Production and characterization of ferulic acid esterase activity in crude extracts by Streptomyces avermitilis CECT 3339

Streptomyces avermitilis CECT 3339 produces extracellular ferulic acid esterase (FAE) activity during growth on a range of lignocellulose substrates. Maximal levels of FAE activity were detected in culture filtrates from S. avermitilis CECT 3339 grown in media containing wheat bran and yeast extract as carbon and nitrogen sources respectively. Biochemical characterization of this enzyme activity revealed that it was 100-fold higher when wheat bran was pretreated with Celluclast (a mix of hydrolytic enzymes). FAE was found to be end-product-inhibited. Characterization of the properties of the enzyme showed that FAE exhibited an activity optimum pH at 6 with pH stability between pH 6 and 8. The optimum temperature was 50 °C while the temperature stability was between 30 °C and 40 °C, with rapid inactivation at 60 °C and above. The characteristics and stability of FAE from S. avermitilis CECT 3339 suggest a potential role for this enzyme in combination with endoxylanases for the upgrading of plant-residue silage and for biopulping. Received: 17 November 1997 / Received revision: 13 March 1998 / Accepted: 13 April 1998     

A type B feruloyl esterase from <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> with broad pH applicability

A hypothetical protein AN1772.2 of Aspergillus nidulans was found to have a 56% identity with a known type C ferulic acid esterase (FAE) from Talaromyces stipitatus. In addition, it contained a 13-amino acid conserved region flanking the characteristic G-X-S-X-G motif of a serine esterase, suggesting a FAE function for the protein. The putative FAE was successfully cloned from the genomic DNA and expressed in Saccharomyces cerevisiae. The recombinant protein exhibited high FAE activities. Therefore, its function as an FAE was unequivocally determined. About 86% of the enzyme activity was found in the growth medium, indicating that the native signal peptide was effective in the yeast expression system. The recombinant FAE was purified to its homogeneity, and subsequently characterized. The FAE is stable over an unusually wide range of pH (4.0–9.5), has a pH optimum of 7.0, and a temperature optimum of 45°C. A substrate specificity profiling reveals that the enzyme is a type B FAE, despite its strong sequence homology with type C FAEs, raising an interesting question on the role of the conserved region in substrate specificity.     

Engineering <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> to produce feruloyl esterase for the release of ferulic acid from switchgrass

The Aspergillus niger feruloyl esterase gene (faeA) was cloned into Saccharomyces cerevisiae via a yeast expression vector, resulting in efficient expression and secretion of the enzyme in the medium with a yield of ~2 mg/l. The recombinant enzyme was purified to homogeneity by anion-exchange and hydrophobic interaction chromatography. The specific activity was determined to be 8,200 U/μg (pH 6.5, 20°C, 3.5 mM 4-nitrophenyl ferulate). The protein had a correct N-terminal sequence of ASTQGISEDLY, indicating that the signal peptide was properly processed. The FAE exhibited an optimum pH of 6–7 and operated optimally at 50°C using ground switchgrass as the substrate. The yeast clone was demonstrated to catalyze the release of ferulic acid continuously from switchgrass in YNB medium at 30°C. This work represents the first report on engineering yeast for the breakdown of ferulic acid crosslink to facilitate consolidated bioprocessing.     

Factors affecting ferulic acid release from Brewer’s spent grain by Fusarium oxysporum enzymatic system

In this study, the factors affecting ferulic acid (FA) release from Brewer’s spent grain (BSG), by the crude enzyme extract of Fusarium oxysporum were investigated. In order to evaluate the importance of the multienzyme preparation on FA release, the synergistic action of feruloyl esterase (FAE, FoFaeC-12213) and xylanase (Trichoderma longibrachiatum M3) monoenzymes was studied. More than double amount of FA release (1 mg g⁻¹ dry BSG) was observed during hydrolytic reactions by the crude enzyme extract compared to hydrolysis by the monoenzymes (0.37 mg g⁻¹ dry BSG). The protease content of the crude extract and the inhibitory effect of FA as an end-product were also evaluated concerning their effect on FA release. The protease treatment prior to hydrolysis by monoenzymes enhanced FA release about 100%, while, for the first time in literature, FA in solution found to have a significant inhibitory effect on FAE activity and on total FA release.     

