THE LAMINARINASE FROM THERMOPHILIC EUBACTERIUM RHODOTHERMUS-MARINUS -CONFORMATION, STABILITY, AND IDENTIFICATION OF ACTIVE-SITE CARBOXYLICRESIDUES BY SITE-DIRECTED MUTAGENESIS

A gene (lamR) encoding laminarinase (LamR) was cloned from the marine thermophilic eubacterium Rhodothermus marinus ITI278, The enzyme purif ied from recombinant Escherichia coli cells hydrolyses mixed 1,3-1,4-b eta-glucans (lichenan, barley and oat beta-glucan) and 1,3-beta-homogl ucans (laminarin, curdlan) by an endo type action pattern. The CD spec trum of laminarinase is characteristic for a protein with prevailing b eta secondary-structural elements, and the fluorescence spectrum point s to a surface localisation of the tryptophan residues. A half-transit ion concentration of 5.4 M guanidinium chloride was measured for the d enaturant-induced unfolding of laminarinase monitoring changes of the ellipticity at 222 nm and the fluorescence, Substitution of acidic res idues Glu129, Asp131 and Gln134, which are invariant in family 16 glyc osyl hydrolases, caused a severe reduction of beta-glucan-hydrolysing activity suggesting their central role in enzymatic hydrolysis, Deleti on of Met133 drastically reduced catalytic activity, Met133 is invaria nt in family 16 laminarinases but not present in the active-site regio n of bacterial 1,3-1,4-beta-glucanases which also belong to glycosyl h ydrolase family 16, Replacement of Met133 by Ala, Cys or Trp did not a ffect activity against 1,3-1,4-beta-polyglucans and 1,3-beta-polygluca ns, but in mutant Met133A the rate of hydrolysis of cellobiosyltriose (G1-4G1-3Gr) was increased about 10 times. Hydrolysis of 1,3-beta-olig osaccharides and 1,4-beta-oligosaccharides (DP 2-7) demonstrated the a bility of the enzyme to cleave 1,3-beta-linkages and 1,4-beta-linkages in low-molecular-mass carbohydrates independent of the structure of n eighbouring linkages. The laminarinase contains five or six subsites f or substrate binding according to the action pattern deduced from hydr olysis of labelled and unlabelled curdlan oligosaccharides of differen t chain length.