Proteolytic Surgery for β-1,3-Glucanase BglH to Improve Thermotolerance
Takashi INAGAKI, Mami YAMAMOTO and Rikizo AONO
Department of Bioengineering, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226, Japan
BglH is one of the extracellular β-1,3-glucanases produced by Bacillus circulans IAM1165. This strain was isolated previously as the first microbe able to lyse fungal cell walls. Molecular mass of this enzyme is 91 kDa. The sequence of the gene coding for BglH shows an open reading frame for 877 amino acids. The catalytic domain of the enzyme is estimated in the central region of the sequence on the basis of comparison of amino acid sequences for β-1,3- or ]3-1,3-1,4-glucanases derived from various bacteria. Three segments consisting of approximately 100 amino acids are repeated at the N-terminus. These segments are probably responsible for the binding to insoluble β-l,3-glucans.
The N-terminal segments can be deprived of by proteolytic digestion to generate artificial β-1,3-glucanases having the hydrolyric activity for soluble β-1,3-glucan. Chymotrypsin treatment generated mainly two products, BglHDN2 and BglHDN3. These products were deprived of two and three N-terminus segments, respectively. BglHDN3 acquired remarkable thermotolerance. Although BglH has comparatively high thermostability, the activity of BglH is lost completely by incubation at 70℃ for 30 min. BglHDN2 showed similar thermostability. BglHDN3 was active after the incubation. Even after incubation at 100℃ for 10 min, this artificial enzyme remained about 40% of the activity.
