Cloning and expression ofClostridium stercorarium cellulase genes inEscherichia coli

Schwarz, Wolfgang H.; Jauris, Sigrid; Kouba, Monika; Bronnenmeier, Karin; Staudenbauer, Walter L.

doi:10.1007/bf01026642

Cited by 21 publications

(10 citation statements)

References 14 publications

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“…The OleR protein resembled different bacterial β‐glucosidases from Mycobacterium tuberculosis 43.4% (Philipp et al ., 1996), from Cellvibrio gilvus 41.9% (Kashigawi et al ., 1993), from Agrobacterium tumefaciens 35.9% (Castle et al ., 1992) and from Clostridium thermocellum 35.4% (Schwarz et al ., 1989). However, the highest score (56.7%) was with the EryBI protein from the erythromycin producer Sacc.…”

Section: Resultsmentioning

confidence: 99%

Two glycosyltransferases and a glycosidase are involved in oleandomycin modification during its biosynthesis by Streptomyces antibioticus

Quirós

Aguirrezabalaga

Olano

et al. 1998

Molecular Microbiology

173

165

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SummaryA 5.2 kb region from the oleandomycin gene cluster in Streptomyces antibioticus located between the oleandomycin polyketide synthase gene and sugar biosynthetic genes was cloned. Sequence analysis revealed the presence of three open reading frames (designated oleI, oleN2 and oleR). The oleI gene product resembled glycosyltransferases involved in macrolide inactivation including the oleD product, a previously described glycosyltransferase from S. antibioticus. The oleN2 gene product showed similarities with different aminotransferases involved in the biosynthesis of 6-deoxyhexoses. The oleR gene product was similar to several glucosidases from different origins. The oleI, oleR and oleD genes were expressed in Streptomyces lividans. OleI and OleD intracellular proteins were partially purified by affinity chromatography in an UDP-glucuronic acid agarose column and OleR was detected as a major band from the culture supernatant. OleI and OleD showed oleandomycin glycosylating activity but they differ in the pattern of substrate specificity: OleI being much more specific for oleandomycin. OleR showed glycosidase activity converting glycosylated oleandomycin into active oleandomycin. A model is proposed integrating these and previously reported results for intracellular inactivation, secretion and extracellular reactivation of oleandomycin.

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Section: Resultsmentioning

confidence: 99%

Two glycosyltransferases and a glycosidase are involved in oleandomycin modification during its biosynthesis by Streptomyces antibioticus

Quirós

Aguirrezabalaga

Olano

et al. 1998

Molecular Microbiology

173

165

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“…Sodium-dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and zymogram analysis of xylanase components SDS-PAGE of the samples was performed according to Laemmli (1970) using concentrated crude culture filtrate as samples. Native polyacrylamide gel electrophoresis (PAGE) using 12% gel was performed for visualization of xylanase activity in situ (Mayorga-Reyes and Noyola 1998; Schwarz et al 1989). Substrate, i.e.…”

Section: Optimum Ph and Stability Of Crude Xylanasementioning

confidence: 99%

Production and biochemical characterization of a novel cellulase-poor alkali-thermo-tolerant xylanase from Coprinellus disseminatus SW-1 NTCC 1165

