Article Figures & Data
Figures
- FIG. 1.
P. amylolyticus strain 27C64 PelA optima for pH (A), temperature (B), and CaCl2 (C). The pH optimum was determined at 40°C with 1 mM CaCl2, the temperature optimum was determined at pH 10.5 in a range of 25 to 55°C, and the CaCl2 concentration optimum was determined at pH 10.5 and 45°C in a range of 0 to 2.5 mM. One unit of enzyme activity was defined as the amount of enzyme that produces 1 μmol Δ4,5-unsaturated product from PGA per minute under assay conditions; specific activity is reported as U/mg protein.
- FIG. 2.
Activity on different pectic substrates for P. amylolyticus strain 27C64 pectate lyase A (PamPelA), P. barcinonensis pectate lyase A (PbaPelA), and B. subtilis pectate lyase C (BsuPelC). a, data from reference 21; b, data from reference 22. P. amylolyticus strain 27C64 PelA, P. barcinonensis PelA, and B. subtilis PelC substrate utilization ranges, with activity on PGA as well as pectin with any degree of methylation, are unique among the pectate lyases described to date.
- FIG. 3.
P. amylolyticus strain 27C64 PelB optima for pH (A), temperature (B), and CaCl2 (C). The pH optimum was determined at 40°C with 1 mM CaCl2, the temperature optimum was determined at pH 9.5 in a range of 15 to 65°C, and the CaCl2 concentration optimum was determined at pH 9.5 and 55°C in a range of 0 to 2.5 mM.
- FIG. 4.
P. amylolyticus strain 27C64 pectate lyase B activity on different pectic substrates. PelB showed the highest activity on 20 to 34% methylated pectin but retained 67%, 51%, 25%, and 1% of its maximum activity on polygalacturonic acid and 8.5%, 55 to 70%, and 90% methylated pectin, respectively, providing evidence that PelB is active on PGA as well as highly methylated pectin.
Tables
- TABLE 1.
Cloning strains and plasmids used in this study
Strain, plasmid, or oligonucleotide Relevant characteristics Reference or source Bacterial strain Escherichia coli DH5α F−endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG φ80dlacZΔM15 Δ(lacZYA-argF)U169 hsdR17(rK− mK+) λ− 16 Plasmids pUC19 lacZα+; Apr Invitrogen (Carlsbad, CA) pEDH13C2 pUC19 derivative; P. amylolyticus DNA fragment with pelA This study pEDH27 pUC19 derivative; pelA+ This study pUC19-19F6 pUC19 derivative; P. amylolyticus DNA fragment with pelB This study pWEB1 pUC19 derivative; pelB+ This study Oligonucleotides PLAscF (for pelA) 5′-GTACAGGGCCCGGATCCTTGACATGATAGAAGCACTCTACTATATTCTAGTGCTTCTACGGTTCTGTGGGACAA-3′ This study PLAscR (for pelA) 5′-CGATCAAGCTTGGGCCCGAGCGGCCGCCTCGAGTCCACATGGTTTGGAGCATTTCG-3′ This study ScpelBF (for pelB) 5′-GCAGTGAGCTCTTGACATGATAGAAGCACTCTACTATATTCTAGTTATACTTATCGGGAGGAATCG-3′ This study ScpelBR (for pelB) 5′-CATGGGATCCCGGAGCGCTTAACTTAGTAACTC-3′ This study
Supplemental material
Files in this Data Supplement:
- Supplemental file 1 -
GenBank accession numbers for similar enzymes (Table S1); amino acid alignment of PelA with pectate lyase class 3 enzymes
by CLUSTAL W (Fig. S1) and that of pectate lyase class 1 enzymes by MUSCLE (Fig. S2).
PDF file, 102K.
- Supplemental file 1 -
GenBank accession numbers for similar enzymes (Table S1); amino acid alignment of PelA with pectate lyase class 3 enzymes
by CLUSTAL W (Fig. S1) and that of pectate lyase class 1 enzymes by MUSCLE (Fig. S2).
