Enzymes From Marine Microorganisms For The Preparation

DIPARTIMENTO DI BIOTECNOLOGIE MEDICHE E MEDICINA TRASLAZIONALEDOTTORATO IN SCIENZE BIOCHIMICHECICLO XXX – BIO/10Enzymes from marine microorganisms for the preparation ofbiologically active moleculesDocente guida: Prof. Patrizia FerraboschiDirettore del corso di dottorato: Prof. Sandro SonninoTesi di Dottorato di:BENEDETTA GUIDIMatricola n. R10868Anno Accademico 2016/2017

A tutte le famiglie che ho avuto in questi tre anni,

« If the doors of perception were cleansed, everything would appear to man as it is, infinite. »W. Blake“In dreams begins responsibility.”W. B. Yeats“Quando la tempesta sarà finita, probabilmente non saprai neanche tu come hai fatto adattraversarla e a uscirne vivo. Anzi, non sarai neanche sicuro se sia finita per davvero. Ma su unpunto non c'è dubbio.Ed è che tu, uscito da quel vento, non sarai lo stesso che vi è entrato.”H. Murakami

ABSTRACTThis PhD project focuses on the identification, isolation and characterization of new biocatalysts ableto generate biologically active molecules with significant enantioselectivity. Through screening, weidentified marine strains, from MaCuMBA (Marine Culturable Microorganism for BiotechnologicalApplications) and BIODEEP (Biotechnologies from the deep) European project collections, whichshow a marked enantioselectivity on intermediates of molecules of biological interest.Biotransformation substrate range included pramipexole, as main target, but it also embraces othercommon building blocks for synthetic industrial preparation.The stereoselective reduction of structurally different ketones using halotolerant marine yeasts(Meyerozyma guilliermondii and Rhodotorula mucilaginosa) was studied using cells grown and bioconverted in seawater. The preparation of valuable chemicals through water-saving (bio)processesbased on the direct exploitation of seawater is a significant step towards sustainable biocatalysis. Bychoosing a suitable strain, high yields and stereoselectivity could be achieved in most cases. Notably,high chemoselectivity and enantioselectivity were observed using R. mucilaginosa in the reductionof aromatic β-ketonitriles, which allowed the recovery of the optically pure corresponding alcohols;notably, reduction with whole cells of yeasts generally gives a mixture of undesired products, asobserved with M. guilliermondii.Keto-reduction potential of thirty-three marine bacterium species was checked and afterwards thepossibility to convert this substrate directly into the optically pure amine was investigated: marinebacteria were screened to identify transaminase activity. Based on the previous results in terms ofhalotolerance and transaminase activity, the marine bacterium strain Virgibacillus pantothenticus21D was selected for the genome sequencing in order to clone and express an ω-transaminase enzyme.A recombinant non-marine ketoreductase from Pichia glucozyma (KRED1-Pglu) was used for theenantioselective reduction of various cyclic ketones including pramipexole ketone intermediate.Thanks to co-factor recycling system, the purified enzyme showed very promising results.The soluble expression of a novel ω-transaminase from a newly isolated halotolerant marinebacterium Virgibacillus pantothenticus was attained. Despite of several standard methodologiesapplied, the marine wild-type enzyme was total insoluble in E. coli host and it was satisfactorilysolubilized by one single-point mutation, allowing the characterization of the new omegatransaminase. The enzyme shows an interesting salt and solvent tolerance, in accordance to its originand it results particularly active on some interesting building block molecules.

