Javier A. Linares-Pastén

Coenzyme A-acylating propionaldehyde dehydrogenase (PduP) from Lactobacillus reuteri: Kinetic characterization and molecular modeling

3-Hydroxypropionic acid (3-HP), an important C3 chemical for a bio-based industry, is natively produced by Lactobacillus reuteri from glycerol. Conversion of glycerol occurs via the intermediate 3-hydroxypropionaldehyde (3-HPA), followed by an ATP-producing pathway initiated by the CoA-acylating propionaldehyde dehydrogenase (PduP). The pduP gene of L. reuteri was cloned and expressed in Escherichia coli and the recombinant enzyme was purified to homogeneity for characterization of its activity and properties. Kinetic studies with propionaldehyde as substrate showed a maximum specific activity of 28.9 U/mg, which is 80-fold higher than that reported previously. Maximum activity of 18 U/mg was obtained at 3-HPA concentration of 7 mM, above which substrate inhibition was observed. Substrate inhibition was also seen with coenzyme A at a concentration above 0.5mM and with NADP(+) above 9 mM. A structure of PduP is proposed based on homology modeling. In silico docking of the co-factors coenzyme A and NAD(+), respectively, showed a common binding site consisting of amino acids Thr145, Ile275, Cys277 and Ser417, which through site-directed mutagenesis to alanine and kinetic studies, were confirmed as essential for the catalytic activity of PduP.

Structural Considerations on the Use of Endo-Xylanases for the Production of prebiotic Xylooligosaccharides from Biomass

Abstract: Xylooligosaccharides (XOS) have gained increased interest as prebiotics during the last years. XOS and arabinoxylooligosaccharides (AXOS) can be produced from major fractions of biomass including agricultural by-products and other low cost raw materials. Endo-xylanases are key enzymes for the production of (A)XOS from xylan. As the xylan structure is broadly diverse due to different substi-tutions, diverse endo-xylanases have evolved for its degradation. In this review structural and functional aspects are discussed, focusing on the potential applications of endo-xylanases in the production of dif-ferently substituted (A)XOS as emerging prebiotics, as well as their implication in the processing of the raw materials. Endo-xylanases are found in at least eight different glycoside hydrolase families (GH), and can either have a retaining or an inverting catalytic mechanism. To date, it is mainly retaining endo-xylanases that are used in applications to produce (A)XOS. Enzymes from these GH-families (mainly GH10 and GH11, and the more recently investigated GH30) are taken as prototypes to discuss substrate preferences and main products obtained. Finally, the need of new and accessory enzymes (new specifici-ties from new families or sources) to increase the yield of different types of (A)XOS is discussed, along with in vitro tests of produced oligosaccharides and production of enzymes in GRAS organisms to fa-cilitate use in functional food manufacturing.

Thermostable glycoside hydrolases in Biorefining

Glycoside hydrolases, which are responsible for the degradation of the major fraction of biomass, the polymeric carbohydrates in starch and lignocellulose, are predicted to gain increasing roles as catalysts in biorefining applications in the future bioeconomy. In this context, thermostable variants will be important, as the recalcitrance of these biomass-components to degradation often motivates thermal treatments. The traditional focus on degradation is also predicted to be changed into more versatile roles of the enzymes, also involving specific conversions to defined products. In addition, integration of genes encoding interesting target activities opens the possibilities for whole cell applications, in organisms allowing processing at elevated temperatures for production of defined metabolic products. In this review, we overview the application of glycoside hydrolases related to the biorefining context (for production of food, chemicals, and fuels). Use of thermostable enzymes in processing of biomass is highlighted, moving from the activities required to act on different types of polymers, to specific examples in todays processing. Examples given involve (i) monosaccharide production for food applications as well as use as carbon source for microbial conversions (to metabolites such as fuels and chemical intermediates), (ii) oligosaccharide production for prebiotics applications (iii) treatment for plant metabolite product release, and (iv) production of surfactants of the alkyl glycoside class. Finally future possibilities in whole cell biorefining are shown.

