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.     

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.     

Hyperthermostable acetyl xylan esterase

An esterase which is encoded within a Thermotoga maritima chromosomal gene cluster for xylan degradation and utilization was characterized after heterologous expression of the corresponding gene in Escherichia coli and purification of the enzyme. The enzyme, designated AxeA, shares amino acid sequence similarity and its broad substrate specificity with the acetyl xylan esterase from Bacillus pumilus, the cephalosporin C deacetylase from Bacillus subtilis, and other (putative) esterases, allowing its classification as a member of carbohydrate esterase family 7. The recombinant enzyme displayed activity with p-nitrophenyl-acetate as well as with various acetylated sugar substrates such as glucose penta-acetate, acetylated oat spelts xylan and DMSO (dimethyl sulfoxide)-extracted beechwood xylan, and with cephalosporin C. Thermotoga maritima AxeA represents the most thermostable acetyl xylan esterase known to date. In a 10 min assay at its optimum pH of 6.5 the enzyme's activity peaked at 90°C. The inactivation half-life of AxeA at a protein concentration of 0.3 µg µl⁻¹ in the absence of substrate was about 13 h at 98°C and about 67 h at 90°C. Differential scanning calorimetry analysis of the thermal stability of AxeA corroborated its extreme heat resistance. A multi-phasic unfolding behaviour was found, with two apparent exothermic peaks at approximately 100–104°C and 107.5°C. In accordance with the crystal structure, gel filtration analysis at ambient temperature revealed that the enzyme has as a homohexameric oligomerization state, but a dimeric form was also found.     

Production and partial characterization of alkaline feruloyl esterases by Fusarium oxysporum during submerged batch cultivation

Production of feruloyl esterases (FAEs) by Fusarium oxysporum was enhanced by optimization of initial pH of the culture medium, the type and concentration of nitrogen and carbon source. Submerged batch cultivation in a laboratory bioreactor (17 l) produced activity at 82 nkat g⁻¹ dry substrate (corn cobs) which compared favorably to those reported for the other microorganisms. Use of de-esterified corn cobs as carbon source decreased FAE production by 5.5-fold compared to untreated corn cobs even though ferulic acid (FA) was added to the concentration found in alkali-extracts of corn cobs. Production of FAE does not therefore, require FA, however, production is diminished by the removal of esterified FA from the growth substrate. Optimal FAE activity was observed at pH 7 and 50 °C with 68 and 55% activity at pH 8 and pH 9, respectively. The esterase was fully stable at pH 5–8 and up to 40 °C and retained 72 and 40% of its activity after 6 h at pH 9 and pH 10, respectively. After separation by isoelectric focusing electrophoresis, a zymogram indicated one major FAE activity exhibiting pI value of 10.5. This revised version was published online in August 2006 with corrections to the Cover Date.     

Purification and properties of a highly enantioselective l-menthyl acetate hydrolase from Burkholderia cepacia

A highly enantioselective l-menthyl acetate esterase was purified to homogeneity from Burkholderia cepacia ATCC 25416, with a recovery of 4.8% and a fold purification of 22.7. The molecular weight of the esterase was found to be 37 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The N-terminal amino acid sequence was “MGARTDA”, and there was no homology in contrast to other Burkholderia sp. esterases. This enzyme preferentially hydrolyzed short-chain fatty acid esters of menthol with high stereospecificity and high hydrolytic activity, while long-chain l-menthyl esters were poor substrates. Considered its substrate specificity and N-terminal sequence, this esterase was concluded as a new enzyme belonging to the carboxylesterase group (EC 3.1.1.1) of esterase family. The optimum temperature and pH for enzyme activity using racemic menthyl acetate as substrate were 30 °C and 7.0, respectively. The esterase was more stable in the pH range of 7.0–9.0 and temperature range of 30–40 °C. Hydrolytic activity was enhanced by Ca²⁺, K⁺ and Mg²⁺, but completely inhibited by Hg²⁺, Cu²⁺, ionic detergents and phenylmethylsulfonyl fluoride (PMSF) at 0.01 M concentration.     

