A cinnamoyl esterase from Aspergillus niger can break plant cell wall cross-links without release of free diferulic acids.

A cinnamoyl esterase, ferulic acid esterase A, from Aspergillus niger releases ferulic acid and 5-5- and 8-O-4-dehydrodiferulic acids from plant cell walls. The breakage of one or both ester bonds from dehydrodimer cross-links between plant cell wall polymers is essential for optimal action of carbohydrases on these substrates, but it is not known if cinnamoyl esterases can break these cross-links by cleaving one of the ester linkages which would not release the free dimer. It is difficult to determine the mechanism of the reaction on complex substrates, and so we have examined the catalytic properties of ferulic acid esterase A from Aspergillus niger using a range of synthetic ethyl esterified dehydrodimers (5-5-, 8-5-benzofuran and 8-O-4-) and two 5-5-diferulate oligosaccharides. Our results show that the esterase is able to cleave the three major dehydrodiferulate cross-links present in plant cell walls. The enzyme is highly specific at hydrolysing the 5-5- and the 8-5-benzofuran diferulates but the 8-O-4-is a poorer substrate. The hydrolysis of dehydrodiferulates to free acids occurs in two discrete steps, one involving dissociation of a monoesterified intermediate which is negatively charged at the pH of the reaction. Although ferulic acid esterase A was able to release monoesters as products of reactions with all three forms of diesters, only the 5-5- and the 8-O-4-monoesters were substrates for the enzyme, forming the corresponding free diferulic acids. The esterase cannot hydrolyse the second ester bond from the 8-5-benzofuran monoester and therefore, ferulic acid esterase A does not form 8-5-benzofuran diferulic acid. Therefore, ferulic acid esterase A from Aspergillus niger contributes to total plant cell wall degradation by cleaving at least one ester bond from the diferulate cross-links that exist between wall polymers but does not always release the free acid product.     

Intestinal release and uptake of phenolic antioxidant diferulic acids.

Diferulic acids are potent antioxidants and are abundant structural components of plant cell walls, especially in cereal brans. As such, they are part of many human and animal diets and may contribute to the beneficial effect of cereal brans on health. However, these phenolics are ester-linked to cell wall polysaccharides and cannot be absorbed in this form. This study provides the first evidence that diferulic acids can be absorbed via the gastrointestinal tract. The 5-5-, 8-O-4-, and 8-5-diferulic acids were identified in the plasma of rats after oral dosing with a mixture of the three acids in oil. Our study also reveals that human and rat colonic microflora contain esterase activity able to release 5-5-, 8-O-4-, and 8-5-diferulic acids from model compounds and dietary cereal brans, hence providing a mechanism for release of dietary diferulates prior to absorption of the free acids. In addition, cell-free extracts from human and rat small intestine mucosa exhibited esterase activity towards diferulate esters. Hence, we have shown that esterified diferulates can be released from cereal brans by intestinal enzymes, and that free diferulic acids can be absorbed and enter the circulatory system. Our results suggest that the phenolic antioxidant diferulic acids are bioavailable.     

Modification of esterified cell wall phenolics increases vulnerability of tall fescue to herbivory by the fall armyworm

Feruloylation of arabinoxylan in grass cell walls leads to cross-linked xylans. Such cross-linking appears to play a role in plant resistance to pathogens and insect herbivores. In this study, we investigated the effect of ferulate cross-linking on resistance to herbivory by fall armyworm (Spodoptera frugiperda) making use of genetically modified tall fescue [Schedonorus arundinaceus (Festuca arundinacea)] expressing a ferulic acid esterase gene. Mature leaves of these plants have significant reduced levels of cell wall ferulates and diferulates but no change in acid detergent lignin. These reduced levels of esterified cell wall ferulates in transgenic plants had a positive effect on all measures of armyworm larval performance examined. More larvae survived (89 vs. 57?%) and grew faster (pupated 2.1?days sooner) when fed transgenic leaves with reduced levels of cell wall ferulates, than when fed control tall fescue leaves where levels of cell wall ferulates were not altered. Overall, mortality, growth and food utilization were negatively associated with level of esterified cell wall ferulates and diferulates in leaves they were fed. This study is the first to use transgenic plants with modified level of cell wall esterified ferulates to test the role of feruloylation in plant resistance to insects. It is concluded that the accumulation of ferulates and the cross-linking of arabinoxylans via diferulate esters in the leaves of tall fescue underlies the physical barrier to insect herbivory. Reducing ferulate cross-linking in grass cell walls could increase susceptibility of these plants to insect folivores.     

