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1.
Recombinant plasmid pM25 containing the celE gene of Clostridium thermocellum, which codes for an enzymatically active endoglucanase, was transformed into Lactobacillus plantarum by electroporation. Strains harboring pM25 expressed thermostable endoglucanase, which was found predominantly in the culture medium. Two other plasmids, pGK12 and pSA3, were transformed into L. plantarum, and the stability of each plasmid was evaluated.  相似文献   

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The endoglucanase gene of Clostridium thermocellum F7 was cloned in Escherichia coli cells using pKM4 vector. Both the physical mapping and analysis of the gene products in the E. coli mini-cells system suggest cloning of a new cel gene different from those described earlier. The activity of endoglucanase in E. coli cells is localized in the periplasm, which correlates with secretion of enzymes of this type in C. thermicellum. Apart from 2 major components with Mr 42.5 and 43 kDa, corresponding to mature protein forms, we observed the formation of minor products of various electrophoretic motilities. Cloning of the endoglucanase gene on bhr vector pBS954 controlled by its own regulatory signal yielded high level of the endoglucanase activity in the recombinant strains of Klebsiella pneumoniae, Serratia marcescens and Erwinia carotovora comparable with the level of the gene expression in E. coli cells.  相似文献   

5.
Six independent and distinct cel genes coding endoglucanases have been selected from C. thermocellum pUC19-based gene bank in E. coli TG1. E. coli-derived Cel-proteins possessing Mr from 39,000 to 61,000 are able to cleave lichenan, as well as xylan and carboxymethyl cellulose. Cel 7- and Cel 8-endoglucanases are characterized by cellobiohydrolase type substrate specificity, being able to cleave model fluorogenic aryldisaccharide substrate MU-G2. The clone pCU110 (cel 7) produces about 10-fold more endoglucanase activity than other clones.  相似文献   

6.
The nucleotide sequence of the CelB gene, encoding the extracellular endoglucanase B of Clostridium thermocellum, is reported. The putative start of the 1689 bp coding sequence was assigned to an ATG codon which is preceded by an AGGAGG sequence typical of ribosomal binding sites in Gram-positive bacteria. The amino-terminal end of the deduced protein sequence is similar to signal peptides described for other bacterial secretory proteins. The carboxy-terminal ends of endoglucanases A and B appear to be remarkably homologous. A striking feature of the conserved region is that both proteins contain two reiterated stretches of 23 aminoacids each, separated by 9 residues.  相似文献   

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Abstract Growth and production of cellulosome by three strains (YS, LQRI and NCIB 10682) of Clostridium thermocellum were compared using Avicel (microcrystalline cellulose) and cellobiose as carbon sources. All three strains grew faster on cellobiose than on Avicel and produced 0.71–0.74 IU of endoglucanase/ml compared to 0.88–1.18 IU/ml on Avicel. Also, the cellulase produced by these strains in the presence of 0.2–1% cellobiose and Avicel, when compared on the basis of equal units of endoglucanase (0.5 IU), degraded cotton almost completely. SDS-PAGE further confirmed the production of cellulosome by all three strains when grown on cellobiose and Avicel. Thus, the cellobiose, like Avicel, acts as a true inducer of cellulosome in C. thermocellum .  相似文献   

9.
An endoglucanase (1,4-beta-D-glucan glucanohydrolase, EC 3.2.1.4) was purified from Clostridium thermocellum by procedures that included centrifugation, ultrafiltration, selective precipitation, ion-exchange Sephadex chromatography and preparative gel electrophoresis. The 22-fold-purified enzyme behaved as a homogeneous protein under non-denaturing conditions. The enzyme represented a significant component (greater than 25%) of total extracellular endoglucanase activity, but was purified in low yield by the procedures employed. The native molecular weight of the endoglucanase was determined by ultracentrifugational analysis, amino acid composition and polyacrylamide-gel electrophoresis, and varied between 83000 and 94000. The enzyme contained 11.2% carbohydrate and was isoelectric at pH 6.72. The pH and temperature optima of the endoglucanase were 5.2 and 62 degrees C respectively. The enzyme lacked cysteine and was low in sulphur-containing amino acids. The purified endoglucanase displayed: high activity towards carboxymethylcellulose, celloheptaose, cellohexaose and cellopentaose; low activity towards Avicel microcrystalline cellulose and cellotetraose; no detectable activity towards cellotriose or cellobiose; increased activity towards cello-oligosaccharides with increasing degree of polymerization. The internal glycosidic bonds of cello-oligosaccharides were cleaved by the enzyme in preference to external linkages. The apparent Michaelis constant ([S]0.5V) and Vmax. for cellopentaose and cellohexaose hydrolysis were 2.30 mM and 39.3 mumol/min per mg of protein, and 0.56 mM and 58.7 mumol/min per mg of protein, respectively.  相似文献   

