Molecular Characterization of a Novel Alkaline Endo-Pectate Lyase from Paenibacillus borealis and Over-Production in Bioreactor Realized by Constructing the Tandem Expression Cassettes in Host Genome

Alkaline pectate lyases hold significant promise for various industrial applications, including the degumming processes in papermaking and textiles. In this study, a novel pectinase, PelA, derived from a strain of <i>Paenibacillus borealis</i>, was characterized both at the molecular level and through enzymatic analysis. This enzyme represents a distinct cluster diverging from the well-characterized <i>Bacillus</i> pectinases and exhibits molecular activity under alkaline conditions, with an optimal pH of 9.5. It can be classified as an endo-(1,4)-pectate lyase, capable of cleaving the α-1,4 glycosidic bonds of polygalacturonic acid via a trans-elimination mechanism. Notably, the addition of the metal ion Ca²⁺ did not enhance enzyme activity. To achieve high-level secretory expression and improve its economic viability for bioapplications, the gene copy number of <i>pelA</i> in the host genome was increased by constructing tandem <i>pelA</i> gene expression cassettes. Following optimization of cultivation conditions and monitoring of cell growth, the recombinant strain harboring the multi-copy <i>pelA</i> gene attained an expression level of 7520 U/mL in a bioreactor. This study successfully achieved high-level secretory expression of an alkaline pectinase, thereby enhancing its potential for industrial bioapplications and providing a reference for future research on the heterologous expression of target genes.