AFM Investigations of Cellulose Fibers in Bintje Potato (Solanum tuberosum L.) Cell Wall Fragments

Atomic force microscopy (AFM) has been used to image the cellulose networks in moist fragments of the cell walls of Bintje potato (Solanum tuberosum L.). The interfiber spacing in hydrated native cell wall fragments was found to be 26.2 nm. This value is consistent with published estimates of the contour length of xyloglucan cross-links determined by transmission electron microscopy (TEM) studies of cell walls. Sequential extraction of the pectin using CDTA and Na₂CO₃ led to shrinkage of the cell wall fragment and a reduction in interfiber spacing to 20.2 nm. Partial extraction of xyloglucan using 1 M KOH caused a small decrease in interfiber spacing to 19.5 nm. Finally, the almost complete removal of xyloglucan with 4 M KOH substantially reduced the interfiber spacing to 11 nm. The results are consistent with a model for the cell wall in which the cellulose–xyloglucan network is immersed in a swollen, hydrated pectin network.     

低温对棉纤维比强度形成的生理机制影响

通过设置播期试验使棉纤维加厚发育过程(铃龄25~50 d)处于不同的温度条件下,研究低温对棉花纤维比强度形成的内在生理机制影响,为采取调控措施解决目前棉花(Gossypium)生产中存在的晚熟劣质问题提供理论依据。两年试验结果表明:棉纤维加厚发育期24.0 ℃左右的日均温是高强纤维形成的最佳温度,其内在生理机制表现为棉纤维蔗糖合成酶活性最高,β_1,3_葡聚糖酶活性最低,纤维素的累积量和累积速率均明显高于其它低温条件,纤维超分子结构取向参数角较小,处于优化状态,最终表现为纤维比强度亦最大;低于21.0 ℃时即对棉纤维加厚发育相关酶活性产生明显影响,纤维比强度降低。当温度降到15.0 ℃左右时,棉纤维蔗糖合成酶活性显著降低,而β-1,3_葡聚糖酶活性显著升高,同时纤维素累积量和累积速率均显著降低,纤维超分子结构取向参数角明显宽化,棉纤维不能正常发育,不利于高强纤维的形成(铃重仅为3.22 g,纤维比强度仅为15.73 cN·tex^-1)。     

嗜热厌氧纤维素降解细菌的分离、鉴定及其系统发育分析

利用纤维素降解细菌和纤维素粘附的方法分别从新鲜牛粪、高温堆肥和本实验室保存的纤维素降解富集物中分离得到4株嗜热厌氧纤维素降解细菌。分离菌株为革兰氏染色阴性,直的或稍弯曲杆菌,菌体大小为0.4μm～0.6μm×3μm～15μm,严格厌氧,不还原硫酸盐,形成芽孢。多数芽孢着生于菌体顶端。分离菌株能利用纤维素滤纸、纤维素粉Whatman CFII、微晶纤维素、纤维素粉MN300和未经处理的玉米秆芯、甘蔗渣、水稻秸杆。分离菌株在pH6.2～8.9、温度45℃～65℃范围内利用纤维素,最适pH为7.0～7.5,最适温度为55℃～60℃,发酵纤维素产生乙醇、乙酸、H2和CO2。分离菌株还可利用纤维二糖、葡萄糖、果糖、麦芽糖、山梨醇作为碳源。部分长度的16S rDNA序列分析表明,分离菌株EVAI与Clostridium thermocellum具有99.8%相似性。     

微紫青霉CBHⅠ酶纤维素结合结构域在大肠杆菌中的分泌型表达及性质研究

为研究纤维素酶纤维素结合结构域的结构与功能 ,进而深入了解天然纤维素的生物降解机制和提高纤维素酶的生物工艺学价值 ,采用 PCR技术体外扩增了携带微紫青霉外切葡聚糖纤维二糖水解酶 ( CBH ) CBD编码区的 DNA片段 ,将 CBD编码区 DNA片段插入带有 Erwiniacarotovora pe1 B前导肽序列的大肠杆菌质粒 p KK- tac- new上进行了表达 .携带微紫青霉 CBDCBH 编码区的大肠杆菌重组菌株 DH5α( p KK- tac- new- 8)产生有活性的分泌型 CBDCBH 蛋白 .SDS-PAGE检测显示所产生的 CBDCBH 蛋白分子量约 1 0 .8k D.在 IPTG诱导下 ,该菌株所产生的CBDCBH 蛋白含量达 45.2 mg/L,且 90 %以上的 CBD蛋白分泌到培养物上清液中 .结晶纤维素 CF-1 1溶液经 CBDCBH 处理后 ,浊度比对照提高了 1 2 8.9% ,天然棉花纤维结构经 CBDCBH 处理后产生一定程度的非水解性降解作用 ,表明微紫青霉 CBDCBH 具有解聚天然结晶纤维素的作用 .     