A Thermostable phytase from <Emphasis Type="Italic">Neosartorya spinosa</Emphasis> BCC 41923 and its expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

A phytase gene was cloned from Neosartorya spinosa BCC 41923. The gene was 1,455 bp in size, and the mature protein contained a polypeptide of 439 amino acids. The deduced amino acid sequence contains the consensus motif (RHGXRXP) which is conserved among phytases and acid phosphatases. Five possible disulfide bonds and seven potential N-glycosylation sites have been predicted. The gene was expressed in Pichia pastoris KM71 as an extracellular enzyme. The purified enzyme had specific activity of 30.95 U/mg at 37°C and 38.62 U/mg at 42°C. Molecular weight of the deglycosylated recombinant phytase, determined by SDS-PAGE, was approximately 52 kDa. The optimum pH and temperature for activity were pH 5.5 and 50°C. The residual phytase activity remained over 80% of initial activity after the enzyme was stored in pH 3.0 to 7.0 for 1 h, and at 60% of initial activity after heating at 90°C for 20 min. The enzyme exhibited broad substrate specificity, with phytic acid as the most preferred substrate. Its K _m and V _max for sodium phytate were 1.39 mM and 434.78 U/mg, respectively. The enzyme was highly resistant to most metal ions tested, including Fe²⁺, Fe³⁺, and Al³⁺. When incubated with pepsin at a pepsin/phytase ratio of 0.02 (U/U) at 37°C for 2 h, 92% of its initial activity was retained. However, the enzyme was very sensitive to trypsin, as 5% of its initial activity was recovered after treating with trypsin at a trypsin/phytase ratio of 0.01 (U/U).     

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     

Purification and biochemical characterization of feruloyl esterases from Aspergillus terreus MTCC 11096

Aspergillus terreus MTCC 11096 isolated from the soils of agricultural fields cultivating sweet sorghum was previously identified to produce feruloyl esterases (FAEs). The enzymes responsible for feruloyl esterase activity were purified to homogeneity and named as AtFAE‐1, AtFAE‐2, and AtFAE‐3. The enzymes were monomeric having molecular masses of 74, 23 and 36 kDa, respectively. Active protein bands were identified by a developed pH‐dependent zymogram on native PAGE. The three enzymes exhibited variation in pH tolerance ranging between pH 5–8 and thermostability of up to 55°C. Inhibition studies revealed that the serine residue was essential for feruloyl esterase activity; moreover aspartyl and glutamyl residues are not totally involved at the active site. Metal ions such as Ca²⁺, K⁺, and Mg²⁺ stabilized the enzyme activity for all three FAEs. Kinetic data indicated that all three enzymes showed catalytic efficiencies (k_cat/K_m) against different synthesized alkyl and aryl esters indicating their broad substrate specificity. The peptide mass fingerprinting by MALDI/TOF‐MS analysis and enzyme affinity toward methoxy and hydroxy substituents on the benzene ring revealed that the AtFAE‐1 belonged to type A while AtFAE‐2 and AtFAE‐3 were type C FAE. The FAEs could release 65 to 90% of ferulic acid from agrowaste substrates in the presence of xylanase. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:924–932, 2013     

Novel bacterial ferulic acid esterase from <Emphasis Type="Italic">Cellvibrio japonicus</Emphasis> and its application in ferulic acid release and xylan hydrolysis

Recent genome sequencing of Cellvibrio japonicas revealed the presence of two highly homologous ferulic acid esterases (FAEs), encoded by fee1A and fee1B. In this work, the putative FAE, Fee1B, was successfully cloned and expressed in an E. coli system and the purified enzyme was characterized as a type-D FAE with a pH and temperature optima of 6.5 and 35−40°C, respectively. Additionally, the two tandem N-terminal carbohydrate binding modules of the multi-domain enzyme were shown to be crucial for optimum enzyme activity. The potential of the enzyme in biomass processing was demonstrated with its high synergy with a xylanase in the release of reducing sugar from arabinoxylan and its ability to liberate ferulic acid from various complex xylan substrates.     

