Study On Properties And Molecular Modification Of Inulin Fructotransferase From Streptomyces Sp.

DFA I(?-D-fructofuranose-2?,1:2,1?-?-D-fructofuranose dianhydride,DFAI)is a cyclic disaccharide composed of two fructose units,which rarely exist in nature.It shows good sweetness but lower energy than sucrose,and it has many potential physiological effects to human health.DFA I and DFA III(?-D-fructofuranose-2?,1:2,3?-?-D-fructofuranose dianhydride,DFAIII)are isomers.So far,there is few report on the physiological effects of DFA I,probably because of the difficulty to obtain DFA I in a large scale.Biological production of DFA I shows some significant advantages,including high conversion rate,less by-products,and economically feasible.The biological production of DFA I may be realized by inulin fructotransferase(IFTase)(DFA I-forming)from inulin as substrate.In this study,a mircrobial strain S.davawensis SK 39.001 was saved by the laboratory,which was able to produce DFA I when using inulin as the sole carbon source.The results showed that S.davawensis genome involved an IFTase(DFA I-forming)encoding gene.The IFTase(DFA I-forming)gene fragment(1179 bp)was extracted by genetic engineering methodology from S.davawensis SK 39.001.The full-length nucleotide sequence was commercially synthesized and was sub-cloned into pET-22b(+)vector.The recombinant plasmid was then transformed into E.coli BL21(DE3)for expression,and Isopropyl ?-D-1-thiogalactopyranoside(IPTG)was used to induce the excessive expression of protein.The recombinant enzyme was proved to be able to convert inulin to DFA I,and then was named S.davawensis IFTase(DFA I-forming),briefly SdIFTase.The enzymatic properties of the recombinant SdIFTase were studied.The enzyme had a monomer molecular weight of 40,000 as assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and showed the maximal activity as 100 U/mg at pH 5.5 and 40°C.Compared with previously reported IFTases(DFA I-forming),the SdIFTase displayed a moderate thermostability and was stable for 30 min when it was incubated at 70°C.The Km was measured to be 2.89 mmol/L and the conversion ratio of inulin to DFA I reached 81%,69% and 51% when inulin concentration was 10,50,and 100 g/L,respectively.The smallest substrate was determined to be nystose(GF3).The SdIFTase could slightly hydrolyse GF3 to DFA I and sucrose.In addition,the SdIFTase was a metal-independent enzyme.In addition,the relationship between the structure and function of IFTase was investigated,using site-directed mutation and computational methodology.First,site-directed mutation was carried out in site D183,E194,and I80,which were related to catalytic activity of enzyme,the variants D183 A,D183N,E194 A,E194N,I80 A,I80E and I80 S had obvious effect on enzyme activity.It indicated that D183,E194,and I80 might be at the active center.Second,the variants G281 S,G121A/T122 L,G236S/G281S/A257S/T313S/A314 S were designed to improve the thermostability.Using NanoDSC to study thermostability,it showed that the Tm values of these variants were improved.Especially,the Tm of G121A/T122 L was enhanced by 4.5°C.Additionally,enzyme activity of G121A/T122 L was highly improved.Therefore,given the increase of enzyme activity and thermalstability of G121A/T122 L,the application of SdIFTase in industry may be more feasible.