A truncated Fibrobacter succinogenes 1,3-1,4-beta-d-glucanase with improved enzymatic activity and thermotolerance.
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.
Wen
Tuan-Nan
TN
Institute of Botany, Academia Sinica, Taipei 115, Taiwan, ROC.
Chen
Jui-Lin
JL
Lee
Shu-Hua
SH
Yang
Ning-Sun
NS
Shyur
Lie-Fen
LF
D002942:Circular Dichroism; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D045848:Fibrobacter / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010843:Pichia / Q000235:genetics; D011489:Protein Denaturation; D015202:Protein Engineering; D017384:Sequence Deletion / Q000235:genetics*; D013050:Spectrometry, Fluorescence; D013696:Temperature
D006026:Glycoside Hydrolases; C028020:licheninase