Isolation of the Volatile Components of Fresh Onion by Thermal Desorption Cold Trap Capillary Gas Chromatography

A new esterase activity from Bacillus licheniformis was characterized from an Escherichia coli recombinant strain. The protein was a single polypeptide chain with a molecular mass of 81 kDa. The optimum pH for esterase activity was 8-8.5 and it was stable in the range 7-8.5. The optimum temperature for activity was 45°C and the half-life was 1 h at 64°C. Maximum activity was observed on p-nitrophenyl caproate with little activity toward long-chainfatty acid esters. The enzyme had a K_M of 0.52 mM for p-nitrophenyl caproate hydrolysis at pH 8 and 37°C. The enzyme activity was not affected by either metal ions or sulfydryl reagents. Surprisingly, the enzyme was only slightly inhibited by PMSF. These characteristics classified the new enzyme as a thermostable esterase that shared similarities with lipases. The esterase might be useful for biotechnological applications such as ester synthesis.     

Molecular cloning, expression and characterization of a novel feruloyl esterase enzyme from the symbionts of termite(Coptotermes formosanus) gut

Termites play an important role in the degradation of dead plant materials and have acquired endogenous and symbiotic cellulose digestion capabilities. The feruloyl esterase enzyme (FAE) gene amplified from the metagenomic DNA of Coptotermes formosanus gut was cloned in the TA cloning vector and subcloned into a pET32a expression vector. The Ft3-7 gene has 84% sequence identity with Clostridium saccharolyticum and shows amino acid sequence identity with predicted xylanase/chitin deacetylase and endo-1,4-beta-xylanase. The sequence analysis reveals that probably Ft3-7 could be a new gene and that its molecular mass was 18.5 kDa. The activity of the recombinant enzyme (Ft3-7) produced in Escherichia coli (E.coli) was 21.4 U with substrate ethyl ferulate and its specific activity was 24.6 U/mg protein. The optimum pH and temperature for enzyme activity were 7.0 and 37oC, respectively. The substrate utilization preferences and sequence similarity of the Ft3-7 place it in the type-D sub-class of FAE.     

Complete Reversal of Coenzyme Specificity of Isocitrate Dehydrogenase from <Emphasis Type="Italic">Haloferax volcanii</Emphasis>

Haloferax volcanii Ds-threo-isocitrate dehydrogenase (ICDH) was highly expressed in bacteria as inclusion bodies. The recombinant enzyme was refolded, purified and characterized, and was found to be NADP-dependent like the wild-type protein. Sequence alignment of several isocitrate dehydrogenases from evolutionarily divergent organisms including H. volcanii revealed that the amino acid residues involved in coenzyme specificity are highly conserved. Our objective was to switch the coenzyme specificity of halophilic ICDH by altering these conserved amino acids. We were able to switch coenzyme specificity from NADP⁺ to NAD⁺ by changing five amino acids by site-directed mutagenesis (Arg291, Lys343, Tyr344, Val350 and Tyr390). The five mutants of ICDH were overexpressed in Escherichia coli as inclusion bodies and each recombinant ICDH protein was refolded and purified, and its kinetic parameters were determined. Coenzyme specificity did not switch until all five amino acids were substituted.     

A thermostable feruloyl-esterase from the hemicellulolytic bacterium <Emphasis Type="Italic">Thermobacillus xylanilyticus</Emphasis> releases phenolic acids from non-pretreated plant cell walls

A gene (Tx-est1) encoding a thermostable feruloyl-esterase was isolated from the genome of the Gram-positive hemicellulolytic thermophilic bacterium Thermobacillus xylanilyticus. This gene contains an open reading frame of 1,020 bp encoding a protein with molecular mass of 37.4 kDa, similar to feruloyl-esterases from cellulolytic bacteria and fungi. The recombinant enzyme Tx-Est1 was expressed and produced in Escherichia coli. Tx-Est1 contains the conserved putative lipase residues Ser 202, Asp 287, and His 322 which act as catalytic triad in its C-terminus part. Purified Tx-Est1 was active against phenolic acid derivatives and stable at high temperatures. Optimal activity was observed at 65 °C and the optimal pH was around 8.5. The kinetic parameters of the esterase were determined on various substrates. The enzyme displayed activity against methyl esters of hydrocinnamic acids and feruloylated arabino-xylotetraose, exhibiting high specificity and affinity for the latter. Our results showed that Tx-Est1 is a thermostable feruloyl-esterase which could be useful to hydrolyze arabinoxylans from graminaceous plant cell walls as the enzyme is able to release phenolic acids from a lignocellulose biomass.     

