【作者】 郭芬芬；

【导师】 赵建；

【作者基本信息】 山东大学 ， 微生物学， 2014， 博士

【摘要】 麻纤维是我国一类重要的传统纤维原料,种类繁多,资源丰富。其中苎麻具有“中国草”之称,由其皮部韧皮纤维加工成的纺织品具有洁白、挺括、冰爽、舒适、抑菌、保健、卫生等优点,深受国内外消费者喜爱。但苎麻韧皮纤维在使用前,必须首先将其中的胶质脱除,使单根纤维之间分离,同时赋予纤维一定的成纺性能。其中酶法脱胶由于具有脱胶专一性强、作用条件温和、所得麻纤维品质好,柔软、蓬松卷曲、手感好、强度高、光泽度好等诸多优点,以及环境污染轻的优势,成为麻纤维脱胶的发展方向。但目前在高效脱胶菌株、主要脱胶酶组分和脱胶机理等方面的研究还有待于深入,以促进酶法脱胶的发展。除了韧皮纤维外,占生物学产量60%左右的麻秆部分,目前除少部分用于压制纤维板或制浆造纸外,大部分未被有效利用,造成资源的极大浪费,因此急需强化该方面的研究,以达到麻类原料全组分利用的目的。由于麻秆中富含纤维素,因此可望用于转化生产燃料乙醇,但目前尚未有利用苎麻秆生产生物乙醇的报道。在生物转化生产燃料乙醇过程中,由于原料中的木质素在纤维素酶解过程中,会与酶产生非反应性的结合,导致对酶的无效吸附,从而影响纤维素的酶解效率。了解木质素与酶的吸附规律和机制对提高纤维素的酶解具有重要意义。由于不同原料来源的木质素的组成和性质等的不同,将会对吸附造成不同的影响,而该方面研究还未见报道。基于以上背景,本论文对苎麻等原料皮部韧皮纤维的高效脱胶酶系、酶法脱胶机制、麻秆部纤维预处理和糖化发酵产燃料乙醇的可行性、木质素对纤维素酶的吸附及机制等进行了研究。本论文的主要研究结果如下：1.苎麻高效脱胶菌株的筛选、酶学性质和果胶酶的生产通过比较不同菌株粗酶液的脱胶能力,发现Bacillus sp. Y1的粗酶液具有快速和高效的脱胶能力,脱胶过程在1h内基本完成,并且几乎不含有纤维素酶。酶学性质研究表明,Bacillus sp. Y1粗酶液中的PGL的最适pH为9.6-10.0,最适温度为60℃,在pH7-10.5和30-50℃具有很好的稳定性,有较宽的pH和温度范围,和很好的过氧化氢耐受性,具有较好的应用于酶法脱胶的潜力。该粗酶液在脱胶过程中与过氧化氢具有协同作用,可明显提高苎麻纤维的脱胶率和纤维白度,为酶法脱胶和过氧化氢漂白工艺相结合提供了理论基础。通过单因子实验、混料设计、部分因子实验和中心组合设计,对Bacillus sp.Y1产酶培养基组分和培养条件进行了优化,以提高其产酶水平。优化后得到最佳产酶条件为：碳源10.5%,(NH4)2SO40.37%(w/v), MgSO40.3%(w/v),Tween-800.1%(w/v),初始pH8.2,每50mL的发酵培养基的接种量为1.3mL(种子培养液的OD600约为5.77),秋千式摇床转速为100rpm,34℃下发酵60h。相比优化前,PGL酶活和PG酶活分别提高了1倍和2.44倍,产酶高峰缩短了12h,由72h提前至60h。2.脱胶机制研究通过Bacillus sp. Y1粗酶液酶系分析和粗酶、纯酶添加实验验证,证明Bacillus sp. Y1酶液中具有的高蛋白酶活力是该酶液有效脱胶的一个主要因素,并发现蛋白酶和果胶酶在脱胶中存在协同作用。为酶法脱胶中的有效脱胶酶组分和脱胶酶系改造提供了新的见解。通过对不同生长期的苎麻的化学成分和脱胶性能进行分析,发现头麻和二麻在化学成分和脱胶效果上几乎没有差别,而三麻脱胶性能最差。对头麻和三麻不同脱胶时间下的样品进行比较研究,发现三麻中果胶和半纤维素的脱除率明显低于头麻,SEM观察也发现三麻表面附着的胶质明显多于头麻,且较难脱除。分析推测可能是半纤维素阻碍了三麻胶质的脱除。3.预处理苎麻秆和红麻秆糖化发酵生产燃料乙醇研究比较了几种不同化学预处理方法对苎麻秆和红麻秆纤维素酶解性能的改善效果以及预处理后秆部纤维糖化发酵产燃料乙醇的性能。研究发现碱法预处理较适用于此类原料。4%NaOH和0.02%蒽醌-2-磺酸钠盐(AQSS),在170℃下处理1h的苎麻秆和红麻秆,木质素脱除率最高,经过分批补料半同步糖化发酵工艺,在补料至底物浓度为20%时,乙醇浓度可达63g/L,转化率分别为77%和79%。红麻秆经5.2%NaHSO3和0.2%H2SO4,在170℃下处理1h,补料至20%的底物浓度时,乙醇浓度可达到65g/L,纤维素转化率为72%。4.不同原料来源木质素的组成和性质及其对纤维素酶吸附的影响和机制研究来源于不同种类原料的木质素对纤维素酶表现出不同的吸附性能,吸附强弱排序为松木木质素>玉米秸木质素>杨木木质素>红麻秆木质素。通过对不同木质素组成和性质的研究,推测木质素的结构特征差异可能是造成吸附差异的潜在机制,如功能团和木质素组成等。具有低S/G比例的木质素对纤维素酶表现出更高的吸附能力。玉米秸木质素中高的酚羟基含量和低的羧酸基团含量是其对纤维素酶吸附能力大于红麻秆木质素和杨木木质素的原因之一。相比玉米秸木质素,松木木质素具有低含量的脂肪羟基,增加了木质素的疏水性,可能是松木木质素对纤维素酶吸附能力大于玉米秸木质素的原因之一。通过酶活测定、纯酶吸附等实验,发现木质素对纤维素酶不同酶组分的吸附力强弱顺序为CBH、木聚糖酶>EG>BG,BG的最弱吸附力表明,CBM在蛋白吸附中发挥重要作用。更多还原

【Abstract】 Ramie is the traditional fiber crops in China. Its fibers are considered as the longest, strongest, and silkiest in plant fibers and have excellent properties as natural textile material, such as preeminent absorption, quick drying, easy dyeing, shrinkage resistance, good bacteria, mildew, and insect resistance. However, decorticated ramie fibers contain20-35%gum, which mainly consists of pectin and hemicellulose. This gum should be removed as much as possible for most applications. Conventional degumming using NaOH solution consumes large amounts of chemicals and energy, and causes serious environmental pollution. Enzymatic degumming is a gentle reaction, with less damage to fibers and flexible operation, as well as easy processing and quality control, and so