脂生产的廉价底物,该底物的使用可以显著降低槐糖脂生产成本,具有重要的实际意义和较大的应用前景。更多还原

【Abstract】 Surfactants or surface active agents are widely applied in a variety of industrial sectors, such as detergent, cosmetic, petroleum, environment, and agriculture, etc. They are called "industrial monosodium glutamate", therefore, they are one of the most important classes of industrial bulk chemicals. With the growing environmental awareness, reducing pollution in origin and avioding generating secondary pollution have become consensus of the people. Biosurfactant has attracted increasing attention. Sophorolipids have attracted much more attention for their application potential in petroleum, environmental industries, cosmetics, detergents, pharmaceuticals and nanotechnology due to their high production yields, biodegradability, low ecotoxicity, biocompatibility, and production based on renewable resources. They are probably one of the most promising biosurfactants which can bring great society, economic and ecological benefits.The main research contents and results are as follows:1. Regulation of nitrogen metabolism of sophorolipids synthesisMetabolic regulation studies of sophorolipids (SLs) synthesis almost focused on effects of carbon sources on the sophorolipid-producing yeast, Candida bombicola ATCC22214. For Wickerhamiella domercqiae var. sophorolipid CGMCC1576, yeast which was screened by our laboratory, few reports on the effects of nitrogen sources on sophorolipid production and composition were available.The effects of different nitrogen sources on growth of W. domercqiae and on production and composition of SLs were studied. Organic nitrogen sources were more favorable for accumulation of biomass than inorganic ones. Presence of ammonium ion from different inorganic nitrogen sources (except NH4HCO3) greatly inhibited the production of lactonic SLs. However, when organic nitrogen sources were used, lactonic SL production was strongly increased. Increase of ammonium content in total N source led to more lactonic SL production rather than more acidic SL and the decrease of yeast biomass and pH value of the broth. Production of biomass and crystalline lactonic SLs from organic/inorganic nitrogen sources was enhanced with the increase of pH value when adjusted by sodium hydroxide or sodium citrate solution. Addition of pH controller of calcium carbonate at the beginning of the cultivation resulted in a slightly increased biomass, but in an obvious decreasing production of crystalline lactonic SL. However, adjustment of pH could not improve the production of total SLs. These results demonstrated that inorganic nitrogen source influenced SL synthesis by changing the pH value of the fermentation broth, but pH value is not the only reason.Fourier-transform infrared (FT-IR), gas chromatography mass spectrometry (GC-MS) and high performance liquid chromatography (HPLC) were employed to compare the composition of SL mixture obtained from yeast extract and ammonium sulfate as nitrogen sources. More than15acidic SL molecules and only4lactonic SL molecules were produced by using ammonium sulphate as nitrogen source, these SL molecules were separated by preparative HPLC and their structures were elucidated by MS and GC-MS. These results suggest extraordinary regulatory effects of nitrogen source on growth and SL synthesis of W. domercqiae.2. Analysis of transcription profiling of gene expression of W. domercqiae using digital gene expression tag profiling (DGE) technologyThe differences on gene expression level of W. domercqiae with two different nitrogen sources, yeast extract and ammonium sulphate were compared through gene expression profiling analyzed by Digital Gene Expression tag profiling (DGE) technology. Two libraries were built from Wickerhamiella domercqiae var. sophorolipid CGMCC1576cultured with yeast extract and ammonium sulphate as nitrogen source, which were named YE library and NS library, respectively. The YE library had6,011,524total tags including64,329distinct tags, and the NS had5,886,784sequenced total tags with72,375distinct tags.4,291differentially expressed distinct tags were detected between the two libraries, including2,020up-and2,271down-regulated distinct tags in the comparison of YE library with NS library. When FDR≤0.001and the absolute value of log2Ratio≥1was used as the threshold to judge the significance of gene expression difference,392tags were up-regulated and317tags were down-regulated significantly under ammonium sulphate condition.The sequencing tags were mapped to the W. domercqiae genome and the result showed that about96%tags could be mapped to the W. domercqiae genome. For tags of library YE and NS,70.73%and67.73%of tags could be mapped to the reference genes from W. domercqiae. After analyzing the distinctly-expressed genes involved in SL synthesis of the two libraries, many glycosyltransferase, acetyltransferase and esterase/lipase genes were up-regulated significantly when yeast extract was used as nitrogen source. These results indicated that co-regulation of these genes involved in SL synthesis. However, under ammonium sulphate condition, only2genes encoding esterase/lipase were up-regulated significantly. Nitrogen source influenced lactonic SL production significantly and it was suggested that esterification reaction of the carboxyl group of the hydroxy fatty acid with a hydroxyl group of sophorose was catalyzed by a specific lactone esterase or lipase. However, neither of both esterases has been found in the W. domercqiae genome or in other SL