Polysaccharide lyase: Molecular cloning, sequencing, and overexpression ofthe xanthan lyase gene of Bacillus sp strain GL1

When grown on xanthan as a carbon source, the bacterium Bacillus sp, strain GL1 produces extracellular xanthan lyase (75 kDa), catalyzing the first st ep of xanthan depolymerization (H, Nankai, W. Hashimoto, H. Miki, S, Kawai, and K, Murata, Appl, Environ. Microbiol, 65:2520-2526, 1999). A gene for t he lyase was cloned, and its nucleotide sequence was determined. The gene c ontained an open reading frame consisting of 2,793 bp coding for a polypept ide with a molecular weight of 99,308, The polypeptide had a signal peptide (2 kDa) consisting of 25 amino acid residues preceding the N-terminal amin o acid sequence of the enzyme and exhibited significant homology with hyalu ronidase of Streptomyces griseus (identity score, 37.7%), Escherichia coli transformed with the gene without the signal peptide sequence showed a xant han lyase activity and produced intracellularly a large amount of the enzym e (400 mg/liter of culture) with a molecular mass of 97 kDa. During storage at 4 degreesC, the purified enzyme (97 kDa) from E. coli was converted to a low-molecular-mass (75-kDa) enzyme with properties closely similar to tho se of the enzyme (75 kDa) from Bacillus sp. strain GL1, specifically in opt imum pH and temperature for activity, substrate specificity, and mode of ac tion. Logarithmically growing cells of Bacillus sp, strain GL1 on the mediu m with xanthan were also found to secrete not only xanthan lyase (75 kDa) b ut also a 97-kDa protein with the same N-terminal amino acid sequence as th at of xanthan lyase (75 kDa). These results suggest that, in Bacillus sp. s train GL1, xanthan lyase is first synthesized as a preproform (99 kDa), sec reted as a precursor (97 kDa) by a signal peptide-dependent mechanism, and then processed into a mature form (75 kDa) through excision of a C-terminal protein fragment with a molecular mass of 22 kDa.