Application of Two Newly Identified and Characterized Feruloyl Esterases from Streptomyces sp. in the Enzymatic Production of Ferulic Acid from Agricultural Biomass

Ferulic acid (FA), a component of hemicellulose in plant cell walls, is a phenolic acid with several potential applications based on its antioxidant properties. Recent studies have shown that feruloyl esterase (FAE) is a key bacterial enzyme involved in FA production from agricultural biomass. In this study, we screened a library of 43 esterases from Streptomyces species and identified two enzymes, R18 and R43, that have FAE activity toward ethyl ferulate. In addition, we characterized their enzyme properties in detail. R18 and R43 showed esterase activity toward other hydroxycinnamic acid esters as well, such as methyl p-coumarate, methyl caffeate, and methyl sinapinate. The amino acid sequences of R18 and R43 were neither similar to each other, nor to other FAEs. We found that R18 and R43 individually showed the ability to produce FA from corn bran; however, combination with other Streptomyces enzymes, namely xylanase and α-l-arabinofuranosidase, increased FA production from biomass such as corn bran, defatted rice bran, and wheat bran. These results suggest that R18 and R43 are effective FAEs for the enzymatic production of FA from biomass.     

Enzymatic hydrolysis of olive wastewater for hydroxytyrosol enrichment

Aspergillus niger broth culture on wheat bran was assessed for olive wastewater (OW) hydrolysis in order to release hydroxytyrosol (HT). The enzyme profiles of this culture broth gave essentially (IU/L): 3000 β-glucosidase and 100 esterase. Hydrolysis activity of A. niger enzyme preparation was evaluated by using three substrates: raw OW, phenolic fraction extracted from OW by ethyl acetate and its corresponding exhausted fraction. Large amounts of free simple phenolics were released from exhausted fraction and raw OW after enzymatic treatment. HPLC analyses show that HT was the main phenolic compound. One step of ethyl acetate extraction of hydrolysed OW allowed the recovery of 0.8 g of HT per litre of OW. The antioxidant activity of extracts from OW and exhausted fraction, measured by DPPH method, was drastically enhanced after hydrolysis treatment. This study demonstrates that hydrolysed OW is a potential source of bioactive phenolic compounds with promising applications in food and pharmaceutical industries.     

p-Nitrophenyl and cholesteryl-N-alkyl carbamates as inhibitors of cholesterol esterase

p-Nitrophenyl and cholesteryl-N-alkyl carbamates are good inhibitors of porcine pancreatic cholesterol esterase-catalyzed hydrolysis of p-nitrophenyl butyrate. p-Nitrophenyl-N-butyl and N-octyl carbamates (compounds 1 and 2, respectively) are potent active site-directed irreversible inhibitors of this enzyme. The inhibition of cholesterol esterase by compound 1 or 2 shows saturation kinetics with increasing inhibitor concentration. The activity of cholesterol esterase in the presence of compound 1 or 2 can be protected by the competitive inhibitor, phenylboronic acid. First-order decreases in cholesterol esterase activity effected by compound 1 or 2 are also observed in the presence of taurocholate/phosphatidylcholine micelles. Dilution of the inhibited enzyme results in a gradual return of activity, the rate of which is increased in the presence of the nucleophile hydroxylamine. Hence, inhibition of cholesterol esterase-catalyzed hydrolysis of p-nitrophenyl butyrate by compound 1 or 2 in the aqueous or micellar phase occurs via a carbamyl-cholesterol esterase mechanism. The turnover of the butyl carbamylenzyme is increased in the presence of micelles, which indicates that the micelles have a direct effect on the catalytic activity of the enzyme. However, this effect is dependent on the structure of the substrate as the turnover of the octyl carbamylenzyme is unaffected in the presence of micelles. A comparison of the second-order rate constants for the inhibition of cholesterol esterase by compound 1 or 2 indicates that the octyl derivative is the more potent inhibitor. Cholesteryl-N-alkyl carbamates do not carbamylate cholesterol esterase but instead act as reversible inhibitors. This is due to the stability of cholesteryl carbamates relative to p-nitrophenyl carbamates.     

