Abstract
Feruloyl esterase (FAE; EC 3.1.1.73) catalyzes the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl group in an esterified sugar to assist in waste biomass degradation or to release ferulic acid (FA). An FAE-producing strain was isolated from humus soil samples and identified as Bacillus pumilus SK52.001. The BpFAE gene from B. pumilus SK52.001 was speculated and heterogeneously expressed in Bacillus subtilis WB800 for the first time. The enzyme exists as a monomer with 303 amino acids and a molecular mass of 33.6 kDa. Its specific activity was 377.9 ± 10.3 U/(mg protein), using methyl ferulate as a substrate. It displays an optimal alkaline pH of 9.0, an optimal temperature of 50 °C, and half-lives of 1434, 327, 235, and 68 min at 50, 55, 60, and 65 °C, respectively. Moreover, the purified BpFAE released 4.98% FA of the alkali-acidic extractable FA from de-starched wheat bran (DSWB). When the DSWB was enzymatically degraded by the synergistic effect of the BpFAE and commercial xylanase, the FA amount reached 49.47%. It suggested that the alkaline BpFAE from B. pumilus SK52.001, which was heterologously expressed in B. subtilis WB800, possesses great potential for biomass degradation and achieving high-added value FA production from food by-products.
Highlights
Feruloyl esterase (FAE; EC 3.1.1.73) is a member of the alpha/beta hydrolase family that cleaves ester or ether linkages between ferulic acid (FA) and sugars in order to facilitate plant cell wall degradation and release FA [1,2]
Positive transformants were cultured overnight in 5 mL LB medium supplemented with ampicillin, and plasmids pMA5-fae were extracted by FastPure Plasmid Mini Kit (Vazyme Biotech Co., Ltd., Nanjing, China) and sequenced by Sangon Biotech Co., Ltd. (Shanghai, China)
The 75 mg de-starched wheat bran (DSWB) was suspended in 3 mL of the Tris-HCl buffer (50 mmol/L, pH 9.0), which was enzymatically degraded by adding 0.18 mg of purified BpFAE, 0.31 mg of commercial xylanase, and 0.18 mg of BpFAE along with 0.31 mg xylanase
Summary
Feruloyl esterase (FAE; EC 3.1.1.73) is a member of the alpha/beta hydrolase family that cleaves ester or ether linkages between ferulic acid (FA) and sugars in order to facilitate plant cell wall degradation and release FA [1,2]. FAE has been used in paper processing and bleaching, and as an additive in animal feed to improve nutrient digestibility [3]. Owing to their utility, strains that secrete FAEs continue to be identified, and some FAEs from fungi have been studied extensively, including their classification [7], biochemical properties, three-dimensional structures, and catalytic mechanisms [9]. An increasing number of researchers have begun to use Bacillus subtilis as a host to express enzymes, mainly because it is a food-grade microorganism and suitable for the industrial production of FAEs. B. subtilis is amenable to a simple fermentation process and lacks obvious codon bias [15]. The studies were conducted to characterize the biochemical properties and liberate FA from de-starched wheat bran (DSWB) by synergy action of BpFAE and commercial xylanase
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