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

Zwane, Eunice Nonhlanhla (2014-12)

Thesis (PhD)--Stellenbosch University, 2014.

Thesis

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.

AFRIKAANSE OPSOMMING: Feruliensuuresterase (FSE) is by die vrystelling van feruliensuur vanaf xilaan betrokke en dus 'n belangrike ensiem vir die onttrekking van feruliensuur uit biomassa, terwyl dit ook plantselwande se weerstandigheid vir biobrandstofproduksie verlaag en die verteerbaarheid van veevoer verbeter. Die produksie van FSE is in stamme van Aspergillus tubingensis, Aspergillus carneus, Aspergillus niger en Rhizopus oryzae ondersoek. Die A. tubingensis T8.4 stam het die hoogste aktiwiteit op korogsemels getoon met die produksie van 'n tipe A FSE aktief teen metiel-p-kumuraat, metielferulaat en metielsinapaat. Die natuurlike A. tubingensis feruliensuuresterase geen (faeA) was onderhewig aan glukose-onderdrukking en substraat-induksie deur mieliesemels. Die resultate het op ‘n samewerking tussen endoglukanase, endoxilanase en feruliensuuresterase vir die benutting van mieliesemels gedui. Die A. niger D15#26 stam, wat oor verlaagde protease-aktiwiteit beskik en nie die groeimedium versuur nie (en dus hoë-vlak uitdrukking van rekombinante ensieme bevorder), is as gasheer vir die uitdrukking van 'n genoom-kopie van die A. tubingensis faeA geen onder transkripsionele beheer van die A. niger gpdA promotor gebruik. Die A. niger D15[AtfaeA] stam het 13.5 U/ml FAEA na 5 dae op ge-outoklaveerde mieliesemels gelewer (3-voudig hoër as die A. tubingensis skenkerstam) en kon 50% van die beskikbare feruliensuur uit onbehandelde mieliesemels onttrek. Die rekombinante AtFAEA is 7-voudig met anioon-uitruilingschromatografie gesuiwer en sy identiteit met peptiedmassavingerafdrukke bevestig. Die fisiese eienskappe van die rekombinante AtFAEA was soortgelyk aan dié van die natuurlike ensiem; die ensiemaktiwiteit was die hoogste by pH 6 en 50°C. Dit was stabiel by pH 3 tot 7 en 30°C tot 60°C, met ‘n Km van 0.43 mM, Kcat van 0.48/min en Kcat/Km van 1.1/min.mM. Hierdie eienskappe het aangedui dat AtFAEA geskik vir die voedsel, pulp en papier, en voerbedrywe kan wees waar belangrike plantchemikalieë uit die hemisellulose-ruggraat vrygestel kan word. Kweking van A. niger D15[AtfaeA] in 'n bioreaktor het FSEA produksie aansienlik verbeter, met voerlot-fermentasie wat 2-voudig meer FAE aktiwiteit as die lot-fermentasie gelewer het. Die A. niger D15[AtfaeA] stam het 'n hoër biomassa-opbrengs, volumetriese produktiwiteit en volumetriese aktiwiteit onder voerlot- as lot-fermentasie getoon, wat daarop dui dat voerlot-toestande meer geskik is vir die grootskaalse produksie van AtFAEA deur A. niger. 'n Ongesuiwerde preparaat van die A. niger D15[AtfaeA] ensiemmengsel het onderskeidelik 531 mg/l en 177 mg/l feruliensuur uit mielie- en korogsemels onttrek, asook 77 mg/l p-kumuraat vanuit korogsemels. Dit bevestig dat AtFAEA die feruliensuur-inhoud en voedingswaarde van mielie- en korogsemels kan verhoog, wat voedingswaarde tot veevoer kan toevoeg. Die ensiemmengsel het ook 0.2 g feruliensuur/100 g oplosbare vastestowwe vanuit Aspalathus linearis (rooibos) blare en stingels onttrek, wat dui op die potensiaal van AtFAEA vir die onttrekking van polifenole uit ander plantsubstrate.

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