Jiang-Ke Yang

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 Paenibacillus borealis, was characterized both at the molecular level and through enzymatic analysis. This enzyme represents a distinct cluster diverging from the well-characterized Bacillus 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 pelA in the host genome was increased by constructing tandem pelA gene expression cassettes. Following optimization of cultivation conditions and monitoring of cell growth, the recombinant strain harboring the multi-copy pelA 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.

Alkaline pectate lyases have great prospects in industry fields such as degumming of papermaking and textile, etc. In this study, a novel pectinase PelA from a Paenibacillus borealis strain was molecular characterized and enzymatically defined. This enzyme represents an important cluster divergent from the well-characterized Bacillus pectinase, shows molecularly active under alkaline condition, and has the optimal pH=9.5. It could be clustered as endo-(1,4)-pectate lyase, and break the α-1,4 glycosidic bonds of the polygalacturonic acid by trans-elimination mode. The extra addition of metal ion Ca²⁺ could not improve the enzyme activity. In order to achieved its high-level secretory expression and improve its economic competence in bioapplication, the gene copy number of PelA in host genome was improved by constructing the tandem PelA gene expression cassettes. After cultivation condition optimization, cell growth monitoring, the recombinant strain carrying the multi-copy pelA gene reached the expression level of 7520 U/mL culture in a bioreactor. This study has fulfilled the high-level secretory expression of an alkaline pectinase, facilitated its industrial bioapplication and also could be a reference for future work on the heterologous expression of target genes.

Chymosin (EC3.4.23.4), primarily sourced from calf abomasum, serves as a conventional coagulant in milk curdling during cheese production. To improve the enzymatic properties and enhance the expression of calf chymosin (BtChy) in Kluyveromyces lactis to meet the demands of the cheese industry, the in silico engineering via hotspot scanning and molecular dynamics analysis was adopted. This approach improved the activity of BtChy on milk curdling and increased its sensitivity at 65°C. Multiple strategies were utilized to develop an environmentally friendly method for chymosin production. These included screening for constitutive promoters and signal peptides, as well as in vitro construction of a concatemer of the BtChy gene. The optimal combination, comprising the P_TDH3 promoter, invertase signal peptide, and a four-copy BtChy gene integrated into the yeast genome, was identified. After high-density cultivation in a 5-L bioreactor, the recombinant yeast achieved an activity of 42,000 SU/mL, a 52.5-fold increase over the original wild-type chymosin gene.