Pectate lyase C from Bacillus subtilis: a novel endo-cleaving enzyme with activity on highly methylated pectin

Abstract

The gene yvpA from Bacillus subtilis was cloned and expressed in Escherichia coli. It encoded a pectate lyase of 221 amino acids that was denominated PelC. The heterologously expressed enzyme was purified by His-tag affinity chromatography and characterized. PelC depolymerized polygalacturonate and pectins of methyl esterification degree from 22 % to 89 %, exhibiting maximum activity on 22 % esterified citrus pectin. It showed an absolute Ca 2+ requirement and the optimum temperature and pH were 65 6C and pH 10, respectively. The deduced amino acid sequence of PelC showed 53 % identity to pectate lyase PelA from Paenibacillus barcinonensis, which was also characterized. Similarly to PelC, purified PelA showed activity on polygalacturonate and pectins with a high degree of methyl esterification. The two enzymes cleaved pectic polymers to a mixture of oligogalacturonates, indicating an endo mode of action. Analysis of activity on trigalacturonate showed that PelC cleaved it to galacturonic acid and unsaturated digalacturonate, whereas PelA did not show activity on this substrate. PelC and PelA showed high homology to a few recently identified pectate lyases of family 3 and form with them a cluster of small-sized pectate lyases from non-pathogenic micro-organisms.

Identification and Characterisation of a Pectinolytic Enzyme from Paenibacillus xylanolyticus

“…4a), in agreement with the optimum temperature found for other bacterial pectate lyases reported in literature (Ouattara et al 2010;Soriano et al 2006). When the effect of pH on the activity of PaenxylPel was investigated (Fig.…”

Biochemical Properties of Pectate Lyases Produced by Three Different Bacillus Strains Isolated from Fermenting Cocoa Beans and Characterization of Their Cloned Genes

“…Pel-22, Pel-66, and Pel-90 proved to be thermophilic, with a high level of activity at 60°C. This is not surprising, since most of the Pels from Bacillus have been reported to show maximum activity at temperatures between 60 and 70°C (12,17,21,40). Furthermore, the Pels studied present strong stability at room temperature in a range of pH 3 to 10.…”

“…In contrast, Pel-22 showed maximum activity near neutral pH (7.5). To date, a microbial Pel has rarely been found with this feature, since most microbial Pels studied have exhibited optimum pHs in the range from 8.5 to 11.5 (2,17,20,21,25,40,42).…”

“…The cell supernatants were adjusted to 1 mM dithiothreitol (DTT), and the resulting extracts were subjected to ammonium sulfate fractionation. Solid ammonium sulfate was gradually added to the clarified supernatant fluid at 20,40,55,70,90, and 100% saturation, with gentle stirring in an ice bath. The resulting fractions were collected by centrifugation at 12,000 ϫ g for 15 min and dissolved in small volumes of buffer A (20 mM Tris-HCl, pH 7.2, 1 mM EDTA, and 1 mM DTT).…”

Complete genome sequence of Planococcus donghaensis JH1T, a pectin-degrading bacterium

“…Pectin is a complex polysaccharide that, together with cellulose and hemicellulose, constitutes the middle lamella and primary cell wall of terrestrial plants (Abbott and Boraston, 2008;Kashyap et al, 2001;Soriano et al, 2006). The pectinaceous nature of plants presents major challenges for industries that use plants as raw materials, such as the juice beverage industry (Jayani et al, 2005).…”