Сarbohydrate binding module CBM28 of endoglucanase Cel5D from Caldicellulosiruptor bescii recognizes crystalline cellulose.

Optimal catalytic activity of endoglucanase Cel5D from the thermophilic anaerobic bacterium Caldicellulosiruptor bescii requires the presence of a carbohydrate-binding module of family 28, CbCBM28. The binding properties of CbСВМ28 with cello-, laminari-, xylo- and chito-oligosaccharides were studied by isothermal titration calorimetry. CbСВМ28 bound only cello-oligosaccharides comprising at least four glucose residues with binding constants of 2.5·104 and 2.2·106M-1 for cellotetraose and cellohexaose, respectively. The interaction between CbСВМ28 and amorphous cellulose is best described by a two-binding-site model with the binding constants of 1.5·105 and 1.9·105M-1. In a competitive binding assay in the presence of a 10-fold excess of cellohexaose the binding constant of CbСВМ28 to amorphous cellulose was 1.9·105M-1. A two-binding-site model also better approximates the binding to Avicel with the binding constants of 8.3·105 and 3.2·104M-1; while in the presence of cellohexaose, the binding is described by a single-binding-site model with the binding constant of 2.3·104M-1. With CbСВМ28 binding to bacterial crystalline cellulose with a constant of 7.4·104M-1, this is the first report of such a strong binding to crystalline cellulose for a module of family 28.

Crystallization and preliminary X-ray diffraction studies of the family 54 carbohydrate-binding module from laminarinase (ß-1,3-glucanase) Lic16A of Clostridium thermocellum.

The crystallization and preliminary X-ray diffraction analysis of the carbohydrate-binding module (CBM) from laminarinase Lic16A of the hyperthermophilic anaerobic bacterium Clostridium thermocellum (ctCBM54) are reported. Recombinant ctCBM54 was prepared using an Escherichia coli/pQE30 overexpression system and was crystallized by the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 2.1 Å resolution using synchrotron radiation. The crystals belonged to space group P6322, with unit-cell parameters a = b = 130.15, c = 131.05 Å. The three-dimensional structure of ctCBM54 will provide valuable information about the structure-function relation of the laminarinase Lic16A and will allow the exploitation of this binding module in biotechnological applications.

Carbohydrate-binding properties of a separately folding protein module from beta-1,3-glucanase Lic16A of Clostridium thermocellum.

The multi-modular non-cellulosomal endo-1,3(4)-beta-glucanase Lic16A from Clostridium thermocellum contains a so-called X module (denoted as CBMX) near the N terminus of the catalytic module (191-426 aa). Melting of X-module-containing recombinant proteins revealed an independent folding of the module. CBMX was isolated and studied as a separate fragment. It was shown to bind to various insoluble polysaccharides, including xylan, pustulan, chitin, chitosan, yeast cell wall glucan, Avicel and bacterial crystalline cellulose. CBMX thus contains a hitherto unknown carbohydrate-binding module (CBM54). It did not bind soluble polysaccharides on which Lic16A is highly active. Ca2+ ions had effects on the binding, e.g. stimulated complex formation with chitosan, which was observed only in the presence of Ca2+. The highest affinity to CBMX was shown for xylan (binding constant K=3.1x10(4) M(-1)), yeast cell wall glucan (K=1.4x10(5) M(-1)) and chitin (K=3.3.10(5) M(-1) in the presence of Ca2+). Lic16A deletion derivatives lacking CBMX had lower affinity to lichenan and laminarin and a slight decrease in optimum temperature and thermostability. However, the specific activity was not significantly affected.

Co-expression of the Thermotoga neapolitana aglB gene with an upstream 3'-coding fragment of the malG gene improves enzymatic characteristics of recombinant AglB cyclomaltodextrinase.

A cluster of Thermotoga neapolitana genes participating in starch degradation includes the malG gene of sugar transport protein and the aglB gene of cyclomaltodextrinase. The start and stop codons of these genes share a common overlapping sequence, aTGAtg. Here, we compared properties of expression products of three different constructs with aglB from T. neapolitana. The first expression vector contained the aglB gene linked to an upstream 90-bp 3'-terminal region of the malG gene with the stop codon overlapping with the start codon of aglB. The second construct included the isolated coding sequence of aglB with two tandem potential start codons. The expression product of this construct in Escherichia coli had two tandem Met residues at its N terminus and was characterized by low thermostability and high tendency to aggregate. In contrast, co-expression of aglB and the 3'-terminal region of malG (the first construct) resulted in AglB with only one N-terminal Met residue and a much higher specific activity of cyclomaltodextrinase. Moreover, the enzyme expressed by such a construct was more thermostable and less prone to aggregation. The third construct was the same as the second one except that it contained only one ATG start codon. The product of its expression had kinetic and other properties similar to those of the enzyme with only one N-terminal Met residue.

Lic16A of Clostridium thermocellum, a non-cellulosomal, highly complex endo-beta-1,3-glucanase bound to the outer cell surface.

