Overexpression of Aspergillus tubingensis faeA in protease-deficient Aspergillus niger enables ferulic acid production from plant material

Ferulic acid is a natural antioxidant found in various plants and serves as a precursor for various fine chemicals, including the flavouring agent vanillin. However, expensive extraction methods have limited the commercial application of ferulic acid, in particular for the enrichment of food substrates. A recombinant Aspergillus tubingensis ferulic acid esterase Type A (FAEA) was expressed in Aspergillus niger D15#26 and purified with anion-exchange chromatography (3487U/mg, Km =0.43mM, Kcat=0.48/min on methyl ferulate). The 36-kDa AtFAEA protein showed maximum ferulic acid esterase activity at 50°C and pH 6, suggesting potential application in industrial processes. A crude AtFAEA preparation extracted 26.56 and 8.86mg/g ferulic acid from maize bran and triticale bran, respectively, and also significantly increased the levels of p-coumaric and caffeic acid from triticale bran. The cost-effective production of AtFAEA could therefore allow for the enrichment of brans generally used as food and fodder, or for the production of fine chemicals (such as ferulic and p-coumaric acid) from plant substrates. The potential for larger-scale production of AtFAEA was demonstrated with the A. niger D15[AtfaeA] strain yielding a higher enzyme activity (185.14 vs. 83.48U/ml) and volumetric productivity (3.86 vs. 1.74U/ml/h) in fed-batch than batch fermentation.

For the first time, the presence of a ferulic acid esterase (FAE) was demonstrated in Streptomyces ambofaciens. This extracellular enzyme was produced on a range of lignocellulosic substrates. The maximal level of activity was detected in the presence of either destarched wheat bran or oat spelt xylan as the sole carbon source. We found that 1% (m/v) of destarched wheat bran was the optimal concentration to induce its production. With this inducer, no ferulic acid dimers were released from the cell wall by the produced FAE. Interestingly, rape cattle cake (Brassica napus), which does not contain esterified ferulic acid, was also shown to induce the production of the FAE from S. ambofaciens. The FAE was partially purified from the culture supernatant. The purified enzyme was optimally active at pH 7 and 40 degrees C. The substrate specificity of the FAE from S. ambofaciens was investigated: the highest activity was determined with methyl p-coumarate, methyl ferulate, and methyl cinnamate. Furthermore, the FAE required a certain distance between the benzene ring and the ester bond to be active. According to these biochemical characteristics, the FAE from S. ambofaciens has been classified as a type B FAE.

Ferulic acid is a known precursor for vanillin production but the significance of agro waste as substrates for its extraction, in combination with microbes is a less explored option. Various lactic acid bacteria were screened for the production of ferulic acid esterase (FAE) and Enterococcus lactis SR1 was found to produce maximum FAE (7.54 ± 0.15IU/ml) in the synthetic medium under submerged fermentation. To make the process cost effective, various lignocellulosic agroresidues were evaluated for the production of FAE from the bacterium. It was found that wheat bran serves as the best substrate for FAE production (4.18 ± 0.12IU/ml) from E. lactis SR1. Further, optimization of fermentation conditions for FAE production from E. lactis SR1 using wheat bran as carbon source led to an increase in the enzyme production (7.09 ± 0.21IU/ml) by 1.5 fold. The FAE produced was used alone or in combination with commercial holocellulase for biological release of FA from the tested agroresidues. The highest release of FA (mg/g) by enzymatic extraction occurred in sugarbeet pulp (2.56), followed by maize bran (1.45), wheat bran (1.39) and rice bran (0.87), when both the enzymes (FAE and holocellulase) were used together. Alkaline extraction and purification of ferulic acid (FA) from these agro residues also showed that sugarbeet pulp contains the highest amount of FA (5.5mg/g) followed by maize bran (3.0mg/g), wheat bran (2.8mg/g) and rice bran (1.9mg/g), similar to the trend obtained in biological/enzymatic extraction of FA from these residues. Furthermore, the substrates were found to release higher reducing sugars when both commercial holocellulase and FAE were used in combination than by the use of holocellulase alone. Thus, FAEs not only release FA but also enabled hemicellulase and cellulase to release more sugars from plant material.

