A trimodular family 16 glycoside hydrolase from the cellulosome of Ruminococcus flavefaciens displays highly specific licheninase (EC 3.2.1.73) activity
| Autor: | Arun Goyal, Carlos M. G. A. Fontes, Abhijeet Thakur, Sunetra Mondal |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
0106 biological sciences
chemistry.chemical_classification 0303 health sciences Dockerin Licheninase Cellulosomes medicine.disease_cause Polysaccharide 01 natural sciences Microbiology Cellulosome 03 medical and health sciences Enzyme chemistry Biochemistry 010608 biotechnology medicine Glycoside hydrolase Escherichia coli 030304 developmental biology |
| Zdroj: | Microbiology. 167 |
| ISSN: | 1465-2080 1350-0872 |
| DOI: | 10.1099/mic.0.001055 |
| Popis: | Cellulosomes are highly complex cell-bound multi-enzymatic nanomachines used by anaerobes to break down plant carbohydrates. The genome sequence of Ruminococcus flavefaciens revealed a remarkably diverse cellulosome composed of more than 200 cellulosomal enzymes. Here we provide a detailed biochemical characterization of a highly elaborate R. flavefaciens 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 Escherichia coli BL21 (DE3) cells. SDS-PAGE analysis of purified His6 tag containing RfGH16_21 showed a single soluble protein of molecular size ~89 kDa, which was in agreement with the theoretical size, 89.3 kDa. The enzyme RfGH16_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 RfGH16_21 revealed maximum activity against barley β-d-glucan (257 U mg−1) followed by lichenan (247 U mg−1), 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 RfGH16_21 hydrolyses barley β-d-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. |
| Databáze: | OpenAIRE |
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