Purification of a membrane-associated serine esterase from murine cytotoxic T lymphocytes by a single reverse-phase column

The plasma and organelle membranes of a cytotoxic T lymphocyte line, CTLL-R8, were isolated by subcellular fractionation. After dissolving in detergent-containing buffer, the membrane proteins were isolated by high-performance liquid chromatography on a single reverse-phase column. The serine esterase activity in the fractions was detected by measuring hydrolysis of the ester compound N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester. A major band was revealed in the fraction with highest serine esterase activity. Under sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this band assumes a molecular weight of about 30 kDa. The amino-terminal sequence of the protein was analyzed and shows 100% identity with that of MCSP-3/granzyme F, a soluble serine esterase previously identified in the cytoplasmic granules of cytotoxic T lymphocytes. Modifications of this reverse-phase column method would thus represent a simple, convenient strategy for obtaining high yields of all the lymphocyte surface proteases, which could then be further characterized for function.     

Development and validation of an improved diced electrophoresis gel assay cutter-plate system for enzymomics studies

Diced electrophoresis gel (DEG) assay is a methodology to identify enzymes with a specified activity in complex cell or tissue lysates by means of two-dimensional separation using isoelectric focusing and native PAGE, followed by dicing of the gel into small pieces that are assayed separately, and digestion and peptide fingerprinting to identify the protein(s) of interest in positive wells. The existing hand-made system has some disadvantages, and here we describe the development and validation of an improved cutter-plate system that enables simple, reliable and reproducible DEG assay in a 384-well plate-based format with signal readout using fluorometric or LC-MS-based reaction monitoring. To illustrate the usefulness of this system, we describe its application to profile esterase activities in ovarian adenocarcinoma SKOV3 cell lysate and mouse liver lysate that activate a fluorogenic substrate, fluorescein dibutyrate (FDBu), as well as esterase activities in mouse liver lysate that activate S-bromobenzylglutathione dicyclopentyl ester (BBGDC), a prodrug of anti-tumor agent S-bromobenzylglutathione. The activity spot patterns detected for FDBu and BBGDC were completely different, indicating that different metabolic systems are involved in hydrolysis of these substrates. The major detected spot in each case was identified. The developed system provides a highly reproducible general assay platform that should be useful for characterizing novel protein functions in complex bio-samples, as well as enzymomics studies.     

Fermentation of Maize Bran, Oat Bran, and Wheat Bran by Bacteroides ovatus V975

Bacteroides ovatus is a Gram-negative obligate anaerobe that was isolated from the human colon and is capable of utilizing xylan. The objective of this study was to evaluate the ability of B. ovatus V975 to digest maize bran, oat bran, and wheat bran as well as the isolated cell walls from each bran source. Strain V975 was incubated in basal medium that contained either 0.1 or 0.3 g of each bran or each bran cell wall for 0, 24, 48, and 72 h. Acetate and succinate were the main products detected from each fermentation; however, less of each end product was produced from the isolated cell walls than from each bran. More of the oat bran was digested (in vitro dry matter disappearance = 74.8%) during the 72 h incubation than any other bran source. While each bran contained arabinose and xylose, more glucose, galactose, and mannose were utilized by strain V975 during the 72-h incubation than either pentose sugar. Compared with each bran, the bran cell walls had lower concentrations of most sugars, and more glucose than any other sugar was utilized by strain V975. These results suggest that strain V975 preferentially utilizes glucose, galactose, and mannose in each bran, while glucose is the main sugar fermented in bran cell walls. Received: 19 June 1997 / Accepted: 31 July 1997     

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.     

