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首页> 外文期刊>Biochemistry >A Truncated Fibrobacter succinogenes l,3-l,4-beta-D-Glucanase with Improved Enzymatic Activity and Thermotolerance
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A Truncated Fibrobacter succinogenes l,3-l,4-beta-D-Glucanase with Improved Enzymatic Activity and Thermotolerance

机译：具有改善的酶活性和耐热性的截短的产琥珀酸纤维杆菌l，3-l，4-beta-D-葡聚糖酶

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摘要

As an approach to improving Fibrobacter succinogenes l,3-l,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 (~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 deg 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 (~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.
机译：作为一种改进产琥珀酸纤维杆菌的方法，工业上使用l，3-l，4-β-D-葡聚糖酶（Fsbeta-葡聚糖酶），并研究该酶中C末端的结构-功能关系，即C末端使用基于PCR的基因截断方法生成截短的（〜10 kDa）Fsbeta-葡聚糖酶，然后在大肠杆菌BL21（DE3）或巴斯德毕赤酵母X-33宿主细胞中过表达。初始速率动力学，蛋白质折叠和热稳定性对野生型和截短型葡聚糖酶的特征进行了表征。通过SDS-PAGE判断，毕赤酵母细胞中表达的截短酶被糖基化并由两个显性多肽带组成。周转率增加约3-4倍（检测到大肠杆菌（称为TF-葡聚糖酶）或毕赤酵母（Pichia）宿主细胞（称为糖基化TF-葡聚糖酶）中产生的纯化的截短的葡聚糖酶，检测到相对于全长酶的k_（cat））。葡聚糖酶是已知最活跃的1,3- 1.4-β-D-葡聚糖酶，比活度为10800±200单位/ mg。检测到全长酶，TF-葡聚糖酶和地衣聚糖具有相似的结合亲和力（K_m = 2.5-2.89 mg / mL）。在90°C下孵育10分钟后，两种形式的截短型葡聚糖酶均保留了其原始酶促活性的80％以上，而全长酶在相同处理后仅具有其原始酶促活性的30％。该报告表明，Fsbeta-葡聚糖酶中C末端区域的缺失（〜10 kDa），由富含丝氨酸的重复序列和富含正电荷氨基酸的基本末端域组成，显着提高了酶的催化效率和耐热性。

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