Determining parameters for the use of a β-galactosidase reporter gene in Schizophyllum commune

✍ Scribed by Angela Mankel; Erika Kothe

• Publisher: John Wiley and Sons
• Year: 1999
• Tongue: English
• Weight: 389 KB
• Volume: 39
• Category: Article
• ISSN: 0233-111X
• DOI: 10.1002/(sici)1521-4028(199906)39:3<169::aid-jobm169>3.0.co;2-z

✦ Synopsis

High b-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 b-galactosidase activities. Two different enzymes exhibiting b-galactosidase activity were purified by affinity adsorption and anion exchange chromatography. Enzyme Gal1 possessed high substrate specificity for b-galactosides. The native enzyme (molecular mass 140 kDa) was a homodimer of subunits with an apparent molecular mass of 66 kDa. Antibodies raised against E. coli b-galactosidase recognized Schizophyllum commune Gal1. The second enzyme, Gal2, had a lower specific activity and hydrolyzed b-glucosides as well as b-galactosides. The previously characterized b-glucosidase II of S. commune (LO 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 b-galactosidase as a reporter gene in S. commune.