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1.
The family II cellulose-binding modules (CBM) from Thermobifida fusca Cel5A and Cel48A were cloned in the Escherichia coli/Streptomyces shuttle vector pD730, and the plasmids were transformed into Streptomyces lividans TKM31. CBM(Cel5A), and CBM(Cel48A), CBM(Cel6B) were expressed and purified from S. lividans. The molecular masses were determined by mass spectrometry, and the values were 10595 +/- 2, 10915 +/- 2, and 11291 +/- 2 Da for CBM(Cel5A), CBM(Cel6B), and CBM(Cel48A), respectively. Three different binding models (Langmuir, Interstice Penetration, and Interstice Saturation) were tested to describe the binding isotherms of these CBMs on bacterial microcrystalline cellulose (BMCC). The experimental binding isotherms of T. fusca family II CBMs on BMCC are best modeled by the Interstice Saturation model, which includes binding to the constrained interstice surface of BMCC as well as traditional Langmuir binding on the freely accessible surface. The Interstice Saturation model consists of three different steps (Langmuir binding, interstice binding, and interstice saturation). Full reversibility only occurred in the Langmuir region. The irreversibility in the interstice binding and saturation regions probably was caused by interstice entrapment. Temperature shift experiments in different binding regions support the interstice entrapment assumption. There was no systematic difference in binding between the two types of exocellulase CBMs--one that hydrolyzes cellulose from the nonreducing (CBM(Cel6B)) end and one that hydrolyzes cellulose from the reducing end (CBM(Cel48A)). 相似文献
2.
The starch-synthase III (SSIII), with a total of 1025 residues, is one of the enzymes involved in plants starch synthesis. SSIII from Arabidopsis thaliana contains a putative N-terminal transit peptide followed by a 557-amino acid SSIII-specific domain (SSIII-SD) with three internal repeats and a C-terminal catalytic domain of 450 amino acids. Here, using computational characterization techniques, we show that each of the three internal repeats encodes a starch-binding domain (SBD). Although the SSIII from A. thaliana and its close homologous proteins show no detectable sequence similarity with characterized SBD sequences, the amino acid residues known to be involved in starch binding are well conserved. 相似文献
3.
Cellulose is a linear homopolymer of beta 1-4 linked glucose residues. Chitin is similar to cellulose in structure, and can be described as cellulose with the hydroxyl group on the C2 carbon replaced by an acetylamine group. Both cellulose and chitin form tightly packed, extensively hydrogen-bonded micro-fibrils. Up to now, binding of cellulase catalytic domains (CDs) to chitin has not been reported. In this article, binding of the CDs of Thermobifida fusca Cel6A, Cel6B, Cel48A, Cel5A, and Cel9A to alpha-chitin was investigated. The CDs of endocellulases, Cel6A and Cel5A did not bind to alpha-chitin; one exocellulase, Cel48A CD bound alpha-chitin moderately well; and the exocellulase Cel6B CD and the processive endocellulase Cel9A CD bound extremely tightly to alpha-chitin. Only mutations of Cel6B W329C, W332A and G234S and Cel9A Y206F, Y206S and D261A/R378K caused weaker binding to alpha-chitin than wild-type, and all these mutations were of residues near the catalytic center. One mutant enzyme, Cel9A D261A/R378K had weak chitinase activity, but no soluble products were detected. Chitotriose and chitotetraose were docked successfully to the catalytic cleft of Cel9A. In general, the positioning of the sugar residues in the model structures matched the cellooligosaccharides in the X-ray structure. Our results show that the binding of chitin by a cellulase can provide additional information about its binding to cellulose. 相似文献
4.