Ferulic acid (FA) production has become a frequent focus on today’s research due to its antioxidant properties. However, there has been little to none studies reported on the usage of mixed culture as inoculum in FA production. This study aims to determine the kinetics FA production in a mixed culture fermentation by applying the Michaelis–Menten kinetic model. In this study, mixed culture fermentation process using oil palm frond bagasse (OPFB) as substrate was applied for FA production improvement. Optimum condition was applied to study ferulic acid esterase (FAE) mechanism for kinetic modelling purposes. The kinetic model used was based on the Michaelis–Menten kinetic model. Runge–Kutta Fourth Order method was used to solve the kinetic model. Maximum FAE activity was achieved at the 28 h of fermentation process at 3.7 × 10−3 mU mL−1. This result proved that enzymatic hydrolysis occurred during fermentation process. Kinetic study was conducted with Michaelis–Menten kinetic model used as a reference kinetic equation. Three kinetic constants, Vmax, Km and Ks were determined as 3.725 × 10−3 g L−1 h−1, 28.231 g L−1 and 1.33 × 10−2 h−1 respectively using Runge–Kutta Fourth Order approach. The outcome of this study confirms that the kinetics of the process fit well with the Michaelis–Menten model. This also suggests that the usage of mixed culture has the potential to induce enzymatic hydrolysis hence improving FA production from OPFB during fermentation process.

A putative alpha/beta hydrolase fold-encoding gene (locus tag TTE1809) from the genome of Thermoanaerobacter tengcongensis was cloned and expressed in Escherichia coli as a possible source of thermostable feruloyl esterase (FAE) for the production of antioxidant phenolic acids from biomass. Designated as TtFAE, the 33-kDa protein was purified to apparent homogeneity. The lipase-like sequence characteristics of TtFAE and its substrate specificity towards methyl ferulate, methyl sinapate, and methyl p-coumarate classify it as a new member of the type A FAEs. At 75 degrees C, the enzyme retained at least 95% of its original activity for over 80 min; at 80 degrees C, its half-life was found to be 50 min, rendering TtFAE a highly thermostable protein. Under different hydrolytic conditions, ferulic acid (FA) was shown to be released from feruloylated oligosaccharides prepared from triticale bran. An estimated recovery of 68 mg FA/100 g triticale bran was demonstrated by a 30% release of the total FA from triticale bran within a 5-h incubation period. Both the oxygen radical absorbing capacity values of the feruloylated oligosaccharides and free FA were also determined. Overall, this work introduces a new bacterial member to the growing family of plant cell wall degrading FAEs that at present is largely of fungal origin, and it benchmarks the bioproduction of FA from triticale bran.

Two forms of ferulic acid esterase from Aspergillus niger have been isolated from a commercial source of pectinase. One, designated I, has a M(r) of 132,000, is probably dimeric, and has a pI of 3.0. The second, designated II, was partially purified and is monomeric (M(r) 29,000), with a pI of 3.6. Both enzymes were free of pectinase and xylanase activity and released ferulic acid from methyl ferulate. In association with a xylanase, they also released ferulic acid from destarched wheat bran. Ferulic acid esterase II released a small amount of ferulic acid (0.09 unit/mg of protein) in the absence of xylanase. The enzymes had different specificities for a range of methyl ester derivatives of cinnamoyl and benzoyl acids, acetylated xylan and p-nitrophenyl acetate.

Oat hulls contain relatively high amounts of hydroxycinnamic acids, mainly ferulic (4-hydroxy-3-methoxycinnamic) and p-coumaric acids (4-hydroxy-cinnamic), which are inhibitory to cell wall biodegradability by rumen microorganisms. In this paper, a study of the interactive effects of enriched sources of Aspergillus ferulic acid esterase (A-FAE) and Trichoderma xylanase (T-XYL) at different levels on the quantitative release of ferulic acid and p-coumaric acid from oat hulls was carried out. The results show that relative to A-FAE alone, the combined action of A-FAE and T-XYL was superior in causing the release of ferulic acid [up to 41.0% (± 2.1%)], indicating that T-XYL is important in acting with A-FAE in the degradation of feruloyl-polysaccharides of oat hulls. There was no effect of A-FAE alone, but a significant effect of A-FAE in combination with T-XYL on the release of p-coumaric acid from oat hulls. However, there was no extensive release of p-coumaric acid [(maximum release of 9.0% (± 0.7%)] by A-FAE in the presence of T-XYL, indicating a specificity of A-FAE for feruloyl groups, which only efficiently releases ferulic acid and not p-coumaric acid from oat hulls. This study suggests that A-FAE with T-XYL has an interactive effect to be able to break the ester linkage between ferulic acid and the attached sugar, releasing a significant proportion of the ferulic acid from oat hulls. This action, which causes disruption of crosslinks, has the potential to improve hydrolysis of the remaining polysaccharides by rumen microorganisms, which, in turn, would improve rumen degradability of oat hulls. Key words: Ferulic acid esterase, oat hulls, hydroxycinnamic acids, biodegradation

