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A highly efficient β-glucosidase from the buffalo rumen fungus Neocallimastix patriciarum W5
Hsin-Liang Chen, Yo-Chia Chen, Mei-Yeh J Lu, Jui-Jen Chang, Hiaow-Ting C Wang, Tzi-Yuan Wang, Sz-Kai Ruan, Tao-Yuan Wang, Kuo-Yen Hung, Hsing-Yi Cho, Huei-Mien Ke, Wan-Ting Lin, Ming-Che Shih, Wen-Hsiung Li
Biotechnology for Biofuels , 2012, DOI: 10.1186/1754-6834-5-24
Abstract: In this study, a cDNA encoding β-glucosidase was isolated from the buffalo rumen fungus Neocallimastix patriciarum W5 and is named NpaBGS. It has a length of 2,331 bp with an open reading frame coding for a protein of 776 amino acid residues, corresponding to a theoretical molecular mass of 85.1 kDa and isoelectric point of 4.4. Two GH3 catalytic domains were found at the N and C terminals of NpaBGS by sequence analysis. The cDNA was expressed in Pichia pastoris and after protein purification, the enzyme displayed a specific activity of 34.5 U/mg against cellobiose as the substrate. Enzymatic assays showed that NpaBGS was active on short cello-oligosaccharides from various substrates. A weak activity in carboxymethyl cellulose (CMC) digestion indicated that the enzyme might also have the function of an endoglucanase. The optimal activity was detected at 40°C and pH 5?~?6, showing that the enzyme prefers a weak acid condition. Moreover, its activity could be enhanced at 50°C by adding Mg2+ or Mn2+ ions. Interestingly, in simultaneous saccharification and fermentation (SSF) experiments using Saccharomyces cerevisiae BY4741 or Kluyveromyces marxianus KY3 as the fermentation yeast, NpaBGS showed advantages in cell growth, glucose production, and ethanol production over the commercial enzyme Novo 188. Moreover, we showed that the KY3 strain engineered with the NpaNGS gene can utilize 2 % dry napiergrass as the sole carbon source to produce 3.32 mg/ml ethanol when Celluclast 1.5 L was added to the SSF system.Our characterizations of the novel β-glucosidase NpaBGS revealed that it has a preference of weak acidity for optimal yeast fermentation and an optimal temperature of ~40°C. Since NpaBGS performs better than Novo 188 under the living conditions of fermentation yeasts, it has the potential to be a suitable enzyme for SSF.
Substrate molecule enhances the thermostability of a mutant of a family 11 xylanase from Neocallimastix patriciarum
C You, H Yuan, Q Huang, H Lu
African Journal of Biotechnology , 2010,
Abstract: Thermophilic xylanases are ideal for many industrial processes operated at elevated temperatures. Here, we report a mutant of a family 11 xylanase (Xyn-CDBFV) from Neocallimastix patriciarum with a mutation (D57N) in the active center could be further stabilized by the substrate. Despite that the thermostability of the two enzymes are almost the same in the absence of the substrate, the mutant displays a 10 - 15oC higher optimal temperature of activity than the wild type. Potential hydrogen bonding interactions predicted by molecular docking between the substrate molecule and residues N57, E202 of the mutant are probably responsible for the stabilizing effect. This suggests that it will be useful to consider the stabilizing effect induced by the substrate in optimization processes of enzyme thermostability.
