【作者】 杜娟；

【导师】 赵建；

【作者基本信息】 山东大学 ， 微生物学， 2010， 硕士

【摘要】 阿魏酸是在植物界中存在最广泛的羟基肉桂酸成分之一,因其具有去除活氧自由基、吸收紫外线、抑制色素生成及抗炎等作用,被广泛的应用于医药、化妆品及化工等行业。另外,阿魏酸与半纤维素、木素通过酯键交联,加强了三维的多糖网状结构,并且限制了纤维素的利用和微生物对于植物纤维类材料的降解。阿魏酸酯酶是半纤维素降解酶系的组成部分之一,它可以打断半纤维素之间的、半纤维素与木素之间交联的酯键,一方面促进半纤维素、木素与纤维素的分离,有利于纤维素的进一步利用；另一方面促进阿魏酸等酚酸类物质的分离。阿魏酸酯酶在生物乙醇生产、制浆造纸、动物饲料生产和生物合成中具有广泛的应用价值。本论文中,采用阿魏酸乙酯为唯一碳源的筛选培养基,根据产阿魏酸酯酶的菌株可在该平板上产生透明圈的特点,从土壤样品和实验室保藏的菌株中筛选到几株产阿魏酸酯酶菌株。进一步通过不断传代培养和液态发酵方式对菌种进行复筛,最终筛选得到一株产阿魏酸酯酶能力强的菌株4-3-a。通过扩增其18S rDNA片段和内转录间隔区序列(ITS)的方法对菌株进行鉴定,并结合菌株形态和菌体形态观察将该菌株鉴定为黄曲霉(Aspergillus flavus)。该菌株是有报道的第二株产阿魏酸酯酶的黄曲霉,其产酶能力与相关文献报道的菌株相比处于中上游水平。对用来测定阿魏酸酯酶酶活的底物去淀粉麦麸(DSWB)的制备方法进行了改进,通过研究证明,采用淀粉酶处理方法代替文献中使用的化学处理方法,可以使得麦麸中残留的淀粉含量达到测定酶活时对底物要求的水平。利用单因子实验、正交实验等统计学方法对于液态发酵培养基成分进行优化,得到较好的产酶培养基组成为：碳源6%,酵母浸粉0.4%,KH2PO40.05%,NaNO30.9%,MgSO40.05%。同时实验结果表明,碳源种类对阿魏酸酯酶的生产影响较大,其中以麦草粉、玉米秸秆粉和玉米芯粉为碳源时产阿魏酸酯酶情况较好。进一步利用混料设计方法对碳源的组成进行了优化,获得了最优的碳源配比,即当碳源总量为6%时,玉米杆和玉米芯的配比为36:64时可以获得最高的产酶效果。通过优化该菌株液体发酵生产阿魏酸酯酶的工艺参数,获得了最佳发酵工艺条件为：初始pH值为7.0,转速为210 rpm,培养温度为32℃,接种量为6%,装液量为100 mL(500 mL三角瓶)。经过对菌株4-3-a的液态发酵培养基成分及培养条件进行优化,使其产阿魏酸酯酶的水平提高2.5倍。利用混料设计实验方法优化混合碳源的组成,在国内尚属首次。对菌株4-3-a所产的阿魏酸酯酶进行酶学性质分析,当以去淀粉麦麸(DSWB)作为反应底物时,该酶的最适反应pH为6.0,最适反应温度为65℃；该酶在pH 5.0～7.0的微酸性环境中和50℃以下时有良好的稳定性；同时,该酶对溶解合成底物用的40%及以下的乙醇具有良好的耐受性。卡伯值测定和红外光谱分析表明麦草经过菌株液态发酵处理后,木素含量有所减少,酯键和木素的特征吸收都有不同程度的降低,证实了阿魏酸酯酶的作用；进一步分析发现,菌液处理后麦秸中阿拉伯糖含量降低显著,说明阿魏酸酯酶可以促进阿拉伯木聚糖的溶出。采用热水处理然后磨浆的方式,可以在大量保留酯键的基础上使麦秸结构变得疏松,有利于后续的酶解进行。纤维素酶解时添加阿魏酸酯酶,可以改善底物的酶解糖化效果,也可以在保持较高纤维素转化率的前提下,通过添加阿魏酸酯酶减少纤维素酶用量。更多还原

