Electroelution as a simple and fast protein purification method: isolation of an extracellular xylanase from Bacillus sp. CCMI 966

Sá–Pereira, Paula; Duarte, Júlio César; Costa‐Ferreira, Maria

doi:10.1016/s0141-0229(00)00185-x

Cited by 32 publications

(16 citation statements)

References 24 publications

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“…However, it was higher than 5.5 kDa as observed by Bastawde (4). In contrast, some workers documented a higher molecular weight than that observed in the present study (5,19,20,38,39). Low molecular weight xylanases are preferred for commercial application in paper and pulp industry as they penetrate throughout the pulp fibres more effectively to hydrolyze xylan making the fiber structure more permeable to extraction of lignin by chemicals (21).…”

Section: Molecular Weight Determinationcontrasting

confidence: 55%

“…(29) and Thermomyces lanuginosus (24). However, some xylanases were found to have lower K m ranging from 0.025-1.7 mg/ml (8,16,23,38).…”

Section: Molecular Weight Determinationmentioning

confidence: 99%

“…Purified xylanases exhibiting optimum temperature in the range of 50-55 ºC have been reported from several Bacillus and Streptomyces sp. (1,6,38,44). However, some xylanases showed higher temperature optima (5,7,16,18,24).…”

Section: Measurement Of Xylanase Activity At Different Temperaturesmentioning

confidence: 99%

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One-step purification and characterization of cellulase-free xylanase produced by alkalophilic Bacillus subtilis ash

Sanghi¹,

Garg²,

Gupta³

et al. 2010

Braz. J. Microbiol.

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The present study describes the one-step purification and characterization of an extracellular cellulase-free xylanase from a newly isolated alkalophilic and moderately thermophilic strain of Bacillus subtilis ASH.Xylanase was purified to homogeneity by 10.5-fold with ~43% recovery using ion-exchange chromatography through CM-Sephadex C-50. The purified enzyme revealed a single band on SDS-PAGE gel with a molecular mass of 23 kDa. It showed an optimum pH at 7.0 and was stable over the pH range 6.0-9.0. The optimum temperature for enzyme activity was 55 °C. The purified xylanase did not lose any activity up to 45 ºC, however, it retained 80% and 51% of its activity after pre-incubation at 55 ºC and 60 ºC, respectively. The enzyme obeyed Michaelis-Menton kinetics towards birch wood xylan with apparent K m 3.33 mg/ml and V max 100 IU/ml. The enzyme was strongly inhibited by Hg 2+ and Cu 2+ while enhanced by Co 2+ and Mn 2+. The purified enzyme could be stored at 4 ºC for six weeks without any loss of catalytic activity. The faster and economical purification of the cellulase-free xylanase from B. subtilis ASH by onestep procedure together with its appreciable stability at high temperature and alkaline pH makes it potentially effective for industrial applications.

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Section: Molecular Weight Determinationcontrasting

confidence: 55%

“…(29) and Thermomyces lanuginosus (24). However, some xylanases were found to have lower K m ranging from 0.025-1.7 mg/ml (8,16,23,38).…”

Section: Molecular Weight Determinationmentioning

confidence: 99%

See 1 more Smart Citation

One-step purification and characterization of cellulase-free xylanase produced by alkalophilic Bacillus subtilis ash

Sanghi¹,

Garg²,

Gupta³

et al. 2010

Braz. J. Microbiol.

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“…Measurement of enzyme activity [260,261] under temperature-controlled conditions and eluted in a continuous flow of buffer (see Section 3.4). Additionally, an on-line detector and fraction collector were also included.…”

Section: Electroelution In Dialysis Bagmentioning

confidence: 99%

Preparative isolation of protein complexes and other bioparticles by elution from polyacrylamide gels

Seelert

Krause

2008

Electrophoresis

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Due to its unmatched resolution, gel electrophoresis is an indispensable tool for the analysis of diverse biomolecules. By adaptation of the electrophoretic conditions, even fragile protein complexes as parts of intracellular networks migrate through the gel matrix under sustainment of their integrity. If the thickness of such native gels is significantly increased compared to the analytical version, also high sample loads can be processed. However, the cage-like network obstructs an in-depth analysis for deciphering structure and function of protein complexes and other species. Consequently, the biomolecules have to be removed from the gel matrix into solution. Several approaches summarized in this review tackle this problem. While passive elution relies on diffusion processes, electroelution employs an electric field to force biomolecules out of the gel. An alternative procedure requires a special electrophoresis setup, the continuous elution device. In this apparatus, molecules migrate in the electric field until they leave the gel and were collected in a buffer stream. Successful isolation of diverse protein complexes like photosystems, ATP-dependent enzymes or active respiratory supercomplexes and some other bioparticles demonstrates the versatility of preparative electrophoresis. After liberating particles out of the gel cage, numerous applications are feasible. They include elucidation of the individual components up to high resolution structures of protein complexes. Therefore, preparative electrophoresis can complement standard purification methods and is in some cases superior to them.

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“…Similar methodology involving electroelution for the purification of a high weight xylanase excreted by a strain of Bacillus sp. was described by Sá-Pereira et al (14).…”

Section: Electroelution Protein Purificationmentioning

confidence: 99%

Production and purification of an Endo1,4-beta-Xylanase from Humicola grisea var. thermoidea by electroelution

Cardello¹,

Custódio²,

Goulart³

et al. 2003

Braz. J. Microbiol.

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Electroelution as a simple and fast protein purification method: isolation of an extracellular xylanase from Bacillus sp. CCMI 966

Cited by 32 publications

References 24 publications

One-step purification and characterization of cellulase-free xylanase produced by alkalophilic Bacillus subtilis ash

One-step purification and characterization of cellulase-free xylanase produced by alkalophilic Bacillus subtilis ash

Preparative isolation of protein complexes and other bioparticles by elution from polyacrylamide gels

Production and purification of an Endo1,4-beta-Xylanase from Humicola grisea var. thermoidea by electroelution

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Electroelution as a simple and fast protein purification method: isolation of an extracellular xylanase from Bacillus sp. CCMI 966

Cited by 32 publications

References 24 publications

One-step purification and characterization of cellulase-free xylanase produced by alkalophilic Bacillus subtilis ash

One-step purification and characterization of cellulase-free xylanase produced by alkalophilic Bacillus subtilis ash

Preparative isolation of protein complexes and other bioparticles by elution from polyacrylamide gels

Production and purification of an Endo1,4-beta-Xylanase from Humicola grisea var. thermoidea by electroelution

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Product

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Production and purification of an Endo1,4-beta-Xylanase from Humicola grisea var. thermoidea by electroelution