Production, characterisation and application of a recombinant ferulic acid esterase from Aspergillus tubingensis

[摘要] ENGLISH ABSTRACT: Ferulic acid esterase (FAE) is involved in the release of ferulic acid from xylan and is animportant enzyme for the extraction of ferulic acid from plant biomass, whilst also reducingplant cell wall recalcitrance for biofuel production and improving the digestibility of animalfeed. The production of FAE was investigated in strains of Aspergillus tubingensis,Aspergillus carneus, Aspergillus niger and Rhizopus oryzae. The A. tubingensis T8.4 strainshowed the highest activity on triticale bran, producing a type A FAE active against methylp-coumarate, methyl ferulate and methyl sinapate. The native A. tubingensis ferulic acidesterase gene (faeA) was subject to glucose inhibition and substrate induction by maize bran.The results also indicated a combined action of endoglucanase, endoxylanase and ferulic acidesterase for the utilisation of maize bran.The A. niger D15#26 strain, which has reduced protease activity and does not acidify thegrowth medium (thus promoting high-level expression of recombinant enzymes) was used ashost for the expression of a genomic copy of the A. tubingensis faeA gene undertranscriptional control of the A. niger gpdA promoter. The A. niger D15[AtfaeA] strainproduced 13.5 U/ml FAEA after 5 days on autoclaved maize bran (3-fold higher than theA. tubingensis donor strain) and was able to extract 50% of the available ferulic acid fromnon-pretreated maize bran.The recombinant AtFAEA was purified 7-fold with anion-exchange chromatography andits identity confirmed with peptide mass fingerprinting. The physical properties of therecombinant AtFAEA were similar to that of the native enzyme; enzyme activity peaked atpH 6 and 50°C. It was stable at pH 3 to 7 and 30°C to 60°C, with a Km of 0.43 mM, Kcat of0.48/min and Kcat/Km of 1.1/min.mM. These properties suggest that AtFAEA would besuitable for the food, pulp and paper, and animal feed industries where importantphytochemicals could be released from the hemi-cellulosic backbone.Culturing A. niger D15[AtfaeA] in a bioreactor significantly improved AtFAEAproduction, with fed-batch fermentation yielding 2-fold higher FAE activity than batchfermentation. Fed-batch conditions resulted in a higher biomass yield, volumetricproductivity and volumetric activity than batch fermentation, suggesting that fed-batchconditions are better suited for large-scale production of AtFAEA in A. niger.A crude preparation of the A. niger D15[AtfaeA] enzyme cocktail extracted 531 mg/l and177 mg/l ferulic acid from maize bran and triticale bran, respectively, as well as 77 mg/lp-coumaric acid from triticale bran. This confirmed that AtFAEA could increase the ferulicacid content and nutritional value of maize and triticale bran, which can add nutritional valueto animal feed. The enzyme cocktail was also able to extract 0.2 g ferulic acid/100 g solublesolids from Aspalathus linearis (rooibos) leaves and stems, indicating the potential ofAtFAEA for the extraction of polyphenolics from other plant substrates.

[发布机构] Stellenbosch University