植物细胞壁蛋白与木质纤维素酶解

木质纤维素是生产生物能源和材料的重要原料.木质纤维素具有高度复杂的结构,其酶解效率除了受自身的凝聚态结构影响外,还受到细胞壁自身组分的影响.本文综述了植物细胞壁中主要蛋白的特征及其与木质纤维素酶解的关系.从植物自身细胞壁蛋白活性出发来研究木质纤维素的酶解,为研究其酶解机制和高效酶解方法提供了新思路.     

植物细胞壁纳米结构与纳米纤维素的化学纯化处理结合机械解纤法制备

植物中含有自然界中储量最为丰富的天然高分子—纤维素.植物具有多层级结构,在微观层面由细胞构成.细胞的主体物质存在于细胞壁中,主要以纤维素纳米纤丝—基体复合结构形式存在,这种结构对植物的性能和功能起到了至关重要的作用.采用化学纯化处理结合机械解纤策略,可以将细胞壁内部起支撑作用的纳米纤维素解离出来.纳米纤维素展现出一维纳米纤维结构、高结晶度以及优异的力学性能和热性能,在许多领域均展现出巨大的应用潜力.本文主要介绍我们课题组近几年对植物多层级结构、细胞特征、细胞壁生物合成以及细胞壁纳米结构的研究和思考,总结了在植物纳米纤维素化学纯化处理结合机械解纤法制备方面的研究进展,并对其存在的问题以及今后重点研究方向进行了总结和展望.     

非离子型表面活性剂对木质纤维素酶催化水解的影响及机理

木质纤维素的酶解糖化过程是纤维素生物质转化中的关键步骤,也是限制纤维素生物转化生产燃料和化学品的主要瓶颈。大量的研究表明,非离子型表面活性剂能够强化木质纤维素酶解过程,显著提高纤维素的酶催化水解效率。本文综述了非离子型表面活性剂对纯纤维素和木质纤维素底物酶解的影响,分析了底物结构特性、水解条件、纤维素酶组成等诸多因素与表面活性剂作用效果之间的关联,并从纤维素酶的吸附特性、纤维素酶组分间的协同作用等方面对非离子表面活性剂的作用机理进行了总结。结合已有的研究进展和存在的问题,提出了今后表面活性剂对于木质纤维素酶催化水解影响的研究重点方向,即系统分析底物结构、水解条件等因素对表面活性剂作用的宏观影响,以及分析这种作用的热力学和动力学特性,而微观上需要从原子和分子层面上解析表面活性剂与底物和纤维素酶之间的相互作用特性。     

木质纤维素酶解糖化*

纤维素水解转化为可发酵糖工艺是纤维素乙醇炼制过程中至关重要的环节。酶法水解工艺具有条件温和、副产物少、环境友好等特点,因而受到广泛重视。目前许多学者已针对如何提高木质纤维素酶解效率、降低纤维素酶成本等问题,开展了多种化学、生物技术及工艺耦合的研究。本文综述了近几年木质纤维素酶解领域取得的最新工艺进展和理论研究成果,对原料预处理、多酶复配优化、酶脱附与重复利用、工艺耦合、高固液比反应等方面的研究情况进行了总结,同时展望了木质纤维素酶解工艺的未来发展方向。     

聚甲基丙烯酸-聚乙烯吡啶复合物固定纤维素酶的研究

<正> 作为植物细胞壁主要成份的纤维素,占植物总重的一半,是地球上最丰富的有机物,是对人类有用而且潜力很大的自然资源。将这些廉价易得的纤维素利用纤维素酶转变成葡萄糖,不仅在食品工业上有现实意义,而且对未来化学工业的发展有着不可估量的影响。但由于酶在水溶液中不稳定,反应后难以分离和回收,只能使用一次,成本较高,使其应用受到一定的限制,固定酶的发展正好弥补了上述不足。     

冬小麦麸皮抗冻蛋白一级结构的研究

冬小麦麸皮抗冻蛋白(TaAFP)的一级结构是研究其高级结构和功能的基础.本研究结合N-末端测序技术和肽指纹图谱技术,测定TaAFP的一级结构,并对其同源性进行分析.MALDI-TOF-MS质谱分析显示TaAFP的分子量为13637.711 Da.使用Trypsin(胰蛋白酶)、Hydroxylamine(羟胺)和Chymotrypsin(胰凝乳蛋白酶)分别对TaAFP酶解,并对各个酶解片段的肽指纹图谱进行解析和连接,最终确定TaAFP的一级结构为MARKVIALAFLLLLTISLSKSNAARVKYNGGESGGGGGGGGGGGGGGNGSG-SGSGYGYNYGKGGGQSGGGQGGGGGGGGGGGSNGSGSGSGYGYGYGQGNGGAQGQGSGGGGGGGGGGGGGGSGQGSGSGYGYGYGKGGGGGGGGGGDGGGGGGGGSAYVGRHE,测定覆盖度达到了100%.序列比对分析以及同源性分析结果显示,TaAFP是一种与植物细胞壁和抵抗寒冷相关的蛋白质.     

