BACKGROUND & AIMS: :Butyrivibrio proteoclasticus is a significant component of the microbial population of the rumen of dairy cattle. It is a xylan-degrading organism whose genome encodes a large number of open reading frames annotated as fiber-degrading enzymes. We have determined the three-dimensional structure of Est2A, an acetyl xylan esterase from B. proteoclasticus, at 2.1 Å resolution, along with the structure of an inactive mutant (H351A) at 2.0 Å resolution. The structure reveals two domains-a C-terminal SGNH domain and an N-terminal jelly-roll domain typical of CE2 family structures. The structures are accompanied by experimentally determined enzymatic parameters against two model substrates, para-nitrophenyl acetate and para-nitrophenyl butyrate. The suite of fiber-degrading enzymes produced by B. proteoclasticus provides a rich source of new enzymes of potential use in industrial settings.

背景与目标： : 酪氨酸弧菌是奶牛瘤胃微生物种群的重要组成部分。它是一种降解木聚糖的生物，其基因组编码大量被注释为纤维降解酶的开放阅读框。我们已经以2.1的分辨率确定了Est2A (一种来自c的乙酰木聚糖酯酶) 的三维结构，以及以2.0的分辨率确定了非活性突变体 (H351A) 的结构。该结构揭示了两个结构域-CE2家族结构典型的C端SGNH结构域和N端jelly-roll结构域。结构伴随着针对两种模型底物 (对硝基苯乙酸酯和对硝基苯丁酸酯) 的实验确定的酶促参数。由B. proteoclasticus产生的纤维降解酶套件提供了在工业环境中潜在使用的新酶的丰富来源。

BACKGROUND & AIMS: :Two anaerobic bacteria, designated strains SYSU GA16112T and SYSU GA16107, were isolated from a hot spring in Tengchong County, Yunnan Province, south-west PR China. Phylogenetic analyses based on 16S rRNA gene sequences showed that strains SYSU GA16112T and SYSU GA16107 belong to the family Dysgonamonadaceae. Cells of strains SYSU GA16112T and SYSU GA16107 were Gram-stain-negative, rod-shaped and non-motile. The major fatty acids (>10 %) of strains SYSU GA16112T and SYSU GA16107 were identified as anteiso-C15 : 0 and anteiso-C17 : 0 3OH. The polar lipid profile of strain SYSU GA16112T was found to consist of phosphatidylethanolamine, two unidentified aminophospholipids, two unidentified phosphoglycolipids, two unidentified aminolipids and one unidentified polar lipid, while that of strain SYSU GA16107 consisted of phosphatidylethanolamine, two unidentified polar lipids, three unidentified aminophospholipids, two unidentified phosphoglycolipids and one unidentified aminolipid. The genomic DNA G+C contents of strains SYSU GA16112T and SYSU GA16107 were determined to be 41.90 and 41.89 %, respectively, and the average nucleotide identity value between them was 99.99 %. Based on their morphological and physiological properties, and results of phylogenetic analyses, strains SYSU GA16112T and SYSU GA16107 are considered to represent a novel species of a novel genus, for which the name Seramator thermalis gen. nov., sp. nov. (type strain SYSU GA16112T=CGMCC 1.5281T=KCTC 15753T) is proposed.

背景与目标： : 从中国西南部云南省腾冲县的一个温泉中分离出两种厌氧菌，分别命名为SYSU GA16112T和SYSU GA16107。基于16S rRNA基因序列的系统发育分析表明，菌株SYSU GA16112T和SYSU GA16107属于Dysgonamonadaceae。菌株SYSU GA16112T和SYSU GA16107的细胞为革兰氏染色阴性，杆状且不活动。菌株SYSU GA16112T和SYSU GA16107的主要脂肪酸 (>10  %) 被鉴定为anteiso-C15  :  0和anteiso-C17  : 0 3OH。发现SYSU GA16112T菌株的极性脂质谱由磷脂酰乙醇胺，两个未鉴定的氨基磷脂，两个未鉴定的磷酸糖脂，两个未鉴定的氨基脂和一个未鉴定的极性脂质组成，而SYSU GA16107菌株的极性脂质由磷脂酰乙醇胺组成，两个未鉴定的极性脂质，三个未鉴定的氨基磷脂，两个未鉴定的磷酸糖脂和一个未鉴定的氨基脂。测定菌株SYSU GA16112T和SYSU GA16107的基因组DNA G + C含量分别为41.90和41.89   %，它们之间的平均核苷酸同一性值为99.99   %。根据其形态和生理特性以及系统发育分析的结果，菌株SYSU GA16112T和SYSU GA16107被认为代表了一个新属的新物种，其名称为Seramator thermalis gen。11月，sp。11月(提出了类型应变SYSU GA16112T = CGMCC 1.5281T = KCTC 15753T)。

