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.