Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576 S379

Conclusion: In conclusion, our results showed that the immo-bilized EH preparation was robust biocatalyst for the preparative-scale production of enantiopure SO and PED.

doi:10.1016/j.jbiotec.2010.09.465

[P-I.100]

Evolution of Chymotrypsin-Like Enzymes for Specific HydrolyticBioconversions of Industrial Interest

Volonté Federica 1,2,∗, Tessaro Davide 1,2, Molla Gianluca 1,2,D’Arrigo Paola 1,2, Servi Stefano 1,2, Pollegioni Loredano 1,2

1 Centro Interuniversitario di Ricerca in Biotecnologie Proteiche “TheProtein Factory”, Politecnico di Milano and Università degli Studidell’Insubria, Varese, Italy2 Flamma S.p.A, via Bedeschi 22, 24040 Chignolo D’isola (BG), ItalyKeywords: Chymotrypsin-like; Bioconversion; Site-saturationmutagenesis; Protein engineering

In order to possess an efficient hydrolytic enzyme active onvarious compounds of pharmaceutical interest and to simulta-neously overcome the European limitations related to the useof enzymes from animal sources, we planned the production ofevolved chymotrypsin-like activities as recombinant proteins in E.coli. Preliminary investigations showed that microbial proteaseswere most efficiently expressed in E. coli as compared to mam-malian ones. In particular the enzyme named MICRO ctr wassuccessfully expressed as a fully soluble and mature, active enzyme.The recombinant enzyme was purified by a single chromatographic(affinity) step; it is active on classical substrates of chymotrypsinsuch as caseine, azocaseine and N-succinyl-L-phenylalanine-p-nitroanilide (SPNA). Noteworthy, MICRO ctr shows a 15-fold lowerKm for SPNA as substrate than bovin chymotrypsin. When therecombinant enzyme is used on racemic mixtures of a chiral�-substituted phenylpropionate methyl ester a 3-fold faster con-version is observed, as well as a comparable e.e. of the final S-acidproduct (≈90%) as compared to the commercial pancreatic chy-motrypsin from bovin.

The active site residues of MICRO ctr putatively involved in theenantioselectivity towards the S-enantiomer were identified by adocking analysis performed using a model of the 3D structure ofthe enzyme. The position of the side chains belonging to the cat-alytic triad resembles that observed in chymotrypsin but pocketS1 for substrate binding seems larger in MICRO ctr. Site-saturationmutagenesis was then performed on seven active site residues ofMICRO ctr and the libraries were screened on various methyl/ethylester derivatives of industrial interest by employing a new colori-metric assay based on the coupling with an alcohol oxidase activeon the alcoholic group released by the serine-protease.

In conclusion, MICRO ctr (and its evolved variants) represents asuitable alternative to animal chymotrypsin and thus an innovativebiotool for biopharmaceutical processes.

Project: Metadistretti Regione Lombardia 2007 “Enzimi per Bio-catalisi”

doi:10.1016/j.jbiotec.2010.09.466

[P-I.101]

Improving Protein Stability And Enzyme Recovery Under StressConditions By Use Of Recombinant Molecular Chaperones

Semra KOCABIYIK ∗, Sema AYGAR, Bilsev KOYUNCU

Middle East Technical University, Department of Biological Sciences,TurkeyKeywords: protein folding; chaperone; heat shock; stability

Molecular chaperones are highly conserved stress proteins thatplay vital roles in mediating polypeptide folding, signalling, chap-eroning and cytoprotection inside cells. The major protein foldingchaperones Hsp70 (Dna K) and Hsp60 (GroEL) in eucarya and ineubacteria play a central role in the heat shock response of thesecells. In most archaea small heat-shock proteins (sHSP) togetherwith thermosomes (possibly in cooperation with the co-factorprefoldin) are the ubiquitous molecular chaperones. Much of thecurrent work is aimed at delineating molecular and cellular mech-anisms by which these protein families function in maintaininghomeostasis and modulating stress response. Thus, the increasedunderstanding of the chaperoning system enables us to utilize themfor enhanced protein quality or quantity control in biotechnologicalapplications.

