β-Galactosidase over-production by a mig1 mutant of Kluyveromyces marxianus KM for efficient hydrolysis of lactose

Abstract

In this study, the MIG1 gene in the industrial yeast Kluyveromyces marxianus KM was disrupted by integrating the HPT gene (encoding hygromycin B phosphotransferase) into ORF (Open Reading Frame) of the MIG1 gene. The disruptant (mig1 mutant) KM-15 obtained could grow in the media containing hygromycin or 2-deoxy-d-glucose, respectively. β-Galactosidase production and expression of its gene in the disruptant KM-15 were significantly enhanced compared to those in K. marxianus KM. This confirmed that Mig1, the transcriptional repressor, indeed regulated expression of the gene and biosynthesis of the enzyme in K. marxianus KM. Under the optimal conditions, the β-galactosidase activity of 121.0U/mL was produced by the disruptant KM-15 within 60h during 2-L fermentation. 99.2% of lactose in 9.0% of lactose solution, 99.0% of lactose in 12.0% of whey powder suspension and 99.3% of lactose in the milk were hydrolyzed by the crude β-galactosidase within 3h, 4h and 3h, respectively. The end products of the lactose hydrolysis were only glucose and galactose.