Molecular cloning and sequencing of the gene encoding an exopolygalacturonase of a Bacillus isolate and properties of its recombinant enzyme.

An exopolygalacturonase (exo-PGase; EC 3.2.1.82) was found in the culture broth of a Bacillus isolate. The gene encoding the exo-PGase, pehK, was cloned by polymerase chain reaction using mixed primers designed from N-terminal and internal amino acid (aa) sequences of the enzyme (PehK). The determined nucleotide (nt) sequence of pehK revealed a 2940 bp open reading frame (980 aa) that encoded a putative signal sequence (27 aa) and a mature protein (953 aa; 103810 Da). The recombinant enzyme was purified to homogeneity from a culture broth of Bacillus subtilis harboring a pehK-containing plasmid. It had a molecular mass of 105 kDa and a pI value of 5.0. The maximum activity was observed at pH 8 and 55 degrees C in Tris-HCl buffer. The degradation products from polygalacturonic or oligogalacturonic acids were digalacturonic acid, like the exo-PGases, PehX of Erwinia chrysanthemi and PehB of Ralstonia solanacearum. The deduced aa sequence of PehK exhibited moderate homology to those of PehX and PehB with approx. 30% identity for both. High homology was observed in a suitably aligned internal region of the three enzymes (65% identity), and some of the conserved aa residues appeared to form the catalytic core of the enzymes.

Purification and properties of a high-molecular-weight, alkaline exopolygalacturonase from a strain of Bacillus.

1An exopolygalacturonase [exo-PG; poly (1,4-alpha-D-galacturonide) digalacturonohydrolase, EC 3.2.1.82] was found in a culture of Bacillus sp. strain KSM-P576. The purified exo-PG had a molecular weight of approximately 115,000 and an isoelectric point of pH 4.6. The N-terminal amino acid sequence was Thr-Glu-Val-Ser-Pro-Lys-Ser-Pro-Ala-Ser-Pro-Val. Maximum activity toward polygalacturonic acid (PGA) was observed at 55 degrees C and pH 8.0 in 100 mM Tris-HCl buffer. The exo-PG was quite stable in various pH buffers between pH 6 and 12 when incubated at 30 degrees C for 1 h. Mg(2+,) Mn(2+,) Pd(2+) and Ca(2+) ions stimulated the enzyme activity. The exo-PG released digalacturonic acid from PGA, tri-, tetra-, and penta-galacturonic acids. The apparent K(m) values for oligogalacturonic acids were almost identical, and k(cat) values increased with the chain length of the substrates.

Purification and properties of a galacturonic acid-releasing exopolygalacturonase from a strain of Bacillus.

An exopolygalacturonase [exo-PGase; poly (1,4-alpha-D-galacturonide) galacturonohydrolase, EC 3.2.1.67] was found to be extracellularly produced by Bacillus sp. strain KSM-P443. The exo-PGase was purified to homogeneity, as judged by polyacrylamide gel electrophoresis, through sequential column chromatographies. The enzyme had a molecular weight of approximately 45,000 and an isoelectric point of pH 5.8. The N-terminal sequence was Ser-Met-Gln-Lys-Ile-Lys-Asp-Glu-Ile-Leu-Lys-Thr-Leu-Lys-Val-Pro-Val-Phe and had no sequence similarity to those of other pectinolytic enzymes reported to date. Maximum activity toward polygalacturonic acid (PGA) was observed at 60 degrees C and at pH 7.0 in 100 mM Tris-HCl buffer without requiring any metal ions. When the chain length of oligogalacturonic acids increased, the apparent Km for them decreased, but the kcat values increased. This is the first bacterial exo-PGase that releases exclusively mono-galacturonic acid from PGA, di-, tri-, tetra-, and penta-galacturonic acids.

Highly alkaline pectate lyase Pel-4A from alkaliphilic Bacillus sp. strain P-4-N: its catalytic properties and deduced amino acid sequence.

The gene for a highly alkaline pectate lyase, Pel-4A, from alkaliphilic Bacillus sp. strain P-4-N was cloned, sequenced, and overexpressed in Bacillus subtilis cells. The deduced amino acid sequence of the mature enzyme (318 amino acids, 34805 Da) showed moderate homology to those of known pectate lyases in the polysaccharide lyase family 1. The purified recombinant enzyme had an isoelectric point of pH 9.7 and a molecular mass of 34 kDa, and exhibited a very high specific activity compared with known pectate lyases reported so far. The enzyme activity was stimulated 1.6 fold by addition of NaCl at an optimum of 100 mM. When Pel-4A was stored at 50 degrees C for 60 h, striking stabilization by 100 mM NaCl was observed in a pH range from 5 to 11.5, whereas it was stable only around pH 11 in the absence of NaCl.

Purification and properties of a low-molecular-weight, high-alkaline pectate lyase from an alkaliphilic strain of Bacillus.

A low-molecular-weight, high-alkaline pectate lyase (pectate transeliminase, EC 4.2.2.2) was found in an alkaline culture of Bacillus sp. strain KSM-P15, purified to homogeneity, and crystallized. The enzyme had a relative molecular weight of approximately 20,300 as measured by sedimentation equilibrium, with a sedimentation coefficient (s20,w0) of 1.73 S. It was a basic protein with an isoelectric point of pH 10.3, and the alpha-helical content was only 6.6%. In the presence of Ca2+ ions, the enzyme degraded polygalacturonic acid in a random manner to yield 4,5-unsaturated oligo-galacturonides and had its optimal activity around pH 10.5 and 50-55 degrees C. It also had a protopectinase-like activity on cotton fibers. The N-terminal amino acid sequences of the intact protein (28 amino acids) and its two lysyl endopeptidase-cleaved peptide fragments (8 and 12 amino acids) had very low sequence similarity with pectate lyases reported to date. These results strongly suggest that the pectate lyase of Bacillus sp. strain KSM-P15 may be a novel enzyme and belongs in a new family.