In this study our purpose is to take the advantage of therecombinant expression of stable archaeal chaperones for potentialbiotechnological applications (e.g. improving the thermostability,disaggregation and proteolysis inhibitions). To this end, we clonedand recombinantly expressed the thermosome (THS), prefoldin(PF), and a small sHSP protein from thermoacidophilic archaeaonThermoplasma volcanium(Tpv). We showed that these chaperonesare both functionally stable and fully functional under stress con-ditions. Recombinant Tpv THS together with tpvPF reduced theaggregation of chemically denatured citrate synthase from pigheart and also promoted its renaturation in an ATP dependent fash-ion. The recovery of the enzyme activity was about 5- fold moreas compared to spontaneous renaturation. The recombinant sHSPprevented the citrate synthase from aggregating under heat-shockand chemical denaturation conditions. In addition, viability of E. colicells over expressing tpv sHSP compared with the control culturewas enhanced about 15-fold after 60 min at 52 ◦C.

This result indicates that tpv chaperones can be potentiallyemployed for improving the utility and stability of enzymes in var-ious applications, including biochips, bioseparation or diagnosticimmunological assays.

doi:10.1016/j.jbiotec.2010.09.467

[P-I.102]

Efficient Immobilization of Epoxide Hydrolase onto Florisil forEnantioselective Resolution of Racemic Styrene Oxide

Deniz Yildirim ∗, S.Seyhan Tükel, Dilek Alagöz, Özlem Alptekin

Cukurova University,Sciences and Letters Faculty,Chemistry Depart-ment, TurkeyKeywords: Epoxide hydrolase; Racemic epoxide; Kinetic Resolu-tion; Florisil

Introduction: Enantiopure epoxides and their vicinal diols arekey intermediates in the preparation of bulk drug substances andfine chemicals because of their versatile reactivity [1]. Asymmet-ric kinetic resolution of racemic epoxide with epoxide hydrolases(EHs; EC 3.3.2.3) gives remaining epoxide and corresponding vic-inal diol in an enantiomerically pure form. Of the methods used,

S380 Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576

EH-catalyzed kinetic resolution of racemic epoxides have muchattention for preparing such substances since EHs are ubiquitousin nature, cofactor-independent enzymes and show high enantio-,chemo- and regioselectivities on a broad variety of substrates [2-3].

Method: In this study, EH from Aspergillus niger was covalentlyimmobilized onto Florisil [4]. The activity of free and immobi-lized EH were determined with HPLC [5]. Kinetic resolution ofracemic styrene oxide was carried out under predetermined opti-mum conditions.Enantiomeric excess (ee) of styrene oxide (SO) and1-Phenyl-1,2-ethanediol (PED) were analyzed by using chiral HPLCequipped with a Shodex ORpak CDC chiral column.

Result: The rate of rac-SO hydrolysis was measured at substrateconcentrations ranging from 1 to 20 mM. The apparent Km and Vmax

values were found as 0.19 mM and 6.12 U mg/prot. and 0.64 mMand 10.4 U mg/prot. for free and immobilized EH preparations,respectively. Catalytic efficiency of immobilized EH was 1.27x104

s-1M-1and this value was 50% of that of the free EH. The ee of (S)-SOand (R)-PED were determined as 99 and 97% for free, 99 and 99% forimmobilized EH, respectively after 2 h reaction time. The reusabil-ity of immobilized EH was evaluated by using batch-type columnreactor (1x5 cm) and it was determined that immobilized EH wasretained about 100% of its initial activity after 10 reuses.

Conclusion: In conclusion, EH from A.niger immobilized ontoFlorisil showed good enzymatic properties that were slightly dif-ferent from those of the free EH.

doi:10.1016/j.jbiotec.2010.09.468

[P-I.103]

Influence of The Immobilized (R)-Hydroxynitrile Lyase FromPrunus pseudoarmenaca on (R)-Cyanohydrins Synthesis

Dilek Alagöz ∗, S.Seyhan Tükel, Deniz Yildirim, Özlem Alptekin

Cukurova University, Sciences and Letters Faculty, Chemistry Depart-ment, TurkeyKeywords: Hydroxynitrile lyase; Prunus pseudoarmeniaca; (R)-cyanohydrins; Eupergit

Hydroxynitrile lyases (HNLs) (EC 4.1.2.x) are one of the keyenzymes in cyanogenic plants, catalyzing the final step in the biode-gredation pathway of cyanogenic glycosides releasing HCN andthe corresponding carbonyl components [1]. In vitro, HNLs catal-yse enantioselective C-C bond formation via the addition of HCN toaldehydes and ketones to yield the corresponding optically activecyanohydrins, which are synthetically versatile building blocks forthe synthesis of fine chemicals, pharmaceuticals and agrochemi-cals [2]. To improve economic feasibility of HNLs in pharmaceutical,industrial and technological processes, soluble HNLs were immo-bilized by using different immobilization procedures [3-7]. In ourprevious study, partially purified HNL from Prunus pseudoarmeni-aca was successfully immobilized onto Eupergit supports and lyaseactivity was also characterized [8].

