Wen Tuan-Nan, Chen Jui-Lin, Lee Shu-Hua, Yang Ning-Sun, Shyur Lie-Fen
Institute of Botany, Academia Sinica, Taipei 115, Taiwan, ROC.
Biochemistry. 2005 Jun 28;44(25):9197-205. doi: 10.1021/bi0500630.
As an approach to improving Fibrobacter succinogenes 1,3-1,4-beta-d-glucanase (Fsbeta-glucanase) for use in industry and to studying the structure-function relationship of the C-terminus in the enzyme, a C-terminally truncated ( approximately 10 kDa) Fsbeta-glucanase was generated using a PCR-based gene truncation method and then overexpressed in either Escherichia coli BL21(DE3) or Pichia pastoris strain X-33 host cells. The initial rate kinetics, protein folding, and thermostability of the wild-type and truncated glucanases were characterized. The truncated enzyme expressed in Pichia cells was found to be glycosylated and composed of two dominant polypeptide bands as judged by SDS-PAGE. An approximate 3-4-fold increase in the turnover rate (k(cat)), relative to that of the full-length enzyme, was detected for the purified truncated glucanases produced in E. coli (designated TF-glucanase) or Pichia host cells (designated glycosylated TF-glucanase). The glycosylated TF-glucanase is the most active known 1,3-1,4-beta-d-glucanase, with a specific activity of 10 800 +/- 200 units/mg. Similar binding affinities for lichenan (K(m) = 2.5-2.89 mg/mL) were detected for the full-length enzyme, TF-glucanase, and glycosylated TF-glucanase. Both forms of truncated glucanase retained more than 80% of their original enzymatic activity after a 10 min incubation at 90 degrees C, whereas the full-length enzyme possessed only 30% of its original enzymatic activity after the same treatment. This report demonstrates that deletion of the C-terminal region ( approximately 10 kDa) in Fsbeta-glucanase, consisting of serine-rich repeats and a basic terminal domain rich in positively charged amino acids, significantly increases the catalytic efficiency and thermotolerance of the enzyme.
为了改进用于工业的琥珀酸纤维杆菌1,3 - 1,4-β-D-葡聚糖酶（Fsβ-葡聚糖酶）并研究该酶C末端的结构-功能关系，使用基于PCR的基因截短方法产生了一种C末端截短（约10 kDa）的Fsβ-葡聚糖酶，然后在大肠杆菌BL21(DE3)或毕赤酵母菌株X-33宿主细胞中进行过表达。对野生型和截短型葡聚糖酶的初始速率动力学、蛋白质折叠和热稳定性进行了表征。通过SDS-PAGE判断，在毕赤酵母细胞中表达的截短酶被糖基化，由两条主要的多肽带组成。相对于全长酶，在大肠杆菌中产生的纯化截短葡聚糖酶（命名为TF-葡聚糖酶）或毕赤酵母宿主细胞中产生的（命名为糖基化TF-葡聚糖酶）的周转速率（kcat）检测到约3 - 4倍的增加。糖基化TF-葡聚糖酶是已知活性最高的1,3 - 1,4-β-D-葡聚糖酶，比活性为10800±200单位/毫克。全长酶、TF-葡聚糖酶和糖基化TF-葡聚糖酶对地衣多糖的结合亲和力相似（Km = 2.5 - 2.89毫克/毫升）。两种形式的截短葡聚糖酶在90℃孵育10分钟后均保留了超过80%的原始酶活性，而全长酶在相同处理后仅保留了30%的原始酶活性。本报告表明，在Fsβ-葡聚糖酶中缺失由富含丝氨酸的重复序列和富含带正电荷氨基酸的碱性末端结构域组成的C末端区域（约10 kDa），可显著提高该酶的催化效率和耐热性。
