A Novel Actinobacterial Cutinase Containing a Noncatalytic Polymer-Binding Domain.

The single putative cutinase-encoding gene from the genome of Kineococcus radiotolerans SRS30216 was cloned and expressed in Escherichia coli as a secreted fusion protein, designated YebF-KrCUT, where YebF is the extracellular carrier protein. The 294-amino-acid sequence of KrCUT is unique among currently characterized cutinases by having a C-terminal extension that consists of a short (Pro-Thr)-rich linker and a 55-amino-acid region resembling the substrate binding domain of poly(hydroxybutyrate) (PHB) depolymerases. Phylogenetically, KrCUT takes a unique position among known cutinases and cutinase-like proteins of bacterial and fungal origins. A modeled structure of KrCUT, although displaying a typical α/ß hydrolase fold, shows some unique loops close to the catalytic site. The 39-kDa YebF-KrCUT fusion protein and a truncated variant thereof were purified to electrophoretic homogeneity and functionally characterized. The melting temperatures (Tm) of KrCUT and its variant KrCUT206 devoid of the putative PHB-binding domain were established to be very similar, at 50 to 51°C. Cutinase activity was confirmed by the appearance of characteristic cutin components, C16 and C18 hydroxyl fatty acids, in the mass chromatograms following incubation of KrCUT with apple cutin as the substrate. KrCUT also efficiently degraded synthetic polyesters such as polycaprolactone and poly(1,3-propylene adipate). Although incapable of PHB depolymerization, KrCUT could efficiently bind PHB, confirming the predicted characteristic of the C-terminal region. KrCUT also potentiated the activity of pectate lyase in the degradation of pectin from hemp fibers. This synergistic effect is relevant to the enzyme retting process of natural fibers. IMPORTANCE To date, only a limited number of cutinases have been isolated and characterized from nature, the majority being sourced from phytopathogenic fungi and thermophilic bacteria. The significance of our research relates to the identification and characterization of a unique member of the microbial cutinases, named KrCUT, that was derived from the genome of the Gram-positive Kineococcus radiotolerans SRS30216, a highly radiation-resistant actinobacterium. Given the wide-ranging importance of cutinases in applications such as the degradation of natural and synthetic polymers, in the textile industry, in laundry detergents, and in biocatalysis (e.g., transesterification reactions), our results could foster new research leading to broader biotechnological impacts. This study also demonstrated that genome mining or prospecting is a viable means to discover novel biocatalysts as environmentally friendly and biotechnological tools.

Structural analysis of Bacillus pumilus phenolic acid decarboxylase, a lipocalin-fold enzyme.

The decarboxylation of phenolic acids, including ferulic and p-coumaric acids, to their corresponding vinyl derivatives is of importance in the flavouring and polymer industries. Here, the crystal structure of phenolic acid decarboxylase (PAD) from Bacillus pumilus strain UI-670 is reported. The enzyme is a 161-residue polypeptide that forms dimers both in the crystal and in solution. The structure of PAD as determined by X-ray crystallography revealed a ß-barrel structure and two α-helices, with a cleft formed at one edge of the barrel. The PAD structure resembles those of the lipocalin-fold proteins, which often bind hydrophobic ligands. Superposition of structurally related proteins bound to their cognate ligands shows that they and PAD bind their ligands in a conserved location within the ß-barrel. Analysis of the residue-conservation pattern for PAD-related sequences mapped onto the PAD structure reveals that the conservation mainly includes residues found within the hydrophobic core of the protein, defining a common lipocalin-like fold for this enzyme family. A narrow cleft containing several conserved amino acids was observed as a structural feature and a potential ligand-binding site.

Thermostable feruloyl esterase for the bioproduction of ferulic acid from triticale bran.

