Expression and characterization of recombinant cazyme in Lactococcus lactis nz9000 to enhance silage quality

GIZZI, F; MARTÍN, M; MAGNI, C; BLANCATO, VS

Mendoza

Congreso; LVIII Reunión Anual de SAIB; 2022

SAIB

The main components of plant cell walls that constitute forages include cellulose, lignin, and hemicellulose (which is primarily formed by xylan). These are the building blocks for livestock feed. Silage fermentation is crucial for agroindustry and society because ruminants can generate meat and milk from plant biomass that is unsuitable for human consumption. However, how efficiently plant cell walls could be digested has a significant impact on how successful this process is. In hardwoods and grasses, xylan, which is composed of -1,4-linked xylopyranosyl residues, is the second most prevalent polysaccharide. It is hydrolyzed by Xylanases (EC 3.2.1.8) present in many fungi, yeasts as well as bacteria. By enhancing fermentation and digestibility, increasing metabolizable energy, and causing a shift in structural carbohydrates, the incorporation of enzymes in the silage promotes the degradation and also is beneficial once the silage reaches the rumen. One of the most widely common lactic acid bacteria used in the manufacturing of fermented foods is Lactococcus lactis, which is generally regarded as safe (GRAS). Thus, its incorporation into biotechnological procedures and the manufacture of commercial enzymes could simplify the downstream processing while reducing contamination hazards. In this context, the aim of this work was the over-expression of the XynA xylanase in L. lactis NZ9000 strain and its biochemical characterization to assess its potential for ensiling improvement. The xynA gene from Bacillus subtilis was codon-optimized, synthesized, and cloned in the pNZ8048 plasmid under the control of the Pnis promoter. Protein over-expression was detected, in medium supernatant. XynA was purified to homogeneity by Ni-affinity chromatography and its biochemical properties were characterized. Xylanase activity was examined by the DNS assay, by measuring the amount of reducing sugars liberated from solubilized beechwood xylan. We found that XynA activity is maximum at 50°C however the enzyme is stable up to 40°C thus defining the optimal temperature to 40°C. Concerning pH dependance, maximum activity was found between pH 5 and 6 with a stability range between pH 4,5 - 8. These characteristics are consistent with what has been reported so far about numerous xylanases (XynA) from several organisms. The measured parameters for the purified XynA protein are consistent with the pH and temperature found in silage practices.