Recombinant CAZyme production in Lactococcus lactis to enhance silage quality

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

Rosario

Workshop; V Encuentro y II Workshop de la Red de Argentina de Tecnología Enzimática; 2023

Red TEZ

One of the most widely common lactic acid bacteria (LAB) 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 downstream processing while reducing contamination hazards. Silage fermentation is crucial for agroindustry and society because ruminants can generate meat and milk from plant biomass that is unsuitable for human consumption. The main components of plant cell walls that constitute forages include cellulose, lignin, and hemicellulose (which is primarily formed by xylan). It is hydrolyzed by Xylanases (EC 3.2.1.8) present in many fungi, yeasts, as well as bacteria. The main objective of this study is enhancing the xylanase activity in silage to improve the digestibility of plant materials for ruminants. This will be achieved by expressing the recombinant enzyme in L. lactis. The ultimate goal is to provide a cost-effective and sustainable strategy to enhance the nutritional value of silage, which could lead to increased animal productivity and improved feed efficiency. In our laboratory the xynA gene from Bacillus subtilis was over-expressed in L. lactis NZ9000, the enzyme was purified and characterized as having an optimal temperature of 40°C, and its maximum activity was found between pH 5 and 6; the Vmax, Hill coefficient, and S0.5 values of the purified protein were 77.02 UI/mg, 1.67, and 1.77 g/L, respectively.In the present work, we continue the characterization of the protein Bsxyn11A by analyzing its activity on plant substrates.The purified enzyme and culture supernatant of LAB carrying pNZ-xynA or pNZ (negative control) plasmids were incubated with birchwood xylan or triturated sorghum in sodium phosphate buffer for 24 hours at 37°C. Subsequently, a thin-layer chromatography (TLC) analysis was performed to visualize the xylooligosaccharides generated by the CAZyme activity.We observed that the purified enzyme acted on the xylan substrate as expected, producing several polymers except for the X1 monomer, which is consistent with the xylanase family 11 and in line with previous results. Additionally, the culture supernatants of LAB expressing Bsxyn11A were able to degrade pure xylan.Then the purified Bsxyn11A was incubated with triturated sorghum: in the TLC analysis we could observe differential signals between enzyme-treated and non-treated substrate. Also, when the ground sorghum was incubated with control culture supernatants of LAB or supernatants of LAB expressing Bsxyn11A we observed modifications on the visible spots. Thus, the enzyme produced changes in the carbohydrate pattern observable by TLC.The results presented indicate that the recombinant xylanase produced in L. lactis is capable to degrade carbohydrates present in both sorghum and xylan. More studies are needed to optimize incubation conditions and identify the carbohydrates produced. Given these results, we conclude that the xylanase secreted by the recombinant L. Lactis is a good candidate to improve silage fermentation.