Cloning, expression, and characterization of two pectate lyases isolated from the sheep rumen microbiome

Pectate lyases (Pels) have a vital function in degradation of the primary plant cell wall and the middle lamella and have been widely used in the industry. In this study, two pectate lyase genes, IDSPel16 and IDSPel17, were cloned from a sheep rumen microbiome. The recombinant enzymes were expressed in Escherichia coli and functionally characterized. Both IDSPel16 and IDSPel17 proteins had an optimal temperature of 60 ?, and an optimal pH of 10.0. IDSPel16 was relatively stable below 60 degrees C, maintaining 77.51% residual activity after preincubation at 60 degrees C for 1 h, whereas IDSPel17 denatured rapidly at 60 degrees C. IDSPel16 was relatively stable between pH 6.0 and 12.0, after pretreatment for 1 h, retaining over 60% residual activity. IDSPel16 had high activity towards polygalacturonic acid, with a V(max )of 942.90 +/- 68.11, whereas IDSPel17 had a V-max of only 28.19 +/- 2.23 mu mol/min/mg. Reaction product analyses revealed that IDSPel17 liberated unsaturated digalacturonate (uG(2)) and unsaturated trigalacturonate (uG(3)) from the substrate, indicating a typical endo-acting pectate lyase (EC 4.2.2.2). In contrast, IDSPel16 initially generated unsaturated oligogalacturonic acids, then converted these intermediates into uG2 and unsaturated galacturonic acid (uG(1)) as end products, a unique depolymerization profile among Pels. To the best of our knowledge, the IDSPel16 discovered with both endo-Pel (EC 4.2.2.2) and exo-Pel (EC 4.2.2.9) activities. These two pectate lyases, particularly the relatively thermo-and pH-stable IDSPel16, will be of interest for potential application in the textile, food, and feed industries.

Automated summary

This study cloned two pectate lyase genes, IDSPel16 and IDSPel17, from a sheep rumen microbiome and characterized their functional properties. The results showed that IDSPel16 had relatively higher stability and activity, making it potentially useful in the textile, food, and feed industries.