Immacolata Venditto Arnaud Basl Ana S. Luís Max J. Temple Luís M. A. Ferreira Carlos M. G. A. Fontes Harry J. Gilbert Shabir Najmudin 《Acta Crystallographica. Section F, Structural Biology Communications》2013,69(2):191-194
The rumen anaerobic cellulolytic bacterium Eubacterium cellulosolvens produces a large range of cellulases and hemicellulases responsible for the efficient hydrolysis of plant cell wall polysaccharides. One of these enzymes, endoglucanase Cel5A, comprises a tandemly repeated carbohydrate‐binding module (CBM65) fused to a glycoside hydrolase family 5 (Cel5A) catalytic domain, joined by flexible linker sequences. The second carbohydrate‐binding module located at the C‐terminus side of the endoglucanase (CBM65B) has been co‐crystallized with either cellohexaose or xyloglucan heptasaccharide. The crystals belong to the hexagonal space group P65 and tetragonal space group P43212, containing a single molecule in the asymmetric unit. The structures of CBM65B have been solved by molecular replacement. 相似文献
5.
Masahiro Watanabe Kazuhiko Ishikawa 《Acta Crystallographica. Section F, Structural Biology Communications》2014,70(12):1664-1667
Feruloyl esterase (FAE; EC 3.1.1.73) catalyzes the cleavage of the ester bond between ferulic acid and polysaccharides in plant cell walls, and thus holds significant potential for the industrial utilization of biomass saccharification. A feruloyl esterase was identified from the genome database of Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus). The gene consists of the catalytic domain and a carbohydrate‐binding module connected through a serine/threonine‐rich linker region. The recombinant enzyme was prepared, purified and crystallized at 293 K using 0.1 M imidazole pH 8.0, 0.2 M calcium acetate, 14% PEG 8000 as the precipitant. The crystal diffracted to 2.6 Å resolution and the crystal system is primitive orthorhombic, with unit‐cell parameters a = 90.9, b = 123.4, c = 135.4 Å. Four molecules are assumed to be present per asymmetric unit, corresponding to a Matthews coefficient of 2.50 Å3 Da−1 and a solvent content of 50.88%(v/v). 相似文献
6.
糖类是自然界数量最多的一类有机化合物,生物对其降解利用是最重要的反应之一。碳水化合物酶是具有降解、修饰和生成糖苷键功能的一大类酶。由于高分子糖类可溶性低,其糖苷键难以触及,因此其被酶作用的效率相对较低。碳水化合物结合结构域能特异性结合多糖底物,对提升糖类底物的酶催化效率起着关键的作用。本文从碳水化合物结合结构域的家族类型、结构类型、结构与功能关系以及与催化结构域的关系几个方面进行了综述,对阐明碳水化合物结合结构域与碳水化合物识别机制,进而将其广泛应用于生物和医疗领域具有重要意义。 相似文献
7.
Recent advances in the use of mass spectrometry for the determination of the molecular weight and sequencing of oligonucleotides
are disscussed. Matrix-assisted laser desorption (MALDI) and electrospray ionization (ESI) mass spectrometry have been shown
to be especially important techniques for both molecular weight assignment and sequencing of oligonucleotides, and are the
focus of this article which covers the literature through early 1996. 相似文献
8.
Notburga Pfabigan Eva Nordberg Karlsson Guenther Ditzelmueller Olle Holst 《Biotechnology letters》2002,24(14):1191-1197
Full-length and truncated forms of a modular thermostable xylanase (EC 3.2.1.8., glycoside hydrolase family 10) were used in bleaching sequences of hardwood and softwood kraft pulps. Enzymatic treatment led to brightness gains of all pulps but the result depended on the pulp source. The presence of the additional domains in the full-length enzyme (including carbohydrate-binding modules) did not improve the bleaching process. No significant change in viscosity was seen after enzyme treatments indicating an unaffected pulp fibre length. 相似文献
9.
