Laccase-catalyzed cross-linking of amino acids and peptides with dihydroxylated aromatic compounds

In order to design potential biomaterials, we investigated the laccase-catalyzed cross-linking between l-lysine or lysine-containing peptides and dihydroxylated aromatics. l-Lysine is one of the major components of naturally occurring mussel adhesive proteins (MAPs). Dihydroxylated aromatics are structurally related to 3,4-dihydroxyphenyl-l-alanine, another main component of MAPs. Mass spectrometry and nuclear magnetic resonance analyses show that the ε-amino group of l-lysine is able to cross-link dihydroxylated aromatics. Additional oligomer and polymer cross-linked products were obtained from di- and oligopeptides containing l-lysine. Potential applications in medicine or industry for biomaterials synthesised via the three component system consisting of the oligopeptide [Tyr-Lys]₁₀, dihydroxylated aromatics and laccase are discussed.     

African grey parrots (Psittacus erithacus) use inference by exclusion to find hidden food

Exclusion allows the detection of hidden food when confronted with the choice between an empty and a potentially baited food location. However, exclusion may be based on avoidance of the empty location without drawing inferences about the presence of the food in the baited location. So far, such inferences have been demonstrated in the great apes only: after seeing an experimenter eating one of two food types, which both had been hidden previously in two boxes, the apes were able to choose the box that still contained the other food type. African grey parrots are capable of exclusion, and we here assessed if they are capable of inference by exclusion. In our task, two different but equally preferred food items were hidden in full view of the birds under two opaque cups. Then, an experimenter secretly removed one food type and showed it to the bird. Similarly to the apes, one out of seven parrots significantly preferred the baited cup; control conditions rule out that its choice was based on associative learning or the use of olfactory cues. Thus, we conclude that-like the apes-some grey parrots are able to infer the location of a hidden food reward.     

Biotransformation and reduction of estrogenicity of bisphenol A by the biphenyl-degrading Cupriavidus basilensis

The biphenyl-degrading Gram-negative bacterium Cupriavidus basilensis (formerly Ralstonia sp.) SBUG 290 uses various aromatic compounds as carbon and energy sources and has a high capacity to transform bisphenol A (BPA), which is a hormonally active substance structurally related to biphenyl. Biphenyl-grown cells initially hydroxylated BPA and converted it to four additional products by using three different transformation pathways: (a) formation of multiple hydroxylated BPA, (b) ring fission, and (c) transamination followed by acetylation or dimerization. Products of the ring fission pathway were non-toxic and all five products exhibited a significantly reduced estrogenic activity compared to BPA. Cell cultivation with phenol and especially in nutrient broth (NB) resulted in a reduced biotransformation rate and lower product quantities, and NB-grown cells did not produce all five products in detectable amounts. Thus, the question arose whether enzymes of the biphenyl degradation pathway are involved in the transformation of BPA and was addressed by proteomic analyses.

     

Formation of coumarines during the degradation of alkyl substituted aromatic oil components by the yeast <Emphasis Type="Italic">Trichosporon asahii</Emphasis>

In this study, we investigated the ability of the yeast Trichosporon asahii SBUG-Y 833 to assimilate phenylalkanes with alkyl chain lengths from 7 to 12 carbon atoms, and we describe for the first time the formation of coumarines via a novel degradation pathway other than the normal terminal and ß-oxidation pathway of the alkyl residues. Besides benzoic acid and its further oxidation products, six new metabolites were identified. These were the three coumarines—4-hydroxycoumarin, 4,6-dihydroxycoumarin, 4,8-dihydroxycoumarin—and the three alkyl substituted aromatic acids—7-phenylheptanoic acid, 2-hydroxyphenylheptanoic acid, and 2-hydroxyphenylpropanoic acid. 4-Hydroxycoumarin was the main extracellular metabolite during the degradation of both odd- and even-chain phenylalkanes and was also produced during further biotransformation of 2-hydroxyphenylpropanoic acid and trans-2-hydroxycinnamic acid. Due to the ability of T. asahii to form hydroxylated coumarines, the transformation of 7-hydroxycoumarin and 2,4-dihydroxyphenylpropanoic acid was investigated. Yeast cells supplemented with 7-hydroxycoumarin formed 6,7-dihydroxycoumarin and 4,7-dihydroxycoumarin. The transformation of 2,4-dihydroxyphenylpropanoic acid yielded to 4,7-dihydroxycoumarin as the main product. All hydroxylated coumarines were continuously accumulated and are very resistant to further oxidation. The high potential of the yeast T. asahii SBUG-Y 833 to form different hydroxylated coumarines from alkylaromatics suggests possible applications in the biotechnological production of coumarine structures with medical potential as anticoagulative and antitumor pharmaceutical.     

