Gene Cloning,Expression And Characterization Of α-Galactosidase From Aspergillus Niger

α-Galactosidase（α-Galactosidase,α-Gal,EC 3.2.1.22）is an exoglycosidase that can catalyze the hydrolysis ofα-galactosidase bond in galacto-oligosaccharides,polysaccharides,glycolipids and glycoproteins.It is widely used in food,feed and medicine.In this study,the c DNA obtained by reverse transcription of total RNA of A.niger was used as the template andα-GalⅠgene andα-GalⅡgene were amplified by PCR,then recombinant plasmid p PICZαA-α-GalⅠand p PICZαA-α-GalⅡwas successfully obtained by connecting target gene to the vector p PICZαA.The recombinant plasmid was integrated into P.pastoris X33,and the P.pastoris expression system X33/p PICZαA-α-GalⅠand X33/p PICZαA-α-GalⅡwas constructed,the separation and purification steps were simplified due to its special expression.The induced expression conditions of recombinant engineered bacteria were optimized to improve enzyme activity ofα-GalⅠandα-GalⅡ.Further study its enzymatic properties and explore its optimal reaction conditions,temperature/p H stability,substrate specificity,metals ion stability,enzymatic kinetics,anti-protease characteristics and storage stability.The main results are as follows:（1）Cloning and heterologous expression of theα-Gal gene from A.nigerThe total length ofα-GalⅠgene is 1554 bp,which encodes 518 amino acids,and the similarity with Gen Bank accession number CAK44933.1（Remove signal peptide）is 97%.The total length ofα-GalⅡgene is 1635 bp,which encodes 545 amino acids,and the similarity with Gen Bank accession number CAA44950.1 is 98%.Recombinant genetically engineered bacteria X33/p PICZαA-α-GalⅠand X33/p PICZαA-α-GalⅡwere constructed.α-GalⅠandα-Gal II crude enzyme obtained by inducing two recombinant engineered bacteria with methanol.（2）Optimization ofα-GalⅠinduced expression conditions,purification and enzymatic properties analysisThe optimal concentration of induced expression was 0.5%for recombinant strain X33/p PICZαA-α-GalⅠ,the optimum temperature and induction time were 26℃and 96 h respectively.The synergistic effect was produced by adding 5 mmol/L glutamic acid,10mmol/L glucose and tea polyphenols,which can promote the expression ofα-GalⅠand make the cumulative enzyme activity reach 7.2 U/m L,it was 65 times higher than that before optimization.Purification ofα-GalⅠcrude enzyme solution using His Trap nickel column,after purification,the specific enzyme activity reached 3.02 U/mg,the purification multiple was 10.79,and the recovery was 79.8%.The optimum temperature of recombinationα-GalⅠis 55℃and the optimum p H is 4.0.It remains stable in the temperature range of-20℃-40℃and the p H range of 3.0-10.0.Its optimum reaction substrate is p-Nitrophenyl-α-D-Galactopyranoside（p-NPG）.The addition of 1 mmol/L Ni²⁺can significantly improve the enzyme activity ofα-GalⅠand it was significantly inhibited by adding 1 mmol/L Ag⁺.α-GalⅠshowed good resistance to pepsin and trypsin.The storage period ofα-GalⅠis less than 10 days,the effect of pure enzyme solution stored at 4℃is the best,which can maintain more than 80%of the enzyme activity.It is better to store it at-20℃with 50%p H 4.0 buffer for more than 10 days,and the enzyme activity ofα-GalⅠcan still maintain more than 65%after 30 days.（3）Optimization ofα-GalⅡinduced expression conditions,purification and enzymatic properties analysisThe optimal concentration of induced expression was 0.5%for recombinant strain X33/p PICZαA-α-GalⅡ,the induction temperature was 26℃and the induction time was 96h.The synergistic effect was produced by the addition of 5 mmol/L ammonium sulfate and 10mmol/L glucose,which promoted the expression ofα-GalⅡand made the cumulative enzyme activity reach 7.4 U/m L,it was 46 times higher than that before optimization.After purification by affinity chromatography,the specific enzyme activity was 3.1 U/mg,the purification multiple was 11.92,and the recovery was 64.36%.The optimum temperature of recombinationα-GalⅡis 55℃and the optimum p H is 3.5.It remains stable in the temperature range of-20℃-45℃and the p H range of 3.0-10.0.Its optimum reaction substrate is p-NPG.The enzyme activity ofα-GalⅡcan be significantly inhibited by adding1 mmol/L Ag⁺,other metal ions have showed different degrees of promoting effect on it.α-GalⅡshowed good resistance to pepsin and trypsin.It is better to store in 20%glycerol solution at-20℃,the enzyme activity ofα-GalⅡcan be maintained at more than 70%after30 days.In this paper,we discuss the enzymatic properties ofα-GalⅠandα-GalⅡ,it is determined that it has good heat resistance,extensive p H stability and good resistance to pepsin and trypsin,which widened application scope ofα-GalⅠandα-GalⅡ,lays a foundation for its application in many fields such as food and medicine.