Biochemical characterization of a halotolerant feruloyl esterase from Actinomyces spp.: refolding and activity following thermal deactivation

Abstract

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.

A detailed overview of xylanases: an emerging biomolecule for current and future prospective

“…Hence, for complete and efficient hydrolysis of xylan into its constituent sugars requires synergistic action of various enzymes with specifically targeting appropriate bonds of xylan. The multifunction xylanolytic system exists in bacteria (Zhang et al 2016a(Zhang et al , 2016b, fungi (Driss et al 2011;) and actinomycetes (Hunt et al 2016) where xylan backbone is randomly cleaved by the action of endo-1, 4-β-d-xylanases; xylose polymer is broken down to its monomeric form by action of β-dxylosidases. Acetyl and phenolic side branches were removed by the action of α-glucuronidase and acetylxylan esterase.…”

A family of archaea-like carboxylesterases preferentially expressed in the symbiotic phase of the mycorrhizal fungus Tuber melanosporum

“…The apparent K m derived from this analysis (0.88 mM) is comparable to the K m values previously reported for true microbial feruloyl esterases such as the prototype Aspergillus niger enzymes AnFAEA and AnFAEB 35 , 36 and the ActOFaeI enzyme from Actinomyces spp . 37 , whereas the estimated V max (1.5 U/mg protein) is approximately 10-fold lower.

Figure 3 Feruloyl ester hydrolysis by TmelEST1.

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Soluble expression of a novel feruloyl esterase from Burkholderia pyrrocinia B1213 in Escherichia coli and optimization of production conditions

“…[13], ActOFaeI from Actinomyces sp. [14], XynZ from Clostridium thermocellum [15], GthFAE from Geobacillus thermoglucosidasius [16], DdFAED and DdFAET from Dickeya dadantii [17] and ScFAE1 and ScFAE2 from Sorangium cellulosum [41]. Pairwise alignment of FAEs showed no greater identity than 20%, and these bacterial FAEs could be divided into several classes as shown in Figure 2.…”

“…[13], Actinomyces sp. [14], Clostridium thermocellum [15], Geobacillus thermoglucosidasius [16] and Dickeya dadantii [17]. Bacterial FAEs were easier to express heterologously and more convenient to apply.…”