Production of 3-hydroxypropionic acid from 3-hydroxypropionaldehyde by recombinant Escherichia coli co-expressing Lactobacillus reuteri propanediol utilization enzymes

3-Hydroxypropionic acid (3-HP) is an important platform chemical for the biobased chemical industry. Lactobacillus reuteri produces 3-HP from glycerol via 3-hydroxypropionaldehyde (3-HPA) through a CoA-dependent propanediol utilization (Pdu) pathway. This study was performed to verify and evaluate the pathway comprising propionaldehyde dehydrogenase (PduP), phosphotransacylase (PduL), and propionate kinase (PduW) for formation of 3-HP from 3-HPA. The pathway was confirmed using recombinant Escherichia coli co-expressing PduP, PduL and PduW of L. reuteri DSM 20016 and mutants lacking expression of either enzyme. Growing and resting cells of the recombinant strain produced 3-HP with a yield of 0.3mol/mol and 1mol/mol, respectively, from 3-HPA. 3-HP was the sole product with resting cells, while growing cells produced 1,3-propanediol as co-product. 3-HP production from glycerol was achieved with a yield of 0.68mol/mol by feeding recombinant E. coli with 3-HPA produced by L. reuteri and recovered using bisulfite-functionalized resin.

A method for rapid screening of ketone biotransformations: detection of whole cell Baeyer-Villiger monooxygenase activity.

A method for screening of ketone biotransformations was developed and applied to the identification of Baeyer-Villiger monooxygenase (BVMO) activity. The method was based on the formation of a purple coloured product on reaction between an enolizable ketone and 3,5-dinitrobenzoic acid in an alkaline solution. Absorbance of the colour decreased with the size of the cycloketone ring. Stoichiometric ratio between cycloketone and 3,5-dinitrobenzoic acid was 1:1 at maximum absorbance. The method was applied for monitoring the consumption of cyclohexanone by bacteria under aerobic conditions, and was found to be potentially useful for both screening assays and quantitative measurements of BVMO activity. Compared to other existing methods, this method is faster, cheaper and amenable for whole cell assays.

Characterization of a family 43 β-xylosidase from the xylooligosaccharide utilizing putative probiotic Weissella sp. strain 92

In this work, we present the first XOS degrading glycoside hydrolase from Weissella, WXyn43, a two-domain enzyme from GH43. The gene was amplified from genomic DNA of the XOS utilizing Weissella strain 92, classified under the species-pair Weissella cibaria/W.confusa, and expressed in Escherichia coli. The enzyme is lacking a putative signal peptide and is, from a homology model, shown to be composed of an N-terminal 5-fold β-propeller catalytic domain and a C-terminal β-sandwich domain of unknown function. WXyn43 hydrolyzed short (1–4)-β-d-xylooligosaccharides, with similar kcat/KM for xylobiose (X2) and xylotriose (X3) and clearly lower efficiency in xylotetraose (X4) conversion. WXyn43 displays the highest reported kcat for conversion of X3 (900 s−1 at 37°C) and X4 (770 s−1), and kcat for hydrolysis of X2 (907 s−1) is comparable with or greater than the highest previously reported. The purified enzyme adopted a homotetrameric state in solution, while a truncated form with isolated N-terminal catalytic domain adopted a mixture of oligomeric states and lacked detectable activity. The homology model shows that residues from both domains are involved in monomer–monomer hydrogen bonds, while the bonds creating dimer–dimer interactions only involved residues from the N-terminal domain. Docking of X2 and X3 in the active site shows interactions corresponding to subsites −1 and +1, while presence of a third subsite is unclear, but interactions between a loop and the reducing-end xylose of X3 may be present.

Effect of Natural and Semisynthetic Pseudoguianolides on the Stability of NF-κB:DNA Complex Studied by Agarose Gel Electrophoresis.

The nuclear factor κB (NF-κB) is a promising target for drug discovery. NF-κB is a heterodimeric complex of RelA and p50 subunits that interact with the DNA, regulating the expression of several genes; its dysregulation can trigger diverse diseases including inflammation, immunodeficiency, and cancer. There is some experimental evidence, based on whole cells studies, that natural sesquiterpene lactones (Sls) can inhibit the interaction of NF-κB with DNA, by alkylating the RelA subunit via a Michael addition. In the present work, 28 natural and semisynthetic pseudoguianolides were screened as potential inhibitors of NF-κB in a biochemical assay that was designed using pure NF-κB heterodimer, pseudoguianolides and a ~1000 bp palindromic DNA fragment harboring two NF-κB recognition sequences. By comparing the relative amount of free DNA fragment to the NF-κB – DNA complex, in a routine agarose gel electrophoresis, the destabilizing effect of a compound on the complex is estimated. The results of the assay and the following structure-activity relationship study, allowed the identification of several relevant structural features in the pseudoguaianolide skeleton, which are necessary to enhance the dissociating capacity of NF-κB–DNA complex. The most active compounds are substituted at C-3 (α-carbonyl), in addition to having the α-methylene-γ-lactone moiety which is essential for the alkylation of RelA.

