The laminarinase from thermophilic eubacterium Rhodothermus marinus--conformation, stability, and identification of active site carboxylic residues by site-directed mutagenesis.

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-beta-glucans (lichenan, barley and oat beta-glucan) and 1,3-beta-homoglucans (laminarin, curdlan) by an endo type action pattern. The CD spectrum of laminarinase is characteristic for a protein with prevailing beta 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 beta-glucan-hydrolysing 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-beta-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-beta-polyglucans and 1,3-beta-polyglucans, but in mutant Met133A the rate of hydrolysis of cellobiosyltriose (G1-4G1-3Gr) was increased about 10 times. Hydrolysis of 1,3-beta-oligosaccharides and 1,4-beta-oligosaccharides (DP 2-7) demonstrated the ability of the enzyme to cleave 1,3-beta-linkages and 1,4-beta-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.