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429 Chem. Listy 97, 363–520 (2003) Chem. Listy 97, 363–520 (2003) Posters P117 APPLICATION OF DEGENERATE REFERENCES OLIGONUCLEOTIDE GENE SHUFFLING FOR CONSTRUCTION OF HYBRID 1. Janssen D. B., Pries F., Ploeg J., Kazemier B., Terpstra P., HALOALKANE DEHALOGENASE Witholt B.: J. Bacteriol. 171, 6791 (1989). 2. Nagata Y., Nariya T., Ohtomo R., Fukuda M., Yano K., ANDREA JESENSKÁa, YUJI NAGATAb, Takagi M.: J. Bacteriol. 175, 6403 (1993). and JIŘÍ DAMBORSKÝc 3. Kulakova A. N., Larkin M. J., Kulakov L. A.: Microbio- logy 143, 109 (1997). a National Centre for Biomolecular Research, Faculty of Science, 4. Jesenska A., Bartos M., Czernekova V., Rychlik I., Masaryk University, Kotlářská 2, Brno, 611 37, Czech Republic, Pavlik I., Damborsky V.: Appl. Environ. Microbiol. 68, e-mail: [email protected]; bDepartment of Environmen- 3724 (2002). tal Life Sciences, Graduate School of Life Sciences, Tohoku 5. Gibbs M. D., Nevalainen K. M., Bergquist P. L.: Gene University, Sendai 980-8577, Japan 271, 13 (2001). Keywords: haloalkane dehalogenase, in vitro recombination, P118 KINETICS AND SPECIFICITY construct, substrate specificity OF HALOALKANE DEHALOGENASE LinB FROM Sphingomonas paucimobilis UT26 Halogenated organic compounds constitute one of the ZBYNĚK PROKOPa, MARTA MONINCOVÁa, largest groups of environmental pollutants as a result of their MARTIN KLVAŇAa, RADKA CHALOUPKOVÁa, widespread use in industry and agriculture. Haloalkane de- DICK B. JANSSENb, YUJI NAGATAc, halogenases hydrolytically convert halogenated aliphatic and JIŘÍ DAMBORSKÝa compounds to corresponding alcohols. Enhancement of cata- lytic properties of environmentally important enzymes using aNational Centre for Biomolecular Research, Masaryk Univer- in vitro evolution techniques is one of the obvious goal of bio- sity, Kotlářská 2, 611 37 Brno, Czech Republic; bUniversity of technology. Groningen, Nijenborgh 4, 9747 AG Groningen, The Nedher- Our study was undertaken to construct and preliminary lands; cDepartment of Life Sciences, Graduate School of Life characterise hybrid haloalkane dehalogenases. Four genes Sciences, Tohoku University, 2-1-1 Katahira, Sendai, 980-8577, cloned from different bacteria were used in this study: dhlA Japan; e-mail: [email protected] cloned from Xanthobacter autotrophicus GJ10 (ref.1), linB from Sphingomonas paucimobilis UT26 (ref.2), dhaA cloned from Rhodoccous erythropolis NCIMB13064 (ref.3) and dhmA Keywords: haloalkane dehalogenase, Sphingomonas pauci- from Mycobacterium avium N85 (ref.4). Considering relatively mobilis, Xanthobacter autotrophicus, reaction pathway, cova- low level of homology among parental genes, Degenerate lent alkyl-enzyme intermediate Oligonucleotide Gene Shuffling techniques was selected as an effective tool for of haloalkane dehalogenase genes5. Altogether twelve different combinations were construc- Steady-state and transient-state kinetic methods were ted using one pair of degenerate oligonucleotides, cloned applied to solve reaction pathway, to identify reaction inter- into pAQN vector and hybrid proteins were overexpressed in mediate and to specify the rate limiting step of catalytic Escherichia coli BL21(DE3). Preliminary screening of deha- action of haloalkane dehalogenase LinB from bacterial strain logenating activity was conducted with resting cells and six Sphingomonas paucimobilis UT26 (ref.1). The steady-state substrates representing different classes of halogenated experiments involved direct monitoring of LinB activity by aliphatic compounds. Four out of twelve hybrid proteins keep isothermal titration calorimetry and initial rate of product good expression and ten proteins showed obvious catalytic formation measurements using gas chromatography. Stopped- activity. Some hybrid proteins showed dehalogenating activity -flow fluorescence and rapid-quench-flow techniques were despite of changes in position of catalytic residues within the applied for the transient-state kinetics measurements. Addi- active site. Comparison of relative activities determined for tionaly, steady-state inhibition experiments and transient- the hybrid enzymes with the activities of wild type enzymes state binding experiments were employed to find out leaving suggests that constructs do not possess novel substrate speci- ability of both products (halide and alcohol) during dehalo- ficities. genation reaction. Work on more efficient expression systems to obtain The results showed that export of products as well as im- higher expression level of hybrid haloalkane dehalogenase is port of substrates into the active site of LinB are fast proces- under progress recently. ses reaching rapid equilibrium. This fast exchange of the ligands between the active site and bulk solvent can be ex- plained by wide opening of the entrance tunnel and