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,

Rhodothermus marimus ATCC 43812, a thermophilic bacterium isolated from marine hot springs, possesses hydrolytic activities for depolymerising substrates such as carob-galactomannan. Screening of expression libraries identified mannanase-positive clones. Subsequently, the corresponding DNA sequences were determined, eventually identifying a coding sequence specifying a 997 amino acid residue protein of 113 kDa. Analyses revealed an N-terminal domain of unknown function and a C-terminal mannanase domain of 550 amino acid residues with homology to known mannanases of glycosidase family 26. Action pattern analysis categorised the R. marinus mannanase as an endo-acting enzyme with a requirement for at least five sugar moieties for effective catalytic activity. When expressed in Escherichia coli, purified gene product with catalytic activity was mainly found as two protein fragments of 45 kDa and 50 kDa. The full-length protein of 113 kDa was only detected in crude extracts of R. marinus, while truncated protein-containing fractions of the original source resulted in a major active protein of 60 kDa. Biochemical analysis of the mannanase revealed a temperature and pH optimum of 85 degrees C and pH 5.4, respectively. Purified, E. coil-produced protein fragments showed high heat stability, retaining more than 70% and 25% of the initial activity after 1 h incubation at 70 degrees C and 90 degrees C, respectively. In contrast, R. marinus-derived protein retained 87% activity after 1 h at 90 degrees C. The enzyme hydrolysed carob-galactomannan (locust bean gum) effectively and to a smaller extent guar gum, but not yeast mannan.

No abstract

The enzymatic hydrolysis of polysaccharides by the 1,3(4)-b-glucanase (LamR) from Rhodothermus marinus has been explored. The enzyme cleaves the 1,3-b-linkages of 3-O-substituted glucose units in 1,3-b-glucans such as laminarin and curdlan, and also the 1,4-b-linkages of 3-O-substituted b-glucose in b-glucans such as lichenin and 1,3-1,4-b-glucan from the cell walls of barley endosperm. The polysaccharide substrates (laminarin, curdlan and barley b-glucan) were characterised using NMR spectroscopy. The reaction of LamR with its substrates was followed by recording one-dimensional and two-dimensional 1 H-NMR and 13 C-NMR spectra at suitable time intervals after addition of the enzyme. It is shown that hydrolysis occurs with retention of the anomeric configuration and that LamR performs transglycosylation to generate both 1,3-b-glycosidic and 1,4-b glycosidic linkages. The transglycosylation results in, e.g. formation of the trisaccharide 4-O-glucosyl-laminaribiose from exclusively 1,3-b-oligoglucosides. When barley 1,3-1,4-b-glucan was incubated with LamR the b-1,4-linkages of 3-O-substituted b-glycosyl residues were rapidly hydrolysed. Simultaneously de novo formation of 1,3-b-glycosidic linkages was observed which, however, were cleaved during prolonged incubations. It is shown that a laminaribiosyl unit is the minimum requirement for formation of an enzyme±substrate complex and subsequent hydrolysis/transglycosylation.