Expression optimization of recombinant xylanase in Lactococcus lactis NZ9000 to enhance silage fermentation

GIZZI, F; MAGNI, C; BLANCATO, VICTOR

Congreso; LVII Reunión Anual de SAIB; 2021

Cellulose, lignin, and hemicellulose (formed mainly by xylan) are among the main constituents of the cell wall of plant cells that make up the basic forages of bovine feed. Xylan, consisting of β-1,4-linked xylopyranosyl residues, is the second most abundant polysaccharide; it is hydrolyzed by Xylanases (EC 3.2.1.8) that are present in many fungi, yeasts as well as bacteria. The ability of ruminants to convert plant biomass unsuitable for human consumption into meat and milk is of great social and agricultural importance. However, the efficiency of this process is highly dependent on the digestibility of plant cell walls. The use of enzymes in the silage contributes to this process in several ways: produces an improvement in fermentation, improves digestibility, increases metabolizable energy, and produces a change in structural carbohydrates, which is beneficial when the silage reaches the rumen. Lactococcus lactis is one of the most commonly used lactic acid bacteria in fermented food production. Because it is considered Generally Recognized As Safe (GRAS), the implementation of this strain in biotechnological processes and industrial enzymes production could simplify the downstream processing and diminish contamination risks. The aim of this work was the over-expression of the XynA xylanase in L. lactis NZ9000 strain. The xynA gene from Bacillus subtilis was codon-optimized, synthesized, and cloned in the pNZ8048 plasmid under the control of the Pnis promoter. Expression and activity were assessed by growing the strain 48 hours in M17-agar plates with 1% xylan and 0, 10, or 50 ng/ml of nisin as inducer. Congo Red stain was used to observe xylan degradation halo, under these conditions both inducer concentrations gave similar results. Then, expression was optimized using M17 liquid media, for L .lactis the best conditions for protein overexpression were 50 ng/ml nisin and 24 hs of induction at 30ºC. Protein over-expression was detected, with the expected molecular weight, in medium supernatant after precipitation with TCA and coomassie staining of SDS-PAGE gels. No intracellular expression of XynA could be observed, indicating that the signal peptide encoded by xynA is functional in L. lactis. Protein presence in medium supernatant was also observed 48 hs after induction suggesting good stability of the protein. Further characterization of enzymatic activity in vitro and in vivo will help to determine potential biotechnological application.