Structure of aBacillus subtilisendo-β-l,4-glucanase gene

Abstract

The nucleotide sequence of the portion of a Bacillus subtilis (strain PAP115) 3 kb Pst I fragment which contains an endo-beta-1, 4-glucanase gene has been determined. This gene encodes a protein of 499 amino acid residues (Mr = 55,234) with a typical B. subtilis signal peptide. Escherichia coli which has been transformed with this gene produces an extracellular endoglucanase with an amino-terminus corresponding to the thirtieth encoded amino acid residue. The gene is preceded by a cryptic reading frame with a rho-independent terminator structure, and itself has such a structure in the immediate 3'-flanking region. We have also identified, in the 5'-flanking region, nucleotide sequences which resemble promoter elements recognized by Bacillus RNA polymerase E sigma 43. Comparison of the encoded amino acid sequence to other known beta-glucanases reveals a small region of similarity to the encoded protein of the Clostridium thermocellum celB gene. These similar regions may contain substrate-binding and/or catalytic sites.

Molecular cloning and nucleotide sequence of the gene encoding an endo-1,4- -glucanase from Bacillus sp. KSM-330

“…KSM-330 may possibly be cleaved by a signal peptidase at the bond between Ala 55 and Val 56 during secretion across the cytoplasmic membrane. However, the site of cleavage is far from the carboxy-terminus of the hydrophobic region and the possible signal peptide of 55 amino acid residues seems somewhat longer than other reported signal peptides of endoglucanases from Bacillus (Fukumori et al, 1986a;MacKay et al, 1986;Robson & Chambliss, 1987). The amino-terminus of Endo-K could, therefore, be processed after secretion by a proteolytic enzyme.…”

“…KSM-330 would show some homology to the alkaline endoglucanase of Bacillus sp. KSM-635 and to other alkaline and neutral endoglucanases of Bacillus reported to date (Fukumori et al, 1986a, b;MacKay et al, 1986;Nakamura et al, 1987;Robson & Chambliss, 1987;Fukumori et al, 1989;Baird et al, 1990). Interestingly, Endo-K exhibits no homology to these alkaline or neutral enzymes.…”

“…KSM-635 . The deduced amino acid sequence of the alkaline endoglucanase protein exhibited significant homology to the animo acid sequences of other alkaline (Fukumori et al, 1986a, b) and neutral (MacKay et al, 1986;Nakamura et al, 1987;Robson & Chambliss, 1987;Zappe et al, 1988) endoglucanases produced by strains of Bacillus and by Clostridium acetobutylicum, with some amino acid residues conserved in either the alkaline enzymes or the neutral enzymes, or in both types of enzyme.…”

“…Cellulolytic enzymes have been reported in several strains of Bacillus (Tewari & Chahal, 1977;Dhillon et al, 1985;Fukumori et al, 1986a, b ;MacKay et al, 1986;Nakamura et al, 1987;Robson & Chambliss, 1987;Au & Chan, 1987;Baird et al, 1990). However, cellulases from Bacillus have received very limited attention, probably because most of these enzymes hydrolyse carboxymethylcellulose (CMC) but barely hydrolyse crystalline forms of cellulose and because they have had no commercial applications until recently.…”

Characterization and Structure of the Cellulase Gene ofBacillus subtilisBSE616

“…Robson and Chambliss,16) however, described how B. subtilis DLG produces a 35,200-dalton exocellular f3-1,4-glucanase, and Escherichia coli transformants carrying the DLG cellulase gene produce a cell-associated f3-1,4-glucanase with a molecular mass of 39,000 ± 400 daltons. Mackay et al 15) described how the molecular masses of active extracellular endoglucanases produced by B. subtilis PAP1l5 and E. coli transformants are 27,000--40,000 daltons and 30,000--40,000 daltons, respectively, and they expected that in both species considerable processing or degradation of the primary translation product will occur, beyond signal peptide cleavage.…”