Sensitive detection of endo-1,4-β-glucanases and endo-1,4-β-xylanases in gels

Biely, Peter; Markovič, Oskar; Mislovičová, Danica

doi:10.1016/0003-2697(85)90096-x

Cited by 128 publications

(64 citation statements)

References 14 publications

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“…The supernatant was heated (70ЊC for 10 min) and cooled to 4ЊC, and denatured proteins were removed by centrifugation (12,000 ϫ g for 20 min). Proteins from the enzyme preparation were separated by nondenaturing polyacrylamide gel electrophoresis, (PAGE), and the xylanase activity was localized by the activity stain method of Biely et al (3). The reactive band was further purified by electroelution using a model 422 Electro-Eluter (Bio-Rad, Hercules, Calif.).…”

Section: Vol 178 1996 S-layer-associated Thermostable Endoxylanase mentioning

confidence: 99%

Cloning, sequencing, and expression of the gene encoding a large S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 in Escherichia coli

et al. 1996

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The gene (xynA) encoding a surface-exposed, S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 was cloned and expressed in Escherichia coli. A 3.8-kb fragment was amplified from chromosomal DNA by using primers directed against conserved sequences of endoxylanases isolated from other thermophilic bacteria. This PCR product was used as a probe in Southern hybridizations to identify a 4.6-kb EcoRI fragment containing the complete xynA gene. This fragment was cloned into E. coli, and recombinant clones expressed significant levels of xylanase activity. The purified recombinant protein had an estimated molecular mass (150 kDa), temperature maximum (80؇C), pH optimum (pH 6.3), and isoelectric point (pH 4.5) that were similar to those of the endoxylanase isolated from strain JW/SL-YS 485. The entire insert was sequenced and analysis revealed a 4,044-bp open reading frame encoding a protein containing 1,348 amino acid residues (estimated molecular mass of 148 kDa). xynA was preceded by a putative promoter at ؊35 (TTAAT) and ؊10 (TATATT) and a potential ribosome binding site (AGGGAG) and was expressed constitutively in E. coli. The deduced amino acid sequence showed 38 to 96% similarity to sequences of family F ␤-glycanases. A putative 32-amino-acid signal peptide was identified, and the C-terminal end of the protein contained three repeating sequences (59, 64, and 57 amino acids) that showed 46 to 68% similarity to repeating sequences at the N-terminal end of S-layer and S-layer-associated proteins from other gram-positive bacteria. These repeats could permit an interaction of the enzyme with the S-layer and tether it to the cell surface.

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Section: Vol 178 1996 S-layer-associated Thermostable Endoxylanase mentioning

confidence: 99%

Cloning, sequencing, and expression of the gene encoding a large S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 in Escherichia coli

et al. 1996

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“…With respect to the endoglucanase components from T. reesei, the existence of two different endoglucanases (I and III) differing in structure and specificity (3,4,10,16,24,27,30), which additionally occur in multiple forms (4,11,12,17,21), has been established. In the present paper we report a reexamination of the control of formation of individual * Corresponding author.…”

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confidence: 99%

Differential regulation of synthesis of multiple forms of specific endoglucanases by Trichoderma reesei QM9414

1988

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A method consisting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent detection of endoglucanases by blotting with a polyclonal antibody against endoglucanase I was used to investigate the effect of induction and carbon catabolite derepression on the synthesis of multiple forms of endoglucanase I by Trichoderma reesei. Five forms appeared upon growth on cellulose, whereas four and only two appeared upon growth on lactose (carbon catabolite derepression) and induction by sophorose in a resting cell system, respectively. All endoglucanases detected resembled endoglucanase I in their specificity, since they exhibited no activity toward xylan or paranitrophenyl-,-D-lactobioside. A small (25-kiodalton) endoglucanase only appeared during growth on cellulose. None of the multiple forms arose by postsecretional modification. The results indicate that sophorose may not be the only compound mediating cellulose induction of the specific endoglucanases in T. reesei.

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“…To follow the enzyme in fractions collected during chromatographic and electrophoretic purification of the enzyme, a cup-plate semiquantitative procedure was used, suggested first for endo-P-1,4-glycanases [17,181. A hot solution of 0.3% RBBA, 0.02% sodium azide and 2% agar (Difco) in 0.025 M sodium phosphate was poured into Petri dishes.…”

Section: Determination Of Alternanase Activitymentioning

confidence: 99%

“…The zymogram technique used was similar to that developed for the detection of endo-P-l,4-glucanases and endo-P-1,4-xylanases [17]. A gel with proteins resolved by PAGE under native conditions was washed twice in 0.1 M sodium phosphate for 10 min, brought into contact with 1 % agarose detection gel containing 0.5% RRBA in 0.025 M sodium phosphate, pH 7, then incubated in a wet chamber at 40°C until a change in the blue background was observed where alternanase hydrolyzed the blue substrate.…”

Section: Location Of Alternanase In Gelsmentioning

confidence: 99%

Purification and Properties of Alternanase, a Novel Endo‐α‐1,3‐α‐1,6‐d‐Glucanase

Biely

Côté

Burgess-Cassler

1994

European Journal of Biochemistry

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A newly isolated soil bacterium strain NRRL B‐21195, tentatively identified as a Bacillus species, was found to be a constitutive producer of a novel type of glycanase that hydrolyses in an endo‐fashion the polysaccharide alternan, an α‐1,3‐α‐1,6‐d‐glucan, referred to in the literature as B‐1355 dextran (fraction S), synthesized from sucrose by alternansucrase of Leuconostoc mesenteroides. The glycanase, named alternanase, has been purified to homogeneity from a cell‐free culture fluid of the bacillus grown in a liquid medium containing d‐glucose, and has been characterized. The enzyme has a molecular mass of 110000 Da (SDS/PAGE) and an isoelectric point of approximately 4.0. Optimum activity occurs at pH 7 and at a temperature of 40°C. The enzyme is stable up to 50°C but loses activity rapidly at 60°C. Its action is inhibited by EDTA and stimulated by Ca2+. The enzyme requires, for its action, d‐glucan chains in which α‐1,3‐linkages alternate with α‐1,6‐linkages; i.e., it is specific for alternan. Monitoring of alternan hydrolysis by determination of liberated reducing sugars pointed to an unusually low extent of hydrolysis and a low specific activity of the enzyme. As shown in the accompanying paper [Côté, G. L. & Biely, P. (1994) Eur. J. Biochem. 226, 641–648] the reason for this finding is that the main hydrolytic products are non‐reducing, novel types of cyclic oligosaccharides.

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Sensitive detection of endo-1,4-β-glucanases and endo-1,4-β-xylanases in gels

Cited by 128 publications

References 14 publications

Cloning, sequencing, and expression of the gene encoding a large S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 in Escherichia coli

Cloning, sequencing, and expression of the gene encoding a large S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 in Escherichia coli

Differential regulation of synthesis of multiple forms of specific endoglucanases by Trichoderma reesei QM9414

Purification and Properties of Alternanase, a Novel Endo‐α‐1,3‐α‐1,6‐d‐Glucanase

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