on. Hence, it is considered a potential alternative to chemical degumming and has attracted wide attention. The research on efficient degumming enzyme, high-efficient degumming enzyme components and degumming mechanism can promote the understanding and development of the enzymatic degumming. Up to now, the ramie stalk and kenaf stalk have not been used effectively. The stalk contains a lot of cellulose and therefore can be used for the production of bioethanol. The research on the pretreatment of the stalks and fermentation for bioethanol production could provide reference for bioethanol production using this kind of raw materials. Lignin has been always deemed to have a negative impact on the saccharification of lignocellulosic feedstocks by physically barring and unproductively adsorbing hydrolytic enzymes. The lignin sources and structural features of lignin affect the inhibition on the saccharification. The characteristics and compositions of lignin are significantly different in diverse types of biomass. The native difference in lignin is the reason for the differences in their adsorption capacity. However, the mechanism on how it affects the inhibitory hydrolysis was not explained. Based on above, the research contents and the main results of the thesis are as follows: 1. Screening of strains capable of producing effective degumming enzyme, enzyme characterization and optimization of enzyme productionIt was found that the crude enzyme from wild-type Bacillus sp. Y1had a powerful and fast degumming ability. Its polygalacturonate lyase (PGL) activity was the highest at pH9.6-10.0and60℃, and stable at pH7.0-10.5and30-50℃. The PGL have a wide scope of pH and temperature, and high H2O2tolerance. The gum loss and brightness of fibers could be significantly improved when H2O2was added during enzymatic degumming with the PGL, showed a synergistic action between the PGL and H2O2on the degumming and bleaching of ramie fibers. It was very suitable for a joint process of enzymatic degumming and H2O2bleaching.The medium components and fermentation conditions for the pectinase production were optimized by some statistical methods, and the activities of polygalacturonate lyase (PGL) and polygalacturonase (PG) were increased to2.00-and3.44-fold respectively, and the fermentation time shortened12hours (from72hrs to60hrs) after optimization. The final optimal medium components and fermentation conditions were as follows:carbon source,10.5%,(NH4)2SO4,0.37%; MgSO4,0.3%; Tween-80,0.1%(w/v); initial pH,8.2; and inoculum number of1.3mL (with the OD600of the seed medium about5.77) per50mL of fermentation medium on a swing shaker (100rpm) at34℃for60h.2. Degumming mechanism with crude enzyme from Bacillus sp. Y1The mechanism about effective degumming with crude enzyme from Bacillus sp. Yl was studied by SDS-PAGE, MS/MS, and the adding experments of enzymes and purified protein. It was found that the crude enzyme of wild-type Bacillus sp. Y1contains more proteins compared with the control