large active site of LinB. In contrary, the release of the halide ion from narrow active site after the reaction was found to be 429 Chem. Listy 97, 363–520 (2003) Posters slow rate limiting step for another haloalkane dehalogenase, All strains were grown in shake flasks with 90 g.l–1 glucose enzyme DhlA from Xanthobacter autotrophicus GJ10 (ref.2). medium and harvested when glucose concentration fell below The actual cleavage of the carbon-halogen bond was found to 10 g.l–1, which was within 10–15 hours. Between 120 and be fast step in both enzymes. Further the results confirmed, 250 U PDC/g dry cell weight were produced. that the reaction proceeds via a covalent alkyl-enzyme inter- PAC production in an aqueous/organic two-phase system mediate. Using bromocyclohexane, chlorocyclohexane and with whole cells at 23 °C resulted in the highest PAC concen- 1-chlorohexane as model substrates, hydrolysis of this inter- tration with C. utilis and in the lowest by-product acetoin for- mediate was found to be the slowest step in the catalytic cycle mation with C. tropicalis. In a single phase system, cell free of LinB. The alkyl-enzyme complex was highly accumulated C. tropicalis PDC also formed the lowest amount of by-pro- due to the fast dehalogenation step following the slow hydro- duct acetoin. Lower acetoin formation in both systems indi- lyses of this intermediate. The study provides a basis for the cates that the formation of this by-product is a characteristic analysis of kinetic steps in hydrolysis of environmentally im- of the PDC itself. portant substrates by the action of LinB. C. utilis and S. cerevisiae PDC were very stable with half- -lives of nearly two weeks at 23 °C. The half-lives were even REFERENCES slightly longer in whole cells. In contrast C. tropicalis PDC was very unstable (half-life 3 days in crude extract and less 1. Nagata Y., Miyauchi K., Damborsky J., Manova K., An- than one day in whole cells). The deactivating effect of sorgova A., Takagi M.: Appl. Environ. Microb. 63, 3707 50 mM benzaldehyde was confirmed, generally reducing the (1997). half-lives by 50 % or more. K. marxianus PDC was stabilized 2. Schanstra J. P., Kingma J., Janssen D. B.: J. Biol. Chem. by the addition of protease inhibitors while these had no in- 271, 14747 (1996). fluence on PDC stability for the other strains. In summary, C. utilis PDC was confirmed to be the best catalyst for PAC production with high stability (half-life of P119 COMPARISON OF FOUR YEAST PYRUVATE nearly two weeks at 23 °C) and highest final PAC concentra- DECARBOXYLASES FOR tions. C. tropicalis PDC had the advantage of lower by-pro- R-PHENYLACETYLCARBINOL PRODUCTION duct acetoin formation but was very unstable and yielded less PAC. GERNALIA SATIANEGARAb, CINDY GUNAWANb, ALLEN K. CHENb, MICHAEL BREUERa, BERNHARD HAUERa, PETER L. ROGERSb, P120 IMPROVED PRODUCTION OF Candida utilis and BETTINA ROSCHEb PYRUVATE DECARBOXYLASE FOR BIOTRANSFORMATION aBASF-AG, Fine Chemicals and Biocatalysis Research, 67056 Ludwigshafen, Germany; bSchool of Biotechnology and Bio- ALLEN K. CHENb, MICHAEL BREUERa, molecular Sciences, University of New South Wales, Sydney BERNHARD HAUERa, PETER L. ROGERSb, NSW 2052, Australia, e-mail: [email protected] and BETTINA ROSCHEb aBASF-AG, Fine Chemicals and Biocatalysis Research, 67056 Keywords: biotransformation, enzyme stability, pyruvate de- Ludwigshafen, Germany; bSchool of Biotechnology and Bio- carboxylase, R-phenylacetylcarbinol molecular Sciences, University of New South Wales, Sydney NSW 2052, Australia, e-mail: [email protected] The chiral intermediate R-phenylacetylcarbinol (PAC) is the precursor in the production of ephedrine and pseudo- Keywords: pyruvate decarboxylase, Candida utilis, pH, ephedrine. Commercially, it is synthesized through biotrans- R-phenylacetylcarbinol formation of benzaldehyde and pyruvate by fermenting yeast Saccharomyces cerevisiae. Alternative use of a cell free sys- tem, e. g. Candida utilis pyruvate decarboxylase (PDC), has Pyruvate decarboxylase (PDC) catalyses the decarboxyla- the advantage of higher PAC yields and concentrations. For tion of the glycolysis end product pyruvate to acetaldehyde both options, issues regarding PDC stability, activity and by- and carbon dioxide as the penultimate reaction of ethanol -product formation are still of key concern. fermentation. This same enzyme is used industrially in the This study investigates PDC from 4 yeast strains i. e. synthesis of enantiomerically pure R-phenylacetylcarbinol C. utilis, C. tropicalis, commercial PAC producer S. cerevisiae (R-PAC), which is the precursor for the chemical synthesis of and thermotolerant Kluyveromyces marxianus. PDC was pro- ephedrine and pseudoephedrine. duced and investigated with respect to PAC production as After a comprehensive
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