BACKGROUND & AIMS: BACKGROUND:Xylan has been identified as a physical barrier which limits cellulose accessibility by covering the outer surface of fibers and interfibrillar space. Therefore, tracking xylan is a prerequisite for understanding and optimizing lignocellulosic biomass processes. RESULTS:In this study, we developed a novel xylan tracking approach using a two-domain probe called OC15 which consists of a fusion of Cellvibrio japonicus carbohydrate-binding domain 15 with the fluorescent protein mOrange2. The new probe specifically binds to xylan with an affinity similar to that of CBM15. The sensitivity of the OC15-xylan detection approach was compared to that of standard methods such as X-ray photoelectron spectroscopy (XPS) and chemical composition analysis (NREL/TP-510-42618). All three approaches were used to analyze the variations of xylan content of kraft pulp fibers. XPS, which allows for surface analysis of fibers, did not clearly indicate changes in xylan content. Chemical composition analysis responded to the changes in xylan content, but did not give any specific information related to the fibers surface. Interestingly, only the OC15 probe enabled the highly sensitive detection of xylan variations at the surface of kraft pulp fibers. At variance with the other methods, the OC15 probe can be used in a high throughput format. CONCLUSIONS:We developed a rapid and high throughput approach for the detection of changes in xylan exposure at the surface of paper fibers. The introduction of this method into the lignocellulosic biomass-based industries should revolutionize the understanding and optimization of most wood biomass processes.

背景与目标：

BACKGROUND & AIMS: :We have successfully conjugated mesalamine (5-aminosalicylic acid, 5-ASA) with xylan, a biopolymer isolated from pineapple stem waste, to form xylan-5-ASA conjugate. The biopolymer was used to provide colon-targeting properties for 5-ASA, a golden standard anti-inflammatory agent commonly used for ulcerative colitis treatment. A series of data from FTIR spectroscopy, UV-Vis spectrophotometry, and HPLC confirmed the xylan-5-ASA conjugate formation. To ensure successful colon targeting properties, in vitro and in vivo drug release studies after oral administration of xylan-5-ASA conjugate to Wistar rats were performed. Xylan-5-ASA conjugate was able to retain 5-ASA release in the upper gastrointestinal tract fluid simulation but rapidly released 5-ASA in the rat colon fluid simulation. In vivo release profile shows a very low peak plasma concentration, reached at 6 h after xylan-5-ASA conjugate administration. The delayed release and the lower bioavailability of 5-ASA from xylan-5-ASA conjugate administration compared to free 5-ASA administration confirmed the successful local colon delivery of 5-ASA using xylan-5-ASA conjugate. The administration of xylan-5-ASA conjugate also exhibited greater efficacy in recovering 2,4,6-trinitrobenzene sulfonic acid-induced colon ulcer compared to free 5-ASA administration. Taken together, xylan isolated from pineapple stem waste is promising to obtain colon targeting property for 5-ASA.

背景与目标： : 我们已经成功地将美沙拉胺 (5-氨基水杨酸，5-ASA) 与木聚糖 (一种从菠萝茎废料中分离出的生物聚合物) 缀合成xylan-5-ASA缀合物。该生物聚合物用于为5-ASA提供结肠靶向特性，5-ASA是一种常用于溃疡性结肠炎治疗的黄金标准抗炎剂。FTIR光谱，UV-Vis分光光度法和HPLC的一系列数据证实了xylan-5-ASA缀合物的形成。为了确保成功的结肠靶向特性，在向Wistar大鼠口服xylan-5-ASA缀合物后进行了体外和体内药物释放研究。Xylan-5-ASA缀合物能够在上胃肠道流体模拟中保留5-ASA释放，但在大鼠结肠流体模拟中迅速释放5-ASA。体内释放曲线显示非常低的峰值血浆浓度，在xylan-5-ASA缀合物施用后6小时达到。与游离的5-ASA施用相比，来自xylan-5-ASA缀合物施用的5-ASA的延迟释放和较低的生物利用度证实了使用xylan-5-ASA缀合物的5-ASA的成功的局部结肠递送。与游离的5-ASA给药相比，xylan-5-ASA缀合物的给药在恢复2,4，6-三硝基苯磺酸诱导的结肠溃疡方面也显示出更大的功效。总之，从菠萝茎废料中分离出的木聚糖有望获得5-ASA的结肠靶向性。

