用于高果糖浆制备的宛氏拟青霉嗜热嗜酸菊粉酶酶学特性分析

Enzymatic Properties of Thermoacidophilic Inulinase from Paecilomyces variotii to be Used in Production of High Fructose Syrup

为实现酶法水解菊糖制备高果糖浆,从宛氏拟青霉(Paecilomyces variotii)XS27发酵液中分离纯化菊粉酶,并对其酶学特性进行研究。发酵液经过硫酸铵盐析、透析、DEAE-Sepharose Fast Flow层析、Sephacry S-100分子筛过滤层析,得到电泳纯的菊粉酶,比活力327.4 U/mg,纯化倍数37.85。十二烷基硫酸钠-聚丙烯酰氨凝胶电泳测得菊粉酶为单一亚基的酶蛋白,分子质量62.0 kDa。菊粉酶能在较宽的pH值范围(3.5～6.5)内保持高活性,最适作用pH 4.0。在温度40～65℃之间,酶活力较高,最适作用温度为60℃。薄层色谱分析显示菊粉酶水解菊糖最终产物为果糖。以菊糖为底物,酶的K_m和V_(max)分别为5.93μmol/L和75.18μmol/(L·min)。Mg2+、Mn2+、Ca2+对酶有显著激活作用,Ba2+、Ni2+和Hg2+对酶有一定抑制作用。β-巯基乙醇、二硫苏糖醇和乙二胺四乙酸对酶有抑制作用,表面活性剂(十二烷基硫酸钠、Tween 80和Trition X100)以及乙醇对酶活力没有影响。从宛氏拟青霉XS27发酵液中分离纯化的菊粉酶在强酸高热的环境下具有强活性和稳定性,对表面活性剂乙醇有高耐受性,适合于果葡糖浆的工业化生产。

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, DEAE-Sepharose 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 K m and V max values were 5.93 μmol/L and 75.18 μmol/(L·min), respectively. The inulinase could be activated by Mg^2+, Mn^2+ and Ca^2+, but inhibited by Ba^2+, Ni^2+ and Hg^2+. 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.