Expression of a novel thermostable Cu,Zn-superoxide dismutase from <Emphasis Type="Italic">Chaetomium thermophilum</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and its antioxidant properties

A new superoxide dismutase (SOD) gene from the thermophilic fungus Chaetomium thermophilum (Ctsod) was cloned and expressed in Pichia pastoris and its gene product was characterized. The specific activity of the purified CtSOD was 2,170 U/mg protein. The enzyme was inactivated by KCN and H₂O₂ but not by NaN₃, confirming that it belonged to the type of Cu, ZnSOD. The amino acid residues involved in coordinating copper and zinc were conserved. The recombinant CtSOD exhibited optimum activity at pH 6.5 and 60°C. The enzyme retained 65% of the maximum activity at 70°C for 60 min and the half-life was 22 and 7 min at 80 and 90°C, respectively. The recombinant yeast exhibited higher stress resistance than the control yeast cells to salt and superoxide-generating agents, such as paraquat and menadione.     

Gene cloning,expression, and characterization of phenolic acid decarboxylase from <Emphasis Type="Italic">Lactobacillus brevis</Emphasis> RM84

Phenolic acid decarboxylase (PAD) catalyzes the synthesis of vinyl phenols from hydroxycinnamic acids. The gene encoding PAD from Lactobacillus brevis was cloned and expressed as a fusion protein in Escherichia coli. The recombinant PAD enzyme is a heat-labile enzyme that functions optimally at 22°C and pH 6.0. The purified enzyme did not show thermostability at temperatures above 22°C. L. brevis PAD is able to decarboxylate exclusively the hydroxycinnamic acids, such as p-coumaric, caffeic, and ferulic acids, with K _m values of 0.98, 0.96, and 0.78 mM, respectively. The substrate specificity exhibited by L. brevis PAD is similar to the PAD isolated from Bacillus subtilis and B. pumilus, but different from that of L. plantarum and Pediococcus pentosaceus. As the C-terminal region may be involved in determining PAD substrate specificity and catalytic capacity, amino acid differences among these proteins could explain the differences observed. The substrate specificity shown by L. brevis PAD shows promise for the synthesis of high-added value products from plant wastes.     

Partial purification and characterization of thermostable esterase from the hyperthermophilic archaeonSulfolobus solfataricus

A thermostable esterase from the hyperthemophilic archaeonSulfolobus solfataricus was partially purified 590-fold with 16.2% recovery. The partially purified esterase had a specific activity of 29.5μmol min⁻¹ mg⁻¹ when the enzyme activity was determined usingp-nitrophenyl butyrate as a substrate. The apparent molecular weight was about 100 kDa, while the optimum temperature and pH for esterase were 75°C and 8.0, respectively. The enzyme showed high thermal stability and solvent tolerance in comparison to its mesophilic counterpart. The enzyme also showed chiral resolution activity for (S)-ibuprofen, indicating thatS. solfataricus esterase can be used for the production of commercially important chiral drugs.     

A cold-active esterase of <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> A3(2): from genome sequence to enzyme activity

The genome sequence of Streptomyces coelicolor A3(2) contains 51 putative lipase and esterase genes mostly of unknown function. The gene estB (locus SCO 6966) was expressed as a His-tagged protein in E. coli. Esterase B was active at low temperatures exerting its maximum activity at 30°C and retaining more than 25% of its activity at 4°C. The optimum pH was 8–8.5. The enzyme was active against short synthetic p-nitrophenylesters (C₂–C₁₀) with maximum activity towards the acetate ester (C₂). The esterase was tested on 13 series of racemic esters of potential interest for the synthesis of chiral pharmaceutical compounds. 4 of the series were substrates and a modest degree of enantioselectivity was observed (enantiomeric ratios of 1.1–1.9).     

