Cloning, Expression And Characterization Of New Feruloyl Esterases From Lactobacillus Johnsonii

Feruloyl esterase are a subclass of the carboxylic acid esterases that are able to hydrolyse the ester bond between hydroxycinnamic acids and sugars present in the plant cell walls to release ferulic acid. Ferulic acid esterase have a widely applications, for example, to release of ferulic acid, the pulp and paper industry, production of fuel ethanol, farming industry, biological synthesis. This paper focused on screening the microorganisms that could produce FAE, cloning and heterologous expression of FAE as well as the biochemical characteristics. The main research results are as follows:1) We screened L. johnsonii JN115 that has the best activity to produce FAE from microorganisms(Bacillus and Lactobacillus) in our Lab. According to the whole genome sequence of L. johnsonii NCC533 and the conserved sequence of FAE, we predicted four gene sequences of coding FAE, they were named as LJ0101(663bp), LJ1610(945bp), LJ0900(777bp), PeNPLJ0054(915bp) respectively. We cloned and expressed four FAE gene successfully. The four recombinant proteins were all well expressed by SDS-PAGE verification. But it’s a pity that PeNPLJ0054 had no FAE activity by enzyme activity validation, the other three had good FAE activity.2) We optimized the induced expression condition of recombinant strains from the four aspects of growth of recombinant strains, add IPTG concentration, start induced time, induced time. The best induced conditions of LJ0101 were added a concentration of 0.3 mmol·L-1 IPTG when OD600 reached 0.4 and induced for 24 h. The best induced conditions of LJ0900 and LJ1610 were added a concentration of 0.5 mmol·L-1 IPTG when OD600 reached 0.6 and induced for 24 h.3) We obtained the pure enzyme after sepration and purification by using Nickel column affinity chromatography and studyed their enzymology properties. The LJ0101 showed the optimal temperature and pH were 50℃ and 5.0. Mg2+ can promote enzyme activity. When with methyl ferulate as the substrate, the minimum Km was 1.50 mmol·L-1, the maximum reaction rate was 26.67 μmol·mg-1·min-1, the kcat/Km was 7.29×104 L·mol-1·s-1. The LJ0900 showed the optimal temperature and pH were 30℃ and 6.0. Mg2+ can promote enzyme activity. When with methyl ferulate as the substrate, the minimum Km was 2.48 mmol·L-1, the maximum reaction rate was 13.54 μmol·mg-1·min-1, the kcat/Km was 2.75×103 L·mol-1·s-1. And LJ1610 showed the optimal temperature and pH were 40℃ and 6.0. Mg2+ can promote enzyme activity. When with ethyl ferulate ester as the substrate, the minimum Km was 2.31 mmol·L-1, the kcat/Km was 2.52×103 L·mol-1·s-1, but the maximum reaction rate was 14.37 μmol·mg-1·min-1 to methyl ferulate. When the tempetature were 30~40℃ and pH were 4-7, the three enzymes were stability. And the enzyme activity were inhibited by mental ions Cu2+å'Œ Fe3+. By the way, we also studied the substrate-specificity, the three enzyme all showed broad sustrate specificity, and they showed high preference and good enzymatic activity to aromatic eaters.