Effect Of Gene Dose And Endoplasmic Reticulum Secretion-associated Protein On Expression Of ?-galactosidase

Alpha-galactosidase is an exo-glucosidase that hydrolyzes alpha-galactoside compounds.The main purpose of this study is to use molecular biology techniques to construct the thermostable and high-expressed conserved?-galactosidase yeast recombinant.In this study,a Neosartorya fischeri P1?-galactosidase Gal A gene was synthesized and expressed in Pichia pastoris.Enzymatic charactering the expression product of the recombinant pAO-Gal A shown that the optimum pH of the expressed?-galactosidase Gal A was pH 4.5 and the optimum temperature was 50?.Salt FeCl₂ with the final concentration of 20 mmol/mL can greatly improve the?-galactosidase enzyme activity.Generally,the average activity of single copy?-galactosidase Gal A recombinant reached55.74 U/mL.We then constructed two copies and three copies of?-galactosidase recombinant expression plasmid,transferred into the yeast and methanol inducible expressed.The average enzyme activity of two copies?-galactosidase was 95.22 U/mL,which was 70.83%higher than that of single copy strain;The average enzyme activity of the three copies?-galactosidase was 35.93 U/mL,which was 35.54%lower than that of the single copy strain.We further constructed four recombinant expression strains by co-expressing the endoplasmic reticulum secreted protein-related gene with?-galactosidase gene.The results showed that the activity of co-expressing recombinant strains HAC1-Gal A,PDI-Gal A,and Ero1-Gal A was 64.24 U/mL,68.7 U/mL,and 70.71 U/mL,respectively.Compared with the single copy recombinant strain,the?-galactosidase activity increased 15.25%,23.25%,and 26.86%,respectively.The average activity of Hsp40-Gal A co-expressed recombinant strain was 30.67 U/mL,which was 44.99%lower than that of the single copy gene recombinant strains.The two-copy recombinant strains of?-galactosidase were methanol-induce expressed in 14L fermentor.After induced for about 90 hours,the enzyme activity of the supernatant was 791.23 U/mL,and the enzyme activity reached 1400 U/mL by incubating in the buffter with 20 mmol FeCl₂.The significance of this study was that we have build a thermal stable?-galactosidase containing yeast recombinant,improved the expression of?-galactosidase,reduced production costs,and provided a excellent candidate enzyme for industrial applications.