A Truncated Fibrobacter succinogenes 1,3-1,4--D-Glucanase with Improved Enzymatic Activ
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- 作者：Tuan-Nan Wen ; Jui-Lin Chen ; Shu-Hua Lee ; Ning-Sun Yang ; and Lie-Fen Shyur
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：June 28, 2005
- 年：2005
- 卷：44
- 期：25
- 页码：9197 - 9205
- 全文大小：182K
- 年卷期：v.44,no.25(June 28, 2005)
- ISSN：1520-4995
文摘
As an approach to improving Fibrobacter succinogenes 1,3-1,4-
-D-glucanase (Fs-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) Fs-glucanase was generated using a PCR-based gene truncationmethod and then overexpressed in either Escherichia coli BL21(DE3) or Pichia pastoris strain X-33 hostcells. The initial rate kinetics, protein folding, and thermostability of the wild-type and truncated glucanaseswere characterized. The truncated enzyme expressed in Pichia cells was found to be glycosylated andcomposed of two dominant polypeptide bands as judged by SDS-PAGE. An approximate 3-4-foldincrease in the turnover rate (kcat), relative to that of the full-length enzyme, was detected for the purifiedtruncated glucanases produced in E. coli (designated TF-glucanase) or Pichia host cells (designatedglycosylated TF-glucanase). The glycosylated TF-glucanase is the most active known 1,3-1,4--D-glucanase, with a specific activity of 10 800 ± 200 units/mg. Similar binding affinities for lichenan (Km= 2.5-2.89 mg/mL) were detected for the full-length enzyme, TF-glucanase, and glycosylatedTF-glucanase. Both forms of truncated glucanase retained more than 80% of their original enzymaticactivity after a 10 min incubation at 90 
C, whereas the full-length enzyme possessed only 30% of itsoriginal enzymatic activity after the same treatment. This report demonstrates that deletion of the C-terminalregion (~10 kDa) in Fs-glucanase, consisting of serine-rich repeats and a basic terminal domain rich inpositively charged amino acids, significantly increases the catalytic efficiency and thermotolerance of theenzyme.
-D-glucanase (Fs-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) Fs-glucanase was generated using a PCR-based gene truncationmethod and then overexpressed in either Escherichia coli BL21(DE3) or Pichia pastoris strain X-33 hostcells. The initial rate kinetics, protein folding, and thermostability of the wild-type and truncated glucanaseswere characterized. The truncated enzyme expressed in Pichia cells was found to be glycosylated andcomposed of two dominant polypeptide bands as judged by SDS-PAGE. An approximate 3-4-foldincrease in the turnover rate (kcat), relative to that of the full-length enzyme, was detected for the purifiedtruncated glucanases produced in E. coli (designated TF-glucanase) or Pichia host cells (designatedglycosylated TF-glucanase). The glycosylated TF-glucanase is the most active known 1,3-1,4--D-glucanase, with a specific activity of 10 800 ± 200 units/mg. Similar binding affinities for lichenan (Km= 2.5-2.89 mg/mL) were detected for the full-length enzyme, TF-glucanase, and glycosylatedTF-glucanase. Both forms of truncated glucanase retained more than 80% of their original enzymaticactivity after a 10 min incubation at 90 C, whereas the full-length enzyme possessed only 30% of itsoriginal enzymatic activity after the same treatment. This report demonstrates that deletion of the C-terminalregion (~10 kDa) in Fs-glucanase, consisting of serine-rich repeats and a basic terminal domain rich inpositively charged amino acids, significantly increases the catalytic efficiency and thermotolerance of theenzyme.