BACKGROUND & AIMS: BACKGROUND:Fermentable sugars are important intermediates in the biological conversion of biomass. Hemicellulose and amorphous cellulose are easily hydrolyzed to fermentable sugars in dilute acid, whereas crystalline cellulose is more difficult to be hydrolyzed. Cellulose fast pyrolysis is an alternative method to liberate valuable fermentable sugars from biomass. The amount of levoglucosan generated from lignocellulose by fast pyrolysis is usually lower than the theoretical yield based on the cellulose fraction. Pretreatment is a promising route to improve the yield of levoglucosan from lignocellulose. RESULTS:The integration of dilute sulfuric acid hydrolysis and fast pyrolysis to obtain fermentable sugars was evaluated in this study. Dilute sulfuric acid hydrolysis could remove more than 95.1 and 93.4 % of xylan (the main component of hemicellulose) from sugarcane bagasse and corncob with high yield of xylose. On the other hand, dilute sulfuric acid hydrolysis was also an effective pretreatment to enhance levoglucosan yield from lignocellulose. Dilute acid hydrolysis could accumulate glucan (the component of cellulose) and remove most of the alkali and alkaline earth metals which were powerful catalysts during fast pyrolysis. Further increase in dilute acid concentration (from 0 to 2 %) in pretreatment could promote the yield of levoglucosan in fast pyrolysis. The acid pretreated sugarcane bagasse and corncob gave levoglucosan yields of 43.8 and 35.2 % which were obvious higher than those of raw sugarcane bagasse (12.0 %) and corncob (7.0 %). CONCLUSIONS:Obtaining fermentable sugars by combination dilute acid hydrolysis of xylan and fast pyrolysis of glucan could make full utilization of biomass, and get fermentable sugars economically from biomass for bio-refinery.

背景与目标：

BACKGROUND & AIMS: :We constructed a recombinant industrial Saccharomyces cerevisiae yeast strain OC2-AXYL2-ABGL2-Xyl2 by inserting two copies of the β-glucosidase (BGL) and β-xylosidase (XYL) genes, and a gene cassette for xylose assimilation in the genome of yeast strain OC-2HUT. Both BGL and XYL were expressed on the yeast cell surface with high enzyme activities. Using OC2-AXYL2-ABGL2-Xyl2, we performed ethanol fermentation from a mixture of powdered cellulose (KC-flock) and Birchwood xylan, with the additional supplementation of a 30-g/l Trichoderma reesei cellulase complex mixture. The ethanol yield (gram per gram of added cellulases) of the strain OC2-AXYL2-ABGL2-Xyl2 increased approximately 2.5-fold compared to that of strain OC2-Xyl2, which lacked β-glucosidase and β-xylosidase activities. Notably, the concentration of additional T. reesei cellulase was reduced from 30 to 24 g/l without affecting ethanol production. The BGL- and XYL-displaying industrial yeast of the strain OC2-AXYL2-ABGL2-Xyl2 represents a promising yeast for reducing cellulase consumption of ethanol fermentation from lignocellulosic biomass by compensating for the inherent weak BGL and XYL activities of T. reesei cellulase complexes.

背景与目标： : 我们通过插入两个拷贝的 β-葡萄糖苷酶 (BGL) 和 β-木糖苷酶 (XYL) 基因，以及在OC-2HUT酵母菌株基因组中用于木糖同化的基因盒，构建了重组工业酿酒酵母酵母菌株OC2-AXYL2-ABGL2-Xyl2。BGL和XYL均在高酶活性的酵母细胞表面表达。使用OC2-AXYL2-ABGL2-Xyl2，我们从粉状纤维素 (KC-flock) 和桦木木聚糖的混合物中进行了乙醇发酵，并补充了30g/l里氏木霉纤维素酶复合混合物。与缺乏 β-葡萄糖苷酶和 β-木糖苷酶活性的OC2-Xyl2菌株相比，该菌株OC2-AXYL2-ABGL2-Xyl2的乙醇产量 (克每克添加的纤维素酶) 增加了约2.5倍。值得注意的是，额外的里氏T纤维素酶的浓度从30g/l降低到24g/l，而不会影响乙醇的生产。OC2-AXYL2-ABGL2-Xyl2菌株的显示BGL和XYL的工业酵母代表了一种有前途的酵母，用于通过补偿里氏T纤维素酶复合物固有的弱BGL和XYL活性来减少木质纤维素生物质中乙醇发酵的纤维素酶消耗。

