Ferulic Acid Esterase Activity from Schizophyllum commune

Schizophyllum commune produced an esterase which released ferulic acid from starch-free wheat bran and from a soluble ferulic acid-sugar ester that was isolated from wheat bran. The preferred growth substrate for the production of ferulic acid esterase was cellulose. Growth on xylan-containing substrates (oat spelt xylan and starch-free wheat bran) resulted in activity levels that were significantly lower than those observed in cultures grown on cellulose. Similar observations were made for endoglucanase, p-nitrophenyllactopyranosidase, xylanase, and acetyl xylan esterase. Of the enzymes studied, only arabinofuranosidase was produced at maximum levels during growth on xylan-containing materials. Ferulic acid esterase that had been partially purified by DEAE chromatography released significant amounts of ferulic acid from wheat bran only in the presence of a xylanase-rich fraction, indicating that the esterase may not be able to readily attack high-molecular-weight substrates. The esterase acted efficiently, without xylanase addition, on a soluble sugar-ferulic acid substrate.     

Influence of ferulic acid on the production of feruloyl esterases by Aspergillus niger

Extracellular feruloyl esterases from the filamentous fungus Aspergillus niger are induced by growth on oat spelt xylan (OSX), which contains no detectable esterified ferulic acid. FAE-III accounted for most of the feruloyl esterase activity. Addition of free ferulic acid to OSX at the start of the culture induced FAE-III secretion a further 2.3-fold, and also induced other feruloyl esterases which could not be ascribed to FAE-III. Wheat bran- (WB)-grown cultures, containing 1% (m/v) ester-linked ferulic acid, gave almost identical FAE-III and total feruloyl esterase activities as the cultures grown on OSX plus ferulic acid. De-esterification of WB yielded less total feruloyl esterase, and 2.4-fold less FAE-III, compared to untreated WB. A slightly modified form of FAE-III was produced on de-esterified WB. These results show that production of FAE-III does not absolutely require ferulic acid. However, production is stimulated by the presence of free ferulic acid through increased expression, and is reduced by the removal of esterified ferulic acid from the growth substrate.     

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     

Production of xylanases from a newly isolated alkalophilic thermophilic Bacillus sp.

A new thermophilic strain of Bacillus SPS-0 which produces thermostable xylanases was isolated from a hot spring in Portugal. Xylanase production was 50 nkat/ml in the presence of wheat bran arabinoxylan. The temperature and pH for optimum activity were 75°C and 6–9, respectively. The hydrolysis patterns demonstrated that crude xylanases yield mainly xylose and xylobiose from xylan, whereas xylose and arabinose were produced from destarched wheat bran. An increase in xylose release was observed when SPS-0 xylanase was supplemented by a ferulic acid esterase. © Rapid Science Ltd. 1998     

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.     

Induction of Cellulolytic and Xylanolytic Enzyme Systems in Streptomyces spp

Extracellular enzyme preparations from Streptomyces flavogriseus and Streptomyces olivochromogenes cultures grown on cellulose contained primarily cellulase activities, but similar preparations from cultures grown on xylan-containing materials possessed high levels of both cellulase and xylanase activities. Growth conditions that gave high endoxylanase levels also resulted in the production of enzymes involved in the hydrolysis of the nonxylose components of xylan. Specific acetyl xylan esterase activities were identified in enzyme preparations from both organisms. Both organisms also produced alpha-l-arabinofuranosidase activity that was not associated with endoxylanase activity. Other activities produced were alpha-l-O-methylglucuronidase and ferulic acid esterase. The latter enzyme was produced only by S. olivochromogenes and is an activity which has not previously been identified as a component of hemicellulase preparations.     

Purification and properties of an acetylxylan esterase from Fibrobacter succinogenes S85.

An acetylxylan esterase (EC 3.1.1.6) was purified to apparent homogeneity from the nonsedimentable extracellular culture fluid of Fibrobacter succinogenes S85 grown on cellulose. This enzyme had an apparent molecular mass of 55 kDa and an isoelectric point of 4.0. The temperature and pH optima were 45 degrees C and 7.0, respectively. The apparent Km and Vmax were 2.7 mM and 9,100 U/mg, respectively, for the hydrolysis of alpha-naphthyl acetate. The enzyme cleaved acetyl residues from birchwood acetylxylan but did not hydrolyze carboxymethylcellulose, larchwood xylan, ferulic acid-arabinose-xylose polymer, p-nitrophenyl-alpha-L-arab-inofuranoside, or longer-chain naphthyl fatty acid esters. The esterase enzyme may play a role in enhancing hemicellulose degradation by F. succinogenes, thereby allowing it greater access to cellulose present in forage cell walls.     