10.
G Joliff  P Bguin    J P Aubert 《Nucleic acids research》1986,14(21):8605-8613
The nucleotide sequence of the celD gene, encoding the previously crystallized endoglucanase D of Clostridium thermocellum, is reported. The enzyme shares a conserved, reiterated domain with the COOH-terminal end of endoglucanases A and B from the same organism. The overexpression in Escherichia coli of celD subcloned in pUC8 appears to result from a translational fusion of the NH2-terminal end of the endoglucanase with the NH2-terminal end of beta-galactosidase.  相似文献   

11.
Summary The native promoter of a xylanase gene isolated from Clostridium thermocellum was replaced with a strong promoter screened from Bacillus subtilis chromosomes. A part of the C-terminal region of the gene which is not related to the xylanase activity was removed. With the modified xylanase gene, B. subtilis was transformed and grown in LB medium. The xylanase gene was expressed well in B. subtilis and extracellular xylanase was produced up to 30 units per ml when the growth reached OD600 of 4.8.  相似文献   

12.
Clostridium thermocellum endoglucanase D (EC 3.2.1.4: EGD), which is encoded by the celD gene, was found to bind Ca2+ with an association constant of 2.03 x 10(6) M-1. Ca2+ stimulated the activity of EGD towards swollen Avicel by 2-fold. In the presence of Ca2+, the Kd of the enzyme towards p-nitrophenyl-beta-D-cellobioside and carboxymethylcellulose was decreased by 4-fold. Furthermore, Ca2+ increased the half-life of the enzyme at 75 degrees C from 13 to 47 min. Since the 3' sequence of celD encodes a duplicated region sharing similarities with the Ca2+-binding site of several Ca2+-binding proteins, a deleted clone was constructed and used to purify a truncated form of the enzyme which no longer contained the duplicated region. The truncated enzyme was very similar to EGD expressed from the intact gene with respect to activity, Ca2(+)-binding kinetics and Ca2+ effects on substrate binding and thermostability. Thus the latter parameters do not appear to be mediated through the duplicated conserved region.  相似文献   

13.
Endoglucanase 7 (EG7) of Clostridium thermocellum was isolated from a recombinant strain of Escherichia coli TG1 cells harbouring recombinant plasmid pCU110 containing the cel7 gene of C. thermocellum. The enzyme was purified to electrophoretic homogeneity and was presented as two components with a molecular mass of 49 and 47 kDa and a pI of 4.35 and 4.30, respectively. The enzyme was shown to have optimum pH of 5.5 and optimum temperature of 55–60° C with carboxymethylcellulose (CMC) as a substrate. EG7 displayed hyper-lichenase, high CMCase, low cellobiosidase and negligibly small activities towards Avicel, amorphous cellulose, laminarin and xylan. The enzyme was shown to be stable at 55° C and within a broad range of pH from 4.5 to 11.0. It is insensitive towards ethanol (up to 5%) and end-product (cellobiose or glucose) inhibition. The hydrophobic nature of the protein, as revealed by retarded elution on gel filtration columns, resulted in an unprecedentedly high yield (about 80%) of purified enzyme. Due to the above-mentioned characteristics, the enzyme should to be quite suitable for use in the mashing process of beer brewing.Correspondence to: N. P. Golovchenko  相似文献   

14.
Two strains of thermophilic cellulolytic anaerobes, Nos. 138 and 183, were isolated from soil and compost, respectively, and identified as Clostridium thermocellum, based on their morphological, physiological and genetic characteristics. These two isolates decomposed cellulose more efficiently than the type strain, C. thermocellum ATCC 27405. The cel A gene of strain No. 138 was cloned onto vector plasmid pBR322 and the hybrid plasmids obtained were introduced into Escherichia coli cells. The cleavage map of the cloned cel A gene and the extent of CM-cellulase expression of the cloned gene in E. coli were the same as those of the cel A gene from ATCC 27405.  相似文献   

