Multiplicity of the endo-1,4-β-<scp>d</scp>-glucanase activity in <i>Cellulomonas flavigena</i>

Sami, Amtul Jamil; Akhter, Mohsina

doi:10.1042/bst0170580

Biochemical Society Transactions

1989

DOI: 10.1042/bst0170580

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Multiplicity of the endo-1,4-β-d-glucanase activity in Cellulomonas flavigena

Amtul Jamil Sami¹,

Mohsina Akhter

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Cited by 3 publications

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“…f7uvigeriu are partly due to the expression o f more than one gene specific for the cellulase activity and partly due t o post-sccretional modifications [ 21. These modifications could be glycosylation, deglycosylation or degradation by proteolytically active enzymes [1][2][3]5,6].In this report we describe the purification and characterization of three extracellular CMCases of C'. J7uvigeriu.…”

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Purification and characterization of three extracellular carboxymethylcellulases of Cellulomonas flavigena

1990

Biochemical Society Transactions

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of' l'iitijuh. Luhore-I. I'ukistuti C'c~llirlottiotius J7uiigeriu produces a complex system of cellulases which is helpful in converting cellulose molecules into glucose molecules. We arc trying to identify and characterize the carboxymethylcellulascs (CMCases) of C. f7uvigetiu after their purification. C'. f7uvigetiu produces multiple forms of CMCase activity which can be separated on non-denaturing gradient 5-20'%, PAGE and identified on carboxymethylcellulose agar plates after electrophoresis by using the zymogram technique [ 1, 21. The multiple forms of cellulases produced by the microbe arc not necessarily the native forms of the enzymes. In the literature, there are different reasons cited for the multiplicity of cellulascs. The multiple forms of CMCase activity in C'. f7uvigeriu are partly due to the expression o f more than one gene specific for the cellulase activity and partly due t o post-sccretional modifications [ 21. These modifications could be glycosylation, deglycosylation or degradation by proteolytically active enzymes [1][2][3]5,6].In this report we describe the purification and characterization of three extracellular CMCases of C'. J7uvigeriu. Three extracellular CMCases of C'. f7uvig~~tiu were purified and characterized from a complex system o f cellulases. The microbe was grown for 72 h of fermentation and extracellular enzymes were isolated by centrifuging the broth at 10 000 g for 30 min [ 1 I. Enzymes were concentrated with 80% (v/v) chilled acetone 11 and passed through a column packed with Sephacryl ( 1.6 cm X 40 cm) at 4°C with 25 mM-Tris/HCI buffer, pH 8.0. This purification step increased the purification factor by three. This partially purified sample was loaded on t o a DEAE-Sephadex column .( 1.6 cm x 20 cm). Proteins were eluted with a linear salt gradient in same buffer. All the fractions after chromatography were analysed for CMCase activity as previously described [ 1 I. Proteins were estimated by reading the absorbance at 280 nm. The first peak of CMCase activity was eluted with a salt concentration of 0.05 M-NaCI. whereas the second peak was eluted with 0.075 ~-NaC1-0.25 M-NaCI. The first peak was called as D1 and second peak as D2. Both the peaks were analysed on non-denaturing gradient 5-20% PAGE and CMCase activity was located on C-methylcellulose agar plates as reported previously [ 2 ] . D1 and D2 showed the presence of at least two bands in each. Efforts were made to separate these bands on a narrow salt gradient but these were not successful. Further purification was carried out on 5 mm thick preparative non-denaturing 5-20% gradient polyacrylamide gel under the conditions previously described [ 1 I .Protein (500 pug) was loaded on to preparative gel and proteins were allowed to elute via diffusion from gel slices in distilled water after electrophoresis. This technique was able to resolve D1 and D2 into CMCase 3, CMCase 4 and CMCase 5 . Each of the enzyme activities appeared as a Abbreviation used: CMC. carboxymethylcellulase.

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“…The first peak was called as D1 and second peak as D2. Both the peaks were analysed on non-denaturing gradient 5-20% PAGE and CMCase activity was located on C-methylcellulose agar plates as reported previously [ 2 ] . D1 and D2 showed the presence of at least two bands in each.…”

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Purification and characterization of three extracellular carboxymethylcellulases of Cellulomonas flavigena

1990

Biochemical Society Transactions

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Purification and characterization of two low-molecular weight endoglucanases of Cellulomonas flavigena

Sami

Akhtar

1993

Enzyme and Microbial Technology

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Degradation of cellulases in cultures ofCellulomonas fimi

Sandercock

Meinke

Gilkes

et al. 1996

FEMS Microbiology Letters

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Endoglucanases CenA, CenB and CenD, cellobiohydrolases CbhA and CbhB, and the mixed function xylanase‐exoglucanase Cex are degraded proteolytically in the supernatants of cultures of Cellulomonas fimi growing with cellulose. All of these polypeptides are modular. The initial sites of proteolysis are within or adjacent to the linkers connecting the modules, leading to the appearance of discrete fragments of the enzymes which retain the functions of the component modules.

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Multiplicity of the endo-1,4-β-d-glucanase activity in Cellulomonas flavigena

Cited by 3 publications

References 0 publications

Purification and characterization of three extracellular carboxymethylcellulases of Cellulomonas flavigena

Purification and characterization of three extracellular carboxymethylcellulases of Cellulomonas flavigena

Purification and characterization of two low-molecular weight endoglucanases of Cellulomonas flavigena

Degradation of cellulases in cultures ofCellulomonas fimi

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