Acetic acid-catalyzed hydrothermal pretreatment of corn stover for the production of bioethanol at high-solids content

Corn stover (CS) was hydrothermally pretreated using CH₃COOH (0.3 %, v/v), and subsequently its ability to be utilized for conversion to ethanol at high-solids content was investigated. Pretreatment conditions were optimized employing a response surface methodology (RSM) with temperature and duration as independent variables. Pretreated CS underwent a liquefaction/saccharification step at a custom designed free-fall mixer at 50 °C for either 12 or 24 h using an enzyme loading of 9 mg/g dry matter (DM) at 24 % (w/w) DM. Simultaneous enzymatic saccharification and fermentation (SSF) of liquefacted corn stover resulted in high ethanol concentration (up to 36.8 g/L), with liquefaction duration having a negligible effect. The threshold of ethanol concentration of 4 % (w/w), which is required to reduce the cost of ethanol distillation, was surpassed by the addition of extra enzymes at the start up of SSF achieving this way ethanol titer of 41.5 g/L.     

Induction, purification, and characterization of two extracellular alpha-L-arabinofuranosidases from Fusarium oxysporum

In the presence of L-arabinose as sole carbon source, Fusarium oxysporum produces two alpha-L-arabinofuranosidases (ABFs) named ABF1 and ABF2, with molecular masses of 200 and 180 kDa, respectively. The two F. oxysporum proteins have been purified to homogeneity. The purified enzymes are composed of three equal subunits and are neutral proteins with pIs of 6.0 and 7.3 for ABF1 and ABF2, respectively. With p-nitrophenyl alpha-L-arabinofuranoside (pNPA) as the substrate, ABF1 and ABF2 exhibited Km values of 0.39 and 0.28 mmol.L(-1), respectively, and Vmax values of 1.6 and 4.6 micromol.min(-1).(mg of protein)(-1), respectively, and displayed optimal activity at pH 6.0 and 50-60 degrees C. ABFs released arabinose only from sugar beet arabinan and not from wheat soluble and insoluble arabinoxylans. The enzymes were not active on substrates containing ferulic acid ester linked to C-5 and C-2 linkages of pNPA showing that phenolic substituents of pNPA sterically hindered the action of ABFs.     

Substrate and positional specificity of feruloyl esterases for monoferuloylated and monoacetylated 4-nitrophenyl glycosides

4-Nitrophenyl glycosides of 2-, 3-, and 5-O-(E)-feruloyl- and 2- and 5-O-acetyl-alpha-L-arabinofuranosides and of 2-, 3-, and 4-O-(E)-feruloyl- and 2-, 3- and 4-O-acetyl-beta-D-xylopyranosides, compounds mimicking natural substrates, were used to investigate substrate and positional specificity of type-A, -B, and -C feruloyl esterases. All the feruloyl esterases behave as true feruloyl esterases showing negligible activity on sugar acetates. Type-A enzymes, represented by AnFaeA from Aspergillus niger and FoFaeII from Fusarium oxysporum, are specialized for deferuloylation of primary hydroxyl groups, with a very strong preference for hydrolyzing 5-O-feruloyl-alpha-L-arabinofuranoside. On the contrary, type-B and -C feruloyl esterases, represented by FoFaeI from F. oxysporum and TsFaeC from Talaromyces stipitatus, acted on almost all ferulates with exception of 4- and 3-O-feruloyl-beta-D-xylopyranoside. 5-O-Feruloyl-alpha-L-arabinofuranoside was the best substrate for both TsFaeC and FoFaeI, although catalytic efficiency of the latter enzyme toward 2-O-feruloyl-alpha-L-arabinofuranoside was comparable. In comparison with acetates, the corresponding ferulates served as poor substrates for the carbohydrate esterase family 1 feruloyl esterase from Aspergillus oryzae. The enzyme hydrolyzed all alpha-L-arabinofuranoside and beta-D-xylopyranoside acetates. It behaved as a non-specific acetyl esterase rather than a feruloyl esterase, with a preference for 2-O-acetyl-beta-D-xylopyranoside.     

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.     

Enzymatic synthesis of butyl hydroxycinnamates and their inhibitory effects on LDL-oxidation

The potential of the Aspergillus niger type A feruloyl esterase (AnFaeA) for the synthesis of various phenolic acid esters was examined using a ternary-organic reaction system consisting of a mixture of n-hexane, 1- or 2-butanol and water. Reaction parameters including the type of methyl hydroxycinnamate, the composition of the reaction media, the temperature, and the substrate concentration were investigated to evaluate their effect on initial rate and conversion to butyl esters of sinapic acids. Optimisation of the reaction parameters lead to 78% and 9% yield for the synthesis of 1-butyl and 2-butyl sinapate, respectively. For the first time, a feruloyl esterase was introduced in the reaction system as cross-linked enzyme aggregates (CLEAs), after optimisation of the immobilisation procedure, allowing the recycling and reuse of the biocatalyst. The inhibition of copper-induced LDL oxidation by hydroxycinnamic acids and their corresponding butyl esters was investigated in vitro. Kinetic analysis of the antioxidation process demonstrates that sinapate derivatives are effective antioxidants indicating that esterification increases the free acid's antioxidant activity especially on dimethoxylated compounds such as sinapic acid compared to methoxy-hydroxy-compounds such as ferulic acid.     

