Genetic Engineering Of Lactoase Production Yeast Strain And The Production Of Lactoase By Fermentation

As Kluyveromyces marxianus has the capacity to assimilate variable substrates;an extremely rapid growth rate, short generation time, thermotolerance, and a highsecretory capacity, it has been widely used in biotechnology. A lot of advantagesmake K. marxianus more suitable to produce enzymes in food industry compared toother microorganisms.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. Twomain proteins involved in glucose repression are Snf1and Mig1. When the glucoseconcentration in the medium is very low or there is no glucose in the growth medium,the Snf1complex is phosphorylated by another unknown kinase in yeast cells. In thiscase, the Snf1complex is active and can catalyze the phosphorylation of Mig1. Thephosphorylated Mig1fails in binding to the promoters of the genes repressed byglucose and is translocated to cytoplasm, leading to the active transcription of thegenes. Therefore, the Mig1protein is a central component for glucose repression inyeasts. The transcriptional repressor was found to play an important role in glucoserepression. In this study, the MIG1gene in Kluyveromyces marxianus KM wasdisrupted by integrating the HPT (hygromycin B phosphotransferase) gene into ORF(Open Reading Frame) of the MIG1gene. The disruptant (mig1) km-15obtainedcould grow in the media containing hygromycin and2-deoxy-D-glucose, respectively.β-galactosidase, inulinase and β-glucosidase production by the disruptant andexpression of their genes in the disruptant km-15were significantly enhanced. Thisconfirms that Mig1, the transcriptional repressor, indeed regulates expression of thegenes and production of the enzymes in K. marxianus km.Then, β-galactosidase production by the mig1mutant of K. marxianus km wasoptimized and the optimal medium was whey powder10.0%(w/v), polypeptone1.0% (w/v), yeast extract1.5%(w/v), MnCl21.0mM. Under the optimal conditions, theβ-galactosidase activity of111.7U/mL was reached within48h at flask level whilethe β-galactosidase activity of121.0U/ml was achieved within60h during2-Lfermentation.99.2%of lactose in9.0%(w/v) of lactose solution was hydrolyzedwithin2.5h when the added β-galactosidase activity was440U/g of lactose whereas98.7%of lactose in12.0%of whey powder suspension was degraded within3.5hwhen the added β-galactosidase activity was440U/g of the whey powder.99.3%oflactose in milk was hydrolyzed within3.0h when the added β-galactosidase activitywas260U/g of lactose. This suggests that the β-galactosidase produced could veryefficiently hydrolyze lactose and whey lactose. The hydrolysates obatimed wereanalyzed by TLC. The results showed that only monosaccharides such as glucose andgalactose existed in the hydrolysates, indicating the lactose was almost completelyhydrolyzed and the lactase had potential applications in food industry.At the same time, inulinase production by K. marxianus km-15was optimizedand the optimal medium was inulin8.0%(w/v), yeast extract3.0%(w/v). Under theoptimal conditions, the inulinase activity of69.0U/mL was reached within42h atflask level while the inulinase activity of79.9U/ml was achieved within60h during2-L fermentation. Over96.2%of inulin (6.0%, w/v) was hydrolyzed in the presenceof the inulinase activity of400U/g of inulin within3.0h. The hydrolysates were alsoanalyzed by TLC. The result showed that main monosaccharides with minoroligosaccharides existed in the final hydrolysates, demontrating the inulinaseproduced by K. marxianus km-15was an exo-inulinase.The psychrotolerant yeast Guehomyces pullulans17-1isolated from seasediment in Antarctica can produce a large amount of cell-bound and extracellularβ-galactosidase, and the yeast strain grew best and produced high level ofβ-galactosidase at15°C. In order to isolate β-galactosidase overproducers of G.pullulans17-1, its cells were mutated by using nitrosoguanidine (NTG). One mutant(NTG-133) with enhanced β-galactosidase production was obtained. Afteroptimization of the medium and cultivation conditions, the mutant could produce38.0U/ml of total β-galactosidase activity within132h at the flask level while the mutant could produce48.1U/ml of total β-galactosidase activity within144h in2-Lfermentor. Over91.8%of lactose solution (9.0%, w/v) was hydrolyzed in thepresence of the β-galactosidase activity of550U/g of lactose within6.0h while over88.3%of lactose in the whey powder was hydrolyzed in the presence ofβ-galactosidase activity of550U/g of lactose within7.5h. The hydrolysates wereanalyzed by TLC. The results showed that main monosaccharides such as glucose andgalactose existed in the hydrolysates.