Oligosaccharide and alkyl β-galactopyranoside synthesis from lactose with Caldocellum saccharolyticum β-glycosidase

The synthetic utility of the thermostable β-glycosidase from Caldocellum saccharolyticum was investigated. The ability of the enzyme to catalyze oligosaccharide and β-galactopyranoside synthesis from lactose was compared with that of the readily commercially available, moderately thermostable β-galactosidase (β- -galactoside galactohydrolase, EC 3.2.1.23) from Aspergillus oryzae. Generally, the C. saccharolyticum enzyme showed significantly greater resistance to inactivation by heat and organic solvent and better yields of product. Although the A. oryzae enzyme gave better oligosaccharide yields at lower lactose concentrations, at higher concentrations (above 50% w/w) the C. saccharolyticum enzyme was significantly better, yielding a sugar mixture containing 42% by weight of tri- plus tetra-saccharides, from a 70% w/w lactose solution, compared with 31% by weight of oligosaccharides with the A. oryzae enzyme. In ethyl galactoside synthesis from ethanol and lactose, neither enzyme appeared to hydrolyze the product to any great extent. Under all conditions tested, the product yield did not peak, even at long reaction times, when most of the lactose had been consumed. The C. saccharolyticum enzyme, however, gave slightly higher product yields and could be used at higher ethanol concentrations without serious loss of activity.     

Structural features of the glycogen branching enzyme encoding genes from aspergilli

A maltose binding protein, p78, was purified to homogeneity from Aspergillus nidulans by a single column chromatography step on cross-linked amylose. The partial amino acid sequence was highly homologous to the glycogen branching enzymes (GBEs) of human and yeast, and p78 did show branching enzyme activity. The genomic gene and its cDNA encoding GBE (p78) were isolated from the A. nidulans genomic and cDNA libraries. Furthermore, a cDNA encoding A. oryzae GBE was entirely sequenced. A. nidulans GBE shared overall and significant amino acid sequence identity with GBEs from A. oryzae (83.9%), Saccharomyces cerevisiae (61.1%) and human (63.0%), and with starch branching enzymes from green plants (55–56%).     

The plant cell wall polysaccharide rhamnogalacturonan II self-assembles into a covalently cross-linked dimer

The location of the 1:2 borate-diol ester cross-link in the dimer of the plant cell wall polysaccharide rhamnogalacturonan II (RG-II) has been determined. The ester cross-links the apiofuranosyl residue of the 2-O-methyl-D-xylose-containing side chains in each of the subunits of the dimer. The apiofuranosyl residue in each of the two aceric acid-containing side chains is not esterified. The site of borate esterification is identical in naturally occurring and in in vitro synthesized dimer. Pb2+, La3+, and Ca2+ increase dimer formation in vitro in a concentration- and pH-dependent manner. Pb2+ is the most effective cation. The dimer accounts for 55% of the RG-II when the monomer (0.5 mM) is treated for 5 min at pH 3.5 with boric acid (1 mM) and Pb2+ (0.5 mM); at pH 5 the rate of conversion is somewhat slower. Hg2+ does not increase the rate of dimer formation. A cation's charge density and its ability to form a coordination complex with RG-II, in addition to steric factors, may regulate the rate and stability of dimer formation in vitro. Our data provide evidence that the structure of RG-II itself determines which apiofuranosyl residues are esterified with borate and that in the presence of boric acid and certain cations, two RG-II monomers self-assemble to form a dimer.     

Enzymatic rearrangement of chitine hydrolysates with β-N-acetylhexosaminidase from Aspergillus oryzae

The role of higher chitooligomers in medical applications is increasing due to their interesting biological activities. Transglycosylation activity of β-N-acetylhexosaminidase from Aspergillus oryzae was employed to produce higher chitooligosaccharides (chitohexaose–chitooctaose) from a mixture of lower chitooligomers prepared by acid hydrolysis of chitin. Enzymatic rearrangement of the chitooligomer mixture was optimized in respect of substrate concentration, presence of inorganic salts, enzyme activity, and reaction time to achieve the highest production of longer chitooligomers.     

