Identification And Exploration Of Biological Characteristics Of Aspergillus Niger Strain ANSTJ01 With High Cellulase Production

Aspergillus niger is a kind of important industrial fermentation strain with fast growth,low nutritional requirements,strong environment adaptability that was usually found in soil,grain and vegetable products.Aspergillus niger was extensively applied in the production of enzymes organic acids,heterologous proteins with the property of producing amylase,acid protease,cellulase,pectinase,glucose oxidase,citric acid,gluconic acid and galic acid,etc.Aspergillus niger could produce high level of amylase and beta-glucosidase considered producing on toxins,and was another kind of cellulase producing fungus with the potential of industrial application after Trichoderma reesei.Cellulase was a compound enzyme that can hydrolyze cellulose into monosaccharides including endoglucanases,exoglucanases and beta-glucosidase.Cellulase was wildly used in food,feed,light industry and biological energy,etc.We isolated a filamentous fungus in the kitchen confirmed as Aspergillus niger,named Aspergillus niger ANSTJ01.Through the morphology and molecular biology identification,using the Aspergillus niger type strain 2485 as controls,we studied the biological characteristics of Aspergillus niger ANSTJ01 including the colony morphology,growth characteristics and enzyme production characteristics.The results showed ANSTJ01 colony had denser orderliness hyphae and the spores appeared after cultured 36 h,the color of the colony was dark black and the back was deep yellow before 120 h and became white after 120 h.The spores were produced at 48 h after the ANSTJ01 strain cultured in PDA liquid medium which was 12 h later than 2485 strain,and ANSTJ01 strain grew faster than 2485 strain,the optimum growth temperature was 30 ?,the optimum growth pH was from 3 to 10.To measure respectively the activity of endoglucanase,exoglucanase,beta-glucosidase filter paper enzyme,pectinase,amylase,xylanase,acid protease,lipase of solid-state fermentation crude enzyme,using the sodium carboxymethyl cellulose solution,microcrystalline cellulosesuspension,salicin solution,filter paper,soluble starch solution,pectin solution,xylan solution,casein solution,palmitic acid p-nitrophenyl acetate solution as the substrate.The results showed the ANSTJ01 strain could produced high acvitivity of amylase of 4 U/g and beta-glucosidase of 2 U/g,and low acvitivity of exoglucanase of 0.01 U/g,and the activity of endoglucanase,exoglucanase,filter paper enzyme,pectinase produced by ANSTJ01 strain was significantly higher than 2485 strain.In order to explore the reason why the cellulase activity of ANSTJ01 strain crude enzyme liquid was higher than 2485 strain,we used the qRT-PCR method to measure the expression levels of endoglucanase gene(eglA,egl B),exoglucanase gene(cbhA,chbB),beta-glucosidase gene(bgl1),cellulase regulation factor gene(XlnR),carbon source metabolic block protein gene(CreA),histone acetylation enzyme gene(Gcn5).The results showed ANSTJ01 strain XlnR gene expression level was 7 times of 2485,eglA gene was 6 times of2485,cbhA gene was 8 times of 2485,Gcn5 gene and cbhB gene were only about 1/5 of 2485,and other cellulase gene expression levels had no significant difference.We measured the characteristic of endoglucanase and exoglucanase,and found out the enzyme characteristic of ANSTJ01 stain and 2485 strain was similar and the the optimum reaction temperature was50? and the optimum reaction pH was weak acid.We cloned and sequenced the eglA gene and cbhA gene,compared and analyzed the amino acid sequences,and we found out the egl A had one amino acid different,and the mutation position was located in the signal peptide,the cbhA had two amino acids different,and the mutation positions were not the active sites.In conclusion,the ANSTJ01 strain cellulase expression level significantly increased regulated by the XlnR factor was the reason why the activity of crude enzyme liquid of ANSTJ01 strain was significantly higher than 2485 strain.We optimized the fermentation conditions of cellulase produced by ANSTJ01 strain in solid-state fermentation conditions including the medium basic composition,medium moisture content,medium inducer,incubation time,incubation temperature,incubation pH.The results showed that the optimization of solid-state fermentation conditions can effectively increased the production of cellulase,and the highest production of cellulase will achievewhen the medium basic composition content of wheat bran and wheat straw powder was from3:2 to 4:1,the medium moisture content was from 75% to 80%,culture temperature was30 ?,culture time was 3 d,and culture pH was 5.