The laminarinase from thermophilic eubacterium Rhodothermus marinus

Abstract

A gene (lamR) encoding laminarinase (LamR) was cloned from the marine thermophilic eubacterium Rhodothermus marinus ITI278. The enzyme purified from recombinant Escherichia coli cells hydrolyses mixed 1,3-1,4-β-glucans (lichenan, barley and oat β-glucan) and 1,3-β-homoglucans (laminarin, curdlan) by an endo type action pattern. The CD spectrum of laminarinase is characteristic for a protein with prevailing β secondary-structural elements, and the fluorescence spectrum points to a surface localisation of the tryptophan residues. A half-transition concentration of 5.4 M guanidinium chloride was measured for the denaturant-induced unfolding of laminarinase monitoring changes of the ellipticity at 222 nm and the fluorescence. Substitution of acidic residues Glu129, Asp131 and Gln134, which are invariant in family 16 glycosyl hydrolases, caused a severe reduction of β-glucanϪhydrolysing activity suggesting their central role in enzymatic hydrolysis. Deletion of Met133 drastically reduced catalytic activity. Met133 is invariant in family 16 laminarinases but not present in the active-site region of bacterial 1,3-1,4-β-glucanases which also belong to glycosyl hydrolase family 16. Replacement of Met133 by Ala, Cys or Trp did not affect activity against 1,3-1,4-β-polyglucans and 1,3-β-polyglucans, but in mutant Met133A the rate of hydrolysis of cellobiosyltriose (G1-4G1-3Gr) was increased about 10 times. Hydrolysis of 1,3-β-oligosaccharides and 1,4-β-oligosaccharides (DP 2-7) demonstrated the ability of the enzyme to cleave 1,3-β-linkages and 1,4-β-linkages in low-molecular-mass carbohydrates independent of the structure of neighbouring linkages. The laminarinase contains five or six subsites for substrate binding according to the action pattern deduced from hydrolysis of labelled and unlabelled curdlan oligosaccharides of different chain length.Keywords : endo-1,3-β-glucanase; conformation; stability ; laminarinase ; Rhodothermus; active-site residue.Mixed linkage β-glucans such as cereal glucan and lichenan containing 1,3-β-linkages and 1,4-β-linkages are attacked by three types of endo-β-glucan hydrolases: endo-1,4-β-glucanases, lichenases, and endo-1,3-β-glucanases. The enzymes are distinguished by their specificities and substrate spectra [1]. Endo-1,4-β-glucanases, or cellulases, hydrolyse substrates containing adjacent glucopyranosyl residues that occur in homo 1,4-β-gluCorrespondence to R. Borriss,

Molecular Cloning and Characterization of a Novel β-Agarase, AgaB, from Marine Pseudoalteromonas sp. CY24

“…AgaB lacks the motif E(I/L/V)D(I/V/A/F)(V/I/L/M/F) (0,1) E, i.e. the catalytic site typical of clan B of glycoside hydrolases (44,45,46) that encompasses families GH 7 and 16 (30). Furthermore, no conserved motifs from other glycoside hydrolases were found in AgaB.…”

TOS1 is circularly permuted 1,3-β-glucanase

“…N-terminal domain is preceded by a signal α-helix specific for secreted, extracellular proteins. 4 The two domains are linked with serine/threonine-rich region, which may be heavily O-glycosylated. Indeed, STRING database 5 rd iteration revealed its weak similarity to some glucanases of glycosyl hydrolases family 16 with scores higher than the given confidence threshold.…”

“…There are substantial differences observed and experimentally studied between active site sequences of 1,3-1,4-β-glucanases and 1,3-β-glucanases (laminarinases). 4 1,3-1,4-β-glucanases share common ExDxE sequence motif and their enzymatic activity is vulnerable to additional residue insertion between conserved Asp and Glu. 9 Laminarinases on the other hand preserve that additional residue (ExDxxE motif), 4 which results in β-bulge formation and leads to β-strand bend.…”

“…4 1,3-1,4-β-glucanases share common ExDxE sequence motif and their enzymatic activity is vulnerable to additional residue insertion between conserved Asp and Glu. 9 Laminarinases on the other hand preserve that additional residue (ExDxxE motif), 4 which results in β-bulge formation and leads to β-strand bend. Deletion mutants lacking the additional residue had reduced enzymatic activity, 4 which may suggest an important role of this residue in the active site formation.…”

“…9 Laminarinases on the other hand preserve that additional residue (ExDxxE motif), 4 which results in β-bulge formation and leads to β-strand bend. Deletion mutants lacking the additional residue had reduced enzymatic activity, 4 which may suggest an important role of this residue in the active site formation.…”