Over-expression Of β-galctosidase Gene In Kluyveromyces Marxianus And High Production Of Lactase By Fermentation

β-galactosidase (commonly also known as lactase) is an enzyme that catalyzesthe hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides. Conventionally, its main application has been in the hydrolysis of lactose in milk orits products. Kluyveromyces marxianus is very suitable for industrial production oflactase with the advantages of high lactase activity, thermotolerance and shortgeneration time. Furthermore, K. marxianus is considered safe and harmless to humanbeings.Many enzymes synthesis and gene expression in microrganisms are repressedwhen high concentration of gluose exists in the medium, and this is called glucoserepression which is a common regulation phenomenon found in microorganisms.Glucose repression is also involved in lactase biosynthesis in K. marxianus, leading tolonger fermentation period, lower lactase production and raw material waste. TheMig1protein encoded by MIG1is a central component for glucose repression inyeasts. It directly controls the transcription of genes with the help of Snf1complex. Inthis study, the MIG1gene in K.marxianus kmi was disrupted in order to obtainglucose derepression. A mutant kmi-17with high lactase activity was obtained. Theresults of PCR and growth on2-deoxy-D-glucose medium showed that glucoserepression happened in the mutant kmi-17. Lactase gene experssion in the mutant wasalso enhanced according to the result of RTFQ PCR. The mutant kmi-17strain wasgrown in the shaking flask and in a10-L fermentor with aeration. The optimalfermentation time was48h with the lactase activity of131.2U/mL at flask levelwhich was62.5%higher than that of the parent strain kmi, while the lactase activity of154.3U/mL which was17.6%higher than that at flask level was achieved within24h during10-L fermentation. These results show that K. marxianus kmi-17strainhad a high potential for industrial production of lactase.Transcriptional activator protein is a special class of trans-acting factors, whichcan recognize, bind specific DNA sequences in the corresponding promoter andactivate its gene expression. It has been reported that the LAC9gene of K. lactisregulates lactase gene expression as the positive factor. Thus, the LAC9p1gene ofK.marxianus was thought to have same functions. In this study, the LAC9p1gene ofK.marxianus was knocked out and lactase activity and lactase gene transcription levelwere tested. The results showed that lactase activity and lactase gene transcriptionlevel had declined compared to those in the parent kmi strain. Thus, Lac9p1proteinencoded by LAC9p1gene played an importangt role in promoting the expression oflactase gene. LAC9p1gene expression vector pMD-rDNA-G418-LAC9wasconstructed, and K. marxianus kmi-17strain was transformed with this vector. Aover-expressing strain kml-1with higher lactase activity than that of K. marxianuskmi-17strain was screened. The result of RTFQ PCR showed that the transcriptionlevel of both LAC9p1gene and lactase gene was enhanced compared to that of thekmi-17strain and parent kmi strain. The kml-1strain was cultivated at flask level andin the10-L fermentor. The optimum fermentation time was48h with the lactaseactivity of150.5U/mL at flask level which is14.4％higher than the kmi-17strain. Inthe10-L fermentation, the maximum lactase production within24h of fermentationwas205.4U/mL,33.1%higher than that of kmi-17strain. Thus, kml-1strain in whichLAC9p1gene was over-expressed had a higher potential for industrial production oflactase than the MIG1mutant and the parent strain.