Fermentation Optimization,Purification And Biochemical Characteristics Of Fructosyltransferase From A New Isolated Penicilliun Griseofulvum

Fructooligosaccharides have received widespread attention and application in life because of their unique physiological effects:regulate the balance of intestinal flora,enhance immune activity,lower cholesterol,reduce blood fat,promote mineral absorption and prevent dental caries.Fructo-oligosaccharides have a wide range of sources.Modern industry mainly produces Fructo-oligosaccharides through fermentation using sucrose as a substrate.Fructosyltransferase（FTase）producing strains are the foundation of the Fructooligosaccharide industry.It is important for the fructo-oligosaccharide industry that screening of FTase-producing strains.In this study,thin-layer chromatography（TLC）and high-performance liquid chromatography（HPLC）were used to analyze the transfructosylation ability and FOS synthesis of wild fungi.As a result,a new FTase-producing strain Penicillium griseofulvum YT126 was obtained.The wild strain Penicillium griseofulvum YT126 showed low FTase activity.To improve this disadvantage,the composition and fermentation conditions of the fermentation medium of Penicillium griseofulvum YT126 were optimized.In order to further understand the enzyme production characteristics of the FTase production strain Penicillium griseofulvum YT126,the fructosyltransferase of Penicillium griseofulvum YT126 was isolated and purified.Finally,some enzymatic properties of the purified FTase were studied.The main research work as follows:（1）To obtain the FTase-producing fungi,the solid medium was applied for cultivation of various samples taken from our campus.To confirm the FTase activity of these fungi,thin-layer chromatography（TLC）was used to analyze the reaction products.The wild type strain YT126 could produce clear bands of fructooligosaccharid（GF₂）on the chromatography plate,which confirmed the transfructosylation activity of the wild type strain YT126.Colonies of YT126 were thicker with radial wrinkles,texture velvety and granular.Meristem structure was abundant.The surface of meristem was gray-green.The amplified nuclear ribosomal internal transcribed spacer（ITS）region of the strain YT126was sequenced and compared with the ITS sequences of micro-organisms represented in the NCBI database gene bank using BLAST.The BLASTn result for its ITS sequence showed 99.46%sequence identity to those of the Penicillium griseofulvum strain.A neighbor-joining phylogenetic tree was constructed by using the ITS sequences of YT126 and other closely related fungal strains according to the Gen Bank database.From the ITS phylogenetic analysis,YT126 shared the same latest common origin and evolutionary lineage with P.griseofulvum and followed by Penicillium citrinum.On the basis of the ITS sequence analysis,together with its morphological characteristics,the strain YT126 was identified as Penicillium griseofulvum and hence named Penicillium griseofulvum YT126.To date,there is no report on the ability of synthesizing FOS by Penicillium griseofulvum and so Penicillium griseofulvum YT126 was used for analysis of FTase production.（2）Based on the single factor optimization results,the response surface method（RSM）was used to systematically optimize the fermentation medium components and culture conditions of Penicillium griseofulcum YT126.Finally,three factors and their optimal values that significantly affect the fermentation results of Penicillium griseum YT126 were obtained.They are the concentration of Cu SO₄,the temperature and the p H.The best technical parameters were obtained as followed:the concentration of Cu SO₄ 0.25 g/L,temperature 24°C and p H7.0.Under the optimal condition,the fructosyltransferase activity produced by strain Penicillium griseum YT126 reached 20.67 U/m L.It is close to the predictive value of 20.35 U/m L.（3）Fructosyltransferase was purified by ammonium sulfate fractionation precipitation,DEAE-sepharose ion exchange chromatography and Sephacryl S-200 HR gel column chromatography from Penicillium griseum YT126.The specific activity of fructosyltransferase purified by Penicillium griseum YT126 was 1463.5 U/mg,the purification rate was42.6 times,and the recovery was 29.6%.Single protein band was obtained from SDS-PAGE,the molecular weight of purified enzyme was approximate 55 k Da by gel filtration chromatography.（4）The characterization of the fructosyltransferase from the strain P.griseofulvum YT126 was studied.The optimal p H and temperature of the enzyme were p H 7.0 and 65°C,respectively.It’s stable in the range of p H5.5-8.0 and 50°C.The addition of most metal ions at a low concentration（1 m M）had no significant influence on activity.The cation Ag⁺and Pb²⁺,however,strongly inhibited enzymatic activity at both low and high concentrations（1 m M and 5 m M）.Conversely,enzymatic activity was slightly enhanced in the experiments using high concentrations（5 m M）of Mg²⁺,Cu²⁺,Fe²⁺as well as Mn²⁺.The kinetics of the enzyme was determined by Lineweaver–Burk plot and K_m of 161.4m M and V_max of 1.8 mmol/（min·L）values were attained.