A new acivicin prodrug designed for tumor-targeted delivery

Acivicin is an antitumor agent known to inhibit cell growth. A new prodrug 9b of acivicin 10 was synthesized, based on a p-hydroxybenzylcarbamate self-immolative spacer capable to release acivicin under esterase activity. The prodrug includes a maleimide-containing arm for linkage with thiol-containing macromolecules such as antibodies. This molecule is intended for the conception of bioconjugates to target an inactive acivicin precursor to tumor cells, when linked to a monoclonal antibody (mAb) which recognizes a tumor-specific antigen. Prodrug cleavage by plasmatic esterases will then restore the acivicin's activity toward tumor cells. We report here the synthesis and the in vitro characteristics of the prodrug. As expected, its inhibitory activity against the gamma-glutamyl transpeptidase (gamma-GT) enzyme and its cytotoxicity towards HL-60 cells were highly reduced compared to the parent drug. The chemical and plasmatic hydrolysis kinetics of the compound was studied by HPLC. The prodrug is stable, being slowly hydrolyzed in pH 7.6 buffer at 37 degrees C with a half-life of 37 h. It is converted into an active acivicin under the effect of pig liver esterase, and its half-life in human plasma is 3 h. These results indicate this compound may be further used as a prodrug-antibody conjugate, to target acivicin to malignant cells.     

Characteristics of acid lipase and acid cholesteryl esterase activity in parenchymal and non-parenchymal rat liver cells

(1) Parenchymal and non-parenchymal cells were isolated from rat liver. The characteristics of acid lipase activity with 4-methylumbelliferyl oleate as substrate and acid cholesteryl esterase activity with cholesteryl[1-14C]oleate as substrate were investigated. The substrates were incorporated in egg yolk lecithin vesicles and assays for total cell homogenates were developed, which were linear with the amount of protein and time. With 4-methylumbelliferyl oleate as substrate, both parenchymal and non-parechymal cells show maximal activities at acid pH and the maximal activity for non-parenchymal cells is 2.5 times higher than for parenchymal cells. It is concluded that 4-methylumbelliferyl oleate hydrolysis is catalyzed by similar enzyme(s) in both cell types. (2) With cholesteryl[1-14C]oleate as substrate both parenchymal and non-parenchymal cells show maximal activities at acid pH and the maximal activity for non-parenchymal cells is 11.4 times higher than for parenchymal cells. It is further shown that the cholesteryl ester hydrolysis in both cell types show different properties. (3) The high activity and high affinity of acid cholesteryl esterase from non-parenchymal cells for cholesterol oleate hydrolysis as compared to parenchymal cells indicate a relative specialization of non-parenchymal cells in cholesterol ester hydrolysis. It is concluded that non-parenchymal liver cells in cholesterol ester hydrolysis. It is concluded that non-parenchymal liver cells possess the enzymic equipment to hydrolyze very efficiently internalized cholesterol esters, which supports the suggestion that these cell types are an important site for lipoprotein catabolism in liver.     

Supplementation of watermelon peels as an enhancer of lipase and esterase production by Yarrowia lipolytica in solid-state fermentation and their potential use as biocatalysts in poly(ethylene terephthalate) (PET) depolymerization reactions

Abstract

Yarrowia lipolytica is a yeast that presents high biotechnological potential due to its ability to produce many metabolites, among them lipases and esterases, which are important industrial biocatalysts. Since Brazil is an agroindustrial country, it generates an enormous diversity of residues or byproducts that can be used as a platform for biomolecules production. This work aims to evaluate lipase and esterase production by Y. lipolytica via solid-state fermentation using soybean bran and soybean bran supplemented with watermelon peels in different contents, and subsequent use of the enzyme extracts for poly(ethylene terephthalate) (PET) hydrolysis. Supplementation of watermelon peels in the lowest content led to an improvement of lipase activity in almost 31%, reaching 75.22?U g^?1. Esterase productivity was 1.5-fold higher when 20?wt% of watermelon peels were added to the media culture. Timecourse evaluation of enzymes production showed a maximum lipase activity in 14?h and similar esterase activity in 14 and 20?h of fermentation. Proteases production were also intensified in supplemented samples. Enzymes produced with 5?wt% watermelon peels supplementation led to higher terephthalic acid concentration (up to 42.02?µmol L^?1) during PET depolymerization. Results suggest a great potential of enzyme production in low cost fermentative media to act as biocatalysts in PET hydrolysis reactions.     

The esterase profile of a lipase from Candida cylindracea

A commercial preparation of a lipase produced by Candida cylindracea catalysed the hydrolysis of both long- and short-chain esters of p-nitrophenol. Six major bands of hydrolytic activity to alpha-naphthyl acetate were detected on polyacrylamide gel electrophoresis and two on isoelectric focusing. The esterase activity fractionated into two major peaks of activity on ion-exchange chromatography and into several peaks of activity on hydrophobic interaction chromatography. These esterase activities showed different substrate specificities to p-nitrophenyl esters, tributyrin and cetyl palmitate.     

