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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.

The possible structural and catalytic functions of the nine tryptophan amino acid residues, including Trp 5 4 , Trp 1 0 5 , Trp 1 1 2 , Trp 1 4 1 , Trp 1 4 8 , Trp 1 6 5 , Trp 1 8 6 , Trp 1 9 8 , and Trp 2 0 3 in Fibrobacter succinogenes 1,3-1,4-β-D-glucanase (Fsβ-glucanase), were characterized using site-directed mutagenesis, initial rate kinetics, fluorescence spectrometry, and structural modeling analysis. Kinetic studies showed that a 5-7-fold increase in K m value for lichenan was observed for W141F, W141H, and W203R mutant Fsβ-glucanases, and approximately 72-, 56-, 30-, 29.5-, 4.9-, and 4.3-fold decreases in k c a t relative to that for the wild-type enzyme were observed for the W54F, W54Y, W141H, W203R, W141F, and W148F mutants, respectively. In contrast, W186F and W203F, unlike the other 12 mutants, exhibited a 1.4- and 4.2-fold increase in k c a t , respectively. W165F and W203R were the only two mutants that exhibited a 4-7-fold higher activity relative to the wild-type enzyme after they were incubated at pH 3.0 for 1 h. Fluorescence spectrometry indicated that all of the mutations on the nine tryptophan amino acid residues retained a folding similar to that of the wild-type enzyme. Structural modeling and kinetic studies suggest that Trp 5 4 , Trp 1 4 1 , Trp 1 4 8 , and Trp 2 0 3 play important roles in maintaining structural integrity in the substrate-binding cleft and the catalytic efficiency of the enzyme.