Polysaccharide lyase: molecular cloning, sequencing, and overexpression of the xanthan lyase gene of Bacillus sp. strain GL1

Abstract

When grown on xanthan as a carbon source, the bacterium Bacillus sp. strain GL1 produces extracellular xanthan lyase (75 kDa), catalyzing the first step of xanthan depolymerization (H. Nankai, W. Hashimoto, H. Miki, S. Kawai, and K. Murata, Appl. Environ. Microbiol. 65:2520-2526, 1999). A gene for the lyase was cloned, and its nucleotide sequence was determined. The gene contained an open reading frame consisting of 2,793 bp coding for a polypeptide 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 amino acid sequence of the enzyme and exhibited significant homology with hyaluronidase of Streptomyces griseus (identity score, 37.7%). Escherichia coli transformed with the gene without the signal peptide sequence showed a xanthan lyase activity and produced intracellularly a large amount of the enzyme (400 mg/liter of culture) with a molecular mass of 97 kDa. During storage at 4 degrees C, the purified enzyme (97 kDa) from E. coli was converted to a low-molecular-mass (75-kDa) enzyme with properties closely similar to those of the enzyme (75 kDa) from Bacillus sp. strain GL1, specifically in optimum pH and temperature for activity, substrate specificity, and mode of action. Logarithmically growing cells of Bacillus sp. strain GL1 on the medium with xanthan were also found to secrete not only xanthan lyase (75 kDa) but also a 97-kDa protein with the same N-terminal amino acid sequence as that of xanthan lyase (75 kDa). These results suggest that, in Bacillus sp. strain GL1, xanthan lyase is first synthesized as a preproform (99 kDa), secreted 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.

FIG. 1

Electrophoretic profile of xanthan lyases. The preparations were subjected to SDS-PAGE. Lane M, synthetic polypeptides with molecular masses of 250, 150, 100, 75, 50, 37, 25, and 15 kDa; lane 1, partially purified xanthan lyase (after Sephacryl S-200HR column chromatography) from Bacillus sp. strain GL1; lane 2, purified xanthan lyase (after QAE-Sephadex A-25 column chromatography) from Bacillus sp. strain GL1; lane 3, cell extract of E. coli transformed with xanthan lyase gene; lane 4, purified xanthan lyase (97 kDa) from E. coli; lanes 5 and 7, processed xanthan lyase (75 kDa) from E. coli; lane 6, conversion of 97-kDa enzyme from E. coli to 75-kDa enzyme. Arrows indicate the positions of xanthan lyases.

FIG. 2

Nucleotide sequence of the xanthan lyase gene and its deduced amino acid sequence. Putative promoters (−35 and −10) and the ribosome-binding site (RBS) are underlined. The N-terminal amino acids of xanthan lyase and internal peptides (P1, P2, and P3) determined by protein sequencing are double underlined. An inverted repeat (possible terminator) is indicated by facing arrows.

FIG. 3

Amino acid sequence alignment of xanthan lyase (XLY) of Bacillus sp. strain GL1 (AB037178) and hyaluronidases (HLY) of Streptomyces griseus (AB028210) and S. pneumoniae (L20670). Five well-conserved regions are boxed. Identical and similar amino acid residues among the three enzymes are marked with asterisks and dots, respectively.

FIG. 4

Southern blot analysis. Genomic DNA from Bacillus sp. strain GL1 was digested with various restriction enzymes and subjected to Southern hybridization using the oligonucleotide coding for the N-terminal amino acids of xanthan lyase (75 kDa) as a probe. Lane 1, BamHI; lane 2, HindIII; lane 3, PstI; lane 4, SmaI; lane 5, SphI; lane 6, KpnI.

FIG. 5

Release of pyruvylated mannose from xanthan by xanthan lyases. Xanthan (0.25%) was incubated for 3 h with 20 U of xanthan lyases from Bacillus sp. strain GL1 and E. coli per μl. Products were analyzed by TLC. Lane 2, product produced by the enzyme from Bacillus sp. strain GL1; lane 3, product produced by the enzyme from E. coli; lane 4, hydrolysate of the product produced by the enzyme from E. coli. Authentic samples: lane 1, xanthan (25 μg); lane 5, d-mannose (45 μg); lane 6, d-glucose (45 μg); lane 7, d-glucuronic acid (45 μg). Pyr-Man indicates pyruvylated mannose.