LAUSR

Pectate lyases play an important role in pectin degradation, and therefore are highly useful in the food and textile industries. Here, we report on the cloning of an alkaline pectate lyase gene (pppel9a) from Paenibacillus polymyxa KF-1. The full-length gene (1350 bp) encodes for a 449-residue protein that belongs to the polysaccharide lyase family 9 (PL9). Recombinant PpPel9a produced in Escherichia coli was purified to electrophoretic homogeneity in a single step using Ni2+-NTA affinity chromatography. The enzyme activity of PpPel9a (apparent molecular weight of 45.3 kDa) was found to be optimal at pH 10.0 and 40 °C, with substrate preference for homogalacturonan type (HG) pectins vis-à-vis rhamnogalacturonan-I (RG-I) type pectins. Using HG-type pectins as substrate, PpPel9a showed greater activity with de-esterified HGs. In addition, PpPel9a was active against water-soluble pectins isolated from different plants. Using this lyase, we degraded citrus pectin, purified fractions using Diethylaminoethyl (DEAE)-sepharose column chromatography, and characterized the main fraction MCP-0.3. High-performance gel permeation chromatography (HPGPC) analysis showed that the molecular mass of citrus pectin (~230.2 kDa) was reduced to ~24 kDa upon degradation. Ultra-performance liquid chromatography - tandem mass spectrometer (UPLC-MS) and monosaccharide composition analyses demonstrated that PpPel9a worked as an endo-pectate lyase, which acted primarily on the HG domain of citrus pectin. In vitro testing showed that the degradation product MCP-0.3 significantly promotes the growth of Lactobacillus plantarum and L. rhamnosus. In this regard, the enzyme has potential in the preparation of pharmacologically active pectin products.