关键词: 菊粉酶, 高果糖浆, 分离纯化, 酶学特性

Abstract: Electrophoretically pure inulinase for use in enzymatic production of high fructose corn syrup was isolated from the fermentation broth of Paecilomyces variotii XS27 by consecutive ammonium sulfate precipitation, dialysis, DEAESepharose Fast Flow chromatography and Sephacry S-100 gel filtration chromatography. The enzyme was purified 37.85 folds with a specific activity. Its relative molecular mass was estimated to be 62.0 kDa by SDS-PAGE, consisting of a single subunit. This inulinase was active over a broad pH range of 3.5–6.5 and the optimum pH was 4.0; it had a high activity at temperatures ranging from 40 ℃ to 65 ℃, and the optimum temperature was 60 ℃. Thin-layer chromatography (TLC) analysis showed that the final hydrolysis product of inulin by this enzyme was fructose. When inulin was used as a substrate, the Km and Vmax values were 5.93 μmol/L and 75.18 μmol/(L·min), respectively. The inulinase could be activated by Mg2+, Mn2+ and Ca2+, but inhibited by Ba2+, Ni2+ and Hg2+. The inulinase activity was inhibited by β-mercaptoethanol, DTT and EDTA but was not affected by surfactants (SDS, Tween 80 and Trition X100) or the organic solvent ethanol. In summary, the strong activity and stability of this inulinase against strong acid and high temperature as well as ethanol makes it suitable for the production of fructose and fructooligosaccharides.

Key words: inulinase, high fructose corn syrup, isolation and purification, enzymatic properties