Comparative study of cellulases and hemicellulases from four fungi: mesophiles Trichoderma reesei and Penicillium sp. and thermophiles Thielavia terrestris and Sporotrichum cellulophilum

The enzymes produced by two thermophilic fungi claimed to produce heat-stable cellulases [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] have been compared to those of two mesophilic fungi on the basis of the following criteria: polysaccharolytic spectrum, heat and pH effects on stability and on activity of the different enzymes, and the ability to hydrolyse raw natural substrates. The cellulases produced by one of the thermophiles, Sporotrichum cellulophilum, appeared to be as heat-labile as those from the mesophile Trichoderma reesei; moreover, the former enzyme preparation is the least efficient of the four tested. Thielavia terrestris enzymes are the most thermostable; on the basis of the other properties tested, T. terrestris enzymes are comparable to, or in some cases better than, those from mesophilic strains. However, the differences are not so great as to compensate for the much lower productivity of T. terrestris compared to the improved T. reesei and Penicillium sp. strains.     

Structure and association of wall fibrils produced by regenerating tobacco protoplasts

A study has been made of the wall fibrils produced by tobacco protoplasts, using scanning electron microscopy in conjunction with negative staining. It has been shown that the fibres seen in scanning electron microscopy correspond to aggregates of microfibrils. These aggregates are only visible where they are lifted clear of the protoplast surface. Negative staining of fixed protoplasts shows that the aggregation of microfibrils into the fibres visible in scanning electron microscopy is probably produced by air-drying. Gentle disruption of microfibrils produces both random broken fragments and bundles of short pieces of fibrillar material about 60 nm in length. This material is present in undisrupted young walls, but not in undisrupted older walls. The microfibrils in young walls seem much more fragile and liable to breakage than those in older walls. These results are discussed in terms of the interpretation of scanning electron microscope images and the mechanism of cellulose microfibril formation by higher plants.Abbreviations SEM Scanning electron microscopy     

纤维二糖脱氢酶的纤维素降解中的作用研究

裂褶菌纤维二糖脱氢酶（ｃｅｌｌｏｂｉｏｓｅ ｄｅｈｙｄｒｏｇｅｎａｓｅ,ＣＤＨ）可以提高纤维素酶对纤维素的降解。以纤维二糖为电子供体,ＣＤＨ作用于羧甲基纤维可降低其溶液的粘度,作用纤维素ＣＦ１１和磷酸膨胀纤维素,分别使其悬浊液的浊度提高７％和１４．４％。ＣＤＨ与纤维二糖水解酶或切纤维素酶在降解棉花纤维素时没有表现出协同作用。但若棉花事先在纤维二糖存在下用ＣＤＨ预处理,则变得易于被水解。     

Comb-shaped graft copolymers with cellulose side-chains prepared via click chemistry

Comb-shaped copolymers with cellobiose acetate or cellulose triacetate (CTA) side-chains, PPMA-g-(CTA2-C15) and PPMA-g-(CTA13-C15), were prepared by grafting N-(15-azidopentadecanoyl)-2,3,6-tri-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl)-β-d-glucopyranosylamine (CTA2-C15-N₃) and N-(15-azidopentadecanoyl)-tri-O-acetyl-β-cellulosylamine (CTA13-C15-N₃, number average degree of polymerization (DP_n) = 13) onto poly(2-propyn-1-yl methacrylate) (PPMA, weight average degree of polymerization (DP_w, X + Y = 5.59 × 10²)) via “click chemistry”. The copolymers were characterized by ¹H, ¹³C and two-dimensional NMR and size exclusion chromatography-multi-angle laser light scattering (SEC-MALS) measurements. The numbers of CTA side-chains (X) of PPMA-g-(CTA2-C15) and PPMA-g-(CTA13-C15) were calculated as 4.03 × 10² and 2.45 × 10², respectively. Copolymers with cellulosic side-chains, PPMA-g-(CELL2-C15) and PPMA-g-(CELL13-C15), were successfully obtained after deacetylation of PPMA-g-(CTA2-C15) and PPMA-g-(CTA13-C15), respectively. X-ray diffraction measurements revealed that PPMA-g-(CELL13-C15) showed crystalline pattern of cellulose II, which is believed to have anti-parallel orientation.     

Establishing in vitro Zinnia elegans cell suspension culture with high tracheary element differentiation

The Zinnia elegans mesophyll cell culture is a useful system for xylogenesis studies. The system is associated with highly synchronous tracheary element (TE) differentiation, making it more suitable for molecular studies requiring larger amounts of molecular isolates, such as mRNA and proteins and for studying cellulose synthesis. There is, however, the problem of non-uniformity and significant variations in the yields of TEs (%TE). One possible cause for this variability in the %TE could be the lack of a standardized experimental protocol in various research laboratories for establishing the Zinnia culture. Mesophyll cells isolated from the first true leaves of Z. elegans var Envy seedlings of approximately 14 days old were cultured in vitro and differentiated into TEs. The xylogenic culture medium was supplied with 1 mg/l each of benzylaminopurine (BA) and α-naphthalene acetic acid (NAA). Application of this improved culture method resulted in stable and reproducible amounts of TE as high as 76% in the Zinnia culture. The increase was mainly due to conditioning of the mesophyll cell culture and adjustments of the phytohormonal balance in the cultures. Also, certain biochemical and cytological methods have been shown to reliably monitor progress of TE differentiation. We conclude that, with the adoption of current improvement in the xylogenic Z. elegans culture, higher amounts of tracheary elements can be produced. This successful outcome raises the potential of the Zinnia system as a suitable model for cellulose and xylogenesis research.