Expression, characterization and structural modelling of a feruloyl esterase from the thermophilic fungus Myceliophthora thermophila

A ferulic acid esterase (FAE) from the thermophilic fungus Myceliophthora thermophila (synonym Sporotrichum thermophile), belonging to the carbohydrate esterase family 1 (CE-1), was functionally expressed in methylotrophic yeast Pichia pastoris. The putative FAE from the genomic DNA was successfully cloned in P. pastoris X-33 to confirm that the enzyme exhibits FAE activity. The recombinant FAE was purified to its homogeneity (39 kDa) and subsequently characterized using a series of model substrates including methyl esters of hydroxycinnamates, alkyl ferulates and monoferuloylated 4-nitrophenyl glycosides. The substrate specificity profiling reveals that the enzyme shows a preference for the hydrolysis of methyl caffeate and p-coumarate and a strong preference for the hydrolysis of n-butyl and iso-butyl ferulate. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 and C-2 linkages of arabinofuranose, whilst it was found capable of de-esterifying acetylated glucuronoxylans. Ferulic acid (FA) was efficiently released from destarched wheat bran when the esterase was incubated together with an M3 xylanase from Trichoderma longibrachiatum (a maximum of 41% total FA released after 1 h incubation). Prediction of the secondary structure of MtFae1a was performed in the PSIPRED server whilst modelling the 3D structure was accomplished by the use of the HH 3D structure prediction server.     

Microbial transformation of ferulic acid to vanillic acid by <Emphasis Type="Italic">Streptomyces sannanensis</Emphasis> MTCC 6637

Streptomyces sannanensis MTCC 6637 was examined for its potentiality to transform ferulic acid into its corresponding hydroxybenzoate-derivatives. Cultures of S. sannanensis when grown on minimal medium containing ferulic acid as sole carbon source, vanillic acid accumulation was observed in the medium as the major biotransformed product along with transient formation of vanillin. A maximum amount of 400 mg/l vanillic acid accumulation was observed, when cultures were grown on 5 mM ferulic acid at 28°C. This accumulation of vanillic acid was found to be stable in the culture media for a long period of time, thus facilitating its recovery. Purification of vanillic acid was achieved by gel filtration chromatography using Sephadex™ LH-20 matrix. Catabolic route of ferulic acid biotransformation by S. sannanensis has also been demonstrated. The metabolic inhibitor experiment [by supplementation of 3,4 methylenedioxy-cinnamic acid (MDCA), a metabolic inhibitor of phenylpropanoid enzyme 4-hydroxycinnamoyl-CoA ligase (4-CL) along with ferulic acid] suggested that biotransformation of ferulic acid into vanillic acid mainly proceeds via CoA-dependent route. In vitro conversions of ferulic acid to vanillin, vanillic acid and vanillin to vanillic acid were also demonstrated with cell extract of S. sannanensis. Further degradation of vanillic acid to other intermediates such as, protocatechuic acid and guaiacol was not observed, which was also confirmed in vitro with cell extract.     