Efficient poly(3-hydroxypropionate) production from glycerol using Lactobacillus reuteri and recombinant Escherichia coli harboring L. reuteri propionaldehyde dehydrogenase and Chromobacterium sp. PHA synthase genes.

Poly(3-hydroxypropionate), P(3HP), is a polymer combining good biodegradability with favorable material properties. In the present study, a production system for P(3HP) was designed, comprising conversion of glycerol to 3-hydroxypropionaldehyde (3HPA) as equilibrium mixture with 3HPA-hydrate and -dimer in aqueous system (reuterin) using resting cells of native Lactobacillus reuteri in a first stage followed by transformation of the 3HPA to P(3HP) using recombinant Escherichia coli strain co-expressing highly active coenzyme A-acylating propionaldehyde dehydrogenase (PduP) from L. reuteri and polyhydroxyalkanoate synthase (PhaCcs) from Chromobacterium sp. P(3HP) content of up to 40% (w/w) cell dry weight was reached, and the yield with respect to the reuterin consumed by the cells was 78%. Short biotransformation period (4.5h), lack of additives or expensive cofactors, and use of a cheap medium for cultivation of the recombinant strain, provides a new efficient and potentially economical system for P(3HP) production.

Extraction of glucuronoarabinoxylan from quinoa stalks (Chenopodium quinoa Willd.) and evaluation of xylooligosaccharides produced by GH10 and GH11 xylanases

Byproducts from quinoa are not yet well explored sources of hemicellulose or products thereof. In this work, xylan from milled quinoa stalks was retrieved to 66% recovery by akaline extraction using 0.5 M NaOH at 80 °C, followed by ethanol precipitation. The isolated polymer eluted as a single peak in size-exclusion chromatography with a molecular weight of >700 kDa. Analysis by Fourier transform infrared spectroscopy and nuclear magnetic resonance (NMR) combined with acid hydrolysis to monomers showed that the polymer was built of a backbone of β(1 → 4)-linked xylose residues that were substituted by 4-O-methylglucuronic acids, arabinose, and galactose in an approximate molar ratio of 114:23:5:1. NMR analysis also indicated the presence of α(1 → 5)-linked arabinose substituents in dimeric or oligomeric forms. The main xylooligosaccharides (XOs) produced after hydrolysis of the extracted glucuronoarabinoxylan polymer by thermostable glycoside hydrolases (GHs) from families 10 and 11 were xylobiose and xylotriose, followed by peaks of putative substituted XOs. Quantification of the unsubstituted XOs using standards showed that the highest yield from the soluble glucuronoarabinoxylan fraction was 1.26 g/100 g of xylan fraction, only slightly higher than the yield (1.00 g/100 g of xylan fraction) from the insoluble fraction (p < 0.05). No difference in yield was found between reactions in buffer or water (p > 0.05). This study shows that quinoa stalks represent a novel source of glucuronoarabinoxylan, with a substituent structure that allowed for limited production of XOs by GH10 or GH11 enzymes.

Arabinoxylanase from glycoside hydrolase family 5 is a selective enzyme for production of specific arabinoxylooligosaccharides.

An arabinose specific xylanase from glycoside hydrolase family 5 (GH5) was used to hydrolyse wheat and rye arabinoxylan, and the product profile showed that it produced arabinose substituted oligosaccharides (AXOS) having 2-10 xylose residues in the main chain but no unsubstituted xylooligosaccharides (XOS). Molecular modelling showed that the active site has an open structure and that the hydroxyl groups of all xylose residues in the active site are solvent exposed, indicating that arabinose substituents can be accommodated in the glycone as well as the aglycone subsites. The arabinoxylan hydrolysates obtained with the GH5 enzyme stimulated growth of Bifidobacterium adolescentis but not of Lactobacillus brevis. This arabinoxylanase is thus a good tool for the production of selective prebiotics.