Mannan-degrading enzymes from Cellulomonas fimi

Abstract

The genes man26a and man2A from Cellulomonas fimi encode mannanase 26A (Man26A) and beta-mannosidase 2A (Man2A), respectively. Mature Man26A is a secreted, modular protein of 951 amino acids, comprising a catalytic module in family 26 of glycosyl hydrolases, an S-layer homology module, and two modules of unknown function. Exposure of Man26A produced by Escherichia coli to C. fimi protease generates active fragments of the enzyme that correspond to polypeptides with mannanase activity produced by C. fimi during growth on mannans, indicating that it may be the only mannanase produced by the organism. A significant fraction of the Man26A produced by C. fimi remains cell associated. Man2A is an intracellular enzyme comprising a catalytic module in a subfamily of family 2 of the glycosyl hydrolases that at present contains only mammalian beta-mannosidases.

FIG. 1

Mannanases from C. fimi analyzed by nonreducing SDS-PAGE using zymograms of culture supernatants. The cultures were grown in minimal medium with no addition (lane 1) or supplemented (0.2%) with LBG (lane 2), mannose (lane 3), glycerol (lane 4), glucose and LBG (lane 5), xylan (lane 6), CM-cellulose (lane 7), or galactose (lane 8). Prestained molecular weight standards are shown in lane M. The cultures were incubated for 2 days (A), 6 days (B), and 11 days (C).

FIG. 2

Alignment of the first 398 amino acids from C. fimi Man26A (Cf Man26A; GenBank AF126471) with the entire sequence of ManA from P. fluorescens subsp. cellulosa (Pf ManA; GenBank X82179). Asterisks indicate identical residues. The catalytic glutamates are in boldface.

FIG. 3

Alignment of the SLH domain repeats of Man26A with the SLH repeats from bacterial proteins. The numbers indicate the positions of the first amino acid residue of each sequence in the different proteins. Cf, C. fimi Man26A; Ta, S-layer protein from Thermus aquaticus (GenBank A47024); Ba, S-layer protein from Bacillus anthracis (GenBank X99724); Ct, cellulosome-anchoring protein from Clostridium thermocellum (GenBank X67506); Tm, outer membrane protein from Thermotoga maritima (GenBank X68276). Asterisks indicate residues that are identical or conserved in all sequences.

FIG. 4

Digestion of Man26A, produced in E. coli, with protease from C. fimi. Man26A was digested with protease (1 μg of protease and 60 μg of Man26A) at 37°C. Samples were removed at intervals and boiled in loading buffer for 2 min to stop digestion. Digestion times were 0, 15, 30, 45, 60, and 90 min (lanes 1 to 6) and 2, 3, 5, 7, 16, and 24 h (lanes 7 to 12). Molecular weight standards are in lanes M1 and M2. The N termini of the polypeptides marked with an asterisk were sequenced.

FIG. 5

Origin of the multiple mannanases from C. fimi analyzed by using nonreducing SDS-PAGE zymograms. Cultures of C. fimi were incubated for 11 days at 30°C in minimal medium supplemented (0.2%) with INM (lane 1), LBG (lane 2), CM-cellulose (lane 3), or LBG and CM-cellulose (lane 4). Samples of the culture supernatants were analyzed. Man26A (0.05 μg) produced in E. coli was analyzed without pretreatment (lane 5), after digestion (1 h, 37°C) with protease from C. fimi (lane 6), or with the supernatant from a culture of C. fimi grown with LBG (lane 7).

FIG. 6

Alignment of the β-mannosidase subfamily in family 2 of the glycosyl hydrolases. Goat Man, Capra hicus β-mannosidase (GenBank U46067); Bovine Man, Bos taurus β-mannosidase (GenBank U17432); Human Man, Homo sapiens β-mannosidase (GenBank U60337); C. fimi Man, C. fimi β-mannosidase (GenBank AF126472). Asterisks indicate identical residues. The putative catalytic glutamates are in boldface.

FIG. 6

Alignment of the β-mannosidase subfamily in family 2 of the glycosyl hydrolases. Goat Man, Capra hicus β-mannosidase (GenBank U46067); Bovine Man, Bos taurus β-mannosidase (GenBank U17432); Human Man, Homo sapiens β-mannosidase (GenBank U60337); C. fimi Man, C. fimi β-mannosidase (GenBank AF126472). Asterisks indicate identical residues. The putative catalytic glutamates are in boldface.