An esterase from the basidiomycete Pleurotus sapidus hydrolyzes feruloylated saccharides

Investigating the secretion of esterases by the basidiomycetous fungus Pleurotus sapidus in a Tween 80-rich nutrient medium, an enzyme was discovered that hydrolyzed the ester bond of feruloylated saccharides. The enzyme was purified by ion exchange and size exclusion chromatography. Polyacrylamide gel electrophoresis analysis showed a monomeric protein of about 55 kDa. The complete coding sequence with an open reading frame of 1,665 bp encoded a protein (Est1) consisting of 554 amino acids. The enzyme showed no significant homology to any published feruloyl esterase sequences, but possessed putative conserved domains of the lipase/esterase superfamily. Substrate specificity studies classified the new enzyme as type-A feruloyl esterase, hydrolyzing methyl ferulate, methyl sinapate, and methyl p-coumarate but no methyl caffeate. The enzyme had a pH optimum of 6 and a temperature optimum at 50 °C. Ferulic acid was efficiently released from ferulated saccharides, and the feruloyl esterase exhibited moderate stability in biphasic systems (50 % toluene or tert-butylmethyl ether).     

Feruloyl esterase activity of the Clostridium thermocellum cellulosome can be attributed to previously unknown domains of XynY and XynZ

The cellulosome of Clostridium thermocellum is a multiprotein complex with endo- and exocellulase, xylanase, beta-glucanase, and acetyl xylan esterase activities. XynY and XynZ, components of the cellulosome, are composed of several domains including xylanase domains and domains of unknown function (UDs). Database searches revealed that the C- and N-terminal UDs of XynY and XynZ, respectively, have sequence homology with the sequence of a feruloyl esterase of strain PC-2 of the anaerobic fungus Orpinomyces. Purified cellulosomes from C. thermocellum were found to hydrolyze FAXX (O-(5-O-[(E)-feruloyl]-alpha-L-arabinofuranosyl)-(1-->3)-O-beta-D- xyl opyranosyl-(1-->4)-D-xylopyranose) and FAX(3) (5-O-[(E)-feruloyl]-[O-beta-D-xylopyranosyl-(1-->2)]-O-alpha-L- arabinofuranosyl-[1-->3])-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose) , yielding ferulic acid as a product, indicating that they have feruloyl esterase activity. Nucleotide sequences corresponding to the UDs of XynY and XynZ were cloned into Escherichia coli, and the expressed proteins hydrolyzed FAXX and FAX(3). The recombinant feruloyl esterase domain of XynZ alone (FAE(XynZ)) and with the adjacent cellulose binding domain (FAE-CBD(XynZ)) were characterized. FAE-CBD(XynZ) had a molecular mass of 45 kDa that corresponded to the expected product of the 1,203-bp gene. K(m) and V(max) values for FAX(3) were 5 mM and 12.5 U/mg, respectively, at pH 6.0 and 60 degrees C. PAX(3), a substrate similar to FAX(3) but with a p-coumaroyl group instead of a feruloyl moiety was hydrolyzed at a rate 10 times slower. The recombinant enzyme was active between pH 3 to 10 with an optimum between pH 4 to 7 and at temperatures up to 70 degrees C. Treatment of Coastal Bermuda grass with the enzyme released mainly ferulic acid and a lower amount of p-coumaric acid. FAE(XynZ) had similar properties. Removal of the 40 C-terminal amino acids, residues 247 to 286, of FAE(XynZ) resulted in protein without activity. Feruloyl esterases are believed to aid in a release of lignin from hemicellulose and may be involved in lignin solubilization. The presence of feruloyl esterase in the C. thermocellum cellulosome together with its other hydrolytic activities demonstrates a powerful enzymatic potential of this organelle in plant cell wall decomposition.