Effects of crude feruloyl and acetyl esterase solutions of Neocallimastix sp. YQ1 and Anaeromyces sp. YQ3 isolated from Holstein steers on hydrolysis of Chinese wildrye grass hay, wheat bran, maize bran, wheat straw and corn stalks

The ferulic acid esterases of Chrysosporium lucknowense C1: Purification, characterization and their potential application in biorefinery

리그닌 분해 세균인 Pseudomonas sp. LG2는 lignocellulose 기질을 분해하여 APPL 화합물을 생성하는 균주이다. 이 균주를 BSG(brewer's spent grain)가 함유된 배지에서 배양한 배양액에서 APPL 화합물을 확인하였다. 세포외 조효소들의 유도에 관한 여러 가지 탄소원의 영향을 조사한 결과 glucose 배지에서는 xylanase의 효소활성만 확인 되었고 xylose, arabinose에서 배양한 조효소에서는 FAE 및 xylanase의 효소활성이 없었다. Oat spelt xylan, HBSG I(hydrolyzed brewer's spent grain I), HBSG II(hydrolyzed brewer's spent grain II) 및 AFBSG(autoclaved fraction from brewer's spent grain)를 탄소원으로 배양한 조효소에서는 FAE 및 xylanase의 효소활성이 확인됐다. Pseudomonas sp. LG2를 oat spelt xylan, HBSG I, HBSG II 및 AFBSG를 탄소원으로 사용하여 14일 동안 배양하면서 배양기간에 따른 세포외 효소들의 FAE와 xylanase 활성을 조사하였다. Xylanase의 최고 활성은 xylan을 탄소원으로 6일간 배양 했을때 5.3 U/mg으로 가장 높았으며, FAE의 최고 활성은 AFBSG를 탄소원으로 배양했을 때 배양 8일째 15.4 mU/mg으로 가장 높았다. Oat spelt xylan, HBSG I, HBSG II 및 AFBSG를 탄소원으로 사용하여 배양한 배지에 분리된 ferulic acid가 확인되었다. 세포외 효소의 FAE 활성은 methyl ferulic acid, methyl caffeic acid, methyl p-coumaric acid에 대해 esterase의 활성을 보였으나, methyl sinapinic acid, methyl vanillic acid 및 methyl gallic acid에 대해서는 esterase의 활성이 없었다. Lignin degrading bacterium Pseudomonas sp. LG2 was able to degrade lignin substrate to a lot of APPL compound. APPL compound was detected in culture supernatants from Pseudomonas sp. LG2 grown with BSC(brewer's spent grain). FAE(ferulic acid esterase) and xylanase are induced from Pseudomonas sp. LG2 in the presence of carbon sources such as oat spelt xylan, HBSG I, II(hydrolyzed brewer's spent grain I, II) and AFBSG(autoclaved fraction from brewer's spent grain). However, xylanase and FAE are not induced by growth of Pseudomonas sp. LG2 on xylose and arabinose. Pseudomonas sp. LG2 is grown on medium containing oat spelt xylan, HBSG I, II and AFBSG and the induction of FAE and xylanase activities of extracellular proteins determined during 14 days. Maximum level of xylanase activity(5.3 U/mg) found at 6 days in culture contained oat spelt xylan as carbon source, whereas maximum level of FAE activity(15.4 mU/mg) was found at 8 days in culture contained AFBSG as carbon source. Most ferulic acid was released in culture supernatants when Pseudomonas sp. LG2 grown on oat spelt xylan, HBSG I, II and AFBSG. FAE of extracellular enzymes was also specific activity on methyl ferulic acid, methyl caffeic acid and methyl p-coumaric acid respectively, but not methyl sinapinic acid, methyl vanillic acid and methyl gallic acid.

