S106 Abstracts / Journal of Bioscience and Bioengineering 108 (2009) S96–S113

References

1. Kojima, T., Takei, Y., Ohtsuka, M., Kawarasaki, Y., Yamane, T. and Nakano, H.: PCRamplification from single DNAmolecules onmagnetic beads in emulsion: applicationfor high-throughput screening of transcription factor targets. Nucleic Acids Res., 33,e150 (2005).

2. Kojima, T., Yamane, T., and Nakano, H.: In vitro selection of DNA binding sites fortranscription factor, PhaR, from Paracoccus denitrificans using genetic library onmicrobeads and flow cytometry. J. Biosci. Bioeng., 101, 440-444 (2006).

doi:10.1016/j.jbiosc.2009.08.308

EP-P20

Construction of fusion proteins consisting of ADH and FDH forNADH regeneration

Xi Wu, Chong Zhang, and Xinhui Xing

Institution of Chemical Engineering, Beijing, Haidian District, China

NAD(P)-dependent dehydrogenases are useful biocatalysts for thesynthesis of chiral compounds. For the applications of NAD(P)-dependent oxidoreductases, the efficient and cost-effective systemcapable of simultaneous regeneration of NAD(P)H is indispensable.

The bifunctional fusion protein systems consisting of ADHoriginated from Rhodococcus erythropolis (READH), FDH originatedfrom Candida boidinii (CbFDH) or maltose-binding protein (MBP) wereconstructed to regenerate the cofactors for the biocatalysis by READH.READH is an (S)-specific nicotinamide adenine dinucleotide(NADH)-dependent alcohol dehydrogenase, meanwhile, CbFDH is a NADH-dependent formate dehydrogenase. The strategies of the differentfusion protein systems included: (1) fusion of the N terminus ofREADH to the C terminus of MBP, (2) fusion of the N terminus ofCbFDH to the C terminus of MBP, (3) fusion of the N terminus ofREADH to the C terminus of CbFDH, (4) fusion of the C terminus ofREADH to the N terminus of CbFDH. A series of plasmids wereconstructed including pMAL-c2X-readh for the fusion expression ofMBP and READH, pMAL-c2X-cbfdh for the fusion expression of MBPand CbFDH, pMAL-c2X-readh-cbfdh for the fusion expression ofCbFDH and READH, pMAL-c2X-cbfdh-readh for the fusion expressionof READH and CbFDH. The activities of READHs were depressed in allfusion strategies to different extent. When the N terminus of READHwas fused to the C terminus of CbFDH, READH reached the highestactivity, but CbFDH showed no activity. In contrast, when the Cterminus of READH was fused to the N terminus of CbFDH, CbFDHshowed the highest activity, and both moieties displayed theiractivities. From this study, we suggest that the rational design of thebifunctional fusion protein systems can improve the biocatalysisefficiency by the simultaneous cofactor regeneration.

This work was supported by the Projects of the National NaturalScience Foundation of China (20836004).

doi:10.1016/j.jbiosc.2009.08.309

EP-P22

The presence of a modified FAD in formate oxidase fromDebaryomyces vanjiriae MH201 and Aspergillus oryzae RIB40

Yoshifumi Maeda,1 Daiju Doubayashi,1 Masaya Oki,1 Hiroaki Nose,1

Yutaka Fujii,2 and Hiroyuki Uchida1

Department of Applied Chemistry and Biotechnology, Graduate School ofEngineering, University of Fukui, Fukui-shi, Japan1 and Department ofMolecular Life Chemistry, Fukui Medical College, University of Fukui,Fukui-shi, Japan2

The amino acid sequence of the unnamed protein from Aspergillusoryzae RIB40 (accession no. XP_001727378) shows the highestsimilarity to those of 3 formate oxidase (FOD) isoforms produced byDebaryomyces vanjiriaeMH201, among those of the proteins classifiedto the glucose-methanol-choline oxidoreductase family. There is aconserved motif of FAD binding site in each of the unnamed proteinand the yeast FOD isoforms. The unnamed protein and one of thefungus FOD isoforms were produced in Escherichia coli under thecontrol of lac- and T7 promoters in C-His6-tagged form. The geneproducts purified by affinity column chromatography catalyzed theoxidation of formate to yield hydrogen peroxide. Their optimum pHand temperature were similar, but the fungus enzyme was morethermo-stable than the yeast enzyme. The purified enzymes showedsimilar UV-visible spectra, whichwere atypical for usual flavoproteins.Point mutation of a glycine residue in the conserved motif of the yeastFOD led to loss of enzyme activity. The extracts obtained by boiling thepurified enzymes showed similar UV-visibler spectra with maxima at358 and 455 nm and similar pH-dependent fluorescence excitationspectra with maxima at 350 and 450 nm when an emission wavelength of 540 nmwas used. In LC/MS analyses (electro spray ionizationmethod with positive ion mode), both the extracts showed apredominant ion at m/z 800, whereas FAD did that at m/z 786. Afterthe fragmentations of the ions at m/z 800 and 786 as precursor ions,fragmented ions at m/z 348 and m/z 453 and those at m/z 348 (AMP)and 439 (flavinic part) were observed, respectively. These resultssuggest that as a cofactor the FODs have same FAD analogue with amodified isoalloxazine ring, molecular mass of which differ by 14 fromthat of the unmodified ring.

doi:10.1016/j.jbiosc.2009.08.310

EP-P23

Leaching of lipoxygenase-1 from soybean flour

Khosrow Rostami, Yasaman Dayani, Soheyla Azarian, and ZehraEsfahanibolandbalaee

Iranian Research Organisation for Science and Technology-BiotechnologyCenter, Tehran, Islamic Republic of Iran

Lipoxygenases have significant physiological functions generatingwide range of interest with respect to their applications, (1, 2).Lipoxygenase -1 was leached from defatted soybean flour usingacetic acid buffer of 0.05 M of pH 5.2. Effect of various temperaturesfrom 4 °C and higher on the enzyme leaching was investigated. Theresult of operational period on leaching of the enzyme activitydepicted that during 10 min considerable amount of enzyme hasbeen leached. The effect of agitator speed on the enzyme leaching asone of the key parameters was studied and impellers speed of 5 rpsresulted in significant lipoxygenase leaching. A vector diagram ofagitated stirred tank and scaled-up at 5 and 1.67 rps using CFD hasbeen presented, respectively. Lsqnonlin Matalb version 6.5 was usedto develop a correlation. To develop a simple and effectiveprocedure for large scale leaching of lipoxygenase-1, stirred tankreactor, which is widely used in several processes [4] was employed.The CFD developed at 1.67 rps shows that axial flow was developed