INDEXABSTRACT . 5INTRODUCTION . 9BLUE FOR GREEN . 10BIOCATALYSIS . 13BIOCATALYTIC APPLICATIONS . 16ENZYMES . 21CLASSIFICATION . 21KINETIC PARAMETERS . 22SPECIFICITY AND SELECTIVITY . 24STABILITY. 24ENZYME ENGINEERING. 25KETO-REDUCTASES. 26TRANSAMINASES . 26TARGET MOLECULES. 27PRAMIPEXOLE . 27REFERENCES . 29AIM OF THE WORK . 33WHOLE CELL SCREENING . 35MARINE YEASTS ACTIVITY . 35MARINE BACTERIA ACTIVITY . 35RECOMBINANT ENZYME SCREENING . 35KETO-REDUCTASE ACTIVITY . 35ESTERASE AND LIPASE ACTIVITY . 35MARINE ω-TRANSAMINASE ACTIVITY. 36VIRGIBACILLUS PANTOTHENTICUS ω-TRANSAMINASE . 36WHOLE CELL SCREENING - MARINE YEASTS . 36BACKGROUND . 37MARINE YEASTS . 37KETO-REDUCTASE . 38PROJECT AIM. 41RESULTS AND DISCUSSION . 42

KETO-REDUCTASE SCREENING . 42HALOTOLERANCE SCREENING . 48MATERIALS AND METHODS . 52MATERIALS . 52CHARACTERISATION . 53MICROORGANISMS. 53GROWING MEDIUM . 55HALOTOLERANCE SCREENING . 55BIOTRANSFORMATIONS . 57PURIFICATION AND CHEMICAL CHARACTERISATION . 58REFERENCES . 60WHOLE CELL SCREENING – MARINE BACTERIA . 62BACKGROUND . 63MARINE BACTERIA. 63MARINE BACTERIA BIOCATALYSIS . 65ω-TRANSAMINASE . 66PROJECT AIM. 68RESULTS AND DISCUSSION . 68KETO-REDUCTASE . 68ω-TRANSAMINASE . 69VIRGIBACILLUS PANTOTHENTICUS . 69MATERIALS AND METHODS . 70MATERIALS . 70CHARACTERISATION . 71MICROORGANISMS. 71MEDIA AND GROWING CONDITIONS . 72BIOTRANSFORMATION. 73PURIFICATION AND CHEMICAL CHARACTERISATION . 73REFERENCES . 74RECOMBINANT ENZYMES . 76BACKGROUND . 77KETO-REDUCTASE . 77ESTERASE AND LIPASE . 78ω-TRANSAMINASE . 79

PROJECT AIM. 80RESULTS . 80KETO-REDUCTASE . 80ESTERASE AND LIPASE . 81ω-TRANSAMINASE . 82MATERIALS AND METHODS . 82MATERIALS . 82CHARACTERISATION . 82KETO-REDUCTASE . 83ESTERASE AND LIPASE . 83ω-TRANSAMINASE . 84PURIFICATION AND CHEMICAL CHARACTERISATION . 84REFERENCES . 85VPTA - Virgibacillus pantothenticus ω-transaminase . 86BACKGROUND . 87ω-TRANSAMINASE . 87SOLUBILITY ISSUE . 88MUTAGENESIS . 89PROJECT AIM. 89RESULTS AND DISCUSSIONS. 90WILD-TYPE VPTA AND SOLUBILISATION STRATEGIES . 90MUTAGENESIS . 92b . Errore. Il segnalibro non è definito.c . Errore. Il segnalibro non è definito.d . Errore. Il segnalibro non è definito.a . Errore. Il segnalibro non è definito.VPTA T16F . 94EFFECT OF pH AND TEMPERATURE . 95EFFECT OF CO-SOLVENTS AND SALTS . 96AMINO DONORS . 97AMINO ACCEPTORS . 98ENZYME KINETICS . 100MATERIALS AND METHODS . 100MARINE MICROORGANISM, GENE IDENTIFICATION AND CLONING . 100

EXPRESSION OF WILD-TYPE VPTA . 101MUTAGENESIS ON VPTA . 101EXPRESSION OF VPTA T16F . 101PURIFICATION . 102SDS-PAGE ANALYSIS . 102SPECTROPHOTOMETRIC ENZYMATIC ASSAY . 102ENZYMATIC REACTION. 103ANALYTICAL METHODS . 104REFERENCES . 104CONCLUSIONS . 108WHOLE CELL SCREENING – MARINE YEASTS . 109WHOLE CELL SCREENING – MARINE

Homologous halophilic and non-halophilic proteins have similar overall structure, secondary structure content, and number of residues involved in the formation of H-bonds. On the other hand, on the halophilic protein