Cloning,Expression And Properties Of Thermal Stable ?-galactosidase

a-galactosidase(EC3.2.1.22)belongs to the class of glycoside hydrolases.It could specifically hydrolyze the a-galactoside bond of non-reducing ends of different substrates,and thus was used in the feed and paper industry to improving the nutrient utilization and pulp bleaching by hydrolyzing the galactosides in the raw materials.In this paper,a a-galactosidase gene TEgal from Talaromyces emersonii was artificially synthesized,and then successfully expressed in Pichia pastoris.The highest activity of the obtained pAO-TEgal recombinant strain was 9.5 U/mL.TEgal has an optimum temperature of 75?,and has optimum pH of 3.5which indicated that TEgal has the thermolstable characteristics.Metal ion Na+and K+could improve the enzyme activity by 5%and 8%,respectively.In order to improve the expression level of TEgal,two-copy and three-copy a-galactosidase recombinant strains were successfully obstained by the method of constructing the tandem expression cassettes.The average enzyme activity of the expressed two-copy pAO-2TEgal recombinant strain was 16 U/mL,which was 97.3%higher than that of the single-copy strain.The average activity of the three-copy strain pAO-3TEgal was 14.9 U/mL,which was 83.7%higher than that of the single-copy recombinant strain.In this paper,10 amino acids around the active sites of a-galactosidase TEgal were mutated by site-directed mutagenesis.The result showed that the enzyme activity of mutant G46W was 13.55 U/mg,which was almost equal to the original enzyme(14 U/mg).The specific activity of mutant C230D was 2.35 U/mg,which was 83.3%lower than that of the original enzyme.Eight mutant strains,such as D77N,D78N and Y121F,have no a-galactosidase activity.By homology modeling of TEgal and its mutants,molecular docking with raffinose,we further analyzed the effect of these mutated amino acids on the structure and the activity of a-galactosidase.It was proved that Asp77,Asp78,Tyrl21,Cys230,Arg249,Asp284,and Met285 are key amino acids to the catalytic activity of a-galactosidase.In this paper,the thermal stable a-galactosidase TEgal gene was successfully cloned and expressed in P.pastoris.The expression level was further improved by constructing the multi-copy expression cassettes.Seven amino acids were detedcted as important to catalytic activity of TEgal enzyme,thus provided a theoretical basis for the molecular modification of a-galactosidase.