Purification and properties of an acetylxylan esterase from Fibrobacter succinogenes S85

An acetylxylan esterase (EC 3.1.1.6) was purified to apparent homogeneity from the nonsedimentable extracellular culture fluid of Fibrobacter succinogenes S85 grown on cellulose. This enzyme had an apparent molecular mass of 55 kDa and an isoelectric point of 4.0. The temperature and pH optima were 45 degrees C and 7.0, respectively. The apparent Km and Vmax were 2.7 mM and 9,100 U/mg, respectively, for the hydrolysis of alpha-naphthyl acetate. The enzyme cleaved acetyl residues from birchwood acetylxylan but did not hydrolyze carboxymethylcellulose, larchwood xylan, ferulic acid-arabinose-xylose polymer, p-nitrophenyl-alpha-L-arab-inofuranoside, or longer-chain naphthyl fatty acid esters. The esterase enzyme may play a role in enhancing hemicellulose degradation by F. succinogenes, thereby allowing it greater access to cellulose present in forage cell walls.     

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

The production of ferulic acid esterase involved in the release of ferulic acid side groups from xylan was investigated in strains of Aspergillus tubingensis, Aspergillus carneus, Aspergillus niger and Rhizopus oryzae. The highest activity on triticale bran as sole carbon source was observed with the A. tubingensis T8.4 strain, which produced a type A ferulic acid esterase active against methyl p-coumarate, methyl ferulate and methyl sinapate. The activity of the A. tubingensis ferulic acid esterase (AtFAEA) was inhibited twofold by glucose and induced twofold in the presence of maize bran. An initial accumulation of endoglucanase was followed by the production of endoxylanase, suggesting a combined action with ferulic acid esterase on maize bran. A genomic copy of the A. tubingensis faeA gene was cloned and expressed in A. niger D15#26 under the control of the A. niger gpd promoter. The recombinant strain has reduced protease activity and does not acidify the media, therefore promoting high-level expression of recombinant enzymes. It produced 13.5 U/ml FAEA after 5 days on autoclaved maize bran as sole carbon source, which was threefold higher than for the A. tubingensis donor strain. The recombinant AtFAEA was able to extract 50 % of the available ferulic acid from non-pretreated maize bran, making this enzyme suitable for the biological production of ferulic acid from lignocellulosic plant material.     

The structure of the feruloyl esterase module of xylanase 10B from Clostridium thermocellum provides insights into substrate recognition.

BACKGROUND: Degradation of the plant cell wall requires the synergistic action of a consortium of predominantly modular enzymes. In Clostridiae, these biocatalysts are organized into a supramolecular assembly termed a "cellulosome." This multienzyme complex possesses, in addition to its well-described cellulolytic activity, an apparatus specific for xylan degradation. Cinnamic acid esterases hydrolyze the ferulate groups involved in the crosslinking of arabinoxylans to lignin and thus play a key role in the degradation of the plant cell wall in addition to having promising industrial and medical applications. RESULTS: We have cloned and overexpressed the feruloyl esterase module from a 5 domain xylanase, Xyn10B from Clostridium thermocellum. The native structure at 1.6 A resolution has been solved with selenomethionine multiple wavelength anomalous dispersion and refined to a final R(free) of 17.8%. The structure of a hydrolytically inactive mutant, S954A, in complex with the reaction product ferulic acid has been refined at a resolution of 1.4 A with an R(free) of 16.0%. CONCLUSIONS: The C. thermocellum Xyn10B ferulic acid esterase displays the alpha/beta-hydrolase fold and possesses a classical Ser-His-Asp catalytic triad. Ferulate esterases are characterized by their specificity, and the active center reveals the binding site for ferulic acid and related compounds. Ferulate binds in a small surface depression that possesses specificity determinants for both the methoxy and hydroxyl ring substituents of the substrate. There appears to be a lack of specificity for the xylan backbone, which may reflect the intrinsic chemical heterogeneity of the natural substrate.     

Synergistic enhancement of cellobiohydrolase performance on pretreated corn stover by addition of xylanase and esterase activities

Significant increases in the depolymerization of corn stover cellulose by cellobiohydrolase I (Cel7A) from Trichoderma reesei were observed using small quantities of non-cellulolytic cell wall-degrading enzymes. Purified endoxylanase (XynA), ferulic acid esterase (FaeA), and acetyl xylan esterase (Axe1) all enhanced Cel7A performance on corn stover subjected to hot water pretreatment. In all cases, the addition of these activities improved the effectiveness of the enzymatic hydrolysis in terms of the quantity of cellulose converted per milligram of total protein. Improvement in cellobiose release by the addition of the non-cellulolytic enzymes ranged from a 13-84% increase over Cel7A alone. The most effective combinations included the addition of both XynA and Axe1, which synergistically enhance xylan conversions resulting in additional synergistic improvements in glucan conversion. Additionally, we note a direct relationship between enzymatic xylan removal in the presence of XynA and the enhancement of cellulose hydrolysis by Cel7A.     

