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Determining parameters for the use of a β ‐galactosidase reporter gene in Schizophyllum commune
Author(s) -
Mankel Angela,
Kothe Erika
Publication year - 1999
Publication title -
journal of basic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.58
H-Index - 54
eISSN - 1521-4028
pISSN - 0233-111X
DOI - 10.1002/(sici)1521-4028(199906)39:3<169::aid-jobm169>3.0.co;2-z
Subject(s) - schizophyllum commune , enzyme , galactosides , molecular mass , biochemistry , chemistry , lactose , alpha galactosidase , affinity chromatography , beta galactosidase , reporter gene , enzyme assay , heterologous , enzyme kinetics , galactosidases , lac operon , gene , gene expression , active site , fabry disease , medicine , disease , pathology
High β ‐galactosidase activities could easily be detected in culture supernatants of Schizophyllum commune grown on lactose and glycerol. The addition of glucose to the growth medium resulted in lower β ‐galactosidase activities. Two different enzymes exhibiting β ‐galactosidase activity were purified by affinity adsorption and anion exchange chromatography. Enzyme Gal1 possessed high substrate specificity for β ‐galactosides. The native enzyme (molecular mass 140 kDa) was a homo‐dimer of subunits with an apparent molecular mass of 66 kDa. Antibodies raised against E. coli β ‐galactosidase recognized Schizophyllum commune Gal1. The second enzyme, Gal2, had a lower specific activity and hydrolyzed β ‐glucosides as well as β ‐galactosides. The previously characterized β ‐glucosidase II of S. commune (L o et al. 1990) was shown to be identical with Gal2. Both enzymes had temperature optima of 40 °C with less than 5% remaining activity at 60 °C which would allow the use of a thermo‐tolerant heterologous β ‐galactosidase as a reporter gene in S. commune .