UTILIZATION OF ACID-STABLE A-AMYLASE FROM THE CITRIC ACID KOJI (Aspergillus usamii var.) AND PREPARATION OF EDIBLE MICROCAPSULE

在鹿儿岛，柠檬酸是在商业基础上由各种usamii曲霉制成的曲酒，使用淀粉生产工厂的甘薯废浆。为进一步利用副产物，采用柱层析法从曲的酸性萃取物中纯化出了酸稳定型a-淀粉酶（UAA）。该酶在pH为3 ~ 5.5（37℃）、温度为60 ~ 70℃（pH4.0）时表现出较普通中性a-淀粉酶（NA）高约15℃的最佳活性。n端前20个氨基酸的序列以及分子量约为10万的NA与普通NA不同。用14c -葡萄糖标记的一系列麦芽糖低聚糖，测定了其裂解速率参数k0/Km和裂解频率。结果表明，活性中心由5个亚位组成，催化位点位于距非还原端3 ~ 4个亚位之间。通过化学修饰来阐明氨基酸在a-淀粉酶活性位点的作用。利用商用G3-CNP的半乳糖基化合成了一种新的底物Gal-G3-CNP，使我们能够在糖淀粉酶存在的情况下区分UAA和NA。发现UAA能提高葡萄糖淀粉酶对生淀粉的消化率。因此，以玉米淀粉为原料，以曲粗提物为消化原料，制备了一种新的可食用微胶囊淀粉材料。消化后淀粉的链长分布等化学结构未发生变化，而α-淀粉酶消化率、淀粉图最大粘度等物理性质发生了显著变化。与消化后的淀粉和染料化合物的掺入实验表明，消化后的淀粉颗粒空腔比阴离子化合物更容易吸收阳离子化合物。

In Kagoshima, citric acid was produced on a commercial basis by a koji of a variety of Aspergillus usamii, using waste pulp of sweet potato from starch production factories. For the utilization of by-product purified enzyme of acid-stable a-amylase (UAA) was obtained from the acidic extract of the koji by column chromatograph. The enzyme exhibited its optimum activity a pH of around 3-5.5 (at 37℃), and a temperature of 60-70℃ (at pH4.0), which is about 15℃ higher than the ordinary neutral a-amylase (NA). The sequence of the first 20 amino acids from the N-terminus as well as a molecular weight of about 100,000 differed from the ordinary NA. Using a series of maltooligosaccharides labeled with 14C-glucose at the reducing end, the rate parameter k0/Km and the cleavage frequency were determined. It was elucidated that the active center is composed of five subsites, with the catalytic site located between the 3-rd and the 4-th subsites from the nonreducing end. Chemical modification was performed to elucidate the role of the amino acids in the active site of a-amylase. A new substrate Gal-G3-CNP was synthesized by galactosylation of commercial G3-CNP, and allowed us to discriminate between UAA and NA even in the presence of glucoamylase. UAA was found to enhance the rate of digestion of raw starch by glucoamylase. Thus, the digested starch was prepared as a new starchy material of edible microcapsule from corn starch by digestion with the crude extract of the koji. No change in chemical structure was found such as distribution of chain length of the digested starch, while the significant change in the physical properties was found such as α-amylase digestibility and the maximum viscosity in amylogram. Incorporation experiment with the digested starch and the dye compounds revealed that the cavity of starch granule produced by the digestion absorbed cationic compounds easier than anionic compounds.