Laccase is a ligninolytic enzyme widely distributed in wood-rotting fungi and which is also found in a variety of molds and insects as well as some plants and bacteria. Its biological roles range from depolmerization of lignin, coal and humic acids via the oxidation of various mono- and diaromatic structures, to polymerization reactions and pigment formation in microbial cells or spores. Apart from its action in catabolic, depolymerizing and polymerizing processes, laccases have also been shown to be powerful enzymes for coupling two different molecules to create new low-molecular-weight products in high yield. In addition to their homomolecular coupling capabilities, laccases are also able to couple a hydroxylated aromatic substrate with a nonlaccase substrate of variable structure to create new heteromolecular hybrid molecules. Thus, laccases are increasingly finding applications in biotechnology in the fields of environment-friendly synthesis of fine chemicals and for the gentle derivatization of biologically active compounds e.g., antibiotics, amino acids, antioxidants, and cytostatics. Finally, oligomerization and polymerization reactions can lead to new homo- or heteropolymers and biomaterials. These may be useful in a wide range of applications including the production of polymers with antioxidative properties, the copolymerizing of lignin components with low-molecular mass compounds, the coating of cellulosic cotton fibers or wool, the coloring of hair and leathers, or the cross-linking and oligomerization of peptides.

There is an urgent need to develop new antimicrobial agents due to increasing bacterial resistance to therapeutically used drugs. Most methicillin-resistent Staphylococcus aureus (MRSA) strains are resistent not only to b-lactams, but also to most other antimicrobial agents.1) Penicillin resistance among Streptococcus pneumoniae strains is widely accepted as a global problem. [2][3][4][5] Bacteria have developed several strategies for escaping the lethal action of b-lactams. It may be expected that specific circumstance will make one the more effective stragegy than the other. 6) Much effort has been devoted to the discovery of drugs which would not be cleaved by b-lactamases of pathogenic strains and which have suitable physicochemical and pharmacodynamic profiles. 7,8) The modifications of b-lactam antibiotics could not keep pace with the development of resistance in the pathogenic microorganisms, so that numerous bacteria, among them multidrug resistant Staphylococcus strains, can no longer be treated with the currently available b-lactam antibiotics. 1,9,10) Besides the modification of existing antibiotics by chemical or biochemical methods the coupling of presently used antibiotics with other bioactive compounds or components from them which are not in use till now is a promising way to generate novel molecules with improved therapeutic properties.Laccase (benzenediol:oxygen oxidoreductase, EC 1.10.3.2), classically considered a hydroquinone oxidizing enzyme, is able to oligomerize molecules. Up to now main application fields of this enzyme are waste detoxification, textile dye transformation, biosensors and diagnostic application, where the capability to catalyze polymerization reactions is used. 11-13)Recently we reported about our synthetic results on coupling reactions with laccase.14-17) Now we have employed laccase to achieve derivatisation of b-lactam antibiotics and to couple them with derivatives of 2,5-dihydroxybenzoic acid. These derivatives are structurally related to the ganomycins, a new chemical class of antibacterial compounds 18) and to other antibacterial active isolates 19,20) therefore interesting as coupling partner for b-lactams using laccase as initiator of the reaction to produce novel hybridantibiotics by biotransformation.The use of laccase for the derivatisation of antibiotics is limited to a few examples including the phenolic oxidation of 7-(4-hydroxyphenylacetamido)cephalosporinic acid, 21) the dimerization of penicillin X 22) and the oxidative coupling of hydroquinone and mithramicine. 23) In the examples realized to date, the sought object of enhancement of the bioactive effect has not been achieved. 21-23)The aim of this study was (i) to investigate whether laccase can be used for the synthesis of novel penicillins by heteromolecular coupling of two different compounds, (ii) to characterize the products of the reaction, and (iii) to analyze the biological activity of the novel penicillins. Results and DiscussionBiotransformation of Amoxicillin and Ampicillin by Laccase of Tr...

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The efficient enzyme-catalysed reaction of morpholines as model structures for bioactive compounds with para-dihydroxylated aromatic systems was carried out using the oxidoreductase laccase and atmospheric oxygen to produce eight novel morpholine-substituted aromatics. The laccase of Myceliophthora thermophila was used for cross-linking morpholines containing primary or secondary amino groups with para-dihydroxylated laccase substrates. We demonstrate that not only primary amino groups, but also secondary amino groups, are able to couple with para-dihydroxylated aromatic systems in laccase-catalysed reactions. The resulting model products (yields up to 80%) were isolated, structurally characterized and tested for their antibacterial, antifungal and cytotoxic activities. Four of the eight products showed low to moderate growth inhibition against several Gram-positive and -negative bacterial strains and against the yeasts Candida maltosa and Candida albicans. The antibacterial and antifungal activities were determined by an agar disc diffusion test and a modified method according to the EUCAST discussion document E.Dis 7.1 [Rodríguez-Tudela et al. (2003) Clin. Microbiol. Infect. 9, i-viii] for the evaluation of MIC (minimal inhibitory concentration). Differences in cytotoxicity against the human urinary bladder carcinoma cell line 5637 are discussed.

Sixteen novel cephalosporins were synthesized by amination of 2,5-dihydroxybenzoic acid derivatives with the aminocephalosporins cefadroxil, cefalexin, cefaclor, and the structurally related carbacephem loracarbef using laccases from Trametes sp. or Myceliophthora thermophila. All products inhibited the growth of several Gram positive bacterial strains in the agar diffusion assay, among them methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci. The products protected mice against an infection with Staphylococcus aureus lethal to the control animals. Cytotoxicity and acute toxicity of the new compounds were negligible. The results show the usefulness of laccase for the synthesis of potential new antibiotics. The biological activity of the new compounds stimulates intensified pharmacological tests.

Corollosporine isolated from the marine fungus Corollospora maritima and N-analogous corollosporines are antimicrobial substances. Owing to the basic structure of the N-analogous corollosporines, they have become an attractive target for laccase-catalyzed derivatisation. In this regard we report on the straightforward laccase-catalyzed amination of dihydroxylated arenes with N-analogous corollosporines. In biological assays the obtained amination products are more active than the parent compounds.

The rapidly increasing problem of antimicrobial-drug resistance requires the development of new antimicrobial agents. The laccase-catalyzed amination of dihydroxy aromatics is a new and promising method to enlarge the range of currently available antibiotics. Thirty-eight potential 1,2- and 1,4-hydroquinoid laccase substrates were screened for their antibacterial and cytotoxic activity to select the best substrates for laccase-catalyzed coupling reaction resulting in potent antibacterial derivatives. As a result, methyl-1,4-hydroquinone and 2,3-dimethyl-1,4-hydroquinone were used as parent compounds and 14 novel cephalosporins, penicillins, and carbacephems were synthesized by amination with amino-β-lactam structures. All purified products were stable in aqueous buffer and resistant to the action of β-lactamases, and in agar diffusion and broth micro-dilution assays, they inhibited the growth of several Gram-positive bacterial strains including multidrug-resistant Staphylococcus aureus and Enterococci. Their in vivo activity and cytotoxicity in a Staphylococcus-infected, immune-suppressed mouse model are discussed.