木质纤维素在球磨过程中的相变行为

用X射线衍射对中国马尾松中的本质纤维素在球磨过程中的降解、解结晶、重结晶和相转变进行了定量的研究。本文的实验证明,球磨木质纤维素有着与球磨纤维素不同的重结晶和相转变行为,木质纤维素中的共存木素对球磨非晶态纤维素向纤维素Ⅱ的转交起着障碍的作用,但对它们向纤维素Ⅰ的重结晶则不存在影响。这个结果支持由Coring和Sarko等先后提出的纤维素Ⅰ向纤维素Ⅱ转变的理论和机理。     

Ce~(3+)对哈茨木霉(Trichoderma harzianum)产漆酶影响的机制研究

通过酶活的测定、菌体内Ca~(2+)离子、细胞壁结构、细胞膜蛋白的荧光分析,对Ce~(3+)影响哈茨木霉产漆酶的机制进行了初步研究。实验得出Ce~(3+)浓度在10~(-8)g·L~(-1)时,酶活可提高87.5%,10~(-1)g·L~(-1)时抑制作用较强,酶活下降75%。高浓度的Ce~(3+)破坏了菌体细胞壁和细胞膜的结构,从而抑制了菌体正常生长和产酶;低浓度Ce~(3+)仅对细胞膜和细胞壁有影响,增大了其通透性,促进了菌体产酶。菌体内Ca~(2+)离子含量的变化表明:稀土可以通过调节菌体内Ca~(2+)离子的含量来影响菌体生长和产酶。     

植物环蛋白研究进展*

植物环蛋白(cyclotides)是一类植物中富含二硫键、由28-37个氨基酸残基组成的大环蛋白,其分子中含有一个结构独特的环胱氨酸结(cyclic cystine knot, CCK)。由于其独特的结构和广泛的生物活性,如子宫收缩、溶血、抗肿瘤、抗微生物等活性,及其能耐常规的高温、酸解和酶解的稳定结构,可作为多肽药物设计中的模板分子进行结构修饰或活性多肽的载体,而在国际上引起广泛的关注。目前从堇菜科、茜草科和葫芦科约30种植物中发现100多个植物环蛋白,研究主要集中在澳大利亚、瑞典和美国等几个研究组,近年我们也在开展相关研究。本文主要从植物环蛋白的提取、分离、检测与结构鉴定方法,结构与性质,序列的同源性及分类,化学合成与生物合成,生物活性以及应用前景等几个方面介绍植物环蛋白的研究进展。     

木质纤维素-蒙脱土纳米复合材料的制备

采用插层复合方法在碱性水溶液中制备了木质纤维素-蒙脱土纳米复合材料,并分析了反应温度、反应时间、木质纤维素与蒙脱土的质量比等因素对纳米复合材料有机化程度的影响,用FT-IR、XRD、TEM、SEM等方法对纳米复合材料的结构进行了表征。结果表明:蒙脱土片层结构在反应过程中被撑开,木质纤维素分散在其中,形成插层-剥离型纳米复合材料,其热稳定性较木质纤维素有了较大提高。     

Effect of sodium dodecyl sulfate and cetyltrimethylammonium bromide catanionic surfactant on the enzymatic hydrolysis of Avicel and corn stover

Nonionic surfactants could effectively improve the enzymatic hydrolysis efficiency of lignocellulose, while small molecule anionic and cationic surfactants usually inhibited the enzymatic hydrolysis. The results showed that the anionic surfactant sodium dodecyl sulfate (SDS) could improve the enzymatic hydrolysis efficiency of Avicel at the concentration range of 0.1–1 mM, but it did inhibit enzymatic hydrolysis at higher concentration. Cationic surfactant cetyltrimethylammonium bromide (CTAB) was used to regulate the surface charge of SDS; thereby catanionic surfactant SDS-CTAB was formed. The effect of SDS-CTAB catanionic surfactant with varied molar ratios on the enzymatic hydrolysis of pure cellulose and corn stover at various enzymatic hydrolysis environments was investigated. SDS-CTAB could increase the enzymatic hydrolysis of corn stover at high solid loading from 33.3 to 42.4%. Using SDS-CTAB could reduce about 58% of the cellulase dosage to achieve 80% of the enzymatic hydrolysis of corn stover. SDS-CTAB catanionic surfactant could regulate the surface charge of cellulase in the hydrolyzate and reduce the non-productive adsorption of cellulase on the lignin, thereby improving the enzymatic hydrolysis efficiency of lignocellulose.     