Microbial Cellulases and Their Industrial Applications  [PDF]
Ramesh Chander Kuhad,Rishi Gupta,Ajay Singh
Enzyme Research , 2011, DOI: 10.4061/2011/280696
Abstract: Microbial cellulases have shown their potential application in various industries including pulp and paper, textile, laundry, biofuel production, food and feed industry, brewing, and agriculture. Due to the complexity of enzyme system and immense industrial potential, cellulases have been a potential candidate for research by both the academic and industrial research groups. Nowadays, significant attentions have been devoted to the current knowledge of cellulase production and the challenges in cellulase research especially in the direction of improving the process economics of various industries. Scientific and technological developments and the future prospects for application of cellulases in different industries are discussed in this paper. 1. Introduction Biotechnological conversion of cellulosic biomass is potentially sustainable approach to develop novel bioprocesses and products. Microbial cellulases have become the focal biocatalysts due to their complex nature and wide spread industrial applications. Cellulases are composed of independently folding, structurally and functionally discrete units called domains or modules, making cellulases module [1]. Cellulases are inducible enzymes synthesized by a large diversity of microorganisms including both fungi and bacteria during their growth on cellulosic materials (Table 1) [2, 3]. These microorganisms can be aerobic, anaerobic, mesophilic or thermophilic. Among them, the genera of Clostridium, Cellulomonas, Thermomonospora, Trichoderma, and Aspergillus are the most extensively studied cellulase producer [4–7]. Table 1: Microorganisms having cellulolytic abilities. Structurally fungal cellulases are simpler as compared to bacterial cellulase systems, cellulosomes [8–10]. Fungal cellulases typically have two separate domains: a catalytic domain (CD) and a cellulose binding module (CBM), which is joined by a short polylinker region to the catalytic domain at the N-terminal. The CBM is comprised of approximately 35 amino acids, and the linker region is rich in serine and threonine. The main difference between cellulosomes and free cellulase enzyme is in the component of cellulosomes-cohesin containing scaffolding and dockerin containing enzyme. The free cellulase contains cellulose binding domains (CBMs), which are replaced by a dockerin in cellulosomal complex, and a single scaffolding-born CBM directs the entire cellulosomes complex to cellulosic biomass [11, 12]. Mechanistically, cellulase is a family of at least 3 groups of enzymes [10, 13–15], endo-(1,4)-β-D-glucanase (EC
Research of W5 Model Based on Dynamic Context Awareness

- , 2016, DOI: 10.3969/j.issn.1001-0548.2016.02.020
Abstract: Twitter、Sina Micro-blog等社交网络应用为基于位置的服务提供了大量的情境信息,如用户ID(who)、签到时间(when)、GPS坐标(where)、微博内容主题词(what)和微博内容诱因词(why)等,简称5W。它们为用户的行为和偏好研究提供了契机。该文提出了基于5W动态情境感知信息的W5概率模型,并采用包含情境信息的联合概率分布分别从时间、空间和活动等方面挖掘用户动态行为,用于用户和位置的预测。该文实验基于两个数据集:Geo-text(GT)和Sina-tweets(ST),在数据集上进行了用户预测(UP)和位置预测(LP)实验。实验结果表明,W5模型在UP和LP两方面准确率均高于W4模型。同时,W5模型在时间误差和空间距离误差两方面也取得了较好的性能。
JCMT HARP CO 3-2 Observations of Molecular Outflows in W5  [PDF]
Adam Ginsburg,John Bally,Jonathan Williams
Physics , 2011, DOI: 10.1111/j.1365-2966.2011.19279.x
Abstract: New JCMT HARP CO 3-2 observations of the W5 star forming complex are presented, totaling an area of 12000 arcmin^2 with sensitivity better than 0.1 K per 0.4 km/s channel. We discovered 55 CO outflow candidates, of which 40 are associated with W5 and 15 are more distant than the Perseus arm. Most of the outflows are located on the periphery of the W5 HII region. However, two outflow clusters are > 5 pc from the ionization fronts, indicating that their driving protostars formed without directly being triggered by the O-stars in W5. We compare the derived outflow properties to those in Perseus and find that the total W5 outflow mass is surprisingly low given the cloud masses. The outflow mass deficiency in the more massive W5 cloud (M(H2) ~ 5 \times 10^4 Msun) can be explained if ionizing radiation dissociates molecules as they break out of their host cloud cores. Although CO J=3-2 is a good outflow tracer, it is likely to be a poor mass tracer because of sub-thermal line excitation and high opacity, which may also contribute to the outflow mass discrepancy. It is unlikely that outflows could provide the observed turbulent energy in the W5 molecular clouds even accounting for undetected outflow material. Many cometary globules have been observed with velocity gradients from head to tail, displaying strong interaction with the W5 HII region and exhibiting signs of triggered or revealed star formation in their heads. Because it is observed face-on, W5 is an excellent region to study feedback effects, both positive and negative, of massive stars on star formation.