Galactosylation of antibiotics using the β-galactosidase from Aspergillus oryzae

The β-galactosidase from Aspergillus oryzae has been shown to catalyze the synthesis of β-galactosides of antibiotics such as chlorphenisin and chloramphenicol using β-lactose as the galactosyl donor. Among the water-miscible organic solvents tested, 20% (v/v) acetonitrile in the reaction mixture gave the highest yield in galactoside synthesis. The products obtained were purified by preparative TLC and liquid chromatography and analyzed by ¹H-and ¹³C-NMR, and MS (FAB). Chlorphenisin and chloramphenicol were galactosylated exclusively at their primary hydroxy groups. The pH optimum for the transgalactosylation reaction was between pH 4–5. Increasing concentrations of galactosyl donor and aglycon caused increasing yields of galactosides. When the resulting galactosylated antibiotic was withdrawn from the sample, further synthesis was observed. This could be accelerated either by withdrawing the resulting monosaccharides (glucose and galactose) or exchanging them for mannose.     

Chemistry and occurrence of hydroxycinnamate oligomers

Hydroxycinnamates such as ferulic acid, sinapic acid and p-coumaric acid ester-linked to plant cell wall polymers may act as cross-links between polysaccharides to each other, but also to proteins and lignin. Although sinapates and p-coumarates also form cell wall cross-links by the formation of radically or photochemically formed dimers, ferulate derivatives are the quantitatively most important cross-links in the plant cell wall. While the first radically generated ferulate dimer was already identified almost 40 years ago, the spectrum of known ferulate dimers was considerably broadened within the last 15 years. Higher ferulate oligomers were generated in model systems, but also isolated from plant materials. Different model systems using either free hydroxycinnamic acids or their esters are reviewed, highlighting a discussion of the relevance of these models for the plant cell wall. The first ferulate trimer from plant material was discovered in 2003 and seven dehydrotrimers of ferulic acid were isolated from maize bran since. Some of these trimers were also identified in other plant materials such as wheat and rye grains, corn stover, sugar beet and asparagus. Formation mechanisms of ferulate trimers and implications for the plant cell wall are discussed. Ferulate tetramers are the highest oligomers isolated from plant materials so far. These compounds can theoretically cross-link up to four polysaccharide chains, assuming all cross-links are formed intermolecularly. Formation of intramolecular versus intermolecular polysaccharide cross-links is a key question to be answered in the future if we want to judge properly the importance of hydroxycinnamate cross-links in the plant cell wall.     

Comparison of mesophilic and thermophilic feruloyl esterases: characterization of their substrate specificity for methyl phenylalkanoates

The active sites of feruloyl esterases from mesophilic and thermophilic sources were probed using methyl esters of phenylalkanoic acids. Only 13 out of 26 substrates tested were significant substrates for all the enzymes. Lengthening or shortening the aliphatic side chain while maintaining the same aromatic substitutions completely abolished activity for both enzymes, which demonstrates the importance of the correct distance between the aromatic group and the ester bond. Maintaining the phenylpropanoate structure but altering the substitutions of the aromatic ring demonstrated that the type-A esterase from the mesophilic fungus Fusarium oxysporum (FoFaeA) showed a preference for methoxylated substrates, in contrast to the type-B esterase from the same source (FoFaeB) and the thermophilic type-B (StFaeB) and type-C (StFaeC) from Sporotrichum thermophile, which preferred hydroxylated substrates. All four esterases hydrolyzed short chain aliphatic acid (C2-C4) esters of p-nitrophenol, but not the C12 ester of laurate. All the feruloyl esterases were able to release ferulic acid from the plant cell wall material in conjunction with a xylanase, but only the type-A esterase FoFaeA was effective in releasing the 5,5' form of diferulic acid. The thermophilic type-B esterase had a lower catalytic efficiency than its mesophilic counterpart, but released more ferulic acid from plant cell walls.     

Cellobiohydrolase I (Cel7A) from Trichoderma reesei has chitosanase activity

Cellobiohydrolase CBH I (Cel7A) from the filamentous fungus Trichoderma reesei (TrCBHI), which is a member of glycoside hydrolase family (GHF) 7, was expressed in Aspergillus oryzae. We found that the recombinant enzyme showed significant chitosanase activity, as well as cellulase activity, and acted in an endo-type manner on soluble polymeric substrate. Furthermore, another GHF7 CBH I from Aspergillus aculeatus (AaCBHI) expressed in A. oryzae also had chitosanase activity, while endoglucanase EG I (Cel7B) from T. reesei had no activity towards chitosan. To our knowledge, this is the first report of GHF7 enzymes possessing chitosanase activity.     