BACKGROUND & AIMS: :In our search for novel anaerobes with potential carbohydrate polymers degrading activity, we have isolated a xylan-degrading bacterial strain SYSU GA17129T from an anammox bacteria dominant wastewater treatment plant. Phylogenetic analysis of the 16S rRNA gene sequence indicated the strain SYSU GA17129T belong to the order Clostridiales and shared highest sequence identity with Caldicoprobacter faecalis DSM 20678T (89.9%). The strain was thermophilic, obligately anaerobic, non-motile and rod shaped. Optimum growth of the strain was observed at 45 °C, pH 8.0 and in the presence of 0.5% NaCl (w/v). The chemotaxonomic features of the strain SYSU GA17129T comprised of C14:0 FAME, iso-C15:0 FAME and C16:0 FAME as the major fatty acids (>10%), diphosphatidylglycerol, phosphatidylinositol mannoside, an unidentified phospholipid, three unidentified polar lipids and two unidentified glycolipids as its polar lipids, and meso-diaminopimelic acid (meso-DAP) as the diamino acid in peptidoglycan. The G + C content of the genomic DNA was 35.9%. The strain could be distinguished from other defined families within the order Clostridiales by the differences in phenotypic and physiological characteristics, distinct phylogenetic lineage in 16S rRNA gene- and genome-based phylogenies and low genomic relatedness index. Based on these distinguishing properties, strain SYSU GA17129T is proposed to represent a new species of a new genus Xylanivirga thermophila gen. nov., sp. nov., within a new family Xylanivirgaceae fam. nov. The type species of the new taxon is SYSU GA17129T (=KCTC 15754T = CGMCC 1.5282T). This strain is characterized within the order Clostridiales, class Clostridia of the phylum Firmicutes.

背景与目标： : 在寻找具有潜在碳水化合物聚合物降解活性的新型厌氧菌时，我们从anammox细菌优势废水处理厂中分离出了降解木聚糖的细菌菌株SYSU GA17129T。对16S rRNA基因序列的系统发育分析表明，SYSU GA17129T菌株属于梭状芽胞杆菌的目的，并且与粪钙菌DSM 20678T (89.9%) 具有最高的序列同一性。该菌株是嗜热的，专性厌氧的，不活动的和杆状的。在45 ℃ 、pH 8.0和0.5% NaCl (w/v) 存在下观察到菌株的最佳生长。SYSU GA17129T菌株的化学分类学特征包括C14:0 FAME，iso-C15:0 FAME和C16:0 FAME作为主要脂肪酸 (>10%)，二磷脂酰甘油，磷脂酰肌醇甘露糖苷，一种未鉴定的磷脂，三种未鉴定的极性脂质和两种未鉴定的糖脂作为其极性脂质，肽聚糖中的二氨基甲酸 (meso-DAP) 作为二氨基酸。基因组DNA的G + C含量为35.9%。通过表型和生理特征的差异，16S rRNA基因和基于基因组的系统发育的独特系统发育谱系以及低基因组相关性指数，可以将该菌株与梭状芽胞杆菌中的其他定义家族区分开。基于这些区别特性，提出了SYSU GA17129T菌株代表新的木聚糖11月属的新物种。11月，在一个新的木聚糖科家族中。11月新分类单元的模式种为SYSU GA17129T (= KCTC 15754T   =  CGMCC 1.5282T)。该菌株的特征是梭状芽胞杆菌的梭状芽胞杆菌类。

