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Volume 167, Issue 7

A trimodular family 16 glycoside hydrolase from the cellulosome of displays highly specific licheninase (EC 3.2.1.73) activity Free

Abstract

Cellulosomes are highly complex cell-bound multi-enzymatic nanomachines used by anaerobes to break down plant carbohydrates. The genome sequence of revealed a remarkably diverse cellulosome composed of more than 200 cellulosomal enzymes. Here we provide a detailed biochemical characterization of a highly elaborate cellulosomal enzyme containing an N-terminal dockerin module, which anchors the enzyme into the multi-enzyme complex through binding of cohesins located in non-catalytic cell-bound scaffoldins, and three tandemly repeated family 16 glycoside hydrolase (GH16) catalytic domains. The DNA sequence encoding the three homologous catalytic domains was cloned and hyper-expressed in BL21 (DE3) cells. SDS-PAGE analysis of purified His tag containing GH16_21 showed a single soluble protein of molecular size ~89 kDa, which was in agreement with the theoretical size, 89.3 kDa. The enzyme GH16_21 exhibited activity over a wide pH range (pH 5.0–8.0) and a broad temperature range (50–70 °C), displaying maximum activity at an optimum pH of 7.0 and optimum temperature of 55 °C. Substrate specificity analysis of GH16_21 revealed maximum activity against barley β--glucan (257 U mg) followed by lichenan (247 U mg), but did not show significant activity towards other tested polysaccharides, suggesting that it is specifically a β-1,3-1,4-endoglucanase. TLC analysis revealed that GH16_21 hydrolyses barley β--glucan to cellotriose, cellotetraose and a higher degree of polymerization of gluco-oligosaccharides indicating an endo-acting catalytic mechanism. This study revealed a fairly high, active and thermostable bacterial endo-glucanase which may find considerable biotechnological potentials.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): barley β-d-glucan , immobilized metal ion affinity chromatography (IMAC) , Ruminococcus flavefaciens , thin-layer chromatography (TLC) and β-1,3-1,4-endoglucanase
© 2021 The Authors
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2021-07-23
2024-04-24
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A trimodular family 16 glycoside hydrolase from the cellulosome of Ruminococcus flavefaciens displays highly specific licheninase (EC 3.2.1.73) activity
Microbiology 167, 001055 (2021); https://doi.org/10.1099/mic.0.001055
/content/journal/micro/10.1099/mic.0.001055
/content/journal/micro/10.1099/mic.0.001055
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