【Abstract】 Ferulic acid is the most abundant, ubiquitous hydroxycinnamic acid distributed widely throughout the plant kingdom. It has a wide variety of applications in pharmaceutical, cosmetic and chemical industry because it has effects of erasing radical and active oxygen, absorbing UV, inhibiting pigment formation and inflammatory. Furthermore, ferulic acid is crosslinking with hemicellulose and lignin by ester, which strengthened the three-dimensional polysaccharide network and inhibiting the utilizaion of cellulose and bio-degradation of plant fiber material by microorganisms.Feruloyl esterase (FAE) is one type of hemicellulose degrading enzymes. FAE hydrolyzed the ester bond between the arabinose substitutions and ferulic acid, which is involved in crosslinking xylan to lignin, and destroyed three-dimensional network structures of plant cell walls. Feruloyl esterase has been widely used in the bio-ethanol production, pulping industry, animal feed production and the biological synthesis process.In the paper, Fungus 4-3-a was screened by selective screening using ethyl ferulate as sole carbon source in medium in the plates, and continuously culturing in liquid fermentation to produce FAE. This fungus has a good ability to produce Feruloyl esterase. The strain was identified to be Aspergillus flavus by analyzing 18S rDNA, ITS fragment and morphological properties. The strain was the second reported Aspergillus flavus strain with the ability to produce feruloyl esterase.Destarched wheat bran (DSWB) was used as substrate to determine FAE enzyme activity. The process of DSWB production was researched and DSWB with lower residual starch content was obtained by amylase treatment substitute for chemical treatment reported in many literatures. The DSWB was suitable for determining enzyme activity of FAE.Compositions of culture mediums in liquid fermentation of strain 4-3-a were optimized by using single factor experiments, orthogonal experiments and statistical methods, and the suitable culture medium was:carbon source of 6%, yeast extract of 0.4%, KH2PO4 0.05%, NaNO3 0.9%, MgSO4 0.05%. It was shown that carbon source has a significant influence on production of FAE. The mixture design method was used to optimize the composition and ratio of carbon sources to obtain high activity enzyme. It was found that enzyme with the highest activity of FAE can be produced when dosage of total carbon source was 6%, and ratio of corn stover to corn cob was 36:64. The culture conditions for liquid fermentation of strain 4-3-a were also optimized in the paper and shown as follows:initial pH 7.0, speed 210 rpm, culture temperature 32℃, inculation of 6%, and culture volume of 100 mL in 500 mL flask. Compared to original medium and culture conditions, the activity of FAE was increased to 2.5 folds when strain 4-3-a was cultured in the optimized cultures and conditions. The experimental method of mixture design was firstly used to optimize carbon source composition of the culture mediums.The properties of feruloyl esterase produced by strain 4-3-a were analyzed and shown that the optimum pH is 6.0 and the optimum temperature of 65℃. It is stable in the range of pH 5.0 to 7.0 and temperature below 50℃. It is also an enzyme with ethanol-tolerance when ethanol consistency was below 40% in enzyme solution to dissolve synthetic substrate for determining enzymatic activity.The lignin content, single sugars contents and morphology of wheat straw were analyzed by Kappa number test, HPLC and infrared spectroscopy after wheat straw was treated with the strain 4-3-a by liquid culture method. It was shown that the lignin and sugar contents were decreased after bio-treatment, especially for the arabinose content; and absorption of ester bond was decreased, which means the FAE produced by the strain 4-3-a breaks ester bond and promotes degradation of arabinoxylan. The saccharification of cellulose in wheat straw pretreated with hot water was improved by adding FAE to cellulase solution and conversion ratio of cellulose was increased. Also, the dosage of cellulase can be decreased by adding FAE to hydrolyzing system when conversion ratio was kept at a similar degree.更多还原