BACKGROUND & AIMS: This is the first report describing the analysis of a gene encoding an alpha-glucuronidase, an enzyme essential for the complete breakdown of substituted xylans. A DNA fragment that carries the gene for alpha-glucuronidase was isolated from chromosomal DNA of the hyperthermophilic bacterium Thermotoga maritima MSB8. The alpha-glucuronidase gene (aguA) was identified and characterized with the aid of nucleotide sequence analysis, deletion experiments and expression studies in Escherichia coli, and the start of the coding region was defined by amino-terminal sequencing of the purified recombinant enzyme. The aguA gene encodes a 674-amino-acid, largely hydrophilic polypeptide with a calculated molecular mass of 78593 Da. The alpha-glucuronidase of T. maritima has a novel primary structure with no significant similarity to any other known amino acid sequence. The recombinant enzyme was purified to homogeneity as judged by SDS-PAGE. Gel filtration analysis at low salt concentrations revealed a high apparent molecular mass (> 630 kDa) for the recombinant enzyme, but the oligomeric structure changed upon variation of the ionic strength or the pH, yielding hexameric and/or dimeric forms which were also enzymatically active. The enzyme hydrolysed 2-O-(4-O-methyl-alpha-D-glucopyranosyluronic acid)-D-xylobiose (MeGlcAX2) to xylobiose and 4-O-methylglucuronic acid. The K(m) for MeGlcAX2 was 0.95 mM. The pH optimum was 6.3. Maximum activity was measured at 85 degrees C, about 25 degrees C or more above the values reported for all other alpha-glucuronidases known to date. When incubated at 55-75 degrees C, the enzyme suffered partial inactivation, but thereafter the residual activity remained nearly constant for several days.

背景与目标： 这是第一份报告，描述了编码 α-葡萄糖醛酸苷酶的基因的分析，该酶是取代木聚糖完全分解所必需的酶。从嗜热细菌Thermotoga maritima msb8的染色体DNA中分离出携带 α-葡萄糖醛酸苷酶基因的DNA片段。借助核苷酸序列分析，缺失实验和在大肠杆菌中的表达研究，对 α-葡萄糖醛酸苷酶基因 (aguA) 进行了鉴定和表征，并通过纯化的重组酶的氨基末端测序来定义编码区的起始。aguA基因编码674氨基酸，主要是亲水性多肽，其计算分子量为78593 Da。maritima的 α-葡萄糖醛酸苷酶具有新颖的一级结构，与任何其他已知的氨基酸序列没有显着相似性。通过sdds-PAGE判断，将重组酶纯化至均一。在低盐浓度下的凝胶过滤分析显示重组酶的高表观分子量 (> 630 kDa)，但是寡聚结构随着离子强度或pH的变化而改变，产生也具有酶活性的六聚和/或二聚体形式。该酶将2-O-(4-O-methyl-alpha-D-glucopyranosyluronic酸)-D-木二糖 (MeGlcAX2) 水解为木二糖和4-o-甲基葡萄糖醛酸。MeGlcAX2的K(m) 为0.95 mM。最适pH为6.3。在85 ℃ 、约25 ℃ 或高于迄今已知的所有其他 α-葡萄糖醛酸糖苷酶报告的值时测量最大活性。当在55-75摄氏度下孵育时，该酶发生部分失活，但此后几天内残留活性几乎保持恒定。

BACKGROUND & AIMS: :A family 2b carbohydrate-binding module from Streptomyces thermoviolaceus STX-II was fused at the carboxyl-terminus of XynB, a thermostable and single domain family 10 xylanase from Thermotoga maritima, to create a chimeric xylanase. The chimeric enzyme (XynB-CBM2b) was purified and characterized. It displayed a pH-activity profile similar to that of XynB and was stable up to 90 degrees C. XynB-CBM2b bound to insoluble birchwood and oatspelt xylan. Whereas its hydrolytic activities toward insoluble xylan and p-nitrophenyl-beta-xylopyranoside were similar to those of XynB, its activity toward soluble xylan was moderately higher than that of XynB.

背景与目标： : 来自thermoviolaceus STX-II的2b族碳水化合物结合模块在XynB的羧基末端融合，XynB是来自Thermotoga maritima的热稳定的单域10族木聚糖酶，以产生嵌合木聚糖酶。对嵌合酶 (XynB-CBM2b) 进行纯化和表征。它显示出与XynB相似的pH-活性曲线，并且在90摄氏度下保持稳定。XynB-CBM2b结合到不溶的桦木和燕麦木聚糖上。尽管其对不溶性木聚糖和对硝基苯基-β-木吡喃糖苷的水解活性与XynB相似，但对可溶性木聚糖的水解活性略高于XynB。