Cloning of fatty acid elongase1 gene and molecular identification of A and C genome in <Emphasis Type="Italic">Brassica</Emphasis> species

The fatty acid elongase 1 (FAE1) genes of Brassic napus were cloned from two cultivars, i.e. Zhongshuan No. 9 with low erucic acid content, and Zhongyou 821 with high erucic acid content, using the degenerate PCR primers. The sequence analysis showed that there was no intron within the FAE1 genes. The FAE1 genes from Zhongyou 821 contained a coding sequence of 1521 nucleotides, and those cloned from Zhongshuan No. 9 contained a 1517 bp coding sequence. Alignment of the FAE1 sequences from Brassica rapa, B. oleracea and B. napus detected 31 single nucleotide polymorphic sites (2.03%), which resulted in 7 amino-acid substitutions. Further analysis indicated that 19 SNPs were genome-specific, of which, 95% were synonymous mutations. The nucleotide substitution at position 1217 in the FAE1 genes led to a specific site of restricted cleavage. An AvrII cleavage site was present only in the C genome genes and absent in the A genome FAE1 genes. Digestion profile of the FAE1 sequences from B. rapa, B. oleracea and B. napus produced with AvrII confirmed that the FAE1 genes of B. oleracea origin was recognized and digested, while that of B. rapa origin could not. The results indicated that by AvrII cleavage it was possible to distinguish B. rapa from B. oleracea and between the A and C genome of B. napus. In addition, the FAE1 genes could be used as marker genes to detect the pollen flow of B. napus, thus providing an alternative method for risk assessment of gene flow.     

Esterification of ferulic acid with polyols using a ferulic acid esterase from Aspergillus niger

Commercially available enzyme preparations were screened for enzymes that have a high ability to catalyze direct ester-synthesis of ferulic acid with glycerol. Only a preparation, Pectinase PL “Amano” produced by Aspergillus niger, feruloylated glycerol under the experimental conditions. The enzyme responsible for the esterification was purified and characterized. This enzyme, called FAE-PL, was found to be quite similar to an A. niger ferulic acid esterase (FAE-III) in terms of molecular mass, pH and temperature optima, substrate specificity on synthetic substrates, and the N-terminal amino acid sequence. FAE-PL highly catalyzed direct esterification of ferulic acid and sinapinic acid with glycerol. FAE-PL could feruloylate monomeric sugars including arabinose, fructose, galactose, glucose, and xylose. We determined the suitable conditions for direct esterification of ferulic acid with glycerol to be as follows: 1% ferulic acid in the presence of 85% glycerol and 5% dimethyl sulfoxide at pH 4.0 and 50 °C. Under these conditions, 81% of ferulic acid could be converted to 1-glyceryl ferulate, which was identified by ¹H-NMR. The ability of 1-glyceryl ferulate to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals was higher than that of the anti-oxidant butyl hydroxytoluene.     

Induction of ferulic acid esterase and xylanase activities in Streptomyces avermitilis UAH30

Ferulic acid esterase activity (FAE) was detected, along with xylanase activity, in culture supernatants from Streptomyces avermitilis UAH30 grown in the presence of the lignocellulosic substrates, oat spelt xylan, wheat bran without starch and sugar cane bagasse. The maximum activity was detected with wheat bran (1.75 mU ml⁻¹). No correlation between FAE activity and the amount of esterified ferulic acid present in the substrate was observed. The addition of either glucose, mannitol or glycerol to the culture medium containing oat spelt xylan resulted in a reduction of 40–75% in the xylanase activity detected in culture supernatants. FAE activity could only be detected in supernatants from cultures grown in the presence of glycerol and mannitol, when commercially available xylanases were added to the assay. These results highlight the importance of assaying for FAE activity in the presence of high levels of xylanase activity.     

Biotransformation of ferulic acid and related compounds by mutant strains of Pseudomonas fluorescens

Pseudomonas fluorescens strain FE2 isolated in the presence of ferulic acid was able to grow on hydroxylated and methoxylated compounds bearing the hydroxyl group in the para position. By ethylmethansulphonate (EMS) and transposon mutagenesis, mutants unable to utilize ferulic acid have been selected. The metabolic characterization of the wild-type strain and its mutants indicates that ferulic acid was degraded through the formation of vanillic acid. Mutant FE2B in co-oxidation experiments with glutamate, is able to transform ferulic and dihydroferulic acid into vanillic acid, 4-hydroxycinnamic acid and 3 (4-hydroxyphenyl)-propanoic acid into 4-hydroxybenzoic acid, and 3-hydroxycinnamic acid into 3-hydroxybenzoic acid. The bioconversion of hydroxylated aromatic substrates by the FE2B mutants suggests that the presence of a hydroxyl group on the aromatic ring is required for deacetylase activity.     

