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.

Purified recombinant cytochrome P450 52A3 and the corresponding NADPH-cytochrome P450 reductase from the alkane-assimilating yeast Candida maltosa were reconstituted into an active alkane monooxygenase system. Besides the primary product, 1-hexadecanol, the conversion of hexadecane yielded up to five additional metabolites, which were identified by gas chromatography-electron impact mass spectrometry as hexadecanal, hexadecanoic acid, 1,16-hexadecanediol, 16-hydroxyhexadecanoic acid, and 1,16-hexadecanedioic acid. As shown by substrate binding studies, the final product 1,16-hexadecanedioic acid acts as a competitive inhibitor of n-alkane binding and may be important for the metabolic regulation of the P450 activity. Kinetic studies of the individual sequential reactions revealed high V max values for the conversion of hexadecane, 1-hexadecanol, and hexadecanal (27, 23, and 69 min ؊1 , respectively), whereas the oxidation of hexadecanoic acid, 1,16-hexadecanediol, and 16-hydroxyhexadecanoic acid occurred at significantly lower rates (9, 9, and 5 min ؊1 , respectively). 1-Hexadecanol was found to be the main branch point between mono-and diterminal oxidation. Taken together with data on the incorporation of 18 O 2 -derived oxygen into the hexadecane oxidation products, the present study demonstrates that a single P450 form is able to efficiently catalyze a cascade of sequential mono-and diterminal monooxygenation reactions from n-alkanes to ␣,-dioic acids with high regioselectivity.The capability of several yeast species to use n-alkanes and other aliphatic hydrocarbons as a sole source of carbon and energy is mediated by the existence of multiple microsomal cytochrome P450 forms. Corresponding P450 genes or cDNAs have been isolated from the alkane-assimilating yeasts Candida maltosa (1-6), C. tropicalis (7-9), and C. apicola (10). The presently available 21 sequences belong to the family CYP52, according to the nomenclature of the P450 superfamily (11).The metabolic functions of P450 in these yeasts have been established thus far in the terminal oxidation of long-chain n-alkanes to fatty alcohols as the first and rate-determining step of the n-alkane degradation pathway and in the -hydroxylation of fatty acids, initiating the diterminal degradation pathway (Refs. 12-14; for a review, see Ref. 15). Sequential gene disruption revealed that in C. maltosa, four of its eight CYP52 genes, namely CYP52A3, CYP52A4, CYP52A5, and CYP52A9, are directly involved in alkane assimilation (16). After heterologous expression in Saccharomyces cerevisiae, each of the corresponding P450 isoenzymes was found to exhibit an individual substrate specificity in terms of the preferred class (n-alkanes or fatty acids) and the chain length of the hydroxylated aliphatic compounds (17,18).In the present study, we addressed the question of whether P450 monooxygenases may be involved not only in the primary hydroxylation reactions mentioned above but also in the subsequent oxidation steps leading to the formation of fatty acids and long-chain ...

Aromatic organic compounds that are present in the environment can have toxic effects or provide carbon sources for bacteria. We report here the global response of Bacillus subtilis 168 to phenol and catechol using proteome and transcriptome analyses. Phenol induced the HrcA, sigmaB and CtsR heat-shock regulons as well as the Spx disulfide stress regulon. Catechol caused the activation of the HrcA and CtsR heat-shock regulons and a thiol-specific oxidative stress response involving the Spx, PerR and FurR regulons but no induction of the sigmaB regulon. The most surprising result was that several catabolite-controlled genes are derepressed by catechol, even if glucose is taken up under these conditions. This derepression of the carbon catabolite control was dependent on the glucose concentration in the medium, as glucose excess increased the derepression of the CcpA-dependent lichenin utilization licBCAH operon and the ribose metabolism rbsRKDACB operon by catechol. Growth and viability experiments with catechol as sole carbon source suggested that B. subtilis is not able to utilize catechol as a carbon-energy source. In addition, the microarray results revealed the very strong induction of the yfiDE operon by catechol of which the yfiE gene shares similarities to glyoxalases/bleomycin resistance proteins/extradiol dioxygenases. Using recombinant His6-YfiE(Bs) we demonstrate that YfiE shows catechol-2,3-dioxygenase activity in the presence of catechol as the metabolite 2-hydroxymuconic semialdehyde was measured. Furthermore, both genes of the yfiDE operon are essential for the growth and viability of B. subtilis in the presence of catechol. Thus, our studies revealed that the catechol-2,3-dioxygenase YfiE is the key enzyme of a meta cleavage pathway in B. subtilis involved in the catabolism of catechol.

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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...