Activity staining of cellulases in polyacrylamide gels containing mixed linkage β-glucans

Schwarz, Wolfgang H.; Bronnenmeier, Karin; Gräbnitz, Folke; Staudenbauer, Walter L.

doi:10.1016/0003-2697(87)90369-1

Cited by 165 publications

(93 citation statements)

References 14 publications

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“…For gel-based CMCase detection 0.1% (w/v), CMC was added to the resolving gel solution 53 . Upon completion of electrophoresis, gels were washed twice for 30 min with 2.5% (w/v) Triton X-100, and re-activation of the enzymes was achieved by incubation in activity buffer (50 mM Tris HCl pH 7.4, 100 mM NaCl, 5 mM CaCl 2 , 10 mM ZnCl 2 , 1% (v/v) Triton X-100, 0.1% NaN 3 ) for 24 h at 42 1C.…”

Section: Methodsmentioning

confidence: 99%

Cellulose degradation and assimilation by the unicellular phototrophic eukaryote Chlamydomonas reinhardtii

et al. 2012

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Section: Methodsmentioning

confidence: 99%

Cellulose degradation and assimilation by the unicellular phototrophic eukaryote Chlamydomonas reinhardtii

et al. 2012

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“…Proteins were separated by denaturing SDS-PAGE (10 % polyacrylamide) and renaturated by three successive washings in 50 mM phosphate-citrate buffer (pH 6?2) with and without i-propanol as described previously (Schwarz et al, 1987). The gel slabs were overlaid with agarose gel containing polymeric substrates (1 %, w/v), incubated at 55˚C and stained with 0?1 % (w/v) Congo Red (Sigma-Aldrich).…”

mentioning

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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“…The detection of carboxymethyl cellulase (CMCase) activity in gels was performed according to the method of Schwarz et al (1987), with some modifications. CBPs were eluted from Avicel cellulose with 1ϫ sample buffer for SDS-PAGE for 10 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Presence of several cellulose-binding proteins in culture supernatant and cell lysate of Eubacterium cellulosolvens 5.

Toyoda¹,

Yoda²,

Nakamura³

et al. 2001

J. Gen. Appl. Microbiol.

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Attempts were made to separate and characterize cellulose-binding proteins (CBPs) from both the culture supernatant and cell lysate of Eubacterium cellulosolvens 5. Once the CBPs were bound to Avicel cellulose, they were then effectively eluted with the solution containing 3.2 or 5% sodium dodecyl sulfate (SDS), but not eluted with the solution containing various kinds of carbohydrates and reagents. Namely, CBPs in both the culture supernatant and cell lysate of the bacterium bound tightly and strongly to cellulose. The SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the eluted CBPs indicated that the CBPs contained the two major proteins having the molecular weights of approximately 160 and 84 kilodaltons (kDa) and one sub-major protein having a molecular weight of approximately 140 kDa. Zymogram analysis after the SDS-PAGE of the eluted CBPs showed that two proteins exhibited the highest levels of carboxymethyl cellulase (CMCase) activity corresponding to the molecular weights of approximately 160 and 90 kDa. A major protein having the molecular weight of approximately 160 kDa exhibited a distinct CMCase activity and was designated as CBPE1. Western immunoblot analysis indicated that the proteins prepared from 16 representative strains of rumen bacteria did not cross-react with rabbit antiserum raised against CBPE1. Thus, CBPE1 may be a unique CBP that plays an important role in the adhesion of the bacterium to cellulose.

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Activity staining of cellulases in polyacrylamide gels containing mixed linkage β-glucans

Cited by 165 publications

References 14 publications

Cellulose degradation and assimilation by the unicellular phototrophic eukaryote Chlamydomonas reinhardtii

Cellulose degradation and assimilation by the unicellular phototrophic eukaryote Chlamydomonas reinhardtii

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

Presence of several cellulose-binding proteins in culture supernatant and cell lysate of Eubacterium cellulosolvens 5.

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