嗜热细菌Clostridium thermocellum阿魏酸酯酶基因的克隆、异源表达及酶学特性分析

【目的】阐明嗜热细菌Clostridium thermocellum Xyn Z蛋白的阿魏酸酯酶催化域的酶学特性,为其在生物质能源及其它发酵工业中的应用奠定基础。【方法】分别构建了C.thermocellum Xyn Z的阿魏酸酯酶催化域(FAE)及该阿魏酸酯酶催化域和碳水化合物结合域(FAE-CBM6)编码基因的原核表达载体,并在大肠杆菌菌株BL21(DE3)中异源表达,在此基础上分析比较了温度、pH、底物、金属离子及CBM6结合域对阿魏酸酯酶活性的影响。【结果】重组FAE酶及FAE-CBM6酶发挥催化活性的适宜pH值为5.0-9.0,适宜温度为50-70°C,它们对不同金属离子的响应有差异。【结论】在同一反应条件下,FAE-CBM6酶的酶活均比FAE高,说明CBM6结合域的存在对于阿魏酸酯酶活性有促进作用。     

Lipase-catalyzed preparation of mono- and diesters of ferulic acid

Lipophilic and stable derivatives of ferulic acid are required to improve its efficacy in fatty foods and to optimize its use in cosmetic and pharmaceutical preparations. We report an improved synthesis of ferulic acid monoesters (ethyl ferulate and lauryl ferulate) using immobilized lipase from Candida antarctica B (CALB) in diisopropyl ether (DIPE). Maximum yields were 89% and 85% in 200 h for ethyl and lauryl ferulate, respectively. Ethyl ferulate was further acylated with vinyl esters to form ferulate diesters. 4-Acetoxy-ethyl ferulate was obtained with the immobilized lipase from Alcaligenes sp. (QLG) with 59% yield in 72 h, whereas 4-dodecanoyloxy-ethyl ferulate (a new compound) was synthesized with 52% yield in 72 h using CALB. DIPE was the best solvent for the transesterifications. Finally, the anti-inflammatory activity of the synthesized derivatives was evaluated in vitro; the compounds bearing a dodecyl chain showed improved anti-inflammatory activity compared with short-chain esters.     

A type D ferulic acid esterase from Streptomyces werraensis affects the volume of wheat dough pastries

A type D ferulic acid esterase (FAE) was identified in the culture supernatant of Streptomyces werraensis, purified, sequenced, and heterologously produced in E. coli BL21(DE3)Star by co-expressing chaperones groES-groEL (69 U L⁻¹). The unique enzyme with a mass of about 48 kDa showed no similarity to other FAEs, and only moderate homology (78.5%) to a Streptomycete β-xylosidase. The purified reSwFAED exhibited a temperature optimum of 40 °C, a pH optimum in the range from pH seven to eight and a clear preference for bulky natural substrates, such as 5-O-trans-feruloyl-l-arabinofuranose (FA) and β-d-xylopyranosyl-(1→2)-5-O-trans-feruloyl-l-arabinofuranose (FAX), compared to the synthetic standard substrate methyl ferulate. Treatment of wheat dough with as little as 0.03 U or 0.3 U kg⁻¹ reSwFAED activity resulted in a significant increase of the bun volume (8.0 or 9.7%, resp.) after baking when combined with polysaccharide-degrading enzymes from Aspergillus. For the first time, the long-standing, but rarely proven positive effect of a FAE in baking was confirmed.

