游离及固定化果糖基转移酶部分酶学性质的比较研究

 从诱变、筛选的米曲霉GX0 0 10菌株所产生的果糖基转移酶 ,经过纯化和固定化操作分别制备游离酶和固定化酶 ,对两者的酶学性质进行了比较研究 .结果表明 ,两者在蔗糖转化为蔗果低聚糖的酶促反应中 ,最适pH为 5 5,在pH5 0～ 7 5之间酶活性相对稳定 .游离酶和固定化酶的适宜温度范围分别是 4 5～ 52℃和 4 0～ 55℃ .在 55℃保温 60min ,酶活性保存率分别是 61 6%和 87 5% .固定化酶的热稳定性提高 .0 1mmol LHg2 +和 1mmol LAg+能完全抑制游离酶的活性 ,但只能部分抑制固定化酶的活性 ,1mmol L的Ti2 +能完全抑制两者的活性 .以蔗糖为底物时 ,游离酶的米氏常数Km=2 15mmol L ,而固定化酶Km =386mmol L .游离酶只能使用一次 ,固定化酶反复使用 54次后 ,剩余活力为 55 2 % .用 55% (W V)蔗糖溶液与固定化酶在pH5 0 ,4 6℃下作用 12h ,可获得61 5% (总低聚糖 总糖 )产物 ,其中蔗果五糖含量达到 7 2 % .

The Comparative Study of Enzymatic Properties between the Free and Immobilized Fructosyltransferase

Fructosyltransferase (FTase;EC 2.4.1.9) converts sucrose effectively to fructooligosaccharide (FOS),a functional sweetener used widely in health food.The enzymatic propertis of immobilized FTase were investigated and compared with those of the free enzyme.It was found that both free and immobilized FTase have the pH optimum of 5 5 and were stable over a pH range of 5.0—7.5.The optimal temperature and thermostability of the immobilized enzyme was improved over that of the free enzyme,resulting in a temperature range for activity of 40—55℃ and a retention of 87 5% of its optimal activity when subjected to 55℃ for 60 min,compared to the free enzyme which exhibited a 45—52℃ activity range and a retention of 61 6% of its optimal activity.The K m of the immobilized FTase (386 mmol/L) was about 1.8\|fold that of the free enzyme (215 mmol/L).The activity of the free enzyme was significantly inhibited by certain heavy metal ions,e.g.0 1 mmol/L Hg 2+ and 1 mmol/L Ag +,but the immobilized enzyme was only partly affected under the same conditions.Both the free and immobilized enzymes were completely inhibited by 1 mmol/L Ti 2+ .Using a sucrose solution (55%, W/V) as a substrate,the production of FOS using the immobilized enzyme was carried out at 46℃ and pH 5 0. After 12 hours,FOS production yielded 61 5% (FOS/total saccharide).The reaction mixture content of 1,1,1,\|kestopentose reached 7 2%.Under the same reaction conditions,1,1,1\|kestopentose was normally less than 4% of the total saccharide.Whereas the free enzyme can only be used once,the immobilized FTase demonstrated excellent repeated use capability and stability during storage.These results suggest that the immobilization of the enzyme effects the interaction between the substrate and active site of the enzyme,the mass transfer of the reactant (sucrose) and products (FOS).The immobilized enzyme is less sensitive to denaturation by high temperature and heavy metal inos and shows great promise for the enzyme\|based production of FOS by industry.