producing species. It was found there that27genes encoding esterase/lipase up-or down-regulated in the two libraries. These results summarized above provide useful information for our further research in constructing gene engineering strain.3. Surface and biological activity of sophorolipid molecules with different structuresSLs produced by several selected yeast strains are mixture of different SL molecules. SL molecules with different structures have been reported to show some differences in their biological and physicochemical properties and can be specifically applied in different fields.Six different SL molecules were prepared from crude SLs produced by W. domercqiae by using glucose and oleic acid as carbon sources. The staictures of the six SL molecules were elucidated by MS and GC/MS. The six SL molecules differ in the acetylation degree of sophorose, carbon chain length, unsaturation degree of fatty acid and lactonic/acidic form. The surface properties of SLs including critical micelle concentration (CMC), minimum surface tension (Min. S.T.), emulsification capacity to hydrocarbon and vegetable oils. Cytotoxicities and biodegradability were also studied and the results demonstrated that SL molecules with different structures exhibited quite different surface properties and biological activities.4. Substrates preparation from delignined corncob residue for sophorolipid synthesisLarge-scale application of sophorolipids was blocked by high production cost. One way to reduce the cost of SL production is to develop other cheap fermentative feedstock. In the dissertation, a lignocellulosic material, delignined corncob residue (DCCR) was used for providing the hydrophilic carbon source (glucose in DCCR hydrolysate), hydrophobic carbon source (single cell oil produced from an oleaginous yeast of Cryptococcus curvatus) and nitrogen source (single cell protein) for SL production.Effects of DCCR saccharification by different commercial cellulase in pH4.8H2O and different buffers including0.2M and0.05M acetate buffer of pH4.8were studied. Finally, KDN cellulase was selected for hydrolysate production.Cell growth and lipid accumulation of the oleaginous yeast of Cryptococcus curvatus from delignined corncob residue hydrolysate (DCCRH) and detoxified DCCRH were studied. In untreated hydrolysate medium, C. curvatus can growth and accumulate lipids except that0.2M acetate DCCRH medium was used. In0.05M acetate and pH4.8H2O DCCRH medium,15.32g/1and17.86g/l of biomass with40.21%and27.44%of lipid contents were obtained, respectively. Furthermore, pretreatment of DCCRH by active carbon absorption (C) and vacuum steam evaporation (Va) led to significant improvement of cell growth and lipid accumulation with highest values of20.49g/1of biomass and46.74%lipid content when cultured in0.05M acetate DCCRH medium. For0.2M acetate DCCRH medium, after detoxification, the produced biomass reached17.36g/1and lipid content increased to44.35%. The accumulated lipids were mainly constituted by neutral lipids with fatty acids as palmitic acid, stearic acid, oleic acid and linoleic acid.Orthogonal test and further optimization were applied to obtain the optimal fermentation medium for C. curvatus growth and lipid production. Under optimal yeast extract and mineral salt solution addition condition, cell growth and lipid production of C. curvatus on different DCCRH medium were compared. The result showed0.05M acetate DCCRH treated by active carbon adsorption and containing5.0g/1yeast extract and20.0ml/1mineral salt solution was the optimal DCCRH medium for SCO production. Under this condition,10.94g/1of SCO and55.41%of lipid content were obtained, respectively. After cell disruption and autoclaving, the cell homogenate containing cell debris and lipids could be served as nitrogen source and a part of lipidic substrate for SL production by W. domercqiae.5. Sophorolipid production from delignined corncob residue hydrolysateSophorolipid production by W. domercqiae from different DCCRH and detoxified DCCRH were investigated. It was found that0.2M acetate DCCRH hydrolyzed by KDN cellulase and treated by active carbon adsorption was more suitable for SL production. DCCRH detoxified by active carbon adsorption to remove the inhibitor of acetic acid increased SL production and proportion of lactonic SL in total SL. The optimizing results indicated that0.2M acetate DCCRH medium containing1.5g/1of yeast extract and60.0g/1of glucose was the optimal DCCRH medium for SL production.Effects of glucose or rapeseed oil supplement and controlling of broth pH in different1.5L bioreactor were studied. Composition of the obtained crude SLs were analyzed by HPLC. SL production from DCCRH was investigated. The results demonstrated that W. domercqiae could utilize the glucose in DCCRH and detoxified DCCRH as hydrophilic carbon source to grow and accumulate SL with relatively high yield. When W. domercqiae were cultivated on DCCRH/detoxified DCCRH and single cell oil as hydrophilic and hydrophobic carbon sources, total SL production of39.08g/l and42.06g/l was obtained. Furthermore, when cell lysate of C. curvatus was used as nitrogen source, oleic acid and detoxified DCCRH or DCCRH as complex carbon sources, production of total SL reached48.97g/l and37.19g/l, respectively. These results demonstrated that renewable DCCRH can be well utilized as low-cost substrate for the production of SL. Exploition of DCCR as substrates for SL production is a practical way to significantly reduce the cost of SL production.更多还原