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Studies on the Synthesis of New Oligosaccharide by Bacillus subtilis Levansucrase and Biological activity of the Synthesized Sugar.

枯草芽孢杆菌左聚蔗糖酶合成新型低聚糖及合成糖生物活性的研究。

基本信息

批准号：
61560105
负责人：
IIZUKA Masaru
金额：
$ 1.15万
依托单位：
Faculty of Science, Osaka City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1986
资助国家：
日本
起止时间：
1986 至 1987
项目状态：
已结题

项目摘要

Levansucrase was obtained as an electrophoretically pure state from cultured cells of Bacillus subtilis grown on sucrose medium using chromatographies on DEAE-Cellulose, hydroxyapatite and Sephadex G-100 after salted out with (NH_4)_2SO_4.The enzyme produced levan for sucrose substrate alone, but it produced hetero oligosaccharide when the reaction was made in the presence of hetero oligosaccharide as an acceptor. The mazimum efficiency of transfructosylation from sucrose to acceptor sugar by the enzyme attained about 80% in the case of the acceptor/donor(molar ratio)= 1 or more. Oligosaccharide formed by levansucrase from sucrose and reducing oligosaccharide was non reducing sugar that C2 of fructosyl residue linked to C1 of reducing end of acceptor sugar. Trehalose which is non reducing oligomer, is also able to be an acceptor although the yield of Trehalose-F is very low. In this case, C2 of the fructosyl residue is linked to C6 of glucose residue in trehalose. Levansucrase linked to CH-Sepharose 4B to produce immobilized enzyme which has increased thermal stability (30゜C->40゜C) was used repeatedly to make maltooligosaccharide-F without loss of the activity. Maltooligosaccharide-F synthesized can be used as substrate for differential determination of amylase activity.Other heterooligosaccharides obtained by levansucrase were tested as substrate for determining enzyme specificity and a new -glucosidase in human urine was found. Heterooligosaccharides produced described above are useful as substrate for screening of new carbohydrase.

枯草芽孢杆菌培养细胞经(NH_4)_2SO_4盐析后，经DEAE-纤维素柱层析、羟基磷灰石柱层析和Sephadex G-100柱层析，得到一种纯电泳态的枯草芽孢杆菌细胞。该酶单独为蔗糖底物产生葡聚糖，但在杂寡糖存在下反应生成杂寡糖。当受体/供体(摩尔比)=1或1以上时，该酶催化蔗糖向受体糖转移的最大效率可达80%左右。低聚糖是由蔗糖的左旋蔗糖酶与还原低聚糖结合而成的非还原糖，其果糖残基的C2与受体糖还原端的C1相连。海藻糖是一种非还原齐聚物，虽然海藻糖-F的产率很低，但它也可以作为受体。在这种情况下，果糖残基的C2与海藻糖中葡萄糖残基的C6相连。将左旋糖苷酶连接到CH-Sepharose4B上，产生具有更高热稳定性的固定化酶(30゜C-&GT；40゜C)，重复使用以制备麦芽低聚糖-F而不损失其活性。以合成的麦芽低聚糖F为底物，差示测定淀粉酶活性。以其他由左旋蔗糖酶得到的杂寡糖为底物进行酶专一性测定，发现了一种新的人尿中的葡萄糖苷酶。上述产生的杂寡糖可作为筛选新碳水化合物酶的底物。