Enantioselective transesterification of secondary alcohols mediated by aminoacylases from Aspergillus species

The aminoacylases (N-acyl- -aminoacid amidohydrolase; E.C. 3.5.1.14) from Aspergillus melleus and Aspergillus oryzae catalyze the enantioselective transesterification of 1-phenylethanol with absolute stereospecificity. Increased catalytic efficiencies were obtained by using solubilized surfactant-coated aminoacylase complexes, which makes them more attractive for industrial application.     

A novel Aspergillus oryzae esterase that hydrolyzes 4-hydroxybenzoic acid esters

In this study we report the biochemical characterization of a hypothetical protein from Aspergillus oryzae exhibiting sequence identity with feruloyl esterase and tannase from the genus Aspergillus. The purified recombinant protein showed a hydrolytic activity toward the ethyl, propyl, or butyl esters of 4-hydroxybenzoic acid, but did not show feruloyl esterase or tannase activity. Finally, the enzyme decreased the antimicrobial activity of parabens against A. oryzae via hydrolysis of the ester bond present in butyl 4-hydroxybenzoic acid.     

Biopreparation of cotton fabric with enzymes produced by solid-state fermentation

Fungal enzyme preparations from Phanerochaete chrysosporium, Aspergillus oryzae, Aspergillus giganteus and Trichoderma virens, produced by solid-state fermentation (SSF) on cotton seed-coat fragment waste as substrate and enzyme inducer were investigated in biopreparation of cotton fabric. Cotton seed-coat fragment is rich in lignin, cellulose and hemicelluloses, therefore enzyme complexes produced by target fungi on such a substrate can be used effectively to degrade impurities in cotton fabrics during biopreparation. Activities of extracellular hydrolytic and ligninolytic enzymes were determined from the SSF extract materials. The potential of the hydrolytic and accompanying oxidative enzymes in the whole SSF cultures was exploited in degradation of seed-coat fragments and other coloring materials of greige cotton fabric. Enzyme assays indicated that many extracellular enzymes have been produced under these conditions including both hydrolytic and oxidative enzymes. A. oryzae NRRL 3485 produced significantly higher amounts of both hydrolytic and oxidative enzymes than other tested fungi. Best results in removal of seed-coat fragments from cotton fabric were obtained by P. chrysosporium NCAIM (=ATCC 34541), P. chrysosporium VKM F-1767 and A. oryzae NRRL 3485 SSF enzyme complexes.     

Asymmetric synthesis of arylselenoalcohols by means of the reduction of organoseleno acetophenones by whole fungal cells

A series of novel organoseleno acetophenones (3a–f) have been synthesized. The microbial reduction of the seleno ketones (3) has been evaluated using whole cells of Rhizopus oryzae CCT 4964, Aspergillus terreus CCT 3320, A. terreus CCT 4083 and Emericella nidulans CCT 3119. These microorganisms showed Prelog and anti-Prelog stereoselectivity, leading to the arylselenoalcohols in moderate to high enantiomeric excesses. The organoselenium compounds were compatible with the biocatalytic conditions employed.     

Production of tannase from Aspergillus ruber under solid-state fermentation using jamun (Syzygium cumini) leaves

Tannase producing fungal strains were isolated from different locations including garbages, forests and orchards, etc. The strain giving maximum enzyme yield was identified to be Aspergillus ruber. Enzyme production was studied under solid state fermentation using different tannin rich substrates like ber leaves (Zyzyphus mauritiana), jamun leaves (Syzygium cumini), amla leaves (Phyllanthus emblica) and jawar leaves (Sorghum vulgaris). Jamun leaves were found to be the best substrate for enzyme production under solid-state fermentation (SSF). In SSF with jamun leaves, the maximum production of tannase was found to be at 30 °C after 96 h of incubation. Tap water was found to be the best moistening agent, with pH 5.5 in ratio of 1:2 (w/v) with substrate. Addition of carbon and nitrogen sources to the medium did not increase tannase production. Under optimum conditions as standardized here, the enzyme production was 69 U/g dry substrate. This is the first report on production of tannase by A. ruber, giving higher yield under SSF with agro-waste as the substrate.     

Specificity and kinetic parameters of recombinant Microdochium nivale carbohydrate oxidase

A new carbohydrate oxidase from Microdochium nivale heterologously expressed in Aspergillus oryzae (rMnO) has been characterized. The carbohydrate oxidase is a flavoenzyme which oxidizes glucose and other mono- or oligosaccharides. It shows a broad substrate specificity towards carbohydrates reacting with aldoses in the 1-position. The rMnO oxidizes the β-form of -glucose, and the product of -glucose oxidation is -gluconic acid.