The purification and characterization of 4-hydroxy-3-methoxycinnamic (ferulic) acid esterase from Streptomyces olivochromogenes.

A 4-hydroxy-3-methoxycinnamic acid (ferulic acid) esterase has been purified from the extracellular broth of cultures of Streptomyces olivochromogenes after growth on oat splet xylan. The purification procedure utilizes ion exchange on DEAE-BioGel A, anion exchange on Mono Q, gel filtration and hydrophobic interaction chromatography. The purified enzyme appeared as a single band on SDS-PAGE, with an apparent Mr of 29,000. Two bands, at pI7.9 and 8.5, were observed on isoelectric focusing. With methyl ferulate as substrate, the pH and temperature optima were 5.5 and 30 degrees C respectively, with a Km of 1.86 mM and Vmax of 0.3 mumols min-1 mg-1. The purfied enzyme released ferulic acid from de-starched wheat bran only in the presence of xylanase.     

Direct production of feruloyl oligosaccharides and hemicellulase inducement and distribution in a newly isolated Aureobasidium pullulans strain

Studies were carried out to screen and identify strains that are able to directly produce ferulic oligosaccharides (FOs) from wheat bran (WB). The inducement and distribution of hemicellulases from strain 2012, which was identified as a non-melanin secreting strain of Aureobasidium pullulans (A. pullulans), were also determined. In a 60 g/L WB solution, A. pullulans could produce 545 nmol/L FOs, 64.12 IU/mL xylanase and 0.14 IU/mL ferulic acid esterase (FAE). A. pullulans was cultivated in media with WB, glucose, xylose, sucrose, lactose or xylan as the carbon source, and hemicellulases were mainly induced by xylan and WB and inhibited by glucose and sucrose. Xylanase and FAE were mainly present in the culture filtrate, xylosidase in the hyphal filaments and arabinofuranosidase was a membrane-bound enzyme. The yield of FOs was positively correlated to the hemicellulases activity, and significantly positively (P < 0.05) correlated to the xylanase activity (r = 0.992).     

Purification and characterization of an extracellular feruloyl esterase from the thermophilic anaerobe Clostridium stercorarium

Feruloyl esterases act as accessory enzymes for the complete saccharification of plant cell wall hemicelluloses. Although many fungal feruloyl esterases have been purified and characterized, few bacterial phenolic acid esterases have been characterized. This study shows the extracellular production of a feruloyl esterase by the thermophilic anaerobe Clostridium stercorarium when grown on birchwood xylan. The feruloyl esterase was purified 500-fold in successive steps involving ultrafiltration, preparative isoelectric focusing and column chromatography by anion exchange, gel filtration and hydrophobic interaction. The purified enzyme released ferulic, rho-coumaric, caffeic and sinapinic acid from the respective methyl esters. The purified enzyme also released ferulic acid from a de-starched wheat bran preparation. At pH 8.0 and 65 degrees C, the Km and Vmax values for the hydrolysis of methyl ferulate were 0.04 mmol l-l and 131 micromol min-1 mg-1, respectively; the respective values for methyl coumarate were 0.86 mmol l-l and 18 micromol min-1 mg-1. The purified feruloyl esterase had an apparent mass of 33 kDa under denaturing conditions and showed optimum activity at pH 8.0 and 65 degrees C. At a concentration of 5 mmol l-l, the ions Ca2+, Cu2+, Co2+ and Mn2+ reduced the activity by 70-80%.     