A putative alpha/beta hydrolase fold-encoding gene (locus tag TTE1809) from the genome of Thermoanaerobacter tengcongensis was cloned and expressed in Escherichia coli as a possible source of thermostable feruloyl esterase (FAE) for the production of antioxidant phenolic acids from biomass. Designated as TtFAE, the 33-kDa protein was purified to apparent homogeneity. The lipase-like sequence characteristics of TtFAE and its substrate specificity towards methyl ferulate, methyl sinapate, and methyl p-coumarate classify it as a new member of the type A FAEs. At 75 degrees C, the enzyme retained at least 95% of its original activity for over 80 min; at 80 degrees C, its half-life was found to be 50 min, rendering TtFAE a highly thermostable protein. Under different hydrolytic conditions, ferulic acid (FA) was shown to be released from feruloylated oligosaccharides prepared from triticale bran. An estimated recovery of 68 mg FA/100 g triticale bran was demonstrated by a 30% release of the total FA from triticale bran within a 5-h incubation period. Both the oxygen radical absorbing capacity values of the feruloylated oligosaccharides and free FA were also determined. Overall, this work introduces a new bacterial member to the growing family of plant cell wall degrading FAEs that at present is largely of fungal origin, and it benchmarks the bioproduction of FA from triticale bran.

Bioproduction of lauryl lactone and 4-vinyl guaiacol as value-added chemicals in two-phase biotransformation systems.

Recombinant Escherichia coli whole-cell biocatalysts harboring either a Baeyer-Villiger monooxygenase or ferulic acid decarboxylase were employed in organic-aqueous two-phase bioreactor systems. The feasibility of the bioproduction of water-insoluble products, viz., lauryl lactone from cyclododecanone and 4-vinyl guaiacol from ferulic acid were examined. Using hexadecane as the organic phase, 10 approximately 16 g of lauryl lactone were produced in a 3-l bioreactor that operated in a semicontinuous mode compared to 2.4 g of product in a batch mode. For the decarboxylation of ferulic acid, a new recombinant biocatalyst, ferulic acid decarboxylase derived from Bacillus pumilus, was constructed. Selected solvents as well as other parameters for in situ recovery of vinyl guaiacol were investigated. Up to 13.8 g vinyl guaiacol (purity of 98.4%) were obtained from 25 g of ferulic acid in a 2-l working volume bioreactor by using octane as organic phase. These selected examples highlight the superiority of the two-phase biotransformations systems over the conventional batch mode.

Practical asymmetric enzymatic reduction through discovery of a dehydrogenase-compatible biphasic reaction media.

[reaction: see text] An enzyme-compatible biphasic reaction media for the asymmetric biocatalytic reduction of ketones with in situ cofactor regeneration has been developed. In this biphasic reaction media, which is advantageous for reactions at higher substrate concentrations, both enzymes (alcohol dehydrogenase and FDH from Candida boidinii) remain stable. The reductions with poorly water-soluble ketones were carried out at substrate concentrations of 10-200 mM, and the optically active (S)-alcohols were formed with moderate to good conversions and with up to >99% ee.

New feruloyl esterases to access phenolic acids from grass biomass.

In the Sorangium cellulosum strain So ce56 genome, two putative esterase-encoding genes (loci sce1896 and sce8927) were cloned, expressed in Escherichia coli, and the resulting enzymes (designated ScFAE1 and ScFAE2) were used to assess the possible release of ferulic acid (FA) from triticale and wheat brans, and an aqueous fraction of steam-exploded wheat straw. The two polypeptides, sharing only 30% sequence identity, exhibit a typical catalytic Ser-Asp-His triad, a characteristic of α/ß-hydrolase fold proteins. Both ScFAE1 (35 kDa) and ScFAE2 (34 kDa) were purified to apparent homogeneity and comparison of their kinetic parameters indicated an apparent higher affinity of ScFAE2 than ScFAE1 towards the various feruloyl substrates. This property was reflected by the observation that ScFAE2 was capable of yielding up to 85% of FA from destarched triticale bran. In the steam-exploded wheat sample, more than 85% yield of FA or p-coumaric acid was also effected by ScFAE2 without the decomposition of valuable chemical such as furfural. The two cloned FAEs represent the first of myxobacterial origin to be characterized and they are classified as new members of the type D family of FAEs.