Two hypothetical proteins XP_001818628 and XP_001819091 (designated AoFaeB and AoFaeC, respectively), showing sequence identity with known type-C feruloyl esterases, have been found in the genomic sequence of Aspergillus oryzae. We cloned the putative A. oryzae feruloyl esterase-encoding genes and expressed them in Pichia pastoris. Both purified recombinant AoFaeB (rAoFaeB) and AoFaeC (rAoFaeC) had apparent relative molecular masses of 61,000 and 75,000, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After N-deglycosylation, both proteins had a relative molecular mass of 55,000. The optimum pH for rAoFaeB was 6.0, although it was stable at pH values ranging from 3.0 to 9.0; rAoFaeC had an optimum pH of 6.0 and was stable in the pH range of 7.0-10.0. Thermostability of rAoFaeC was greater than that of rAoFaeB. Whereas rAoFaeC displayed hydrolytic activity toward methyl caffeate, methyl p-coumarate, methyl ferulate, and methyl sinapate, rAoFaeB displayed hydrolytic activity toward methyl caffeate, methyl p-coumarate, and methyl ferulate but not toward methyl sinapate. Substrate specificity profiling of rAoFaeB and rAoFaeC revealed type-B and type-C feruloyl esterases, respectively. Ferulic acid was efficiently released from wheat arabinoxylan when both esterases were applied with xylanase from Thermomyces lanuginosus. Both recombinant proteins also exhibited hydrolytic activity toward chlorogenic acid.

Oat hulls, an agricultural byproduct, contain a relatively high amount of ferulic acid (FA; 4-hydroxy-3-methoxycinnamic acid), which is believed to be inhibitory to oat hull biodegradability by rumen microorganisms. In this paper, Aspergillus ferulic acid esterase (FAE) was investigated for its ability to release FA from oat hulls. The objectives were to determine the effects of particle size of oat hulls (ground to pass through 1 mm and 250 microm screens and a 100 microm sieve) on release of FA by FAE both in the presence and in the absence of Trichoderma xylanase. The results show that the release of FA by FAE was dependent upon the particle size of oat hulls (< or = 250 microm). In the absence of Trichoderma xylanase, little FA was released by FAE. In the presence of Trichoderma xylanase, there was a significant release of FA by FAE, indicating a synergistic interaction between FAE and Trichoderma xylanase on release of FA from oat hulls. These results indicate that FAE is able to break the ester linkage between FA and the attached sugar, releasing FA from oat hulls. This may leave the remainder of the polysaccharides open for further hydrolytic attack by rumen microorganisms. It is likely that removing FA from oat hulls could improve rumen biodegradability, thus improving the nutritional value of oat hulls.

Ferulic acid (3-methoxy-4-hydroxycinnamic acid), present in complex plant cell walls, is covalently cross-linked to polysaccharides by ester bonds and to components of lignin mainly by ether bonds. Ferulic acid has also been shown to occur in dimer- and trimerized forms through oxidative coupling between esterified and/or etherified ferulic acid residues. These cross-links are among the factors most inhibitory to digestion of complex plant cell walls in ruminants. Recently obtained information on ferulic acid and ferulic acid esterases in relation to complex plant cell wall biodegradation is reviewed. A focus of the review is on structural characteristics of plant cell walls associated with ferulic acid, physicochemical properties of ferulic acid esterase and synergistic interaction between ferulic acid esterase and other accessary cell wall degrading enzymes on the release of ferulic acid and plant cell wall biodegradation. Key words: Ferulic acid, hydroxycinnamic acid, feruloyl esterase, interaction effects, polysaccharide, feruloyl-polysaccharides, plant cell walls, biodegradation

Degradation of feruloylated oligosaccharides from sugar-beet pulp and wheat bran by ferulic acid esterases from Aspergillus niger

Dyadic announces OPTIBIOCAT Project aimed to develop biocatalysts for cosmetic industry