Regulation of protein secretion by mycelial culture of the mushroom Termitomyces clypeatus

Termitomyces clypeatus secreted a 24-kDa xylanase constitutively in xylan medium, but required a gluconeogenic amino acid or Krebs cycle acid for the secretion of a 56-kDa amyloglucosidase in dextrin medium. Aspartate, glutamate, succinate and fumarate all increased secretion of amyloglucosidase from 50% to >90% and enzyme production by 10-fold with little effect on xylanase production. Glutamate or succinate stimulated in vitro release of intracellular amyloglucosidase from washed mycelia in the presence of cycloheximide. Amyloglucosidase accumulated in the absence of glutamate was a high-molecular-mass protein that did not migrate in PAGE. Cellular regulation by the fungus of the secretion of amyloglucosidase is indicated.     

Addition of substrate-binding domains increases substrate-binding capacity and specific activity of a chitinase from Trichoderma harzianum

Chitinase Chit42 from Trichoderma harzianum CECT 2413 is considered to play an important role in the biocontrol activity of this fungus against plant pathogens. Chit42 lacks a chitin-binding domain (ChBD). We have produced hybrid chitinases with stronger chitin-binding capacity by fusing to Chit42 a ChBD from Nicotiana tabacum ChiA chitinase and the cellulose-binding domain from cellobiohydrolase II of Trichoderma reesei. The chimeric chitinases had similar activities towards soluble substrate but higher hydrolytic activity than the native chitinase on high molecular mass insoluble substrates such as ground chitin or chitin-rich fungal cell walls.     

梭热杆菌纤维小体研究进展

梭热杆菌(Clostridium thermocellum)是一种嗜热厌氧细菌,通过分泌大量纤维素酶高效降解纤维素.根据作用纤维素的不同部位,梭热杆菌分泌的纤维素酶分为内切纤维素酶和外切纤维素酶.纤维小体是由支架蛋白、锚定元件、黏合蛋白、纤维素结合域和催化单位组成的复合体,其独特的结构,使得它可以比真菌纤维素酶更紧密地结合到纤维素表面,这个复合结构结合着多种催化单位,而此特殊的结构是梭热杆菌高效降解纤维素的必要条件.近年来,为更深入透彻地了解纤维小体的结构与功能进行了大量的研究工作,现对相关研究进展进行综述,并给出了未来可能的发展方向.     

Cloning of a Clostridium thermocellum DNA fragment encoding polypeptides that bind the catalytic components of the cellulosome

A test based on the binding of 125I-labelled endoglucanase CelD was used to clone a DNA region encoding at least two different polypeptides that interact with the conserved reiterated segment present in many catalytic components of the Clostridium thermocellum cellulosome. One of the polypeptides corresponds to the COOH-terminal region of the SL (or S1) component of the cellulosome (U.T. Gerngross and A.L. Demain, personal communication). It comprises repeated domains that are responsible for binding 125I-labelled CelD, and presumably represent anchoring sites for the various catalytic components of the cellulosome. The other polypeptide is encoded by a gene that has not yet been described.     

Identification of the cellulose-binding domain of the cellulosome subunit S1 from Clostridium thermocellum YS

The 3' region of a gene designated cipB, which shows strong homology with cipA that encodes the cellulosome SL subunit of Clostridium thermocellum ATCC 27405, was isolated from a gene library of C. thermocellum strain YS. The truncated S1 protein encoded by the cipB derivative bound tightly to cellulose. The cellulose-binding domain in this polypeptide consisted of a C-terminal proximal 167 residue sequence which showed complete identity with residues 337-503 of mature SL from C. thermocellum strain ATCC 27405. The cellulose-binding domain interacted with both crystalline and amorphous cellulose, but not with xylan.     

Cellulases and hemicellulases of the anaerobic fungus Piromyces constitute a multiprotein cellulose-binding complex and are encoded by multigene families

Abstract More than 80% of the extracellular Avicelase, endoglucanase, xylanase and mannanase activities of the anaerobic fungus Piromyces were associated with a cellulose-binding complex. The complex was composed of at least 10 polypeptides ranging in size from 190 kDa to 50 kDa, and contained numerous endoglucanases, xylanases and mannanases. Multiple genes encoding each of these activities were isolated from an expressing cDNA library.     

Cellulose degradation by Clostridium thermocellum: From manure to molecular biology

Clostridium thermocellum, a Gram-positive, thermophilic anaerobe produces a highly active cellulase system. This system, termed the cellulosome, is a complex composed of at least 14-18 different types of components organized around a large, cellulose-binding protein. Combining recombinant DNA technology and protein biochemistry has proved to be a successful approach in unravelling some important features of the system.     

Homology modeling of the cellulose-binding domain of a pollen allergen from rye grass: structural basis for the cellulose recognition and associated allergenic properties

A three-dimensional model of the cellulose-binding domain of the rye-grass pollen allergen Lol pI built by homology modeling is proposed as a structural scaffold for expansins and other expansin-related proteins. A groove and an extended strip of aromatic and polar residues presumably account for the cellulose-binding properties of the protein domain. Two of the four predicted T-cell epitopes readily exposed on the surface of the cellulose-binding domain match with previously reported IgE-binding regions. A close structural relationship occurs between the cellulose-binding and allergenic properties.     

The effect of the cellulose-binding domain from Clostridium cellulovorans on the supramolecular structure of cellulose fibers

The cellulose-binding domain (CBD) is the second important and the most wide-spread element of cellulase structure involved in cellulose transformation with a great structural diversity and a range of adsorption behavior toward different types of cellulosic materials. The effect of the CBD from Clostridium cellulovorans on the supramolecular structure of three different sources of cellulose (cotton cellulose, spruce dissolving pulp, and cellulose linters) was studied. Fourier-transform infrared spectroscopy (FTIR) was used to record amides I and II absorption bands of cotton cellulose treated with CBD. Structural changes as weakening and splitting of the hydrogen bonds within the cellulose chains after CBD adsorption were observed. The decrease of relative crystallinity index of the treated celluloses was confirmed by FTIR spectroscopy and X-ray diffraction (XRD). X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) were used to confirm the binding of the CBD on the cellulose surface and the changing of the cellulose morphology.     

Studies on the anaerobic degradation of crystalline cellulose by Clostridium thermocellum using a new assay

Abstract The anaerobic degradation of microcrystalline cellulose by thermostable cellulolytic enzyme complexes from Clostridium thermocellum JW20 (ATCC 31449) was monitored. For quantitative investigations as enzyme-coupled spectrophotometric assay has been developed. The assay allows for the evaluation of the release of cellubiose-/glucose-units from native cellulose. Kinetic studies revealed that the anaerobic breakdown of crystalline cellulose (CC) at 60°C follows Michaelis-Menten kinetics K _m CC values have been determined for different aggregation states of the cellulolytic complex. The presented assay seems well suited to screen for CC-degrading enzymes of various sources, and to further explore the mechanism of CC-breakdown.