Regulation of the xylan-degrading apparatus of Cellvibrio japonicus by a novel two-component system

The microbial degradation of lignocellulose biomass is not only an important biological process but is of increasing industrial significance in the bioenergy sector. The mechanism by which the plant cell wall, an insoluble composite structure, activates the extensive repertoire of microbial hydrolytic enzymes required to catalyze its degradation is poorly understood. Here we have used a transposon mutagenesis strategy to identify a genetic locus, consisting of two genes that modulate the expression of xylan side chain-degrading enzymes in the saprophytic bacterium Cellvibrio japonicus. Significantly, the locus encodes a two-component signaling system, designated AbfS (sensor histidine kinase) and AbfR (response regulator). The AbfR/S two-component system is required to activate the expression of the suite of enzymes that remove the numerous side chains from xylan, but not the xylanases that hydrolyze the beta1,4-linked xylose polymeric backbone of this polysaccharide. Studies on the recombinant sensor domain of AbfS (AbfS(SD)) showed that it bound to decorated xylans and arabinoxylo-oligosaccharides, but not to undecorated xylo-oligosaccharides or other plant structural polysaccharides/oligosaccharides. The crystal structure of AbfS(SD) was determined to a resolution of 2.6A(.) The overall fold of AbfS(SD) is that of a classical Per Arndt Sim domain with a central antiparallel four-stranded beta-sheet flanked by alpha-helices. Our data expand the number of molecules known to bind to the sensor domain of two-component histidine kinases to include complex carbohydrates. The biological rationale for a regulatory system that induces enzymes that remove the side chains of xylan, but not the hydrolases that cleave the backbone of the polysaccharide, is discussed.     

Constitutive expression, purification and characterization of a phosphoglucomutase from Fusarium oxysporum

The phosphoglucomutase gene from a wild type Fusarium oxysporum strain (F3), was homologously expressed, under the control of the constitutive promoter of gpdA of Aspergillus nidulans. The transformant produced elevated levels of phosphoglucomutase activity compared to the wild type, a fact that facilitated the subsequent purification procedure. The enzyme (FoPGM) was purified to homogeneity applying three anion exchange and one gel filtration chromatography steps. The native enzyme revealed a monomeric structure with a molecular mass of 60 kDa, while the isoelectric point was 3.5. FoPGM was active in pH ranged from 6.0 to 8.0, with an optimum using 3-(N-morpholino)propanesulfonic acid buffer at 7.0, while loss of activity was observed when phosphate buffer was used in the above mentioned pH range. The optimal temperature for activity was 45°C but the enzyme became unstable at temperatures above 40°C. FoPGM requires the presence of a divalent cation for its function with maximum activity being obtained with Co(2+). The apparent K(m) for Co(2+) was found to be 10 μM. The enzyme was also active with other divalent metal ions such as Mn(2+), Mg(2+), Ni(2+) and Ca(2+) but to a lesser extent. The following kinetic constants were determined: v(max), 0.74 μmol mg(protein)(-1)min(-1); k(cat), 44.2 min(-1); K(m)(G1P), 0.10mM; K(m)(G1,6 diP), 1.03 μM; k(cat)/K(m)(G1P), 443 mM(-1)min(-1) and k(cat)/K(m)(G1,6 diP), 42,860 mM(-1)min(-1). The enzyme was considered to follow a Ping Pong substituted enzyme or enzyme isomerization mechanism.     

Chemoenzymatic synthesis of feruloyl d-arabinose as a potential anti-mycobacterial agent

The feruloyl esterase (StFaeC) produced by Sporotrichum thermophile transfered the feruloyl group to d-arabinose using a mixture of n-hexane, t-butanol and water. About 45% conversion of d-arabinose to the feruloylated derivative was achieved. The compound had an MIC value against Mycobacterium bovis BCG of 25 μg/ml.     

The feruloyl esterase system of Talaromyces stipitatus: determining the hydrolytic and synthetic specificity of TsFaeC

The active site of the recombinant Talaromyces stipitatus type-C feruloyl esterase (TsFaeC) was probed using a series of C1-C4 alkyl ferulates and methyl esters of phenylalkanoic and cinnamic acids. The enzyme was active on 23 of the 34 substrates tested. Lengthening or shortening the aliphatic side chain while maintaining the same aromatic substitutions completely abolished the enzyme activity. Maintaining the phenylpropenoate structure but altering the substitutions of the aromatic ring demonstrated the importance of hydroxyl groups on meta and/or para position of the benzoic ring. The highest catalytic efficiency of TsFaeC for methyl cinnamates was shown on methyl 3,4-dihydroxy cinnamate and on its hydro form (3,4-dihydroxy-phenyl-propionate). Maintaining the ferulate structure but altering the esterified alkyl group, the comparison of k(cat) and k(cat)/K(m) values showed that the enzyme hydrolysed faster and more efficiently than ethyl ferulate. Alkyl ferulates were applied also for substrate selectivity mapping of feruloyl esterase to catalyze feruloyl group transfer to l-arabinose, using as a reaction system a ternary water-organic mixture consisting of n-hexane, t-butanol and water. The reaction parameters affecting the feruloylation rate and the conversion of the enzymatic synthesis, such as the composition of the reaction media, temperature, substrate and enzyme concentration have been investigated.