Functional Analysis Of The ?-fructofuranosidase Gene From Aspergillus Niger And Study On The Synthesis Of Fructooligosacharides

As a kind of functional prebiotics,fructooligosaccharides(FOS)have been widely used in food,medicine and other fields because of their physiological functions such as regulating human intestinal flora balance,promoting mineral absorption,lowering blood fat and other physiological functions.Industrial production of FOS is usually carried out by using microbial enzymatic methods,Aspergillus niger ATCC 20611 is the main strain currently producing industrially fructooligosaccharides.Under sucrose-inducing conditions,the strain produces fructosyltransferase FopA with high fructosyltransferase activity,which could convert high concentration sucrose to fructooligosaccharides,but due to the reversible reaction process,the final concentration of fructooligosaccharide can only reach 50%-60%.Since 1988,the strain was screened and applied to the industrial production of FOS.People's research focused on optimizing the culture conditions and improving the enzyme activity.The strain's molecular genetic manipulation has hardly been reported.In particular,it is not clear whether ?-fructofuranosidase is responsible for the use of sucrose in strains,and the sucrose-inducing property of this gene has not been fully exploited.Compared with constitutive promoters,inducible promoters can achieve better regulation of gene expression,and can be used to control the expression of the target gene by means of environmental responses or chemical induction.The promoter of the fopA gene of Aspergillus niger ATCC 20611 is a sucrose-inducible promoter,and this strain has the advantages of being able to rapidly achieve high cell density and a low content of extracellular miscible protein,and it does not contain virus inclusions,pyrogens and pathogens.In addition,a FOS-synthesizing strain is screened out in the special habitat riched in sucrose molasses which is also a by-product with sucrose producing process industrially.In this dissertation,the Aspergillus niger ATCC 20611 was used as the research object.First,the ?-fructofuranosidase encoding gene fopA was knocked out,and the carbon source utilization and enzyme production characteristics of the knockout strains were analyzed.It was found that this strain cannot grow in Cassava's medium and fermentation medium where sucrose is a carbon source,nor can it produce?-fructofuranosidase.Therefore,the conclusion that the fopA gene is the key gene for sucrose utilization in Aspergillus niger ATCC 20611 was concluded.After further use of the knockout strain that can use glucose,fructose but can not use sucrose and fructooligosaccharides,the knockout strain is applied to the optimization of the 55%fructooligosaccharides.Finally,the fructooligosaccharides concentration was increased to 87%to achieve high concentrations of fructooligosaccharides production.Afterwards,the efficient protein glucose oxidase was expressed in Aspergillus niger ATCC 20611 to eliminate glucose for enhance the content of FOS.First,select the green fluorescent protein as a model protein to test whether the promoter can successfully perform foreign protein expression.The figure results observed under the fluorescence microscope showed that the fopA gene promoter can successfully express the foreign protein under sucrose induction conditions.Then select the secreted exogenous protein ?-glucosidase BGL1 as a model protein to detect whether the gene can successfully express exogenous secretory proteins.The results of esculin tablet showed that the color reaction of the transformants was very obvious,even higher than that of the positive control;The results of BGL1 enzyme activity assay showed that the enzyme activity in the transformant strain expressing BGL1 was up to 70 U/ml,but the starting strain had no enzyme activity;The extracellular protein bands of the transformed strains are single and distinct in the results of protein electrophoresis.In order to realize the co-expression of glucose oxidase GOX and FopA dual enzymes in this strain to achieve the inhibitory effect of simultaneous release of by-product glucose in fructo-oligosaccharide synthesis,the promoter is further used to express GOX protein,The GOX activity of the transformant FGX55 was 120 U/g.The application of the transformant to the synthesis of fructooligosaccharides showed that the glucose content in the product was reduced by 32%compared to the original strain and the fructooligosaccharides content was increased by 70%.At the same time,a filamentous fungus was successfully screened for its ability to synthesize fructooligosaccharides from the habitat riched in sugar molasses.The morphological identification and phylogenetic tree analysis of the ITS sequence showed that the fungus was Aspergillus tauringii XG21.The strain can use glucose,fructose,xylose,glycerol,sucrose and maltose,but it grows slowly when glycerol is the carbon source.Among the above carbon sources,the strain producing?-fructofuranosidase was only induced under sucrose,and the highest enzyme activity was 280 U/g in 48 hours.When the sucrose concentration was low,the strain showed hydrolytic activity,and the product was mainly glucose and fructose.At high sucrose concentration,fructosyltransferase activity was increased,FOS was continuously generated,and FOS concentration was approximately 56%at equilibrium.The enzyme production was optimized using cane molasses as a carbon source.When the molasses molasses concentration was 20%(equivalent to 8%sucrose),the enzyme activity was 580 U/g,which was nearly doubled when compared with 8%sucrose fermentation.