ANTIMICROBIALS, PATHOGENS ANDDISEASE New Biotechnology · Volume 31S · July 2014

Antimicrobials, pathogens and disease

PS-01

Genomics-based discovery of macrolide glycosyltrans-ferase from Bacillus sp.

Won-Gon Kim ∗, Ji Yeong Park, Hyun Ju Kim

Korea Research Institute of Bioscience and Biotechnology, SouthKorea

Glycosylation of pharmacologically active secondary metabo-lites is an attractive method to improve their biological activityas well as pharmacokinetics. The availability of suitable glycosyl-transferases for naturally unglycosylated compounds, however, islimited because of substrate specificity. Recently, Bacilli have beenused as a source for the isolation of GT enzymes involved in themodification of aromatic or bulky substrates. In an effort to dis-cover enzymes for the glycosylation of a macrolide compound, weidentified putative glycosyltransferases by the genomic analysisof Bacillus sp. that produces various glycosylated macrolides. Theselected glycosyltransferases were screened, leading to the iden-tification of a glycosyltransferase that could catalyze the transferof a glucose residue from UDP-�-D-glucose to the macrolide com-pound. Using NMR and MS analysis, we determined the chemicalstructures of the new products to be a new glucosylated macrolidewhich exhibited improved water solubility. These data showedthat genomics-based discovery of glycosyltransferase from Bacillussp. was an effective strategy for the isolation of a suitable glycosyl-transferase for naturally unglycosylated compounds.

http://dx.doi.org/10.1016/j.nbt.2014.05.901

PS-02

Antifungal protein of seed coat extracts of Theobromacacao L. during fermentation

Fahrurrozi Fahrurrozi1,∗, Claudia Bahmann1, Nicolas Niemenak2,Reinhard Lieberei1, Bernward Bisping1

1 University of Hamburg, Germany2 University of Yaounde I, Cameroon

Seed coat is an important tissue for the regulation of imbibitionand maintenance of the integrity of seed, and it is also the first seedbarrier encountered by pests and pathogens. Seed cotyledons con-tain an array of proteins that may be involved in the protectionof quiescent seeds against fungi. In a previous study (Fahrurroziet al., 2013) we found that the seed coat from Theobroma cacaoL. seeds contains an antifungal activity. Seed coat extract caninhibit growth of fungi (e.g. Aspergillus niger, Penicillium citrinum,Penicillium purpurogenum, Penicillium roquefortii) and yeasts (e.g.Candida lipolytica, Candida krusei, Rhodotorula rubra, Rhodotorulamucilaginosa, Saccharomyces cerevisiae). 25 and 10 mg/mL of seedcoat extract can inhibit growth of fungi and yeasts respectively.By separation of seed coat proteins using SDS-PAGE 17 proteinsbands were found. Analysis of the bands using mass spectrometryshowed that 3 proteins have antifungal effect namely: glucanase,chitinase and osmotin (Bahmann, 2014). Further Niemenak (per-

sonal communication) found 4 proteins that have antifungal effectnamely: glucan endo-1,3-beta-glucosidase, chitinase, 2S albuminstorage protein, nonspecific lipid-transfer protein. During the fer-mentation process the seed coat proteins seem to be degraded,probably caused by an increase in temperature and by activity ofproteases produced by microbes during the fermentation. To findout more details about the degradation of proteins during the fer-mentation, we are currently analyzing 28 protein bands, which weextracted during the fermentation. Temperature characterizationshowed that the seed coat protein is not stable at 40 and 50 ◦C for24 h.

http://dx.doi.org/10.1016/j.nbt.2014.05.902

PS-03

Inactivation of microbial biofilms by visible light witha porphyrinic photosensitizer

Sandra Beirão, Sara Fernandes, Joel Coelho, Adelaide Almeida,Maria da Graca Neves, Maria do Amparo Faustino, João Tomé,Angela Cunha ∗

University of Aveiro, Portugal

Biofilms are aggregates of microbial cells imbedded in amatrix composed essentially by water and extracellular poly-meric substances (EPS). The matrix provides a first line of defenseagainst biological attack, environmental stress, and biocide diffu-sion, making biofilms a challenge to conventional antimicrobialapproaches.

The photodynamic inactivation (PDI) of microorganismsrelies on the interaction of a non-toxic photosensitizer, molec-ular oxygen and light. This study aimed the assessmentof the photodynamic effect on the matrix of model Pseu-domonas aeruginosa biofilms, using the tetra-cationic porphyrinderivative tetra-iodide 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin (Tetra-Py+-Me) as photosensitizer (PS) and white light(380–700 nm) at an irradiance of 40 W m−2. The photodynamicinactivation of imbedded cells, in single-species or mixed biofilmsof Staphylococcus aureus, Pseudomonas aeruginosa and Candida albi-cans was also determined.

A reduction of 81% in the polysaccharides content of matrix ofP. aeruginosa biofilms was observed after treatment with a light doseof 64.8 J cm−2 and 20 �M of Tetra-Py+-Me. The photosensitizationwith Tetra-Py+-Me also caused inactivation of cells in all testedbiofilms. The maximum reduction factors were 3, 7 and 6 logs, forbiofilms of P. aeruginosa, S. aureus and C. albicans, respectively. Inmixed biofilms, the inactivation of S. aureus was as efficient as insingle-strain biofilms (7 log reduction in colony counts) but wasless efficient (5 log) for the yeast.

Photosensitization with Tetra-Py+-Me caused EPS destructionand a significant inactivation of cells. PDI may, therefore, beregarded as a promising approach for biofilm control, even in casesof bacteria-yeast mixed assemblages.

http://dx.doi.org/10.1016/j.nbt.2014.05.903

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