Purification and cellulosomal localization ofClostridium thermocellum mixed linkage ?-glucanase LicB (1,3?1,4-?-D-glucanase)

Zverlov, Vladimir V.; Fuchs, Klaus-Peter; Schwarz, Wolfgang H.; Velikodvorskaya, G. A.

doi:10.1007/bf01022619

Cited by 23 publications

(12 citation statements)

References 16 publications

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“…XynD was detected exclusively on cellobiose even though it had been discovered on cellulose by MS (50), and ManA and LicB were detected exclusively on Avicel, whereas they had previously been observed on cellobiose by Western blot analysis (15,49). These discrepancies could be explained by the differences between the protein identification methods used in the previous studies and the method used in the present work.…”

Section: Discussioncontrasting

confidence: 54%

Global View of the Clostridium thermocellum Cellulosome Revealed by Quantitative Proteomic Analysis

2007

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A metabolic isotope-labeling strategy was used in conjunction with nano-liquid chromatography-electrospray ionization mass spectrometry peptide sequencing to assess quantitative alterations in the expression patterns of subunits within cellulosomes of the cellulolytic bacterium Clostridium thermocellum, grown on either cellulose or cellobiose. In total, 41 cellulosomal proteins were detected, including 36 type I dockerin-containing proteins, which count among them all but three of the known docking components and 16 new subunits. All differential expression data were normalized to the scaffoldin CipA such that protein per cellulosome was compared for growth between the two substrates. Proteins that exhibited higher expression in cellulosomes from cellulose-grown cells than in cellobiose-grown cells were the cell surface anchor protein OlpB, exoglucanases CelS and CelK, and the glycoside hydrolase family 9 (GH9) endoglucanase CelJ. Conversely, lower expression in cellulosomes from cells grown on cellulose than on cellobiose was observed for the GH8 endoglucanase CelA; GH5 endoglucanases CelB, CelE, CelG; and hemicellulases XynA, XynC, XynZ, and XghA. GH9 cellulases were the most abundant group of enzymes per CipA when cells were grown on cellulose, while hemicellulases were the most abundant group on cellobiose. The results support the existing theory that expression of scaffoldin-related proteins is coordinately regulated by a catabolite repression type of mechanism, as well as the prior observation that xylanase expression is subject to a growth rate-independent type of regulation. However, concerning transcriptional control of cellulases, which had also been previously shown to be subject to catabolite repression, a novel distinction was observed with respect to endoglucanases.Clostridium thermocellum, a thermophilic, strictly anaerobic gram-positive bacterium, has the highest rate of cellulose utilization of any bacterium, and for this reason it is deemed of great significance to the pursuit of biofuel production from the cellulosic materials in plant biomass (3,6,20,32). The organism achieves hydrolysis of crystalline cellulose by virtue of a large cell surface-bound protein complex known as the cellulosome, the structure of which consists of a central noncatalytic scaffoldin protein (CipA) bearing up to nine catalytic subunits (44). The attachment of a given subunit is mediated by the interaction of its type I dockerin (Doc1) domain with one of the nine cohesin type I domains of CipA (26). CipA is, in turn, bound to the cell surface by virtue of the interaction of its type II dockerin domain with the type II cohesin domain of one of three S-layer anchor proteins, SdbA, Orf2p, or OlpB (6). CipA also contains a type III cellulose-binding module for attachment of the complex to cellulose (13).Previous studies have shown that cellulolytic activity in C. thermocellum is regulated by either carbon source or growth rate (or both) and that changes with respect to one or the other are reflected in overall cellulase ...

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Section: Discussioncontrasting

confidence: 54%

Global View of the Clostridium thermocellum Cellulosome Revealed by Quantitative Proteomic Analysis

2007

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“…The modular structure of the licA gene Orf5 encodes laminarinase A and is called licA due to the high activity of the gene product on lichenan which it shares with LicB (Zverlov et al, 1994). A putative ribosome-binding site is located at base 4632 upstream of a coding sequence starting at ATG (base 4645) and leading to a large protein of 1323 aa (148 116 Da).…”

Section: Other Reading Framesmentioning

confidence: 99%

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

Fuchs¹,

Zverlov

Velikodvorskaya

et al. 2003

Microbiology

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Clostridium thermocellum produces one major b-1,3-glucanase. Genomic DNA fragments containing the gene were cloned from two strains, DSM1237T (6848 bp) and F7 (9766 bp).Overlapping sequences were 99?9 % identical. The nucleotide sequences contained reading frames for a putative transposase, endo-b-1,3-1,4-glucanase CelC, a putative transcription regulator of the LacI type, b-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)-b-glucanase with a specific activity of 2680 and 340 U mg 21 and a K m of 0?94 and 2?1 mg ml 21 towards barley b-glucan and laminarin, respectively. It was specific for b-glucans containing b-1,3-linkages with an optimum temperature of 70˚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 b-1,3-or b-1,3-1,4-glucan.

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“…If eventually two cuts are near enough to each other, a short oligosaccharide is produced which is soluble and can diffuse away, thus removing the noncellulosic matrix from the surface of the cellulose crystals and allowing access of the cellulases. It is therefore no surprise that the cellulosome of C. thermocellum contains a number of non-cellulolytic enzymes, namely xylanases as well as 1,3-1,4-b-glucanases, a mannanase and a chitinase, which make up about half of the cellulosomal protein (Hayashi et al, 1997(Hayashi et al, , 1999Hallstead et al, 1999;Blum et al, 2000;Kurokawa et al, 2001;Zverlov et al, 1994Zverlov et al, , 2002Zverlov et al, , 2005.…”

Section: Introductionmentioning

confidence: 99%

Two new major subunits in the cellulosome of Clostridium thermocellum: xyloglucanase Xgh74A and endoxylanase Xyn10D

et al. 2005

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The structure and enzymic activity of xyloglucanase Xgh74A and endoxylanase Xyn10D, components in the cellulosomes of cellulose-grown Clostridium thermocellum, were determined. Xyn10D is a thermostable endo-1,4-β-xylanase with a module composition identical to Xyn10C (CBM22-GH10-Doc). It hydrolyses xylan and mixed-linkage 1,3-1,4-β-glucan with a temperature optimum of 80 °C. Xyloglucanase Xgh74A contains a catalytic module of GHF74 in addition to a C-terminal dockerin module. It hydrolyses every fourth β-1,4-glucan bond in the xyloglucan backbone, thus producing decorated cellotetraose units. Its low activity on CMC and lack of activity on amorphous cellulose indicates recognition of the xylosidic side chains present in xyloglucan, which is readily hydrolysed (295 U mg−1). The pattern of the hydrolysis products from tamarind xyloglucan resembles that of other GHF74 xyloglucan endoglucanases. The data indicate that Xgh74A and Xyn10D contribute to the in vivo degradation of the hemicelluloses xyloglucan and xylan by the cellulosome of C. thermocellum. Xgh74A is the first xyloglucanase identified in C. thermocellum and the only enzyme in the cellulosome that hydrolyses tamarind xyloglucan.

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Purification and cellulosomal localization ofClostridium thermocellum mixed linkage ?-glucanase LicB (1,3?1,4-?-D-glucanase)

Cited by 23 publications

References 16 publications

Global View of the Clostridium thermocellum Cellulosome Revealed by Quantitative Proteomic Analysis

Global View of the Clostridium thermocellum Cellulosome Revealed by Quantitative Proteomic Analysis

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

Two new major subunits in the cellulosome of Clostridium thermocellum: xyloglucanase Xgh74A and endoxylanase Xyn10D

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