A new phenol oxidase produced during melanogenesis and encystment stage in the nitrogen-fixing soil bacterium Azotobacter chroococcum

Laccases are copper-containing phenol oxidases that are commonly found in many types of plant, insect, fungi and bacteria. Whilst phenol oxidases have been well characterized in fungal species, laccase-type enzymes originating from bacteria have been much less well defined. Bacteria belonging to the family Azotobacteraceae share many morphological characteristics with strains already known to exhibit polyphenol and phenol oxidase activity; and hence the aim of this work was to identify and characterize a novel laccase from the isolated strain Azotobacter chroococcum SBUG 1484 in an attempt to provide further understanding of the roles such enzymes play in physiological development. Laccase activity was clearly observed through oxidation of 2,6-dimethoxyphenol, other typical substrates including: methoxy-monophenols, ortho- and para-diphenols, 4-hydroxyindole, and the non-phenolic compound para-phenylenediamine. A. chroococcum SBUG 1484 showed production of a cell-associated phenol oxidase when grown under nitrogen-fixing conditions, and was also observed when cells enter the melanogenic and encystment stages of growth. Catechol which is structurally related to melanin compounds was also released from Azotobacter cells into the surrounding culture medium during nitrogen-fixing growth. From our results we propose that a membrane-bound laccase plays an important role in the formation of melanin, which was monitored to correlate with progression of A. chroococcum SBUG 1484 cells into the encystment stage of growth.     

Cleavage and synthesis function of high and low redox potential laccases towards 4-morpholinoaniline and aminated as well as chlorinated phenols

Laccases are able to mediate both cleavage and synthesis processes. The basis for this dual reaction capability lies in the property of the enzyme laccase to oxidize phenolic, and to some extent non-phenolic substances, to reactive radicals which can undergo on the one hand separations of small substitutents or large molecule parts from the parent compound and on the other hand coupling reactions with other radicals or molecules which are not themselves oxidizable by laccase. The cleavage of the non-phenolic compound 4-morpholinoaniline as well as the deamination of 4-aminophenol and the dechlorination of 4-chlorophenol resulted in the formation of 1,4-hydroquinone which is immediately oxidized by laccase to 1,4-benzoquinone. The formation of the 1,4-hydroquinone/1,4-benzoquinone is the rate limiting step for the synthesis of the heteromolecular dimers and trimers composed of 1,4-benzoquinone and one or two molecules of morpholine. In addition to the synthesis of new compounds from the cleavage products, 4-morpholinoaniline polymerized probably via azo groups and C-N bonds to a homomolecular dimer and trimer. Similarities and differences in cleavage and synthesis reactions catalyzed by the low redox potential laccase of Myceliophthora thermophila (0.46 V) and the high redox potential laccase of Pycnoporus cinnabarinus (0.79 V) were determined. In addition, the dependency of the cleavage and synthesis efficiencies on the (a) structure and redox potential of the laccase, (b) structure and redox potential of the substrate, (c) pH value of the buffer used, (d) incubation temperature, (e) solvent concentration, and (f) laccase activity is discussed in general.     

