Enzymatic Properties And Applications Of The Inulin Fructotransferase From Nocardiaceae Family

Difructose anhydride Ⅰ（α-D-fructofuranose-2′,1:2,1′-β-D-fructofuranose dianhydride,DFA Ⅰ）is a kind of annular disaccharides consisted of two fructose units.DFA Ⅰ and functional disaccharides,difructose anhydride ⅠII（α-D-fructofuranose-2’,1:2,3’-β-D-fructofuranose dianhydride,DFA ⅠII）are isomers.However,reports about DFA Ⅰ were quite few.This paper synthesized three recombined inulin fructotransferase（IFTase）from Nocardiaceae family:Nocardioides luteus（Nolu IFTase）,Nocardioides sp.JS614（NospIFTase）and Nocardioides bacterium Broad-1（NobaIFTase）and researched their enzymatic properties.This paper also designed a novel method to produce DFA Ⅰ.The inulosucrase origined from Lactobacillus gasseri DSM 20604 and Nosp IFTase were combined to catalyse sucrose into DFA Ⅰ,and optimized the experiment conditions to improve utilization ratio of raw material.Results of sequence alignment for various IFTases showed that the hydrophobicity difference of 133,292,313,315 amino-acid residues between DFA Ⅰ-forming IFTases and DFA ⅠII-forming IFTase could be the reason for forming different products.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE）showed that the monomer molecular mass of three enzymes ranged from 41,000 to 42,000 Da,their whole molecular mass was detected to be ranged from 50,000 to 62,000 Da by gel column,the two results showed that three enzymes were monomers.The enzymatic properties of three recombinant enzymes were investigated by high performance liquid chromatography（HPLC）.They had similar optimum pH and temperature.NospIFTase had the best thermal stability,it was stable for 187 min when it was incubated at 70°C.NobaIFTase had the closed combination with inulin and the highest catalytic efficiency.Nosp IFTase had the highest conversion ratio of inulin to DFA Ⅰ when inulin concentration was 100 g/L,the value was up to 85.3%after 2-hours reaction.The smallest substrate was determined as nystose GF₃.The recombinant enzymes can hydrolyze GF₃ to DFAI and sucrose.On the other hand,optimum pH was determined to be 6.0 for double-enzymes coupled reaction and inulosucrase originated from Lactobacillus gasseri DSM 20604showed max activity of transferring sucrose into inulin at 45°C.The conversion ratio of sucrose to DFA Ⅰ was 26.5%.At present,no DFA Ⅰ-forming IFTases from Nocardiaceae has been reported.The conclusion of this paper is of great significance for expanding the research scope of DFA Ⅰ IFTase,and provides a theoretical basis for the analysis of the crystal structure of DFA Ⅰ-forming IFTase.The double enzyme method provides a new way for the synthesis of DFA Ⅰ and reduces the cost of DFA Ⅰ synthesis.