An Aspergillus oryzae acetyl xylan esterase: molecular cloning and characteristics of recombinant enzyme expressed in Pichia pastoris

We screened 20,000 clones of an expressed sequence tag (EST) library from Aspergillus oryzae (http://www.nrib.go.jp/ken/EST/db/index.html) and obtained one cDNA clone encoding a protein with similarity to fungal acetyl xylan esterase. We also cloned the corresponding gene, designated as Aoaxe, from the genomic DNA. The deduced amino acid sequence consisted of a putative signal peptide of 31-amino acids and a mature protein of 276-amino acids. We engineered Aoaxe for heterologous expression in P. pastoris. Recombinant AoAXE (rAoAXE) was secreted by the aid of fused alpha-factor secretion signal peptide and accumulated as an active enzyme in the culture medium to a final level of 190 mg/l after 5 days. Purified rAoAXEA before and after treatment with endoglycosidase H migrated by SDS-PAGE with a molecular mass of 31 and 30 kDa, respectively. Purified rAoAXE displayed the greatest hydrolytic activity toward alpha-naphthylacetate (C2), lower activity toward alpha-naphthylpropionate (C3) and no detectable activity toward acyl-chain substrates containing four or more carbon atoms. The recombinant enzyme catalyzed the release of acetic acid from birchwood xylan. No activity was detectable using methyl esters of ferulic, caffeic or sinapic acids. rAoAXE was thermolabile in comparison to other AXEs from Aspergillus.     

Extracellular Carbohydrate Esterase from the Basidiomycete Coprinopsis cinerea Released Ferulic and Acetic Acids from Xylan

cDNA encoding an extracellular carbohydrate esterase (CcEst1) was cloned from the basidiomycete Coprinopsis cinerea. The recombinant CcEst1 expressed in Pichia pastoris acted on p-nitrophenyl acetate, α-naphthyl acetate, and methyl hydroxycinnamic acids, except for methyl sinapic acid. The enzyme released ferulic and acetic acids from wheat arabinoxylan and acetylated xylan respectively. Activity increased on the addition of endo-β-1,4-xylanase.     

Ferulic acid: an antioxidant found naturally in plant cell walls and feruloyl esterases involved in its release and their applications

Ferulic acid is the most abundant hydroxycinnamic acid in the plant world and maize bran with 3.1% (w/w) ferulic acid is one of the most promising sources of this antioxidant. The dehydrodimers of ferulic acid are important structural components in the plant cell wall and serve to enhance its rigidity and strength. Feruloyl esterases are a subclass of the carboxylic acid esterases that hydrolyze the ester bond between hydroxycinnamic acids and sugars present in plant cell walls and they have been isolated from a wide range of microorganisms, when grown on complex substrates such as cereal brans, sugar beet pulp, pectin and xylan. These enzymes perform a function similar to alkali in the deesterification of plant cell wall and differ in their specificities towards the methyl esters of cinnamic acids and ferulolylated oligosaccharides. They act synergistically with xylanases and pectinases and facilitate the access of hydrolases to the backbone of cell wall polymers. The applications of ferulic acid and feruloyl esterase enzymes are many and varied. Ferulic acid obtained from agricultural byproducts is a potential precursor for the production of natural vanillin, due to the lower production cost.     

白色链霉菌乙酰木聚糖酯酶基因的克隆及生物信息学分析

乙酰木聚糖酯酶可以水解乙酰化木聚糖中的O-乙酰取代基团,消除该基团对木聚糖酶水解的空间阻碍作用,增强木聚糖酶对木聚糖的亲和力和降解能力。以白色链霉菌基因组为模板,利用简并PCR和TAIL-PCR扩增获得长约741 bp阅读框片段,编码247个氨基酸。生物信息学分析表明,该多肽片段具有AXE1家族蛋白保守区域;与已知的乙酰木聚糖酯酶蛋白C端区相比,相似性较高,二级和三级结构空间排布特点极为相似;初步判定该多肽片段为白色链霉菌乙酰木聚糖酯酶的C端区域。     

Ferulic Acid: An Antioxidant Found Naturally in Plant Cell Walls and Feruloyl Esterases Involved in its Release and Their Applications

ABSTRACT

Ferulic acid is the most abundant hydroxycinnamic acid in the plant world and maize bran with 3.1% (w/w) ferulic acid is one of the most promising sources of this antioxidant. The dehydrodimers of ferulic acid are important structural components in the plant cell wall and serve to enhance its rigidity and strength. Feruloyl esterases are a subclass of the carboxylic acid esterases that hydrolyze the ester bond between hydroxycinnamic acids and sugars present in plant cell walls and they have been isolated from a wide range of microorganisms, when grown on complex substrates such as cereal brans, sugar beet pulp, pectin and xylan. These enzymes perform a function similar to alkali in the deesterification of plant cell wall and differ in their specificities towards the methyl esters of cinnamic acids and ferulolylated oligosaccharides. They act synergistically with xylanases and pectinases and facilitate the access of hydrolases to the backbone of cell wall polymers. The applications of ferulic acid and feruloyl esterase enzymes are many and varied. Ferulic acid obtained from agricultural byproducts is a potential precursor for the production of natural vanillin, due to the lower production cost.