Location: Renewable Product Technology ResearchTitle: Novel feruloyl esterase from Lactobacillus fermentum NRRL B-1932 and analysis of the recombinant enzyme produced in Escherichia coli.
Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/24/2015
Publication Date: 1/20/2016
Citation: Liu, S., Bischoff, K.M., Anderson, A.M., Rich, J.O. 2016. Novel feruloyl esterase from Lactobacillus fermentum NRRL B-1932 and analysis of the recombinant enzyme produced in Escherichia coli. Applied and Environmental Microbiology. 82(17):5068-5076. doi: 10.1128/AEM.01029-16.
Interpretive Summary: We identified a highly active bacterial feruloyl esterase (FE) from a Lactobacilli strain. FE catalyzes the breakdown of ester linkages in the hemicelluloses portion of the lignocellulosic biomass. By using molecular biology techniques, the DNA sequence encoding this enzyme was cloned and moved in E. coli host for increased production and rapid purification. This study will be useful in biochemical studies of FE as well as applications such as biomass degradation and production of high valued ferulic acid or hydroxycinnamates.
Technical Abstract: Using agar plates containing ethyl ferulate as the sole carbon source, 33 Lactobacillus strains were screened for feruloyl esterase (FE) activity. Among a dozen species showing a clearing zone on the opaque plate containing ethyl ferulate, Lactobacillus fermentum NRRL B-1932 demonstrated the strongest FE activity. FE activities were monitored using HPLC with an acetonitrile-trifluoroacetic acid gradient. To produce sufficient purified FE from Lactobacillus fermentum strain NRRL B-1932 (LfFE), the cDNA encoding LfFE (Lffae) was isolated by using available closely related genome sequences and over-expressed in Escherichia coli. A 29.6 kDa protein LfFE was detected from protein extract of E. coli BL21pLysS carrying pET28bLffae upon IPTG induction. The recombinant LfFE containing a polyhistidine tag was purified by Ni-NTA affinity chromatography. The purified LfFE showed strong activities against several artificial substrates including p-nitrophenyl acetate and 4-methylumbelliferyl p-trimethylammoniocinnamate chloride. The pH and temperature optima of the recombinant LfFE is around 6.5 and 37 deg C, respectively, as determined using either crude or purified recombinant LfFE. This study will be useful in biochemical studies of LfFE as well as increased production of the recombinant LfFE.