Studies on Bacillus licheniformis endo-\-1,3-1,4-d-glucanase: characterization and kinetic analysis |
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Authors: | A Planas M Juncosa A Cayetano E Querol |
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Institution: | (1) Institut de Biologia Fonamental V, Villar Palasi and Departament de Bioquímica i Biologia Molecular, Universitat Autonoma de Barcelona, E-08193 Bellaterra, Barcelona, Spain;(2) CETS Institut Químic de Sarrià, Universitat Ramón Llull, E-08017 Barcelona, Spain |
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Abstract: | Summary A new purification procedure for endo-\-1,3-1,4-d-glucanase from Bacillus licheniformis is described. The secreted enzyme was purified both from B. licheniformis and from recombinant Escherichia coli harbouring the cloned gene by ion exchange chromatography on a CM-Sepharose matrix at pH 5.6. The mature enzyme was resistant to proteolysis by trypsin and chymotrypsin but it was slowly digested by protease V8. It showed a continuous trimming where no large-limit polypeptides were noticeable thus supporting a monodomain structure. Former appearing peptides have been assigned theoretically according to the protein sequence and predictive methods of accessible areas. Kinetic parameters for the hydrolysis of barley \-glucan and lichenan by measuring the net release of reducing sugars at the optimum pH (7.02) and temperature (55° C) are k
cat=3500 ±800 s–1 (turnover number) and K
m=1.45±0.21 mg/ml for barley \-glucan and k
cat=3000±750 s–1 and K
m=1.98±0.40 mg/ml for lichenan.
Correspondence to: E. Querol |
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