Construction Of Lactase Producing Gene Engineering Strain And Optimization Of Fermentation Conditions

Lactase??-D-galactosidase?is able to hydrolyze lactose to produce glucose and galactose.Moreover,lactase can transfer galactoside groups.Lactase belongs to bio-enzyme preparation and it can be mainly used to treat“lactose intolerance”in pharmaceuticals industry.In food industry,lactase can be used to produce low-lactose milk.Some problems exist in large-scale industrial production of lactaseas follows:low production of enzymes,endocrine,low recovery,etc.Therefore,modern genetic engineering technology was adopted in the thesis.The gene sequence of Aspergillus oryzae lactase was selected as target gene.Kluyveromyces lactis GG799 was used as the host cell.A new strain with exocytosis of lactase,excellent enzymatic properties,and high expression level was constructed.A.oryzae lactase gene was used as template.The gene sequence of A.oryzae lactase was synthesized and optimized artificially according to the codon preference of K.lactis without changing the original amino acid sequence.The full-lengthlactase gene lacA after optimization was 2982 bp,and the GC content decreased from 51.4%to 39.2%,678 bases were altered.The level of high frequency codons was improved.The optimized lactase gene sequence lacA was inserted between Xho I site and Bgl II site of pKLAC1 vector to construct the vector pKLAC1-lacA.Then the recombinant plasmid linearized by Sac II was transformed into GG799 by electric pulse method.The transformants were enriched in Yeast Carbon Base?YCB?nitrogen basic medium.After several rounds of electroporation,a strain lacA-1 with high expression level of lactase was selected.LacA-1 with multicopy was identified by whole-cell PCR.The recombinant strain could secrete extracellular lactase identified by SDS-PAGE.Thin-layer Chromatography?TLC?analysis indicated thatthe lactase could hydrolyze lactose into glucose and galactose single-factor and orthogonal test were used to optimize the medium.The optimum components of fermentation medium were as follows:lactose 4%,yeast powder 6%,cornsteep liquor 5%,MgSO₄ 0.1%,MnSO₄ 0.03%.The optimum conditions were as follows:inoculum size is 10%,pH is the initial value,temperature is 28?,rotational speed is 200 r/min,culture volume is 50 mL in 250 mL erlenmeyer flasks,incubation time is 120 h.Under the conditions,the lactase enzyme activity is 142.57 U/mL,which is about 5.81 times more than that obtained by fermentation using initial fermentation medium.The results show that the optimum reaction temperature and pH of the recombinantlactase are 60?and 5.0,respectively.The residual enzyme activity after cryopreservation at 4?for 30 days is 82.73%of original enzyme activity.The residual enzymeactivity was95%of the original enzyme activity when the reaction temperaturewas 70?.The enzymatic properties are better than those of lactase produced by A.oryzae.In conclusion,a new engineering strain with high level extracellular secretion,high lactase activity,simple purification,and safe production process was successfully screened by genetic engineering method,which laid a foundation for the application of lactase in food,medicine,and other industries.