In this study, partially purified HNL from the seeds of Prunuspseudoarmeniaca (a wild apricot) was immobilized onto Euper-git C 250L. Carboligation activities of free and immobilized HNLwere determined according to Bhunya et.al.[9]. The yield and ena-tiomeric excess (ee) of synthesized cyanohydrins were analyzed byHPLC equipped with a UV dedector and ORPak CDC 453-HQ chiralcolumn (4.6 x 150 mm) at 220 nm.

The immobilized HNL onto Eupergit C 250L was applied in theenantioselective addition of HCN to benzaldehyde, acetophenone,ethylmethylketone and isobuthylmethylketone to give their (R)-cyanohydrins. The maximum yield and enantiomeric excess werecalculated as >98% and >99%, respectively for all (R)-cyanohydrins.

Our results showed that free and immobilized HNLs were verypromising catalysts in the synthesis of (R)-cyanohydrins.

doi:10.1016/j.jbiotec.2010.09.469

[P-I.104]

Immobilization and Characterization of Hydroxynitrile LyaseFrom Prunus armeniaca onto Eupergit Supports

Dilek Alagöz ∗, Deniz Yildirim, Özlem Alptekin, S.Seyhan Tükel

Cukurova University, Sciences and Letters Faculty, Chemistry Depart-ment, TurkeyKeywords: hydroxynitrile lyase; Prunus armeniaca; Eupergit; (R)-Mandelonitrile

During the last decades, there has been increased interestin using enzymes for asymetric synthesis and kinetic resolutionto obtain pure enantiomers [1]. Despite the natural function ofHydroxynitrile lyases (HNLs) (EC 4.1.2.x) as metabolic enzymes-cleavage of C-C bond- they are powerful in vitro catalyst for thecyanohydrin reaction – carboligation process. Cyanohydrins arebifunctional molecules and therefore constitute a particularly use-ful class of compounds for synthetic purpose. In order to improvethe HNLs robustness, cross-linked crystals of HNL’s, immobilizedHNL onto Eupergit supports and HNL’s adsorbed on solid supportsor encapsulated in sol–gels and PVA-gels have been used as cata-lysts in the synthesis of optically active cyanohydrins [2-8].

In this study, HNL from seeds of apricot (Prunus armeniaca) waspartially purified by (NH4)2SO4 fractionation and immobilized ontoEupergit C and Eupergit C 250L for the first time. Lyase activities ofHNLs were determined by measuring the amount of benzaldehydefrom rac-mandelonitrile at 250 nm. Carboligation activities of HNLswere determined according to Bhunya et.al. [9].

Km and Vmax values for free HNL, immobilized HNL onto EupergitC and immobilized HNL Eupergit C 250L were calculated as 21 mMand 23.2 U/mg prot., 28.7 mM and 4.5 U/mg prot. and 17.6 mM and2.6 U/mg prot. at predetermined conditions respectively. The resid-ual activities of both immobilized HNLs after 20 reuses in a batchtype reactor were nearly 100% of their initial activities. The ther-mal stabilities of immobilized HNLs were higher than that of freeHNL at 50 ◦C. In this study, we have also demonstrated that immo-bilized HNLs could be used for the enantioselective synthesis of(R)-Mandeonitrile.

doi:10.1016/j.jbiotec.2010.09.470

[P-I.105]

Characterisation of volatile organic compounds (VOC) in differ-ent lignin and their reduction by enzymatic modification for theproduction of fibre-reinforced biopolymers

S. Koenig 1, M. Bremer 2, J. Appelt 2, A.-C. Bansleben 3, H.Unbehaun 2, G. Kerns 1,∗

1 Saxon Institute for Applied Biotechnology, Germany2 Dresden University of Technology, Germany3 Anhalt University of Applied Scienes, GermanyKeywords: lignin; VOC; laccase; biopolymer

Products made of residual lignin and lignocellulosic materialsemit volatile organic compounds (VOC). These emissions are causedby the thermal and chemical decomposition. The main sourcesfor these emissions are low molecular parts of lignin and hemi-cellulose. The emissions contain monoterpenes, sesquiterpenes,