Clostridium thermocellum produces one major beta-1,3-glucanase. Genomic DNA fragments containing the gene were cloned from two strains, DSM1237(T) (6848 bp) and F7 (9766 bp). Overlapping sequences were 99.9 % identical. The nucleotide sequences contained reading frames for a putative transposase, endo-beta-1,3-1,4-glucanase CelC, a putative transcription regulator of the LacI type, beta-1,3-glucanase Lic16A and a putative membrane protein. The licA genes of both strains encoded an identical protein of 1324 aa with a calculated molecular mass of 148 kDa. Lic16A is an unusually complex protein consisting of a leader peptide, a threefold repeat of an S-layer homologous module (SLH), an unknown module, a catalytic module of glycosyl hydrolase family 16 and a fourfold repeat of a carbohydrate-binding module of family CBM4a. The recombinant Lic16A protein was characterized as an endo-1,3(4)-beta-glucanase with a specific activity of 2680 and 340 U mg(-1) and a K(m) of 0.94 and 2.1 mg ml(-1) towards barley beta-glucan and laminarin, respectively. It was specific for beta-glucans containing beta-1,3-linkages with an optimum temperature of 70 degrees C at pH 6.0. The N-terminal SLH modules were cleaved from the protein as well in Escherichia coli as in C. thermocellum, but nevertheless bound tightly to the rest of the protein. Lic16A was located on the cell surface from which it could be purified after fractionated solubilization. Its inducible production allowed C. thermocellum to grow on beta-1,3- or beta-1,3-1,4-glucan.

Two new cellulosome components encoded downstream of celI in the genome of Clostridium thermocellum: the non-processive endoglucanase CelN and the possibly structural protein CseP.

Clostridium thermocellum produces a great number of extracellular cellulases which are free or cellulosome-bound. The nucleotide sequence of a gene cluster containing the genes celI, celN and cseP was determined from C. thermocellum strain F7. Gene products Cel9I and Cel9N are structurally related enzymes having a glycosyl hydrolase family 9 and a carbohydrate-binding module (CBM3c), but show characteristic differences: Cel9I is a non-cellulosomal protein with an additional CBM (CBM3b), whereas Cel9N contains a cellulosomal dockerin module and no additional CBM. Although Cel9I is a processive endoglucanase, Cel9N is non-processive. Both enzymes hydrolyse phosphoric acid swollen cellulose, but the products of hydrolysis are different. The CseP protein encoded in the gene cluster is the first component attached to the cellulosomal scaffoldin for which no catalytic activity could be detected. It was shown to be present in the cellulosome. Its sequence is homologous to the spore-coat assembly protein CotH of Bacillus subtilis, suggesting a structural role of CseP in the cellulosome.

A newly described cellulosomal cellobiohydrolase, CelO, from Clostridium thermocellum: investigation of the exo-mode of hydrolysis, and binding capacity to crystalline cellulose.

The sequence of the celO gene from Clostridium thermocellum F7 was determined. The gene product, cellulase CelO (Ct-Cel5F), had a modular structure consisting of a carbohydrate-binding module of the CBM3 family and a catalytic domain of the glycosyl hydrolase family 5. The presence of the dockerin module indicated that the enzyme was a component of the cellulosome complex. The thermostable recombinant gene product was active on cellodextrins, barley beta-glucan, carboxymethylcellulose and insoluble cellulose. Cellobiose was the only product released from amorphic and crystalline cellulose, cellotetraose and higher cello-oligosaccharides, identifying CelO as a cellobiohydrolase. The cleavage pattern of p-nitrophenyl beta-D-cellotetraoside, blockage of the hydrolysis of NaBH(4)-reduced cellopentaose and the reduction in substrate viscosity suggested activity from the reducing end in a processive mode after making random cuts. Binding to insoluble, i.e. amorphous, and crystalline cellulose was mediated by the carbohydrate-binding module CBM3b, with a preference for the crystalline substrate.

The binding pattern of two carbohydrate-binding modules of laminarinase Lam16A from Thermotoga neapolitana: differences in beta-glucan binding within family CBM4.

Carbohydrate-binding modules (CBMs) are often part of the complex hydrolytic extracellular enzymes from bacteria and may modulate their catalytic activity. The thermostable catalytic domain of laminarinase Lam16A from Thermotoga neapolitana (glycosyl hydrolase family 16) is flanked by two CBMs, 148 and 161 aa long. They share a sequence identity of 30%, are homologous to family CBM4 and are thus called CBM4-1 and CBM4-2 respectively. Recombinant Lam16A proteins deleted for one or both binding modules and the isolated module CBM4-1 were characterized. Proteins containing the N-terminal module CBM4-1 bound to the soluble polysaccharides laminarin (1,3-beta-glucan) and barley 1,3/1,4-beta-glucan, and proteins containing the C-terminal module CBM4-2 bound additionally to curdlan (1,3-beta-glucan) and pustulan (1,6-beta-glucan), and to insoluble yeast cell wall beta-glucan. The activity of the catalytic domain on soluble 1,3-beta-glucans was stimulated by the presence of CBM4-1, whereas the presence of CBM4-2 enhanced the Lam16A activity towards gelatinized and insoluble or mixed-linkage 1,3-beta-glucan. Thermostability of the catalytic domain was not affected by the truncations. Members of family CBM4 can be divided into four subfamilies, members of which show different polysaccharide-binding specificities corresponding to the catalytic specificities of the associated hydrolytic domains.