     

Enhanced vanillin production from ferulic acid using adsorbent resin

High vanillin productivity was achieved in the batch biotransformation of ferulic acid by Streptomyces sp. strain V-1. Due to the toxicity of vanillin and the product inhibition, fed-batch biotransformation with high concentration of ferulic acid was unsuccessful. To solve this problem and improve the vanillin yield, a biotransformation strategy using adsorbent resin was investigated. Several macroporous adsorbent resins were chosen to adsorb vanillin in situ during the bioconversion. Resin DM11 was found to be the best, which adsorbed the most vanillin and the least ferulic acid. When 8% resin DM11 (wet w/v) was added to the biotransformation system, 45 g l⁻¹ ferulic acid could be added continually and 19.2 g l⁻¹ vanillin was obtained within 55 h, which was the highest vanillin yield by bioconversion until now. This yield was remarkable for exceeding the crystallization concentration of vanillin and therefore had far-reaching consequence in its downstream processing.     

Alternaria alternata as a new fungal enzyme system for the release of phenolic acids from wheat and triticale brans

This study describes the release of antioxidant ferulic acid from wheat and triticale brans by mixtures of extracellular enzymes produced in culture by a strain FC007 of Alternaria alternata, a dark mold originally isolated from Canadian wood log. The genus of the mold was confirmed as Alternaria by 18S ribosomal DNA characterization. Enzyme activities for feruloyl esterase (FAE) and polysaccharide hydrolyzing enzymes were measured, and conditions for release of ferulic acid and reducing sugars from the mentioned brans were evaluated. The highest level of FAE activity (89 ± 7 mU ml⁻¹ fermentation culture) was obtained on the fifth day of fermentation on wheat bran as growth substrate. Depending on biomass and processing condition, up to 91.2 or 72.3% of the ferulic acid was released from wheat bran and triticale bran, respectively, indicating the proficiency of A. alternata extracellular enzymes in plant cell wall deconstruction. The apparent high extraction of ferulic acid from wheat and triticale brans represents a potential advantage of using a whole fungal cell enzyme complement over yields reported previously through an artificial assembly of cloned FAE with a particular xylanase in a cocktail format.     

Synthesis of glyceryl ferulate by immobilized ferulic acid esterase

Glyceryl ferulate was synthesized by the condensation of ferulic acid with glycerol using Pectinase PL “Amano” from Aspergillus niger, which contained ferulic acid esterase, to improve the water-solubility of ferulic acid. The optimum reaction medium was glycerol/0.1 M acetate buffer, pH 4.0, (98:2 v/v). The enzyme immobilized onto Chitopearl BCW3003 exhibited the highest activity among the those immobilized onto various kinds of Chitopearl BCW resins. The optimum temperature for the immobilized enzyme was 50°C, and it could be reused at least five times without a significant loss in activity for the synthesis of glyceryl ferulate in batch reaction. Storage of the reaction mixture at 25°C improved the molar fraction of glyceryl ferulate relative to the dissolved ferulic residues.     

Thermostable feruloyl esterase for the bioproduction of ferulic acid from triticale bran

A putative α/β 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°C, the enzyme retained at least 95% of its original activity for over 80 min; at 80°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.     

Microbial production of 4-vinylguaiacol from ferulic acid by Bacillus cereus SAS-3006

Abstract

Ferulic acid is an abundant cinnamic acid derivative found in the plant kingdom. It is a commercially available substrate utilized to produce flavor compounds such as 4-vinylguaiacol (4-VG), vanillin, and vanillic acid. The isolate Bacillus cereus SAS-3006 was screened and selected based on its ability to produce 4-VG upon ferulic acid biotransformation. It was identified based on morphological and physiochemical characteristics and its 16S ribosomal DNA sequence (GenBank accession number: KF699134). A maximum amount (79.4 mg/L) of 4-VG accumulation was observed on the 5th day of incubation when the culture was grown on 2.5 mM ferulic acid as sole carbon source. Further conversion of 4-VG to other intermediates such as vanillin, vanillic acid, protocatechuic acid, acetovanillone, and vanillyl alcohol was not observed. In-vitro conversion of ferulic acid to 4-VG was also studied with cell extracts of B. cereus SAS-3006. The present study provides the first evidence for production of 4-VG as the sole product using B. cereus SAS-3006.     