The mechanism of carbohydrate oxidation by oxygen and artificial electron acceptors has been described by a ping-pong scheme. Compared to Aspergillus niger glucose oxidase (GOx) the reactivity of rMnO at pH 7.0 is significantly lower; k_cat is 20, k_ox 11 and k_red 22 times less, using oxygen as electron acceptor. Also with other two electron acceptors, like DPIP, the activity is low. However, compared to oxygen the rMnO shows 2–10 times higher activity towards some artificial single electron acceptors (AAs). The enzyme activity increases at higher ionic strength of the solution, if positively-charged AAs are used.

The high activity towards AAs and low rate for oxygen as well as broad specificity to carbohydrates indicates that rMnO may have some advantages compared to the most used GOx in connection with enzyme use for analytical devices and for biotechnological purposes.     

Cloning, sequencing, and expression of the cellulase genes of Humicola grisea var. thermoidea

We have cloned an endoglucanase (EGI) gene and a cellobiohydrolase (CBHI) gene of Humicola grisea var. thermoidea using a portion of the Trichoderma reesei endoglucanase I gene as a probe, and determined their nucleotide sequences. The deduced amino acid sequence of EGI was 435 amino acids in length and the coding region was interrupted by an intron. The EGI lacks a hinge region and a cellulose-binding domain. The deduced amino acid sequence of CBHI was identical to the H. grisea CBHI previously reported, with the exception of three amino acids. The H. grisea EGI and CBHI show 39.8% and 37.7% identity with the T. Reesei EGI, respectively. In addition to TATA box and CAAT motifs, putative CREA binding sites were observed in the 5′ upstream regions of both genes. The cloned cellulase genes were expressed in Aspergillus oryzae and the gene products were purified. The optimal temperatures of CBHI and EGI were 60 °C and 55–60 °C, respectively. The optimal pHs of these enzymes were 5.0. CBHI and EGI had distinct substrate specificities: CBHI showed high activity toward Avicel, whereas EGI showed high activity toward carboxymethyl cellulose (CMC).     

Expression of a fungal ferulic acid esterase increases cell wall digestibility of tall fescue (Festuca arundinacea)

In the cell walls of forage grasses, ferulic acid is esterified to arabinoxylans and participates with lignin monomers in oxidative coupling pathways to generate ferulate–polysaccharide–lignin complexes that cross-link the cell wall. Such cross-links hinder cell wall degradation by ruminant microbes, reducing plant digestibility. In this study, genetically modified Festuca arundinacea plants were produced expressing an Aspergillus niger ferulic acid esterase (FAEA) targeted to the vacuole. The rice actin promoter proved to be effective for FAEA expression, as did the cauliflower mosaic virus (CaMV) 35S and maize ubiquitin promoters. Higher levels of expression were, however, found with inducible heat-shock and senescence promoters. Following cell death and subsequent incubation, vacuole-targeted FAEA resulted in the release of both monomeric and dimeric ferulic acids from the cell walls, and this was enhanced several fold by the addition of exogenous endo-1,4-β-xylanase. Most of the FAEA-expressing plants showed increased digestibility and reduced levels of cell wall esterified phenolics relative to non-transformed plants. It is concluded that targeted FAEA expression is an effective strategy for improving wall digestibility in Festuca and, potentially, other grass species used for fodder or cellulosic ethanol production.     

Hydrophilization of immobilized model enzymes suggests a widely applicable method for enhancing protein stability in polar organic co-solvents

β-Galactosidases from Escherichia coli, Kluyveromyces lactis and Aspergillus oryzae were used to characterize the potential for enzyme stabilization of a two-step strategy: (i) immobilization on glutaraldehyde-agarose (Glut90), (ii) subsequent generation of a hydrophilic nano-environment by reaction with polyaldehyde-dextran polymer (Glut90-Pal), followed by polyamine-dextran polymer (Glut90-Pal-Pam). The derivatives were characterized by kinetics parameters, co-solvent (ethanol and acetone) and temperature stability. Hydrophilization achieved important co-solvent stabilization in all cases. One of the most remarkable results obtained was a 25-fold increase in the half-life of the A. oryzae Glut90-Pal-Pam derivative in 50% (v/v) acetone. Stabilization achieved in very drastic co-solvent concentrations is directly related to the hydrophilization of the nano-environment. The K_M values show that the hydrophilic shell appears to behave as an open structure and may create a “partition effect” that protects the enzymes from denaturation. These results show the potential of hydrophilization for building up additional stabilization of immobilized enzymes which would make possible the development of industrial applications.