SEQUENCING OF A 1,3-1,4-BETA-D-GLUCANASE (LICHENASE) FROM THE ANAEROBIC FUNGUS ORPINOMYCES STRAIN PC-2 - PROPERTIES OF THE ENZYME EXPRESSEDIN ESCHERICHIA-COLI AND EVIDENCE THAT THE GENE HAS A BACTERIAL ORIGIN

A 971-bp cDNA, designated licA, was obtained from a library of Orpinom yces sp, strain PC-2 constructed in Escherichia coli. It had an open r eading frame of 738 nucleotides encoding LicA (1,3-1,4-beta-D-glucanas e; lichenase) (EC 3.2.1.73) of 245 amino acids with a calculated molec ular mass of 27,929 Da, The deduced amino acid sequence had high homol ogy with bacterial beta-glucanases, particularly in the central region s and toward the C-terminal halves of bacterial enzymes. LicA had no h omology with plant beta-glucanases, The genomic DNA region coding for LicA was devoid of introns. More than 95% of the recombinant beta-gluc anase produced in E. coli cells was found in the culture medium and pe riplasmic space. A N-terminal signal peptide of 29 amino residues was cleaved from the enzyme secreted from Orpinomyces, whereas 21 amino, a cid residues of the signal peptide were removed when the enzyme was pr oduced by E. coli. The beta-glucanase produced by E. coli was purified from the culture medium, IC had a molecular mass of 27 kDa on sodium dodecyl sulfate-polyacrylamide gels. The K-m and V-max values with lic henin as the substrate at pH 6.0 and 40 degrees C were 0.75 mg/ml and 3,790 mu mol/min/mg, respectively, With barley beta-glucan as the subs trate, the corresponding values were 0.91 mg/ml and 5,320 mu mol/min/m g. This enzyme did not hydrolyze laminarin, carboxymethylcellulose, pu stulan, or xylan. The main products of lichenin and barley beta-glucan hydrolysis were triose and tetraose, LicA represented the first 1,3-1 ,4-beta-D-glucanase reported from fungi. The results presented suggest that licA of Orpinomyces had a bacterial origin.