pectinase, and protease component in it was further substantiated to play an important role in the degumming process. There was a synergistic action between protease and pectinase during degumming.Chemical composition and degumming performance of ramie from different growth period were analyzed. Few differences were found in chemical composition and degumming performance between the first ramie and second ramie, and the degumming performance of the third ramie is the worst. The chemical composition analysis showed that the third ramie contains more hemicellulose. It was found from comparative study on the ramie degumming in different time that the degumming process was finished after1h, and the removal of pectin and hemicellulose in the third ramie was obviously lower than that in the first ramie. SEM observation showed that the gum adhesions on the surface of the third ramie was significant more than that on the first ramie, and more difficult to be removed.3. Pretreatment of ramie stalk and kenaf stalk and Q-SSF for bioethanol productionThe effects of different chemical pretreatment on enzymatic digestibility of ramie stalk and kenaf stalk were studied. Ramie and kenaf stalks pretreated with alkali were chosen to produce ethanol using quasi-simultaneous saccharification and fermentation (Q-SSF) process. The results show that for the stalk pretreated with4%NaOH and0.02%anthraquinone-2-sulfonic acid sodium salt (AQSS) as catalyzer at170℃for1h, the ethanol concentration could reach51g/L after fermentation for168h at18%of solid substrate concentration. By fed-batch to20%of solid substrate concentration, the ethanol concentration could reach63g/L, and the cellulose conversion was77%and79%for ramie stalk and kenaf stalk, respectively. For kenaf stalk pretreated with5.2%NaHSO3and0.2%H2SO4at170℃for1h, the ethanol concentration and cellulose conversion could reach to65g/L and72%, respectively.4. Differences in the adsorption of enzymes onto lignins from diverse types of lignocellulosic biomass and the underlying mechanismIt was found that lignin sources affected enzyme adsorption using structural features, such as functional groups and lignin composition. G lignin had a higher adsorption capacity on enzymes than S lignin. The adsorption capacity of cellulase onto lignin decreased in the order:pine lignin (PL)> corn stover lignin (CL)> aspen lignin (AL)> kenaf lignin (KL). The lignin with low S/G ratio has higher adsorption capacity on enzyme. A higher content of phenolic hydroxyl groups and a lower content of carboxylic acid groups resulted in stronger adsorption affinity for CL than for KL and AL. The lower amount of aliphatic hydroxyls that increased hydrophobic interactions could explain the higher adsorption capacity of PL than CL. For different mono-component enzymes, the adsorption ability decreased in the order: cellobiohydrolase (CBH), xylanase> endoglucanase (EG)> β-glucosidase (BG), indicating the important role of carbohydrate-binding module (CBM) in protein adsorption.更多还原