BACKGROUND & AIMS: :The molecular architecture of plant secondary cell walls is still not resolved. There are several proposed structures for cellulose fibrils, the main component of plant cell walls and the conformation of other molecules is even less well known. Glucuronic acid (GlcA) substitution of xylan (GUX) enzymes, in CAZy family glycosyl transferase (GT)8, decorate the xylan backbone with various specific patterns of GlcA. It was recently discovered that dicot xylan has a domain with the side chain decorations distributed on every second unit of the backbone (xylose). If the xylan backbone folds in a similar way to glucan chains in cellulose (2-fold helix), this kind of arrangement may allow the undecorated side of the xylan chain to hydrogen bond with the hydrophilic surface of cellulose microfibrils. MD simulations suggest that such interactions are energetically stable. We discuss the possible role of this xylan decoration pattern in building of the plant cell wall.

背景与目标： : 植物次生细胞壁的分子结构仍未解决。纤维素原纤维有几种提议的结构，植物细胞壁的主要成分和其他分子的构象甚至鲜为人知。在CAZy家族糖基转移酶 (GT)8中，木聚糖 (GUX) 酶的葡萄糖醛酸 (GlcA) 取代用各种特定的GlcA模式装饰木聚糖主链。最近发现双子叶木聚糖有一个结构域，侧链装饰分布在主干 (木糖) 的每第二个单元上。如果木聚糖骨架以与纤维素中的葡聚糖链相似的方式折叠 (2-fold helix)，则这种排列可能使木聚糖链的未修饰侧与纤维素微纤维的亲水表面氢键。MD模拟表明，这种相互作用在能量上是稳定的。我们讨论了这种木聚糖装饰模式在植物细胞壁构建中的可能作用。

BACKGROUND & AIMS: Background:Domain of Unknown Function 231-containing proteins (DUF231) are plant specific and their function is largely unknown. Studies in the model plants Arabidopsis and rice suggested that some DUF231 proteins act in the process of O-acetyl substitution of hemicellulose and esterification of pectin. However, little is known about the function of DUF231 proteins in woody plant species. Results:This study provides evidence supporting that one member of DUF231 family proteins in the woody perennial plant Populus deltoides (genotype WV94), PdDUF231A, has a role in the acetylation of xylan and affects cellulose biosynthesis. A total of 52 DUF231-containing proteins were identified in the Populus genome. In P. deltoides transgenic lines overexpressing PdDUF231A (OXPdDUF231A), glucose and cellulose contents were increased. Consistent with these results, the transcript levels of cellulose biosynthesis-related genes were increased in the OXPdDUF231A transgenic lines. Furthermore, the relative content of total acetylated xylan was increased in the OXPdDUF231A transgenic lines. Enzymatic saccharification assays revealed that the rate of glucose release increased in OXPdDUF231A transgenic lines. Plant biomass productivity was also increased in OXPdDUF231A transgenic lines. Conclusions:These results suggest that PdDUF231A affects cellulose biosynthesis and plays a role in the acetylation of xylan. PdDUF231A is a promising target for genetic modification for biofuel production because biomass productivity and compositional quality can be simultaneously improved through overexpression.

背景与目标：

BACKGROUND & AIMS: :The thermotolerant yeast Hansenula polymorpha ferments glucose and xylose to ethanol at high temperatures. However, H. polymorpha cannot utilize starchy materials or xylans. Heterologous amylolytic and xylanolytic enzymes have to be expressed in this yeast to provide for utilization and growth on starch and xylan. Genes SWA2 and GAM1 from the yeast Schwanniomyces occidentalis, encoding alpha-amylase and glucoamylase, respectively, were expressed in H. polymorpha. The expression was achieved by integration of the SWA2 and GAM1 genes under the strong constitutive promoter of the H. polymorpha glyceraldehyde-3-phosphate dehydrogenase gene (HpGAP) into H. polymorpha genome. Resulting transformants acquired the ability to grow on a minimal medium containing soluble starch as a sole carbon source. Ethanol production at high-temperature fermentation from starch by the recombinant strains was up to 10 g/L. The XYN2 gene encoding endoxylanase of the fungus Trichoderma reseei was expressed in H. polymorpha. Co-expression of xlnD gene coding for beta-xylosidase of the fungus Aspergillus niger and the XYN2 gene in H. polymorpha was achieved by integration of these genes under control of the HpGAP promoter. Resulting transformants were capable of growth and alcoholic fermentation on a minimal medium supplemented with birchwood xylan as a sole carbon source at 48 degrees C.