Genomic and Secretomic Analyses Reveal Unique Features of the Lignocellulolytic Enzyme System of Penicillium decumbens  [PDF]
Guodong Liu, Lei Zhang, Xiaomin Wei, Gen Zou, Yuqi Qin, Liang Ma, Jie Li, Huajun Zheng, Shengyue Wang, Chengshu Wang, Luying Xun, Guo-Ping Zhao, Zhihua Zhou, Yinbo Qu
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0055185
Abstract: Many Penicillium species could produce extracellular enzyme systems with good lignocellulose hydrolysis performance. However, these species and their enzyme systems are still poorly understood and explored due to the lacking of genetic information. Here, we present the genomic and secretomic analyses of Penicillium decumbens that has been used in industrial production of lignocellulolytic enzymes in China for more than fifteen years. Comparative genomics analysis with the phylogenetically most similar species Penicillium chrysogenum revealed that P. decumbens has evolved with more genes involved in plant cell wall degradation, but fewer genes in cellular metabolism and regulation. Compared with the widely used cellulase producer Trichoderma reesei, P. decumbens has a lignocellulolytic enzyme system with more diverse components, particularly for cellulose binding domain-containing proteins and hemicellulases. Further, proteomic analysis of secretomes revealed that P. decumbens produced significantly more lignocellulolytic enzymes in the medium with cellulose-wheat bran as the carbon source than with glucose. The results expand our knowledge on the genetic information of lignocellulolytic enzyme systems in Penicillium species, and will facilitate rational strain improvement for the production of highly efficient enzyme systems used in lignocellulose utilization from Penicillium species.
Paul Ander,Lars Hildén,Geoffrey Daniel
BioResources , 2008,
Abstract: A new pulp fibre testing procedure called the HCl method was used to compare different spruce and pine fibres and mixtures of these fibres to calculate number of fibre cleavages in dislocations and other weak points. This method was compared with treatment of softwood kraft pulp fibres using different cellulase mixtures. The HCl method can distinguish between mill- and laboratory-made softwood kraft pulp fibres from the same wood batch. The sugar release is characterized by xylose and other hemicellulose sugars and little glucose. This is in contrast to cellulases, which despite strong fibre cleavage, did not distinguish between mill- and laboratory-made pulp fibres and released large amounts of glucose from the fibres. Hemicellulose degradation by HCl and deep penetration of the acid into the primary and secondary fibre cell walls at 80°C seems to be of major importance for the differentiation between mill and laboratory pulp fibres. Cellulases, in contrast, act mostly on the fibre surfaces, and deep penetration only takes place in amorphous regions of dislocations.
Location, formation and biosynthetic regulation of cellulases in the gliding bacteria Cytophaga hutchinsonii  [PDF]
Clifford Louime,Michael Abazinge,Elijah Johnson
International Journal of Molecular Sciences , 2006, DOI: 10.3390/i7010001
Abstract: An analysis of the recently published genome sequence of Cytophagahutchinsonii revealed an unusual collection of genes for an organism that can attackcrystalline cellulose. Consequently, questions were being raised by cellulase scientists, as towhat mechanism this organism uses to degrade its insoluble substrates. Cellulose, being ahighly polymeric compound and insoluble in water, cannot enter the cell walls ofmicroorganisms. Cellulose-degrading enzymes have therefore to be located on the surface ofthe cell wall or released extracellularly. The location of most cellulase enzymes has beenstudied. However, basic information on C. hutchinsonii cellulases is almost non-existent. Inthe present study, the location, formation and biosynthetic regulation of cellulases in C.hutchinsonii were demonstrated on different substrates. Various fractions isolated from C.hutchinsonii after cell rupture were assayed for carboxymethyl-cellulase activity (CMC).The cellulases were found to be predominantly cell-free during active growth on solka-flok,although 30% of activity was recorded on cell-bound enzymes. Relatively little CM-cellulase was formed when cells were grown on glucose and cellobiose. Apparently glucoseor labile substrates such as cellobiose seem to repress the formation of CM-cellulase. Thesefindings should provide some insight into possible hydrolysis mechanisms by C.hutchinsonii.
