Biotechnological potential of VanY, a D,D-carboxypeptidase from Nonomuraea sp. ATCC 39727

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Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576

properties similar to the commercial Kitozyme biopolymer, which is used for cosmetic and pharmaceutical applications. doi:10.1016/j.jbiotec.2010.09.473 [P-I.108] Enzymatic Resolution of cis-Dimethyl 1-Acetylpiperidine-2,3Dicarboxylate for Fine Chemicals Preparation Stefano Fogal 1,∗ , Riccardo Motterle 2 , Giancarlo Arvotti 2 , Marco Galvagni 2 , Andrea Castellin 2 , Elisabetta Bergantino 1 1

University of Padova, Italy Fabbrica Italiana Sintetici, Italy Keywords: enzyme; resolution; moxifloxacin 2

Biocatalysis has become recently a promising tool for chemical synthesis. In particular, enzymes as lipases and esterases are often used to resolve racemic mixture of esters. We propose a enantiospecific enzymatic resolution in order to produce enatiopure synthons precursor of pharmaceutical active substances. The process consists in the enantiospecific hydrolysis of a water-soluble racemic mixture of cis-dimethyl 1-acetylpiperidine2,3-dicarboxylate catalyzed by Candida antarctica lipase B. The separation of product in (2R,3S) configuration from unreacted substrate in (2S,3R) configuration is easily obtained by organic phase extraction. This latter form could be used as enantiomerically pure precursor in the (4aS,7aS)-octahydro-1H-pyrrolo[3,4-b]pyridine synthesis, an expensive building block of the antibiotic Moxifloxacin. By the other hand, the hydrolysed form with (2R,3S) configuration could be used for the preparation of neuromediator analogues. Studies concerning hydrolysis and use of immobilized enzyme has been carried out in order to evaluate its industrial application. The enzyme demonstrate high enantiospecificity, concerning the (2R,3S) enantiomer, and regioselectivity, concerning the ester in position 3. The process has been subject for patent application. doi:10.1016/j.jbiotec.2010.09.474 [P-I.109] Biotechnological potential of VanY, a D,D-carboxypeptidase from Nonomuraea sp. ATCC 39727 E. Binda 1 , G.L. Marcone 1,∗ , F. Beltrametti 3 , L. Pollegioni 1,2 , G. Molla 1,2 , S. Servi 2,4 1

Dipartimento di Biotecnologie e Scienze Molecolari, Università degli Studi dell’Insubria, Italy 2 Centro di Ricerca Interuniversitario in Biotecnologie Proteiche “The Protein Factory”, Politecnico di Milano and Università degli Studi dell’Insubria, Italy 3 Actygea S.r.l, Italy 4 Dipartimento di Chimica Materiali e Ingegneria Chimica G. Natta, Politecnico di Milano, Italy Keywords: D,D-carboxypeptidase; actinomycetes; biocatalysis; heterologous expression In recent years, we have described glycopeptide self-resistance in Nonomuraea sp. ATCC 39727 the producer of A40926, which differently from other glycopeptide producing microorganisms does not contain vanHAX genes usually involved in conferring resistance. We demonstrated the involvement of the vanY gene, usually considered an ancillary and not essential resistance gene in enterococci, in determining resistance of Nonomuraea sp. to gly-

copeptides. This novel mechanism is based on the cleavage of the terminal d-alanine residue from the peptidoglycan precursor. The tetrapeptide produced by the action of the VanY carboxypeptidase is a poor substrate for glycopeptide binding, thus reducing the level of susceptibility to glycopeptides. In this work we report on the heterologous expression of VanY and its biochemical and structural characterization. A biotechnological potential of this protein is represented by the hydrolysis and reversed synthesis of D,D- and D,L-peptides and depsipeptides. In particular VanY might be used in kinetic or dynamic resolution of D,L-amino acid ester solutions, as well as in the synthesis of peptides composed of L-amino acid moieties. The VanY synthetic gene, which codon usage was optimized for E. coli expression, was subcloned into a pET24b(+) vector carrying six additional histidines (to facilitate protein identification and purification). Protein expression was carried out in BL21(DE3)Star E. coli strain and purification of the soluble protein was performed by affinity chromatography: the overall yield was of 3 mg of pure protein/L of fermentation broth. The determination of D,D-carboxypeptidase activity was carried out measuring the release of d-Ala from the D-alaD-ala or Acetyl-Lys-D-Ala-D-Ala substrate using a coupled assay based on d-amino acid oxidase and o-dianisidine. The characterization of kinetic properties of recombinant VanY from Nonomuraea on various compounds of biotechnological interest will be presented. * COST Action CM0701 “Cascade Chemoenzymatic Processes – New Synergies between Chemistry and Biochemistry” doi:10.1016/j.jbiotec.2010.09.475 [P-I.110] Effect of medium composition on the proliferation, morphology and exopolysaccharides production of Aureobasidium pullulans S.Y. Yoon 1,∗ , E.S. Hong 1 , S.H. Kim 1 , P.C. Lee 1 , M.S. Kim 2 , Y.W. Ryu 1 1

Ajou University, Korea, Republic of Korea Research Institute of Bioscience and Biotechnology, Korea, Republic of Keywords: Aureobasidium pullulans; exopolysaccharides; ashbya gossypii; morphology 2

Aureobasidium pullulans has been known as microorganism that produce a large amount of EPS (exopolysaccharides) used for health foods and cosmetics. For the purpose of production of higher EPS from A. pullulans, medium composition was investigated. Especially, changes of morphology and proliferation by medium composition are important parameters to improve EPS production of A. pullulans. Therefore, various carbon and nitrogen sources were examined. As a result, there was a distinguishable morphological change in mycelium grown between organic and inorganic nitrogen sources. Furthermore, organic nitrogen sources played an important role in a lot of EPS production and morphological change. To investigate the best nitrogen source, several nitrogen sources, namely extract of Ashbya gossypii, beef extract, malt extract, corn steep powder, corn steep liquor, yeast extract and soytone peptone, were examined. As a result, extract of A. gossypii was good substitute to yeast extract as organic nitrogen source in morphology to produce EPS. And, after adaptive one-factor-at-atime (OFAT) method experimentation was carried out by previous study, the relative importance of medium components for maximum glucan production was evaluated by using Plackett-Burman design (PBD). Also, on the basis of PBD analysis, Box-Behnken design and Response Surface Method (RSM) were carried out to