背景与目标： : 耐热酵母汉逊酵母在高温下将葡萄糖和木糖发酵为乙醇。但是，H。多形体不能利用淀粉材料或木聚糖。必须在该酵母中表达异源淀粉分解酶和木聚糖分解酶，以提供在淀粉和木聚糖上的利用和生长。来自酵母Schwanniomyces occidentalis的基因SWA2和GAM1分别编码 α-淀粉酶和葡糖淀粉酶，在H. polymorpha中表达。通过将SWA2和GAM1基因在H.多态性glyceraldehyde-3-phosphate脱氢酶基因 (HpGAP) 的强组成型启动子下整合到H.多态性基因组中实现表达。所得的转化体具有在含有可溶性淀粉作为唯一碳源的基本培养基上生长的能力。重组菌株从淀粉高温发酵产生的乙醇产量高达10g/L。编码木霉木霉木聚糖内切酶的XYN2基因在H. polymorpha中表达。编码黑曲霉的 β-木糖苷酶的xlnD基因和H.多态性的XYN2基因的共表达是通过在HpGAP启动子的控制下整合这些基因来实现的。产生的转化体能够在48 ℃ 下在补充有桦木木聚糖作为唯一碳源的基本培养基上生长和酒精发酵。

BACKGROUND & AIMS: :A series of lipid-accumulating yeasts was examined for their potential to saccharify xylan and accumulate triglyceride. Of the genera tested, including Candida, Cryptococcus, Lipomyces, Rhodosporidium, Rhodotorula, and Trichosporon, only Cryptococcus and Trichosporon isolates saccharified xylan. All of the strains could assimilate xylose and accumuate triglyceride under nitrogen-limiting conditions. Strains of Cryptococcus albidus were found to be especially useful for a one-step saccharification of xylan coupled to triglyceride synthesis. Cryptococcus terricolus, a strain constitutive for lipid accumulation, lacked extracellular xylanase, but did assimilate xylose and xylobiose and was able to continuously convert xylan to triglyceride if the culture medium was supplemented with xylanase.

背景与目标： : 检查了一系列积累脂质的酵母糖化木聚糖和积累甘油三酸酯的潜力。在测试的属中，包括念珠菌，隐球菌，脂菌，红孢子虫，红孢子虫和毛孢菌素，只有隐球菌和毛孢菌素分离出糖化木聚糖。在氮限制条件下，所有菌株都可以吸收木糖并积累甘油三酸酯。发现白隐球菌菌株对木聚糖的一步糖化与甘油三酸酯合成特别有用。Terricoccus terricolus是一种脂质积累的菌株，缺乏细胞外木聚糖酶，但确实吸收了木糖和木糖，并且如果培养基中补充了木聚糖酶，则能够连续将木聚糖转化为甘油三酸酯。

BACKGROUND & AIMS: :The thermophilic actinomycete Thermomonospora fusca produced endoxylanase, alpha-arabinofuranosidase, beta-xylosidase, and acetyl esterase activities maximally during growth on xylan. Growth yields on glucose, xylose, or arabinose were comparable, but production of endoxylanase and beta-xylosidase was not induced on these substrates. The crude xylanase activity was thermostable and relatively resistant to end product inhibition by xylobiose and xylan hydrolysis products. Six proteins with xylanase activity were identified by zymogram analysis of isoelectric focusing gels, but only a 32-kDa protein exhibiting three isomeric forms could be purified by fast protein liquid chromatography. Endoglucanases were also identified in carboxymethylcellulose-grown cultures, and their distinction from endoxylanases was confirmed. alpha-Arabinofuranosidase activity was due to a single dimeric protein of 92 kDa, which was particularly resistant to end product inhibition by arabinose. Three bands of acetyl esterase activity were detected by zymogram analysis, and there was evidence that these mainly consisted of an intracellular 80-kDa protein secreted to yield active 40-kDa subunits in the culture supernatant. The acetyl esterases were found to be responsible for acetyl xylan esterase activity in T. fusca, in contrast to the distinction proposed in some other systems. The addition of purified betaxylosidase to endoxylanase increased the hydrolysis of xylan, probably by relieving end product inhibition. The enhanced saccharification of wheat straw caused by the addition of purified alpha-arabinofuranosidase to T. fusca endoxylanase suggested a truly synergistic relationship, in agreement with proposals that arabinose side groups on the xylan chain participate in cross-linking within the plant cell wall structure.

