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The conjugation of Au complexes with proteins and enzymes, generating new types of artificial metalloenzymes, has proven to be interesting and effective to obtain materials with improved properties such as higher stability, catalytic activity and selectivity. In this work, a novel method has been developed for the synthesis and design of artificial gold metalloenzymes at 50ºC in aqueous media, using two genetically modified variants of the alkalophilic lipase Geobacillus thermocatenulatus (GTL). The only difference between these two enzymatic variants is the possible coordination of the Au via active site (GTL-114) or Lid site (GTL-193). TEM analysis of the metalloenzymes revealed the formation of Au (0) nanoparticles with different structures (nanowires, nanorods, nanoshells, nanoclusters) and sizes depending on the mutant and the pH used during the synthesis. Characterisation by fluorescence spectroscopy demonstrated that conjugation of the enzyme to Au altered the tertiary structure of the protein. On the other hand, all metalloenzymes showed excellent reductase like activity. Finally, the selectivity of the enzyme-Au bioconjugates was tested in the asymmetric reduction of acetophenone to 1-phenylethanol in aqueous medium at room temperature. The protein environment played a key role in the reactivity and selectivity of the metalloenzymes, obtaining chiral nanoparticles with an enantiomeric excess of up to 39% towards (R)-1-phenylethanol after two hours of reaction using GTL-114 pH 10 as catalyst.

The ability to degrade ochratoxin A was studied in different bacteria with a well-known capacity to transform aromatic compounds. Strains belonging to Rhodococcus, Pseudomonas, and Brevibacterium genera were grown in liquid synthetic culture medium containing ochratoxin A. Brevibacterium spp. strains showed 100% degradation of ochratoxin A.Ochratoxin ¿ was detected and identified by high-performance liquid chromatographymass spectrometry (HPLC-MS) as a degradation product in the cellfree supernatants. The degradation of ochratoxin A is of public concern for food and environmental safety, because it could contribute to the development of new biological ochratoxin A detoxification systems in foodstuffs. In this study, the degradation of ochratoxin A by bacteria belonging to the food chain was demonstrated for the first time., This work was supported by the “ASER” grant (D.M 16101/7301/08) from the Italian Ministero delle Politiche Agricole Alimentari e Forestali and by grants RM2008-00002 (Instituto Nacional de Investigación Agraria y Alimentaria), AGL2008-01052, Consolider INGENIO 2010 CSD2007-00063 FUN-C-FOOD (Comisión Interministerial de Ciencia y Tecnología), and S2009/AGR-1469 (ALIBIRD) (Comunidad de Madrid).

A new strategy has been developed for site-directed immobilization/rigidification of genetically modified enzymes through multipoint covalent attachment on bifunctional disulfide-glyoxyl supports. Here the mechanism is described as a two-step immobilization/rigidification protocol where the enzyme is directly immobilized by thiol-disulfide exchange between the β-thiol of the single genetically introduced cysteine and the few disulfide groups presented on the support surface (3 μmol/g). Afterward, the enzyme is uniquely rigidified by multipoint covalent attachment (MCA) between the lysine residues in the vicinity of the introduced cysteine and the many glyoxyl groups (220 μmol/g) on the support surface. Both site-directed immobilization and rigidification have been possible only on these novel bifunctional supports. In fact, this technology has made possible to elucidate the protein regions where rigidification by MCA promoted higher protein stabilizations. Hence, rigidification of vicinity of position 333 from lipase 2 from Geobacillus thermocatenulatus (BTL2) promoted a stabilization factor of 33 regarding the unipunctual site-directed immobilized derivative. In the same context, rigidification of penicillin G acylase from E. coli (PGA) through position β201 resulted in a stabilization factor of 1069. Remarkably, when PGA was site-directed rigidified through that position, it presented a half-life time of 140 h under 60% (v/v) of dioxane and 4 °C, meaning a derivative eight times more stable than the PGA randomly immobilized on glyoxyl-disulfide agarose. Herein we have opened a new scenario to optimize the stabilization of proteins via multipoint covalent immobilization, which may represent a breakthrough in tailor-made tridimensional rigidification of proteins.

Aroma is an important sensory parameter of food products. Lactic acid bacteria have enzymatic activities that could be important in the modification of food aroma. The complete genome sequence from Lactobacillus plantarum WCFS1 shows a gene (lp_3054) putatively encoding a protein with benzyl alcohol dehydrogenase activity. To confirm its enzymatic activity lp_3054 from this strain has been overexpressed and purified. Protein alignment indicated that lp_3054 is a member of the family of NAD(P)-dependent long-chain zinc-dependent alcohol dehydrogenases. In lp_3054 all of the residues involved in zinc and cofactor binding are conserved. It is also conserved the residue that determines the specificity of the dehydrogenase toward NAD+ rather than NADP+ and, therefore, L. plantarum benzyl alcohol dehydrogenase is less active in the presence of NADP+ than in the presence of NAD+. The purified enzyme exhibits optimal activity at pH 5.0 and 30 °C. The kinetic parameters Km and Vmax on benzyl alcohol as a substrate were, respectively, 0.23 mM and 204 μmol h-1 mg-1. Besides its activity toward benzyl alcohol, it showed activity against nerol, geraniol, phenethyl alcohol, cinnamyl alcohol, and coniferyl alcohol, all of which are volatile compounds involved in determining food aroma. The biochemical demonstration of a functional benzyl alcohol dehydrogenase activity in this lactic acid bacteria species should be considered when the influence of bacterial metabolism in the aroma of food products is determined, L. plantarum WCFS1 strain was kindly provided by M. Kleerebezem from the Wageningen Centre for Food Sciences, NIZO Food Research. The technical assistance of M. V. Santamaría is greatly appreciated

It was previously reported that cell cultures from Lactobacillus plantarum CECT 748T were able to decarboxylate phenolic acids, such as p-coumaric, m-coumaric, caffeic, ferulic, gallic, and protocatechuic acid. The p-coumaric acid decarboxylase (PDC) from this strain has been overexpressed and purified. This PDC differs at its C-terminal end when compared to the previously reported PDC from L. plantarum LPCHL2. Because the C-terminal region of PDC is involved in enzymatic activity, especially in substrate activity, it was decided to biochemically characterize the PDC from L. plantarum CECT 748T. Contrarily to L. plantarum LPCHL2 PDC, the recombinant PDC from L. plantarum CECT 748T is a heat-labile enzyme, showing optimal activity at 22 °C. This PDC is able to decarboxylate exclusively the hydroxycinnamic acids p-coumaric, caffeic, and ferulic acids. Kinetic analysis showed that the enzyme has a 14-fold higher KM value for p-coumaric and caffeic acids than for ferulic acid. PDC catalyzes the formation of the corresponding 4-vinyl derivatives (vinylphenol and vinylguaiacol) from p-coumaric and ferulic acids, respectively, which are valuable food additives that have been approved as flavoring agents. The biochemical characteristics showed by L. plantarum PDC should be taken into account for its potential use in the food-processing industry, The technical assistance of M. V. Santamaría is greatly appreciated.