Mechanism of autolysis of Neisseria gonorrhoeae.

The major autolysin(s) of Neisseria gonorrhoeae was solubilized from envelopes by extraction with 2% Triton X-100 containing 0.5 M NaCl. Neither Triton X-100 nor NaCl alone could effectively release the autolysin(s). The major autolysin is N-acetylmuramyl-L-alanine amidase (EC 3.5.1.28). The pH optimum for this reaction was broad, ranging from 5.5 to 8.5. Optimal hydrolysis of peptidoglycan occurred in 2% Triton X-100 in 0.1 M KCl. Attempts to purify the autolysin were unsuccessful. A rapid assay for enzyme activity was developed using radioactive cell walls as a substrate ([3H]diaminopimelic acid).     

Synthesis of methyl 5-O-trans-feruloyl-alpha-L-arabinofuranoside and its use as a substrate to assess feruloyl esterase activity

A synthetic scheme was developed for the production of methyl 5-O-trans-feruloyl-alpha-L-arabinofuranoside (FA-Ara) in gram quantities. This molecule accurately models the chemical attachment of ferulic acid to polysaccharides found in cell walls of plants in the Gramineae family. It is therefore a realistic substrate that can be used to monitor feruloyl esterase activity. Ultraviolet spectral analysis indicated that FA-Ara has an absorption maximum distinct from the hydrolytic product, ferulic acid (FA), over a wide range of solution pH values. The log molar extinction coefficient ranges from 4.16 to 4.36 for FA-Ara and 4.16 to 4.33 for FA depending upon the pH of the buffered solution. Consequently a convenient spectrophotometric assay can be utilized to monitor esterase activity. Three different methods were developed for using this model substrate to assess esterase activity, including thin-layer chromatography, a spectrophotometric assay, and the use of high-performance liquid chromatography.     

Purification and Properties of a Polyester Polyurethane-Degrading Enzyme from Comamonas acidovorans TB-35

A polyester polyurethane (PUR)-degrading enzyme, PUR esterase, derived from Comamonas acidovorans TB-35, a bacterium that utilizes polyester PUR as the sole carbon source, was purified until it showed a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This enzyme was bound to the cell surface and was extracted by addition of 0.2% N,N-bis(3-d-gluconamidopropyl)deoxycholamide (deoxy-BIGCHAP). The results of gel filtration and SDS-PAGE showed that the PUR esterase was a monomer with a molecular mass of about 62,000 Da. This enzyme, which is a kind of esterase, degraded solid polyester PUR, with diethylene glycol and adipic acid released as the degradation products. The optimum pH for this enzyme was 6.5, and the optimum temperature was 45 degrees C. PUR degradation by the PUR esterase was strongly inhibited by the addition of 0.04% deoxy-BIGCHAP. On the other hand, deoxy-BIGCHAP did not inhibit the activity when p-nitrophenyl acetate, a water-soluble compound, was used as a substrate. These observations indicated that this enzyme degrades PUR in a two-step reaction: hydrophobic adsorption to the PUR surface and hydrolysis of the ester bond of PUR.     

Cell wall structure regulates the autolytic process throughout growth of Cicer arietinum epicotyls

The autolytic process in epicotyl cell walls of Cicer arietinum L. cv. Castellana, and also the hydrolysis of heat-inactivated cell walls as mediated by a cell wall β-galactosidase (EC 3.2.1.23) (named βIII and previously characterized as responsible for the autolysis), are maximal on the fourth day of germination and coincide with the maximal growth capacity. They decrease during the following days, in which the growth rate diminishes. In both cases, no differences were observed in the percentages of the different sugars released, galactose being the principal one. The βIII fraction from aged epicotyl cell walls hydrolyzed young walls in proportion to its specific activity, and more efficient than when cell walls from aged material were used as the substrate. The βIII fraction from 4 day-old epicotyls (the time for maximal autolysis) was incapable of hydrolyzing aged epicotyl cell walls to the same extent as young ones. These results, together with the levels and activity of the enzyme throughout growth, allow the assumption that the variations in the autolysis and hydrolysis caused by βIII during growth processes are due to structural modifications in the cells walls, modifications that would limit access of the enzyme to its substrate, thus impeding the release of galactose, even though the enzyme is present.     

