Biochemical characterization of a halotolerant feruloyl esterase  from Actinomyces spp.: refolding and activity following thermal  deactivation.
Ferulic acid esterases (FAE, EC. 3.1.1.73) hydrolyse the linkage between hemicellulose and lignin and thus have potential for use in mild enzymatic pretreatment of biomass as an alternative to thermochemical approaches. Here, we report the characterization of a novel FAE (ActOFaeI) obtained from the bacterium, Actinomyces sp. oral which was recombinantly expressed in Escherichia coli BL21 in two forms: with and without its putative signal peptide. The truncated form was found to have <10 % relative activity compared to the full length and was more prone to aggregation after purification. The enzyme with retained peptide demonstrated 2 to 4-fold higher activity against methyl caffeate and methyl p-coumarate, with specific activities of 477.6 and 174.4 U mg(-1) respectively, than the equivalent activities of the benchmark FAE from Aspergillus niger A and B. ActOFaeI retained activity over a broad pH range with a maximum at 9 but >90 % relative activity at pH 6.5 and an optimum reaction temperature of 30 °C. ActOFaeI increased activity by 15% in high salt conditions (1000 mMNaCl) and its thermal unfolding temperature improved from 41.5 °C in standard buffer to 74 °C in the presence of 2500 mM sodium malonate. ActOFaeI also released ferulic acid from destarched wheat bran when combined with a xylanase preparation. After treatment above the thermal denaturation temperature followed by cooling to room temperature, ActOFaeI demonstrated spontaneous refolding into an active state. ActOFaeI displays many useful characteristics for enzymatic pretreatment of lignocellulose and contributes to our understanding of this important family.
Department of Chemical Engineering, Monash University, Clayton, VIC, 3800, Australia.
Cameron J
CJ
Hunt
Department of Chemical Engineering, Monash University, Clayton, VIC, 3800, Australia.
Akshat
A
Tanksale
Department of Chemical Engineering, Monash University, Clayton, VIC, 3800, Australia. victoria.haritos@monash.edu.
Victoria S
VS
Haritos
D000190:Actinomyces / Q000201:enzymology* / Q000235:genetics; D002109:Caffeic Acids / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003373:Coumaric Acids / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
D002109:Caffeic Acids; D003373:Coumaric Acids; D011994:Recombinant Proteins; C042405:methyl caffeate; D002265:Carboxylic Ester Hydrolases; C070196:feruloyl esterase