背景与目标： : 嗜热放线菌Thermomonospora fusca在木聚糖上生长过程中最大程度地产生木聚糖酶，α-阿拉伯呋喃糖苷酶，β-木糖苷酶和乙酰酯酶活性。葡萄糖，木糖或阿拉伯糖的生长产量相当，但在这些底物上未诱导内切木聚糖酶和 β-木糖苷酶的产生。粗木聚糖酶活性是热稳定性的，并且相对抵抗木糖和木聚糖水解产物对终产物的抑制。通过等电聚焦凝胶的酶谱分析鉴定了六种具有木聚糖酶活性的蛋白质，但是通过快速蛋白质液相色谱法只能纯化表现出三种异构体形式的32 kDa蛋白质。在羧甲基纤维素生长的培养物中也鉴定了内切葡聚糖酶，并确认了它们与内切木聚糖酶的区别。Α-阿拉伯呋喃糖苷酶的活性归因于92 kDa的单个二聚体蛋白，该蛋白特别能抵抗阿拉伯糖的终产物抑制。通过酶谱分析检测到三个乙酰酯酶活性带，有证据表明，这些带主要由分泌的细胞内80 kDa蛋白组成，在培养上清液中产生活性40 kDa亚基。与其他一些系统中提出的区别相反，发现乙酰酯酶负责T. fusca中的乙酰木聚糖酯酶活性。在木聚糖内切糖苷酶中添加纯化的甜蜜糖糖苷酶可能通过减轻终产物的抑制作用而增加了木聚糖的水解。通过向T. fusca内切木聚糖酶中添加纯化的 α-阿拉伯呋喃糖苷酶引起的小麦秸秆糖化作用增强，这与木聚糖链上的阿拉伯糖侧基参与植物细胞壁内交联的建议一致结构。

BACKGROUND & AIMS: :We engineered an acetyl xylan esterase (AwaxeA) gene from Aspergillus awamori into a heterologous expression system in Pichia pastoris. Purified recombinant AwAXEA (rAwAXEA) displayed the greatest hydrolytic activity toward alpha-naphthylacetate (C2), lower activity toward alpha-naphthylpropionate (C3) and no detectable activity toward acyl-chain substrates containing four or more carbon atoms. Putative catalytic residues, Ser(119), Ser(146), Asp(168) and Asp(202), were substituted for alanine by site-directed mutagenesis. The biochemical properties and kinetic parameters of the four mutant enzymes were examined. The S119A and D202A mutant enzymes were catalytically inactive, whereas S146A and D168A mutants displayed significant hydrolytic activity. These observations indicate that Ser(119) and Asp(202) are important for catalysis. The S146A mutant enzyme showed lower specific activity toward the C2 substrate and higher thermal stability than wild-type enzyme. The lower activity of S146A was due to a combination of increased K(m) and decreased k(cat). The catalytic efficiency of S146A was 41% lower than that of wild-type enzyme. The synthesis of ethyl acetate was >10-fold than that of ethyl n-hexanoate synthesis for the wild-type, S146A and D168A mutant enzymes. However, the D202A showed greater synthetic activity of ethyl n-hexanoate as compared with the wild-type and other mutants.

背景与目标： : 我们将来自泡盛曲霉的乙酰木聚糖酯酶 (AwaxeA) 基因工程化到巴斯德毕赤酵母的异源表达系统中。纯化的重组AwAXEA (rAwAXEA) 对 α-萘乙酸酯 (C2) 表现出最大的水解活性，对 α-萘基丙酸酯 (C3) 表现出较低的活性，对含有四个或更多碳原子的酰基链底物没有可检测的活性。通过定点诱变将推定的催化残基Ser(119) 、Ser(146) 、Asp(168) 和Asp(202) 取代丙氨酸。检查了四种突变酶的生化特性和动力学参数。S119A和D202A突变酶在催化上无活性，而S146A和D168A突变体显示出显着的水解活性。这些观察结果表明Ser(119) 和Asp(202) 对于催化是重要的。与野生型酶相比，S146A突变酶对C2底物的比活较低，热稳定性较高。S146A的较低活性是由于K(m) 增加和k(cat) 降低的组合。S146A的催化效率41% 低于野生型酶。对于野生型，S146A和D168A突变酶，乙酸乙酯的合成比正己酸乙酯的合成多10倍。然而，与野生型和其他突变体相比，D202A显示出更高的正己酸乙酯的合成活性。