15.
The use of thermostable cellulases is advantageous for the breakdown of lignocellulosic biomass toward the commercial production of biofuels. Previously, we have demonstrated the engineering of an enhanced thermostable family 8 cellulosomal endoglucanase (EC 3.2.1.4), Cel8A, from Clostridium thermocellum, using random error-prone PCR and a combination of three beneficial mutations, dominated by an intriguing serine-to-glycine substitution (M. Anbar, R. Lamed, E. A. Bayer, ChemCatChem 2:997-1003, 2010). In the present study, we used a bioinformatics-based approach involving sequence alignment of homologous family 8 glycoside hydrolases to create a library of consensus mutations in which residues of the catalytic module are replaced at specific positions with the most prevalent amino acids in the family. One of the mutants (G283P) displayed a higher thermal stability than the wild-type enzyme. Introducing this mutation into the previously engineered Cel8A triple mutant resulted in an optimized enzyme, increasing the half-life of activity by 14-fold at 85°C. Remarkably, no loss of catalytic activity was observed compared to that of the wild-type endoglucanase. The structural changes were simulated by molecular dynamics analysis, and specific regions were identified that contributed to the observed thermostability. Intriguingly, most of the proteins used for sequence alignment in determining the consensus residues were derived from mesophilic bacteria, with optimal temperatures well below that of C. thermocellum Cel8A.  相似文献   

16.
Yao Q  Sun T  Chen G  Liu W 《Biotechnology letters》2007,29(8):1243-1247
The endoglucanase CelA from Clostridium thermocellum was strongly expressed in Bacillus subtilis. The enzyme was purified by Ni2+-affinity chromatography. Site-directed substitution of D278 with an asparagine or an alanine residue surprisingly did not decrease the apparent k cat value. Further substitutions of two other potentially critical residues, Y215 and D152, resulted in a 2-fold decrease in apparent k cat value for Y215P and complete loss of activity for D152N.  相似文献   

17.
The Janthinobacterium lividum chi69 chitinase gene linked to the Kluyveromyces lactis killer toxin secretion signal was ligated to the galactose-inducible CYC-GAL hybrid promoter of pEMBLyex4 and transferred directly into Saccharomyces cerevisiae DY-150. Exogenous chitinase activity assayed with 4-methylumbelliferyl--chitotrioside reached a maximum of 0.7 U/ml in the growth medium after 24 h galactose induction without any apparent deleterious effects on the yeast expression host.  相似文献   

18.
Cellulosomes are cellulolytic complexes produced by anaerobic bacteria, and are composed of a scaffolding protein and several catalytic components. The complexes are formed by highly specific interactions of one of the reiterated cohesin modules of the scaffolding protein with a dockerin module of the catalytic components. The affinities of a dockerin module of Clostridium thermocellum CelJ (Cel9D-Cel44A) for several cohesin modules from C. thermocellum and Clostridium josui scaffolding proteins were quantitatively measured by surface plasmon resonance analysis. The recombinant CelJ dockerin-containing protein interacted with three recombinant C. josui cohesin proteins as well as recombinant C. thermocellum cohesin proteins beyond the so-called 'species specificity' of the dockerin and cohesin interactions. However, this protein did not recognize a second cohesin module from the C. josui scaffolding protein, suggesting that the catalytic components are not necessarily arranged randomly on a scaffolding protein in native cellulosomes.  相似文献   

19.
Endoglucanase D, a cellulose degradation enzyme from Clostridium thermocellum has been cloned in Escherichia coli, purified and crystallized. The crystals are trigonal, space group P3(1)12 (or P3(2)12) with a = 57.7 (+/- 0.1) A, c = 192.1 (+/- 0.2) A, and diffract X-rays to a resolution of 2.8 A. They are suitable for a high-resolution X-ray diffraction analysis.  相似文献   

20.
A subunit with carboxymethyl cellulase (CMCase) activity was isolated from the cellulosomes of Clostridium thermocellum after dissociation of the cellulosomes by a mild sodium dodecyl sulfate (SDS) treatment. The subunit displayed only one protein band of 51 kDa on SDS-polyacrylamide gel electrophoresis (SDS-PAGE), but after boiling with SDS it had 3 bands of 60, 56, and 48 kDa. Prolonged incubation with SDS changed the subunit to display exclusively the 48-kDa band after boiling. The 51-kDa subunit was presumably a partially denatured form, and differentiated into 3 species with apparent M(r) of 60, 56, and 48 k through deglycosylation in SDS solution. Enzymatic properties of the 51-kDa subunit resembled those of the endoglucanase A which was purified from the culture fluid and from a E. coli clone with exceptions of temperature and pH optima.  相似文献   

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