As an approach to improving Fibrobacter succinogenes 1,3—1,4-β-D-glucanase (Fsβ-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) Fsβ-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 (kcat) 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-/3-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 glycosylated TF-glucanase. Both forms of truncated glucanase retained more than 80% of their original enzymatic activity after a 10 mm incubation at 90 °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 Fsβ-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. [ABSTRACT FROM AUTHOR]