Separation of the lignocellulose of nonwoody plants into its main components and study of their properties

It has been shown that as a result of preliminary acid hydrolysis followed by alkaline treatment there is a separation of the lignocellulose of nonwoody plants (cotton plant stems, kenaf tow) into the structural substances of the cell wall. The nature of the separated components has been established as cellulose and a lignocarbohydrate mixture.Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 118–122, January–February, 1998.     

pH within pores in plant fiber cell walls assessed by Fluorescence Ratio Imaging

The pH within cell wall pores of filter paper fibers and hemp fibers was assessed by Fluorescence Ratio Imaging (FRIM). It was found that the Donnan effect affected the pH measured within the fibers. When the conductivity of the added liquid was low (0.7 mS), pH values were lower within the cell wall than in the bulk solution. This was not the case at high conductivity (22 mS). The occurrence of the Donnan effect allowed the pH values within pores in normal regions of the cell wall to be compared to the pH in regions with misaligned microfibrils (dislocations) when FRIM was carried out in a low conductivity solution. Surprisingly, no pH difference was observed between normal regions and dislocations, suggesting that pore sizes within the two different regions are approximately the same. In another experiment the Donnan effect was shown to have an effect on hydrolysis of hydrothermally pretreated wheat straw only when conducted in a low conductivity solution and only for xylanase, not cellulases. The hydrolysis experiments indicate that under typical conditions where conductivity is high, the Donnan effect does not lower the pH close to the substrate to an extent that affects enzymatic activity during hydrolysis of lignocellulose.     

Obtaining Mannanoligosaccharide Preparations by Means of the Mechanoenzymatic Hydrolysis of Yeast Biomass

A new method of obtaining biologically active mannanoligosaccharides is proposed. It involves mechanical activation of the enzymatic hydrolysis of components forming the supramolecular structure of the cell wall. Processes that take place during mechanical treatment and enzymatic hydrolysis of yeast biomass and lead to an increase in the availability of mannanoligosaccharides of the yeast cell walls are investigated. The efficiency of the use of mechanoenzymatic approach to obtaining mannanoligosaccharide preparations is evaluated.     

Inhibition Performance of Lignocellulose Degradation Products on Industrial Cellulase Enzymes During Cellulose Hydrolysis

This study examined the inhibition performance by the major lignocellulose degradation products, including organic acids, furan derivatives, lignin derivatives, and ethanol, on a broadly used commercial cellulase enzyme Spezyme CP (Genencor International, Rochester, NY, USA) to cellulose hydrolysis at both the well-mixing state (shaking flask) and the static state (test tube). The cellulase activity in the cellulase complex of Spezyme CP was assumed to be one single “cellulase”, and the apparent kinetic parameters of this cellulase enzyme were measured as an approximate index of the inhibitory effect to the industrial cellulase enzyme. The inhibition performance of these degradation products was compared and analyzed using the determined apparent kinetic parameters. All the degradation products strongly inhibit the cellulose hydrolysis by cellulase enzyme, and the inhibitions on cellulase were all competitive type. The order of the inhibition strength by the lignocellulose degradation products to cellulase is lignin derivatives > furan derivatives > organic acids > ethanol. This study gave a quantitative view to the enzymatic hydrolysis of lignocellulose under the inhibition performance of the lignocellulose degradation products and will help to understand the lignocellulose recalcitrance to enzyme hydrolysis.     