Embedded Stellar Clusters in the W3/W4/W5 Molecular Cloud Complex  [PDF]
John M. Carpenter,Mark H. Heyer,Ronald L. Snell
Physics , 2000, DOI: 10.1086/317352
Abstract: We analyze the embedded stellar content in the vicinity of the W3/W4/W5 HII regions using the FCRAO Outer Galaxy 12CO(J=1-0) Survey, the IRAS Point Source Catalog, published radio continuum surveys, and new near-infrared and molecular line observations. Thirty-four IRAS Point Sources are identified that have far-infrared colors characteristic of embedded star forming regions, and we have obtained K' mosaics and 13CO(J=1-0) maps for 32 of them. Ten of the IRAS sources are associated with an OB star and 19 with a stellar cluster, although three OB stars are not identified with a cluster. Half of the embedded stellar population identified in the K' images is found in just the 5 richest clusters, and 61% is contained in IRAS sources associated with an embedded OB star. Thus rich clusters around OB stars contribute substantially to the stellar population currently forming in the W3/W4/W5 region. Approximately 39% of the cluster population is embedded in small clouds with an average mass of ~130 Mo that are located as far as 100 pc from the W3/W4/W5 cloud complex. We speculate that these small clouds are fragments of a cloud complex dispersed by previous episodes of massive star formation. Finally, we find that 4 of the 5 known embedded massive star forming sites in the W3 molecular cloud are found along the interface with the W4 HII region despite the fact that most of the molecular mass is contained in the interior regions of the cloud. These observations are consistent with the classical notion that the W4 HII region has triggered massive star formation along the eastern edge of the W3 molecular cloud.
Star formation in bright-rimmed clouds and cluster associated with W5 E H{\sc ii} region  [PDF]
Neelam Chauhan,A. K. Pandey,K. Ogura,J. Jose,D. K. Ojha,M. R. Samal,H. Mito
Physics , 2011, DOI: 10.1111/j.1365-2966.2011.18742.x
Abstract: The aim of this paper is to present the results of photometric investigations of the central cluster of the W5 E region as well as a follow-up study of the triggered star formation in and around bright-rimmed clouds (BRCs). We have carried out wide field $UBVI_c$ and deep $VI_c$ photometry of the W5 E H{\sc ii} region. A distance of $\sim$2.1 kpc and a mean age of $\sim$1.3 Myr have been obtained for the central cluster. The young stellar objects (YSOs) associated with the region are identified on the basis of near-infrared and mid-infrared observations. We confirmed our earlier results that the average age of the YSOs lying on/inside the rim are younger than those lying outside the rim. The global distribution of the YSOs shows an aligned distribution from the ionising source to the BRCs. These facts indicate that a series of radiation driven implosion processes proceeded from near the central ionising source towards the periphery of the W5 E H{\sc ii} region. We found that, in general, the age distributions of the Class II and Class III sources are the same. This result is apparently in contradiction with the conclusion by Bertout, Siess & Cabrit (2007) and Chauhan et al. (2009) that classical T Tauri stars evolve to weak-line T Tauri stars. The initial mass function of the central cluster region in the mass range $0.4 \le M/M_\odot \le 30$ can be represented by $\Gamma = -1.29 \pm 0.03$. The cumulative mass functions indicate that in the mass range $0.2 \le M/M_\odot \le 0.8$, the cluster region and BRC NW have more low mass YSOs in comparison to BRCs 13 and 14.
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