Abstract
The Pol6 mutant of Penicillium occitanis fungus is of great biotechnological interest since it possesses a high capacity of cellulases and β-glucosidase production with high cellulose degradation efficiency (Jain et al., Enzyme Microb Technol, 12:691–696, 1990; Hadj-Taieb et al., Appl Microbiol Biotechnol, 37:197–201, 1992; Ellouz Chaabouni et al., Enzyme Microb Technol, 16:538–542, 1994; Ellouz Chaabouni et al., Appl Microbiol Biotechnol, 43:267–269, 1995). In this work, two forms of β-glucosidase (β-glu 1 and β-glu 2) were purified from the culture supernatant of the Pol6 strain by gel filtration, ion exchange chromatography, and preparative anionic native electrophoresis. These enzymes were eluted as two distinct species from the diethylamino ethanol Sepharose CL6B and anionic native electrophoresis. However, both behaved identically on sodium dodecyl sulfate polyacrylamide gel electrophoresis (MW, 98 kDa), shared the same amino acid composition, carbohydrate content (8%), and kinetic properties. Moreover, they strongly cross-reacted immunologically. They were active on cellobiose and pNPG with Km values of 1.43 and 0.37 mM, respectively. β-glu 1 and β-glu 2 were competitively inhibited by 1 mM of glucose and 0.03 mM of δ-gluconolactone. They were also significantly inhibited by Hg2+ and Cu2 at 2 mM. The addition of purified enzymes to the poor β-glucosidase crude extract of Trichoderma reesei increased its hydrolytic efficiency on H3P04 swollen cellulose but had no effect with P. occitanis crude extract. Besides their hydrolytic activities, β-glu 1 and β-glu 2 were endowed with trans-glycosidase activity at high concentration of glucose.
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Abbreviations
- X-glu:
-
5-bromo,4-chloro,3-indolyl,1,4β-glucopyranoside
- Tris:
-
trishydroxymethylaminomethane
- EDTA:
-
ethylene-diaminetetraacetic acid
- p-NP:
-
p-nitrophenol
- pNPG:
-
p-nitrophenyl-β-d-glucopyranoside
- PMSF:
-
phenylmethylsulfonyl fluoride
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Acknowledgements
The authors thank Prof. G. Tiraby and Dr. H. Durand (Cayla Company, France) for kindly supplying the P. occitanis strain used in this work. Prof. R. Ellouz is thanked for his continual support and his interest for the subject.
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Bhiri, F., Chaabouni, S.E., Limam, F. et al. Purification and Biochemical Characterization of Extracellular β-Glucosidases from the Hypercellulolytic Pol6 Mutant of Penicillium occitanis . Appl Biochem Biotechnol 149, 169–182 (2008). https://doi.org/10.1007/s12010-008-8146-y
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DOI: https://doi.org/10.1007/s12010-008-8146-y