Production of ferulic acid from lignocellulolytic agricultural biomass by Thermobifida fusca thermostable esterase produced in Yarrowia lipolytica transformant

A gene (axe) encoding the AXE thermostable esterase in Thermobifida fusca NTU22 was cloned into a Yarrowia lipolytica P01g host strain. Recombinant expression resulted in extracellular esterase production at levels as high as 70.94 U/ml in Hinton flask culture broth, approximately 140 times higher than observed in a Pichia pastoris expression system. After 72 h of fermentation by the Y. lipolytica transformant in the fed-batch fermentor, the fermentation broth accumulated 41.11 U/ml esterase activity. Rice bran, wheat bran, bagasse and corncob were used as hydrolysis substrates for the esterase, with corncob giving the best ferulic acid yield. The corncob was incubated with T. fusca xylanase (Tfx) for 12 h and then with the AXE esterase for an additional 12 h. Ferulic acid accumulated to 396 μM in the culture broth, a higher concentration than with esterase alone or with Tfx and esterase together for 24 h.     

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.     

Glucuronoxylan xylanohydrolase. A unique xylanase with the requirement for appendant glucuronosyl units

A new category of beta-(1----4)-xylan xylanohydrolases that exhibit a specific capacity to hydrolyze glucuronoxylans was characterized using heteroxylans prepared from Vigna (Vigna angularis Ohwi et Ohashi cv. Takara) and maize (Zea mays L.) cell walls together with appropriate derivatives as substrates. Glucuronopyranosyl moieties, as side chains, were prerequisite for enzyme-mediated hydrolysis of the beta-(1----4)-xylosyl linkages. The enzyme degraded glucuronoxylans derived from Vigna cell walls to yield a major oligomeric species (formula; see text) where Xyl represents xylose and GlcA represents glucuronic acid. The enzyme also degraded glucuronoarabinoxylans derived from maize cell walls to yield a major oligomeric species containing a single glucuronosyl side chain and a single unsubstituted beta 1----4Xyl pendant terminal. These results indicate that this xylanohydrolase recognizes glucuronosyl moieties inserted as monomeric side chains along the xylan backbone and mediates the hydrolysis of the beta-(1----4)-xylosyl linkage of the adjacent unsubstituted xylosyl residue in heteroxylans. This enzyme is the first xylanohydrolase identified that recognizes distinctly different sugars constituting side chains. We propose to designate this new enzyme as a glucuronoxylan xylanohydrolase to be abbreviated as glucuronoxylanase. Use of this unique enzyme demonstrated the presence of repeating units in heteroxylans in cell walls of higher plants.     

Enterochelin System of Iron Transport in Escherichia coli: Mutations Affecting Ferric-Enterochelin Esterase

Three mutant strains of Escherichia coli have been isolated which are lacking ferric-enterochelin esterase activity. This enzyme catalyzes the hydrolysis of the enterochelin moiety of ferric-enterochelin to yield ultimately three molecules of N-2,3-dihydroxybenzoylserine. The mutants (designated fes(-)) were shown to be unaffected in enterochelin biosynthesis, capable of enterochelin-mediated iron uptake, and able to utilize ferric-dihydroxybenzoylserine complexes normally. When grown under iron-deficient conditions, however, they showed an absolute requirement for added iron or citrate, a phenotype characteristic of mutants defective in some part of the enterochelin system of iron uptake. These results support the theory that iron, taken up by the cell as ferric-enterochelin is only available for general cell metabolism after hydrolysis of the ligand by enterochelin esterase. The three fes(-) strains were shown to be affected in the B component of enterochelin esterase. The fesB gene which is probably the structural gene coding for component B of the esterase, was shown to be located at about minute 14 on the E. coli chromosome together with seven other genes involved in the enterochelin system of iron transport.     

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%.