Gene Cloning And Biochemical Characterization Of Endo-1,3(4)-β-glucanases From Aspergillus Niger

β-Glucanases play an increasingly important role in beer brewing industy,feed processing,functional food manufacturing,environmental protection and textile industry.However,the applications of most β-glucanases were largely limited by their low activities and heat instability.The genes encoding β-glucanases from Aspergillus niger were expressed in Pichia pastoris.and the enzymological property of β-Glucanases was studied.Firstly,based on the genome annotation information of A.niger CBS 513.88,three endo-β-1,3(4)-glucanase(en3gA,en3gB and en3gC)were cloned from A.niger CICIM F0510 via RT-PCR.The complete ORFs of these genes were predicted to be 1298,1191,and 1533 bp,respectively.The nucleotide and amino acid sequences of en3gA and en3gB were the same as those of A.niger CBS 513.88.However,en3gC had superflouous a fifth introns and 101 amino acid.Secondly,according to Pichia pastoris Expression Kit,three recombinant expression vectors(pPIC9K-en3gA,pPIC9K-en3gB,pPIC9K-en3gC)were constructed and three recombinant p-glucanase were obtained(GS115-En3gA,GS115-En3gB,GS115-En3gC).On the other hand,CMC was used as a substrate to assay the activity of GS115-en3gA,GS115-en3gB and GS115-en3gC.The activity of recombinant enzymes GS115-en3gA,GS115-en3gB and GS115-en3gC reached 1.3 U/mL,8.5 U/mL and 10.1 U/mL,respectively.SDS-PAGE showed that the recombinant enzymes are 44.4 kD,43.6 kD and 53.0kD,respectively,consistent with the predicted sizes.GS115-en3gA exhibited optimum temperature and pH at 65℃ and 5.5,which is stable at the range of 55～70℃ and pH 4.0～5.5.GS115-en3gB exhibited optimum temperature and pH at 50℃ and 5.0,which is stable at the range of 45～70 0C and pH 4.5～5.5.GS115-en3gC exhibited optimum temperature and pH at 550C and 3.5,which is stable at the range of 40～45 0C and pH 2.0～4.5,and has a bad temperature stability.Lastly,three recombinant enzymes had a typical endo hydrolysis CMC and curdlan.Meanwhile,GS115-en3gA had a different way to hydrolysis curdian containing glucose unlike the other two recombinant enzymes.