Molecular tagging of erucic acid trait in oilseed mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>) by QTL mapping and single nucleotide polymorphisms in<Emphasis Type="Italic"> FAE1</Emphasis> gene

Molecular mapping and tagging of the erucic acid trait (C22:1) in Brassica juncea was done by a candidate gene approach. Two QTLs underlying the variation of seed erucic acid content were assigned to two linkage groups of a B. juncea map using a doubled haploid (DH) mapping population derived from high × low erucic acid F₁ hybrid. Two consensus primers corresponding to the full-length Fatty Acid Elongase 1 (FAE1) gene, reported to be involved in the elongation of C18:1 to C22:1, were designed. PCR amplification and subsequent cloning and sequencing identified two FAE1 genes (FAE1.1 and FAE1.2) in both high and low erucic acid mustard lines. Sequence alignment of corresponding FAE1 genes between high and low erucic acid mustard lines identified four substitution type single nucleotide polymorphisms (SNPs) in FAE1.1 and three in FAE1.2. Using the SNuPE method of SNP genotyping, these two genes were mapped to two independent loci that co-segregated with the two QTLs governing the erucic acid trait. Association of wild (E1E2) and mutant (e1e2) haplotypes of two FAE1 genes with erucic acid variation in two segregating populations revealed that the e1e1e2e2 genotype identified low erucic acid individuals (<2%) and E1E1E2E2 identified individuals with highest erucic acid content (>40%). The E1e1E2e2 heterozygote was found to be intermediate in phenotype. The applicability of these SNPs in marker-assisted manipulation of the erucic acid trait was verified by genotyping a set of contrasting germplasm of B. juncea belonging to two distinct gene pools (Indian and east European) and other oil-yielding Brassica species.Communicated by C. Möllers     

Novel ferulic acid esterases are induced by growth of Aspergillus niger on sugar-beet pulp

 A ferulic acid esterase (FAE-III), which was induced by growth of Aspergillus niger CBS 120.49 on oat-spelts xylan, was capable of releasing ferulic acid from wheat bran but not from sugar-beet pulp (SBP) [Faulds CB, Williamson G (1994) Microbiology 140:779–787]. Growth of this strain on SBP gave low levels of ferulic acid esterase activity (using methyl ferulate as substrate). A similar growth with a different A. niger strain (CS 180) gave tenfold higher levels of esterase activity. Assaying culture filtrates obtained from A. niger CS 180 grown on SBP over a 3 to 10-day period against four simple phenolic methyl esters demonstrated that at least two esterases were produced, and, by comparison of substrate specificity, FAE-III was either absent or present only at low levels. Furthermore, immunodetection of proteins did not detect the presence of FAE-III in culture supernatants of SBP-grown cultures, whereas it did in cultures grown on oat-spelts xylan. These results show that SBP does not contain the inducer for FAE-III, but does induce novel esterases. When A. niger CS 180 cultures were grown on different carbon sources, esterase activity was induced on SBP, sugar-beet arabinan and oat-spelts xylan, but not on simple sugars or de-esterified sugar-beet pectin. Further, SBP-grown cultures co-inoculated with arabinanase, galactanase or xylanase did not exhibit increased levels of extracellular FAE activity or an earlier appearance of esterase activity, although there was an increase in esterase activity with added polygalacturonase. These results show that novel esterases are induced by growth of A. niger on SBP. Received: 11 September 1995/Received revision: 5 December 1995/Accepted: 11 December 1995