Development Of A Method For Efficient Cost-effective Screening Of Aspergillus Niger Mutants Having Increased Production Of Glucoamylase And Glucose Oxidase

Aspergillus niger is the one of the most important filamentous fungi used for biotechnological purposes.It has been utilized in industrial fermentations for more than 80 years because of its ability to accumulate and secrete large quantities of metabolites,such as organic acids,and large amounts of heterologous and homologous proteins.Glucoamylase is one of the extracellular products of A.niger.Strains are the basis of fermentation.By the mutagenesis and high throughput screening,high-yield industrial strains can be obtained quickly.In this paper,we developed an efficient cost-effective screening process to improve production of glucoamylase in A.niger.The cultivation of A.niger was achieved with well-dispersed morphology in 48-deep-well microtiter plates,which increased the throughput of the samples compared to traditional flask cultivation.There was a close negative correlation between glucoamylase and its pH of the fermentation broth.A novel high-throughput analysis method using Methyl Orange was developed.When compared to the conventional analysis method using 4-nitrophenyl a-D-glucopyranoside as substrate,a correlation coefficient of 0.96 by statistical analysis was obtained.Using this novel screening method,three genetic stable mutants of glucoamylase producer strain with more than 50%increase in yield were picked.Compared to the parent strain with an activity of 1.31×103 AGI ml-1 in flasks,the glucoamylase activity of mutants?-F-6(2.23×103 AGI ml-1),?-F-2(2.09×103 AGI ml-1)and ?-D-1(1.97×103 AGI ml-1)were increased by 70%,59%and 50%respectively.Besides,four heredity stable mutants of glucose oxidase producer strain were selected for further investigation.Compared to the parent strain with a activity of 1.6×102 U/ml in flasks for the extracellular activity and 2.4×102 U/ml,the GOD activity of mutants E1(2.1×102 U/ml and 3.9×102 U/ml),G1(2.6×102 U/ml and 3.8×102 U/ml)and H1(2.4×102 U/ml and 2.7×102U/ml)were increased by 68,53 and 45%respectivelyThe results emphasize the importance of high-throughput strains screening and its extensive applicability.After the discovery of upgraded strains,the analysis of different expression patterns and comparative genomics approach will be useful to conduct the directional optimization in aspect of genetic recombination and metabolic regulation.