| β-galactosidase is often referred to as lactase which has potential applications in food, medicines, health products industry. Increased lactase activity, the present study mainly proceed from the following aspects:1, from the industrial fermentation medium optimized, inducible factor added, the optimization of fermentation patterns, conditions and the extraction process, separation and purification;2, screen high-yield strains by mutation breeding;3, the modified enzyme;4, looking for more efficient expression system. Currently prokaryotic expression systems and eukaryotic expression systems is commonly used, such as Torres Bacillus subtilis KL88β-galactosidase gene fragment was cloned into E.coli system, the β-galactosidase enzyme production was25-143times higher than the natural β-galactosidase;5, genetically engineered technology, including recombinant DNA technology, transformation of the lactase gene at the molecular level, and thus directed screening of the target protein, the ideal variation. Commonly used research methods are error-prone PCR, DNA Shuffling and site-directed mutagenesis and so on; such as Jiang Shimin maked heterologous DNA homologous recombination in vitro evolution of E. coli P-galactosidase. Domestic began to study on lactase late at the1980s. So far have been reported some of producing lactase strains, including fungi, bacteria, yeast and some plants and animals. Abroad in recent years has more reseach on molecular biology of lactase, as the genome of Lactobacillus acidophilus NCFM strain has been sequenced, and lay the foundation for genetic engineering to improve the activity of lactase.The topics selected recombinant ways to improve the β-galactosidase activity, first in the traditional fermentation conditions optimized for Lactobacillus acidophilus production of β-galactosidase, and then proceed to DNA Shuffling, directional filter to improve activity. Activity results obtained by gene recombination pathway, analysis of the difference between P-galactosidase activity of wild-type strains and the activity of the mutant strains. Blast the amino acid sequence though bioinformatics methods to find the enzyme activity differences.The optimal fermentation conditions with culture medium and fermentation conditions for Lactobacillus acidophilus were:beef extract lOg/L, casein peptone lOg/L, sodium acetate7.5g/L, magnesium sulfate150mg/L, manganese sulfate35mg/L, K2HPO4 3g/L, glucose5g/L, calcium carbonate lOg/L, citric acid triamide2.35g/L, yeast extract6.22g/L, and5%of the inoculum size, the fermentation temperature of36℃, fermentation time of24h, low-speed50r/min. The experiments prove that enzyme activity at the nutritional conditions can be up to3.98U/L, which is2.9times of before optimization strain.Screened out of a high-yield strains that L.acidophilus-F2-10with higher activity by the reorganization of the genome of Lactobacillus acidophilus CICC6075. The reaction substrate of lactase produced by L.acidophilus-F2-10is nitrodiphenol-β-D-galactoside (ONPG), the enzyme activity was significantly increased compared with the wild type strain, enzyme activity reached7.56U/L and lactose were increased by4.6times. The optimum temperature of L.acidophilus-F2-10was45℃. The optimal pH was7.0. The P-galactosidase has good stability within the range of40℃-50℃and the pH6.0-9.0. Mg2+, Fe2+, Fe3+, Mn2+and Al3+can promote the activities of β-galactosidase while Cu2+, Zn2+, Ca2+can inhibit the activities of (3-galactosidase. The K+almost has no effect on the enzyme.The P-galactosidase gene of L.acidophilus-F2-10strains were cloned by PCR and sequencing referred as biological databases, the length of beta-galactosidase gene lacLM is2834bp, collaborative translation encoding the two subunits, respectively for628aa and316aa. Compared to wild-type strains, blast the gene and amino acids, gene mutations sites was5, lacL gene encoding the large subunit which has4amino acid mutation sites, three synonymous mutations, number the first of ATG coding M amino acid as standard, in addition to the512amino acid was Râ†'H mutations,72amino acid was Sâ†'S mutation,245amino acid was Qâ†'Q mutation,247amino acid was Gâ†'G mutation, these utations are silent mutations. lacM gene encoding the small subunit amino acid have a mutation site in the Yâ†'H. The experimental analysis of the gene mutations in the relationship among the mutation site, the secondary structure of amino acids and conserved domain, suggesting a change of β-galactosidase activity may be related to the structural domain of Tim-barrel fold class-related protein mutations, which has a mutation site in the512amino acids with Râ†'H mutation. |
PDF Full Text Request