Effect Of Gene Dose And Gene Mutation On The Expression Of ?-galactosidase

?-galactosidase(EC3.2.1.23)is a type of glycoside hydrolase,and could hydrolyze lactose to produce galactose and glucose.It is a safe and non-toxic biological enzyme recognized by authoritative organizations.It is widely used in medicine,food,environmental protection,genetic engineering and other fields.In the fields of food and medichine,it can alleviate the symptoms of lactose intolerance by hydrolyzing lactose,which has greatly helped people to improve their living standards.In order to obtain a high-yield strain of ?-galactosidase,and facilitate it use in industry fieldsy,the gene Aogal derived from Aspergillus oryzae was artificially synthesized in this study and heterologously expressed it in Pichia pastoris;Its activity was measured by the ONPG method,and the highest activity of the obtained p AO815-Aogal expression strain was 89.16 U/m L;the determination of enzymatic properties showed that the optimum temperature of p AO815-Aogal was 60 ° C,and the optimum of p H was 5.0.After incubating at 30 °C,40 °C and 50 °C for 30 minutes,the activity retention are all more than 80 %.In order to increase the expression leveal of Aogal,this study reconstructed the expression vector p AO815-Aogal-1 after analyzing the codon preference of Aogal.After codon preference optimization,the highest enzyme activity of Aogal-1 was 196.76 U/m L,which was about two times higher than the original gene,and the optimal temperature and the optimal p H of Aogal-1 have not changed.After optimizing the codon preference,we tried to carry out site-directed mutagenesis of Aogal-1 to obtain a higher-yield strain.But after fermentation induction,although the target protein was successfully expressed,the protein expression level was not significantly increased,and the enzyme activity was as low as 12.90 U/m L.We further increased the expression level by constructing the tandem expression cassettes of Aogal-1.A two-and three-copy recombinant expression vector of?-galactosidase Aogal-1 was constructed using the isozyme method.The average enzyme activity of the two-copy recombinant expression strain was 210.07 U/m L,which was44.28 % higher than the single-copy strain;the average enzyme activity of the three-copystrain p AO815-Aogal-3 was 403 U/m L,which was 70.96 % higher than the single-copy recombinant expression strain.High-density fermentation of two copies of recombinant yeast strain B-3,the enzyme activity increased rapidly between 24-96 h and then gradually stabilized;after 72 h of induction,the enzyme activity was 4044.61 U/m L,which was11.02 times of the original shake flask fermentation at 72 h;after induction,the strain's fermentation enzyme activity reached 6353.39 U/m L.