Browsing by Author "Zwane, Eunice Nonhlanhla"
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- ItemProduction, characterisation and application of a recombinant ferulic acid esterase from Aspergillus tubingensis(Stellenbosch : Stellenbosch University, 2014-12) Zwane, Eunice Nonhlanhla; Viljoen-Bloom, M.; Van Zyl, Willem Heber; Rumbold, K.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: Ferulic acid esterase (FAE) is involved in the release of ferulic acid from xylan and is an important enzyme for the extraction of ferulic acid from plant biomass, whilst also reducing plant cell wall recalcitrance for biofuel production and improving the digestibility of animal feed. The production of FAE was investigated in strains of Aspergillus tubingensis, Aspergillus carneus, Aspergillus niger and Rhizopus oryzae. The A. tubingensis T8.4 strain showed the highest activity on triticale bran, producing a type A FAE active against methyl p-coumarate, methyl ferulate and methyl sinapate. The native A. tubingensis ferulic acid esterase 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 acid esterase for the utilisation of maize bran. The A. niger D15#26 strain, which has reduced protease activity and does not acidify the growth medium (thus promoting high-level expression of recombinant enzymes) was used as host for the expression of a genomic copy of the A. tubingensis faeA gene under transcriptional control of the A. niger gpdA promoter. The A. niger D15[AtfaeA] strain produced 13.5 U/ml FAEA after 5 days on autoclaved maize bran (3-fold higher than the A. tubingensis donor strain) and was able to extract 50% of the available ferulic acid from non-pretreated maize bran. The recombinant AtFAEA was purified 7-fold with anion-exchange chromatography and its identity confirmed with peptide mass fingerprinting. The physical properties of the recombinant AtFAEA were similar to that of the native enzyme; enzyme activity peaked at pH 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 of 0.48/min and Kcat/Km of 1.1/min.mM. These properties suggest that AtFAEA would be suitable for the food, pulp and paper, and animal feed industries where important phytochemicals could be released from the hemi-cellulosic backbone. Culturing A. niger D15[AtfaeA] in a bioreactor significantly improved AtFAEA production, with fed-batch fermentation yielding 2-fold higher FAE activity than batch fermentation. Fed-batch conditions resulted in a higher biomass yield, volumetric productivity and volumetric activity than batch fermentation, suggesting that fed-batch conditions 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 and 177 mg/l ferulic acid from maize bran and triticale bran, respectively, as well as 77 mg/l p-coumaric acid from triticale bran. This confirmed that AtFAEA could increase the ferulic acid content and nutritional value of maize and triticale bran, which can add nutritional value to animal feed. The enzyme cocktail was also able to extract 0.2 g ferulic acid/100 g soluble solids from Aspalathus linearis (rooibos) leaves and stems, indicating the potential of AtFAEA for the extraction of polyphenolics from other plant substrates.