Within the CAZy database, there are 81 carbohydrate‐binding module (CBM) families. A CBM represents a non‐catalytic domain in a modular arrangement of glycoside hydrolases (GHs). The present in silico study has been focused on starch‐binding domains from the family CBM41 that are usually part of pullulanases from the α‐amylase family GH13. Currently there are more than 1,600 sequences classified in the family CBM41, almost exclusively from Bacteria, and so a study was undertaken in an effort to divide the members into relevant groups (subfamilies) and also to contribute to the evolutionary picture of family CBM41. The CBM41 members adopt a β‐sandwich fold (～100 residues) with one carbohydrate‐binding site formed by the side‐chains of three aromatic residues that interact with carbohydrate. The family CBM41 can be divided into two basic subdivisions, distinguished from each other by a characteristic sequence pattern or motif of the three essential aromatics as follows: (i) “W‐W‐～10aa‐W” (the so‐called Streptococcus/Klebsiella‐type); and (ii) “W‐W‐～30aa‐W” (Thermotoga‐type). Based on our bioinformatics analysis it is clear that the first and second positions of the motif can be occupied by aromatic residues (Phe, Tyr, His) other than tryptophan, resulting in the existence of six different carbohydrate‐binding CBM41 groups, that reflect mostly differences in taxonomy, but which should retain the ability to bind an α‐glucan. In addition, three more groups have been proposed that, although lacking the crucial aromatic motif, could possibly employ other residues from remaining parts of their sequence for binding carbohydrate. Proteins 2017; 85:1480–1492. © 2017 Wiley Periodicals, Inc. 相似文献
10.
《Bioscience, biotechnology, and biochemistry》2013,77(3):530-535
Chitinase J from alkaliphilic Bacillus sp. J813 comprises a glycoside hydrolase (GH) family 18 catalytic domain (CatD), a fibronectin type III like domain, and a carbohydrate-binding module (CBM) family 5 chitin-binding domain (ChBD). It has been suggested that the ChBD binds to insoluble chitin and enhances its degradation by the CatD. To investigate the roles of two aromatic residues (Trp541 and Trp542), which are exposed on the surface of the ChBD, mutational analysis was performed. Single and double mutations of the two aromatic residues decreased binding and hydrolyzing abilities toward insoluble chitin. This result suggests that the ChBD binds to chitin by hydrophobic interactions via two surface-exposed aromatic residues. However, the double mutant, which has no such aromatic residue, bound to chitin at pH 5.2, probably by electrostatic interactions. Moreover, the ChBD bound to insoluble chitosan by electrostatic interactions. 相似文献
11.
12.
Shabir Najmudin Catarina I. P. D. Guerreiro Luís M. A. Ferreira Maria J. C. Romo Carlos M. G. A. Fontes Jos A. M. Prates 《Acta Crystallographica. Section F, Structural Biology Communications》2005,61(12):1043-1045
Clostridium thermocellum produces a highly organized multi‐enzyme complex of cellulases and hemicellulases for the hydrolysis of plant cell‐wall polysaccharides, which is termed the cellulosome. The bifunctional multi‐modular cellulase ctCel9D‐Cel44A is one of the largest components of the C. thermocellum cellulosome. The enzyme contains two internal catalytic domains belonging to glycoside hydrolase families 9 and 44. The C‐terminus of this cellulase, comprising a polycystic kidney‐disease module (PKD) and a carbohydrate‐binding module (CBM44), has been crystallized. The crystals belong to the tetragonal space group P43212, containing a single molecule in the asymmetric unit. Native and seleno‐l ‐methionine‐derivative crystals diffracted to 2.1 and 2.8 Å, respectively. 相似文献
13.