Kinetic Studies on the Product Inhibition of Enzymatic Lignocellulose Hydrolysis

In order to understand the product inhibition of enzymatic lignocellulose hydrolysis, the enzymatic hydrolysis of pretreated rice straw was carried out over an enzyme loading range of 2 to 30 FPU/g substrate, and the inhibition of enzymatic hydrolysis was analyzed kinetically based on the reducing sugars produced. It was shown that glucose, xylose, and arabinose were the main reducing sugar components contained in the hydrolysate. The mass ratio of glucose, xylose, and arabinose to the total reducing sugars was almost constant at 52.0?%, 29.7?% and 18.8?%, respectively, in the enzyme loading range. The reducing sugars exerted competitive inhibitory interferences to the enzymatic hydrolysis. Glucose, xylose, and arabinose had a dissociation constant of 1.24, 0.54 and 0.33?g/l, respectively. The inhibitory interferences by reducing sugars were superimposed on the enzymatic hydrolysis. The enzymatic hydrolysis could be improved by the removal of the produced reducing sugars from hydrolysate.     

Effects of SPORL and Dilute Acid Pretreatment on Substrate Morphology, Cell Physical and Chemical Wall Structures, and Subsequent Enzymatic Hydrolysis of Lodgepole Pine

The effects of pretreatment by dilute acid and sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) on substrate morphology, cell wall physical and chemical structures, along with the subsequent enzymatic hydrolysis of lodgepole pine substrate were investigated. FE-SEM and TEM images of substrate structural morphological changes showed that SPORL pretreatment resulted in fiber separation, where SPORL high pH (4.2) pretreatment exhibited better fiber separation than SPORL low pH (1.9) pretreatment. Dilute acid pretreatment produced very poor fiber separation, consisting mostly of fiber bundles. The removal of almost all hemicelluloses in the dilute acid pretreated substrate did not overcome recalcitrance to achieve a high cellulose conversion when lignin removal was limited. SPORL high pH pretreatment removed more lignin but less hemicellulose, while SPORL low pH pretreatment removed about the same amount of lignin and hemicelluloses in lodgepole pine substrates when compared with dilute acid pretreatment. Substrates pretreated with either SPORL process had a much higher cellulose conversion than those produced with dilute acid pretreatment. Lignin removal in addition to removal of hemicellulose in SPORL pretreatment plays an important role in improving the cellulose hydrolysis of the substrate.     

Enhancing enzymatic hydrolysis of crystalline cellulose and lignocellulose by adding long-chain fatty alcohols

The effects of long-chain fatty alcohols (LFAs) on the enzymatic hydrolysis of crystalline cellulose by two commercial Trichoderma reesei cellulase cocktails (CTec2 and Celluclast 1.5L) were studied. It was found that n-butanol inhibited the enzymatic hydrolysis, but n-octanol, n-decanol and n-dodecanol had strong enhancement on enzymatic hydrolysis of crystalline cellulose in the buffer pH range from 4.0 to 6.0. LFAs can increase the hydrolysis efficiency of crystalline cellulose from 37 to 57 % at Celluclast 1.5L loading of ten filter paper units (FPU)/g glucan. LFAs have similar enhancement on the enzymatic hydrolysis of crystalline cellulose mixed with lignin or xylan. The enhancement of LFAs increased with the decrease of the crystallinity index. LFAs not only enhanced the high-solid enzymatic hydrolysis of lignocellulose, but also improved the rheological properties of high-solid lignocellulosic slurries by decreasing the yield stress and complex viscosity. Meanwhile, LFAs can improve the enzymatic hydrolysis of cellobiose to glucose, especially at low cellulase loading.     

Cellulose solvent-based pretreatment for corn stover and avicel: concentrated phosphoric acid versus ionic liquid [BMIM]Cl

Since cellulose accessibility has become more recognized as the major substrate characteristic limiting hydrolysis rates and glucan digestibilities, cellulose solvent-based lignocellulose pretreatments have gained attention. In this study, we employed cellulose solvent- and organic solvent-based lignocellulose fractionation using two cellulose solvents: concentrated phosphoric acid [~85?% (w/w) H₃PO₄] and an ionic liquid Butyl-3-methylimidazolium chloride ([BMIM]Cl). Enzymatic glucan digestibilities of concentrated phosphoric acid- and [BMIM]Cl-pretreated corn stover were 96 and 55?% after 72?h at five filter paper units of cellulase per gram of glucan, respectively. Regenerated amorphous cellulose by concentrated phosphoric acid and [BMIM]Cl had digestibilities of 100 and 92?%, respectively. Our results suggested that differences in enzymatic glucan digestibilities of concentrated phosphoric acid- and [BMIM]Cl-pretreated corn stover were attributed to combinatory factors. These results provide insights into mechanisms of cellulose solvent-based pretreatment and effects of residual cellulose solvents and lignin on enzymatic cellulose hydrolysis.