Agnihotri

Dutt

Tyagi

et al. 2010

World J Microbiol Biotechnol

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Fungi producing xylanases are plentiful but alkali-thermo-tolerant fungi producing cellulase-poor xylanase are rare. Out of 12 fungal strains isolated from various sources, Coprinellus disseminatus SW-1 NTCC 1165 yielded the highest xylanase activity (362.1 IU/ml) with minimal cellulase contamination (0.64 IU/ml). The solid state fermentation was more effective yielding 88.59% higher xylanase activity than that of submerged fermentation. An incubation period of 7 days at 37°C and pH 6.4 accelerated the xylanase production up to the maximum level. Among various inexpensive agro-residues used as carbon source, wheat bran induced the maximum xylanase titres (469.45 IU/ml) while soya bean meal was the best nitrogen source (478.5 IU/ml). A solid substrate to moisture content ratio of 1:3 was suitable for xylanase production while xylanase titre was repressed with the addition of glucose and lactose. The xylanase and laccase activities under optimized conditions were 499.60 and 25.5 IU/ml, respectively along with negligible cellulase contamination (0.86 IU/ml). Biochemical characterization revealed that optimal xylanase activity was observed at pH 6.4 and temperature 55°C and xylanase is active up to pH 9 (40.33 IU/ml) and temperature 85°C (48.81 IU/ml). SDS-PAGE and zymogram analysis indicated that molecular weight of alkali-thermo-tolerant xylanase produced by C. disseminatus SW-1 NTCC 1165 was 43 kDa.

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“…This fact persuaded us to screen hydrolytic genes from a genomic library of C. stercorarium DNA 21 . A library of 1139 clones were obtained by the partial digest of genomic DNA with different restriction enzymes.…”

Section: Polysaccharide Degradation By Clostridium Stercorariummentioning

confidence: 99%

“…This fact persuaded us to screen hydrolytic genes from a genomic library of C. stercorarium DNA. 21 A library of 1139 clones were obtained by the partial digest of genomic DNA with different restriction enzymes. Cell-free extracts were prepared and screened with 14 different substrates indicative for enzyme activities involved in cellulose and hemicellulose hydrolysis (TABLE 2).…”

Section: Polysaccharide Degradation By Clostridium Stercorariummentioning

confidence: 99%

Bacterial Cellulose Hydrolysis in Anaerobic Environmental Subsystems—Clostridium thermocellumandClostridium stercorarium, Thermophilic Plant‐fiber Degraders

Zverlov

Schwarz

2008

Annals of the New York Academy of Sciences

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Cellulose degradation is a rare trait in bacteria. However, the truly cellulolytic bacteria are extremely efficient hydrolyzers of plant cell wall polysaccharides, especially those in thermophilic anaerobic ecosystems. Clostridium stercorarium, a thermophilic ubiquitous soil dweller, has a simple cellulose hydrolyzing enzyme system of only two cellulases. However, it seems to be better suited for the hydrolysis of a wide range of hemicelluloses. Clostridium thermocellum, an ubiquitous thermophilic gram-type positive bacterium, is one of the most successful cellulose degraders known. Its extracellular enzyme complex, the cellulosome, was prepared from C. thermocellum cultures grown on cellulose, cellobiose, barley beta-1,3-1,4-glucan, or a mixture of xylan and cellulose. The single proteins were identified by peptide chromatography and MALDI-TOF-TOF. Eight cellulosomal proteins could be found in all eight preparations, 32 proteins occur in at least one preparation. A number of enzymatic components had not been identified previously. The proportion of components changes if C. thermocellum is grown on different substrates. Mutants of C. thermocellum, devoid of scaffoldin CipA, that now allow new types of experiments with in vitro cellulosome reassembly and a role in cellulose hydrolysis are described. The characteristics of these mutants provide strong evidence of the positive effect of complex (cellulosome) formation on hydrolysis of crystalline cellulose.

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Cloning and expression ofClostridium stercorarium cellulase genes inEscherichia coli

Cited by 21 publications

References 14 publications

Two glycosyltransferases and a glycosidase are involved in oleandomycin modification during its biosynthesis by Streptomyces antibioticus

Two glycosyltransferases and a glycosidase are involved in oleandomycin modification during its biosynthesis by Streptomyces antibioticus

Production and biochemical characterization of a novel cellulase-poor alkali-thermo-tolerant xylanase from Coprinellus disseminatus SW-1 NTCC 1165

Bacterial Cellulose Hydrolysis in Anaerobic Environmental Subsystems—Clostridium thermocellumandClostridium stercorarium, Thermophilic Plant‐fiber Degraders

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