聚对苯二甲酸乙二醇酯(polyethylene terephthalate,PET)是全球产量最大的聚酯之一,但由于其废弃量大且难降解,环境污染严重。酶解法处理PET是目前的研究热点,碳水化合物结合模块(carbohydrate binding module,CBM)的加入可以增加酶与底物之间的亲和力,提高酶的降解能力。为了开发更高效的PET水解酶,本研究在PET降解酶LCC-ICCG中引入来源于不同家族具有不同底物亲和力的碳水化合物结合结构域(CBM),并以高结晶度PET粉末和无定型PET膜为底物,对改造后酶的降解效率进行了表征,以探寻降解能力最优的酶。结果表明,融合B型CBM后降低了酶对PET的降解率和Tm值,而引入A、C型CBM后,酶对膜状底物的降解率得到了显著提高,PET的降解速率和Tm值也得到了提升,尤其是融合酶LCC-ICCG-CBM9-2对膜状PET底物的降解率对比原始酶LCC-ICCG提高了10倍以上。本研究的结果表明,通过引入A、C两型CBM的方式,可以提高LCC-ICCG对膜状PET降解率和热稳定性,改善其活性低的问题,从而更有效地降解PET。本研究可为塑料降解提供技术支持,有利于推进环保事业。 相似文献
14.
Spirochaeta thermophila secretes seven glycoside hydrolases for plant biomass degradation that carry a carbohydrate‐binding module 64 (CBM64) appended at the C‐terminus. CBM64 adsorbs to various β1‐4‐linked pyranose substrates and shows high affinity for cellulose. We present the first crystal structure of a CBM64 at 1.2 Å resolution, which reveals a jelly‐roll‐like fold corresponding to a surface‐binding type A CBM. Modeling of its interaction with cellulose indicates that CBM64 achieves association with the hydrophobic face of β‐linked pyranose chains via a unique coplanar arrangement of four exposed tryptophan side chains. Proteins 2016; 84:855–858. © 2016 Wiley Periodicals, Inc. 相似文献
15.
Structural characterization of peptides in the range of 500–5000 Da, using fast atom bombardment (FAB) and Cs+ ion liquid secondary ion mass spectrometry (SIMS), is reviewed. These include syntheitc peptides Kemptamide (mol wt 1516); GIF-C15 (mol wt 1875), an isolated natural product as an acylated pentapeptide; and polypeptides generated from enzymatic digests of proteins. MS data is shown to reveal molecular weight and sequence information as well as determine disulfide bonds between cysteine residues and glycosylation sites in the case of a glycopeptide. The complementarity of MS technique to classical biochemical methods for peptide characterization is highlighted. The reader is briefly acquainted with two newer ionization techniques namely, electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). Synthetic chemists and biochemists can refer to the in-depth review articles that are cited throughout this article. 相似文献
16.
Immacolata Venditto Helena Santos James Sandy Juan Sanchez‐Weatherby Luis M. A. Ferreira Kazuo Sakka Carlos M. G. A. Fontes Shabir Najmudin 《Acta Crystallographica. Section F, Structural Biology Communications》2014,70(12):1628-1630
Cellulases catalyze the hydrolysis of cellulose, the major constituent of plant biomass and the most abundant organic polymer on earth. Cellulases are modular enzymes containing catalytic domains connected, via linker sequences, to noncatalytic carbohydrate‐binding modules (CBMs). A putative modular endo‐β‐1,4‐glucanase (BhCel5B) is encoded at locus BH0603 in the genome of Bacillus halodurans. It is composed of an N‐terminal glycoside hydrolase family 5 catalytic module (GH5) followed by an immunoglobulin‐like module and a C‐terminal family 46 CBM (BhCBM46). Here, the crystallization and preliminary X‐ray diffraction analysis of the trimodular BhCel5B are reported. The crystals of BhCel5B belonged to the orthorhombic space group P2121 2 and data were processed to a resolution of 1.64 Å. A molecular‐replacement solution has been found. 相似文献
17.
