Department of Extremophilic Bacteria, Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria.
Appl Biochem Biotechnol. 2011 Nov;165(5-6):1285-95. doi: 10.1007/s12010-011-9346-4. Epub 2011 Sep 6.
A new cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) from an alkaliphilic halotolerant Bacillus pseudalcaliphilus 8SB was studied in respect to its γ-cyclizing activity. An efficient conversion of a raw corn starch into only two types of cyclodextrins (β- and γ-CD) was achieved by the purified enzyme. Crude enzyme obtained by ultrafiltration was purified up to fivefold by starch adsorption with a recovery of 62% activity. The enzyme was a monomer with a molecular mass 71 kDa estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE. The CGTase exhibited two pH optima, at pH 6.0 and 8.0, and was at most active at 60 °C and pH 8.0. The enzyme retained more than 80% of its initial activity in a wide pH range, from 5.0 to 11.0. The CGTase was strongly inhibited by 15 mM Cu(2+), Fe(2+), Ag(+), and Zn(2+), while some metal ions, such as Ca(2+), Na(+), K(+), and Mo(7+), exerted a stimulating effect in concentration of 5 mM. The important feature of the studied CGTase was its high thermal stability: the enzyme retained almost 100% of its initial activity after 2 h of heating at 40-60 °C; its half-life was 2 h at 70 °C in the presence of 5 mM Ca(2+). The achieved 50.7% conversion of raw corn starch into 81.6% β- and 18.4% γ-CDs after 24 h enzyme reaction at 60 °C and pH 8.0 makes B. pseudalcaliphilus 8SB CGTase industrially important enzyme for cyclodextrin production.
从嗜碱耐盐芽孢杆菌 8SB 中分离得到一种新的环糊精葡萄糖基转移酶（CGTase，EC 2.4.1.19），研究了其γ-环化活性。通过纯化酶，可将生玉米淀粉高效转化为仅两种类型的环糊精（β-和γ-CD）。通过淀粉吸附进行超滤获得的粗酶经过纯化，酶活回收率为 62%，达到 5 倍。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）和天然聚丙烯酰胺凝胶电泳估计，该酶为单体，分子量为 71 kDa。CGTase 表现出两个 pH 最适值，分别在 pH 6.0 和 8.0，最适温度为 60°C 和 pH 8.0。该酶在 pH 5.0 至 11.0 的较宽范围内保留了超过 80%的初始活性。CGTase 被 15 mM Cu(2+)、Fe(2+)、Ag(+)和 Zn(2+)强烈抑制，而一些金属离子，如 Ca(2+)、Na(+)、K(+)和 Mo(7+)，在 5 mM 浓度下表现出刺激作用。研究的 CGTase 的一个重要特点是其具有较高的热稳定性：在 40-60°C 加热 2 小时后，酶保留了几乎 100%的初始活性；在 5 mM Ca(2+)存在下，70°C 时半衰期为 2 小时。在 60°C 和 pH 8.0 下酶反应 24 小时后，生玉米淀粉转化率达到 50.7%，生成 81.6%的β-CD 和 18.4%的γ-CD，这使得芽孢杆菌 8SB CGTase 成为生产环糊精的一种具有工业重要性的酶。