Identification of phenylalkane derivatives when Mycobacterium neoaurum and Rhodococcus erythropolis were cultured in the presence of various phenylalkanes

Phenylalkanes are ubiquitously found in nature as pollutants originating from oil, gas oil and petrol. Rising commercial demand for mineral oil fractions has led to the increased prevalence of environmental contamination, whereby these particular hydrocarbons are encountered by bacteria which have subsequently developed sophisticated metabolic routes for purposes of degradation. Herein a detailed analysis of these metabolic pathways in the degradation of phenylalkanes by Mycobacterium neoaurum and Rhodococcus erythropolis highlighted preponderance for the formation of certain metabolites of which 17 were identified and whereby striking differences were noticed depending specifically upon the length of the substrate’s alkyl chain. Although the degradation of even-numbered phenylalkane substrates was assumed to result in the generation of phenylacetic acid formed due to substrate terminal oxidation and subsequent β-oxidation, cultures of M. neoaurum and R. erythropolis were determined in an extracellular accumulation of odd-numbered acidic metabolites, suggesting a simultaneous presence of sub-terminal degradation mechanisms. However, results obtained from biotransformation assays containing even-chained phenylalkanoic acid intermediates as substrates revealed exclusive β-oxidative mechanisms and no generation of odd-numbered degradation products. R. erythropolis in contrast to M. neoaurum also proved viable for hydroxylation of the aromatic ring of metabolites. Interestingly, the generation of phenylacetic acid and subsequently 2-hydroxyphenyl acetic acid was monitored and entailed the presence of the lactone intermediate 2-coumaranone. These results enhance our understanding of the degradation of phenylalkanes and illustrate the potential application of such species in the bioremediation of these common environmental pollutants and in the strains’ diverse abilities to transform mineral oil compounds to new valuable products.     

Synthesis of imidazol-2-yl amino acids by using cells from alkane-oxidizing bacteria

Sixty-one strains of alkane-oxidizing bacteria were tested for their ability to oxidize N-(2-hexylamino-4-phenylimidazol-1-yl)-acetamide to imidazol-2-yl amino acids applicable for pharmaceutical purposes. After growth with n-alkane, 15 strains formed different imidazol-2-yl amino acids identified by chemical structure analysis (mass and nuclear magnetic resonance spectrometry). High yields of imidazol-2-yl amino acids were produced by the strains Gordonia rubropertincta SBUG 105, Gordonia terrae SBUG 253, Nocardia asteroides SBUG 175, Rhodococcus erythropolis SBUG 251, and Rhodococcus erythropolis SBUG 254. Biotransformation occurred via oxidation of the alkyl side chain and produced 1-acetylamino-4-phenylimidazol-2-yl-6-aminohexanoic acid and the butanoic acid derivative. In addition, the acetylamino group of these products and of the substrate was transformed to an amino group. The product pattern as well as the transformation pathway of N-(2-hexylamino-4-phenylimidazol-1-yl)-acetamide differed in the various strains used.     

Laccase-induced cross-coupling of 4-aminobenzoic acid with para-dihydroxylated compounds 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide and 2,5-dihydroxybenzoic acid methyl ester

A fungal laccase from Trametes villosa (EC 1.10.3.2 p-phenoloxidase) was used to mediate the oxidation and cross-coupling of two para-dihydroxylated benzoic acid derivatives with 4-aminobenzoic acid. The incubation of 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide and 4-aminobenzoic acid with laccase under oxygen conditions resulted in the formation of 2-(4′-carboxy-anilino)-N-(2″-hydroxyethyl)-3,6-dioxo-1,4-cyclohexadien-1-carboxamide as the main product (yield > 85%). When 2,5-dihydroxybenzoic acid methyl ester was a co-substrate of 4-aminobenzoic acid, 2-(4′-carboxy-anilino)-N-(2″-hydroxyethyl)-3,6-dioxo-1,4-cyclohexadien-1-carboxy methyl ester was produced (yield > 75%). Both products were N–C coupling dimers consisting of para-quinone and benzoic acid moieties. The formation of quinone structures in the presence of T. villosa laccase may be useful in pharmaceutical synthesis. Because of high product yields and low amount of by-products laccase of T. villosa seems to be a suitable enzyme among laccases acting at pH 5 for the synthesis of heterologous dimers.