The Research Of Mutagenesis Breeding And Gene Cloning And Expression Of High Yielding Glucoamylase

Glucoamylase,also known as Glucoamylase EC.3.2.1.3.,the molecular weight between 50 KD and 112 KD.It has exonuclease activity and can hydrolyze starch into glucose.Glucoamylase is the enzyme product with the largest yield and the widest application range in China,which has great industrial value.Aspergillus niger is the main production strain in its industry.In order to obtain the high-yield strain of glucoamylase,the mutagenesis breeding methods was used to mutagenesis the A.niger MS1 with UV and DES.After primary screening,rescreening and 8 passage tests,a high-yielding strain of A.niger MS 1-2 with stable heredity was found,whose enzyme activity reached 35523.2u/mL,which was 57.3%higher than the original strain.In order to optimize the enzyme production conditions of A.niger MS 1-2 by liquid fermentation and expand the industrial production,single factor experiment and orthogonal test were conducted to study the composition and conditions of fermentation medium.The results showed that the optimal conditions for producing enzyme by liquid fermentation of A.niger MS 1-2 were:2%Starch,6%bean cake powder,2%corn steep liquor,0.2%(NH4)2SO4,Initial pH of 5,quantity of 5%,fluid volume 50 mL conical flask(250 mL),temperature 30?,speed 200 r/min,fermentation time,144h(6d),the activity of glucoamylase was 41214.1U/mL,which was 15.6%higher than that of the original strain.The enzymatic properties of MS 1-2 glucoamylase were studied,found that the optimal reaction temperature of the glucoamylase was 55 ?,the optimum pH value of 5;In 70 ?heat preservation 1 h,enzyme activity was more than 51.9%In this paper,primers were designed according to the gene sequence of A.niger glucoamylase published on NCBI,by PCR from A.niger MS 1-2 chromosome amplification have been saccharifying enzyrme gene(gla)and non-coding regions of 5' 900bp upstream of fragments,and sequencing analysis respectively.Results:the gene size of A.nigerr MS 1-2 glucoamylase was 2172bp,gla didn't mutate,but the 5'upstream region has seven mutations:This indicated that the increase of MS 1-2 glucoamylase expression in A.niger was not related to its structural genes,and might be related to the mutation of these 7 bases in the upstream segment.After double enzyme digestion,the recombinant plasmid pPIC9K-gla was constructed.It was integrated into Pichia pastoris GS115 by electroporation to screen recombinants.When recombinant Pichia pastoris was induced with 1%methanol for 72h,the maximum saccharification enzyme activity was 23.8U/mL.Restructuring the optimum reaction temperature and pH value of the expression of enzyme was 50 ? and 5,respectively.