Tae‐Wan Kim Harshal A. Chokhawala Dana Nadler Harvey W. Blanch Douglas S. Clark 《Biotechnology and bioengineering》2010,107(4):601-611
Improving the catalytic activity of cellulases requires screening variants against solid substrates. Expressing cellulases in microbial hosts is time‐consuming, can be cellulase specific, and often leads to inactive forms and/or low yields. These limitations have been obstacles for improving cellulases in a high‐throughput manner. We have developed a cell‐free expression system and used it to express 54 chimeric bacterial and archaeal endoglucanases (EGs), with and without cellulose binding modules (CBMs) at either the N‐ or C‐terminus, in active enzyme yields of 100–350 µg/mL. The platform was employed to systematically study the role of CBMs in cellulose hydrolysis toward a variety of natural and pretreated solid substrates, including ionic‐liquid pretreated Miscanthus and AFEX‐pretreated corn stover. Adding a CBM generally increased activity against crystalline Avicel, whereas for pretreated substrates the effect of CBM addition depended on the source of cellulase. The cell‐free expression platform can thus provide insights into cellulase structure‐function relationships for any substrate, and constitutes a powerful discovery tool for evaluating or engineering cellulolytic enzymes for biofuels production. Biotechnol. Bioeng. 2010;107:601–611. © 2010 Wiley Periodicals, Inc. 相似文献
18.
Bruna Medeia Campos Marcelo Vizona Liberato Igor Polikarpov Ana Carolina de Mattos Zeri Fabio Marcio Squina 《Acta Crystallographica. Section F, Structural Biology Communications》2015,71(3):311-314
In recent years, biofuels have attracted great interest as a source of renewable energy owing to the growing global demand for energy, the dependence on fossil fuels, limited natural resources and environmental pollution. However, the cost‐effective production of biofuels from plant biomass is still a challenge. In this context, the study of carbohydrate‐binding modules (CBMs), which are involved in guiding the catalytic domains of glycoside hydrolases to polysaccharides, is crucial for enzyme development. Aiming at the structural and functional characterization of novel CBMs involved in plant polysaccharide deconstruction, an analysis of the CAZy database was performed and CBM family 64 was chosen owing to its capacity to bind with high specificity to microcrystalline cellulose and to the fact that is found in thermophilic microorganisms. In this communication, the CBM‐encoding module named StX was expressed, purified and crystallized, and X‐ray diffraction data were collected from native and derivatized crystals to 1.8 and 2.0 Å resolution, respectively. The crystals, which were obtained by the hanging‐drop vapour‐diffusion method, belonged to space group P3121, with unit‐cell parameters a = b = 43.42, c = 100.96 Å for the native form. The phases were found using the single‐wavelength anomalous diffraction method. 相似文献
19.
20.
Schulz DM Kalkhof S Schmidt A Ihling C Stingl C Mechtler K Zschörnig O Sinz A 《Proteins》2007,69(2):254-269
During the past few years, the structural analysis of proteins and protein complexes by chemical crosslinking and mass spectrometry has enjoyed increasing popularity. With this approach we have investigated the quaternary structure of the complex between annexin A2 and p11, which is involved in numerous cellular processes. Although high-resolution data are available for both interaction partners as well as for the complex between two p11 subunits and two annexin A2 N-terminal peptides, the structure of the complete annexin A2/p11 heterotetramer has not yet been solved at high resolution. Thus, the quaternary structure of the biologically relevant, membrane-bound annexin A2/p11 complex is still under discussion, while the existence of a heterotetramer or a heterooctamer is the prevailing opinion. We gained further insight into the spatial organization of the annexin A2/p11 heterotetramer by employing chemical crosslinking combined with high-resolution mass spectrometry. Furthermore, tandem mass spectrometry served as a tool for an exact localization of crosslinked amino acid residues and for a confirmation of crosslinked product assignment. On the basis of distance constraints from the crosslinking data we derived structural models of the annexin A2/p11 heterotetramer by computational docking with Rosetta. We propose an octameric model for the annexin A2/p11 complex, which exerts annexin A2 function. The proposed structure of the annexin A2/p11 octamer differs from so far suggested models and sheds new light into annexin A2/p11 interaction. 相似文献