Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576 S351

were determined as 2.78 mM and 3125 U/ml respectively. The cat-alytic function of the enzyme was accelerated in the presence of0.1 mM Zn2+, Co2+, Mg2+, Mn2+, Ca2+and was strongly inhibited by0.1 mM EDTA. This enzyme showed broad specificity for phosphatecontaining substrates like pNPP, GMP, AMP, ADP, PEP, ATP, G-1-Pand G-6-P. The activity of enzyme with monoester-phosphorouscompounds was found to be more than diester or trimester com-pounds which suggested its phosphomonoesterase nature.

doi:10.1016/j.jbiotec.2010.09.391

[P-I.26]

Stability and activity of lipases on exposure to oxidative envi-ronments

U. Törnvall ∗, M. Hedström, R. Hatti-Kaul

Department of Biotechnology, Center for Chemistry and ChemicalEngineering, Lund University, Lund, SwedenKeywords: Stability; Oxidation; Structural changes; Lipase

The access to suitable biocatalysts with respect to stability andactivity under process conditions is a major obstacle to the imple-mentation of biocatalysis on a broad front for industrial productionof specialty chemicals. One example is the production of epoxides,where lipases provide an environmentally friendly alternative tothe traditional production processes with the advantage of reducedby-product formation. We have performed the chemo-enzymaticepoxidation in a solvent-free process using renewable raw materi-als such as rapeseed methyl ester and tall oil derivatives, reachingmore than 90% conversion within 6 hours. However, the biocatalyst,Candida antarctica lipase B, is not stable enough for a cost-effectiveindustrial process.

In order to enhance the stability of the lipase, we have developeda mass spectrometry based method enabling the direct study of themodifications occurring on the immobilized enzyme during usage.Oxidation of methionine and tryptophan residues was observed,as well as disruption of disulphide bridges. The findings are layingthe basis for mutagenesis experiments, which has already providedsome answers to the importance of certain amino acids in the stabi-lization of the native lipase. Combined with activity measurements,and studies on the secondary and tertiary structure of the lipaseusing electrospray ionization mass spectrometry, circular dichro-ism and dynamic light scattering, we are deducing a mechanismbehind the oxidative deactivation of the enzyme.

doi:10.1016/j.jbiotec.2010.09.392

[P-I.27]

Characterization of biotin metabolism in Corynebacterium glu-tamicum

C. Stansen, P. Peters-Wendisch ∗, J. Schneider, S. Goetker, V.F.Wendisch

Bielefeld University, GermanyKeywords: biotin; C. glutamicum; global gene expression

Corynebacterium glutamicum is a biotin auxotrophic bacterium,used for large-scale production of amino acids. In order to anal-yse biotin metabolism, the global gene expression pattern in wildtype upon the influence of biotin was measured. Primarily, threegene cluster were identified, which showed an increased expres-sion level upon biotin limitation and a decreased level upon biotinexcess: the strongest regulated genes showed high identity on

protein level to a biotin transport system (BioYMN) from R. etli,and two genes of biotin synthesis (bioA, bioB) were differentiallyexpressed as well. Further analysis of the bioYMN cluster revealedan operon structure, and the transcriptional start site could be iden-tified.

Whereas C. glutamicum possesses the genes bioA, bioD and bioB[1], a bioF homologue encoding KAPA synthase catalysing the firststep of biotin synthesis from pimeloyl-CoA is missing [2]. Heterolo-gous complementation experiments with bioF from E. coli was notsufficient to cure C. glutamicum biotin auxotrophy, except whenpimelic acid was supplemented, indicating that the initial part ofthe biotin synthesis pathway providing pimelic acid is also likelyabsent.

In E. coli the biotin genes are regulated by the bifunctional BirAprotein, which is active as biotin-protein ligase and transcriptionalrepressor of the bio-genes [3]. The effect of birA expression onthe transcriptome of C. glutamicum was analysed and revealed nosignificant changes on mRNA level, indicating that birA from C. glu-tamicum, which lacks an N-terminal DNA-binding domain is notthe regulator of biotin metabolism in this organism. However, birAexpression was analysed in regard to growth and lysine produc-tion, and it could be shown, that overexpression of birA resulted ina significant growth advantage, both, on glucose and lactate as solecarbon source and resulted in a higher lysine yield on glucose ascompared to the control strain.

References

Hatakeyama et al., DNA Seq., 1993.www.coryneregnet.de.Rodionov, Chem. Rev., 2007.

doi:10.1016/j.jbiotec.2010.09.393

[P-I.28]

Effect of nutrients limitation on the macromolecular composi-tion of Pichia angusta

Romina Alvarez ∗, Fernando Acevedo

Pontificia Universidad Católica de Valparaíso, ChileKeywords: Methylotrophic yeast; Nutrients limitation; Dilutionrate; Salmon food

In Chile, the demand for fishmeal and fish oil for the salmonfeed industry is increasing, but their availability is limited. Amongpossible substitutes is microbial biomass. Yeasts are a good sourceof protein, but their lipid content is low in relation to the salmonneeds.

The objective of this research was to use the chemostat as a toolfor altering the lipid to protein ratio of yeast cells as a function ofthe limiting nutrient and the dilution rate.

Continuous cultures of methylotrophic yeast Pichia angustawere performed in a 2-liter chemostat. All cultures were per-formed at 37 ◦C, 200 rpm and pH 4.5. Limiting nutrients werecarbon (CH3OH), nitrogen ((NH4)2SO4), phosphorus (KH2PO4) andmagnesium source (MgSO4 x 7 H2O). We evaluated lipid and pro-tein content at different dilution rates (0.03, 0.05, 0.07, 0.09 and0.11 h-1). The results were statistically analyzed.

The highest protein value was obtained with carbon limitationat 0.03 h-1 (54.5%). This value is significantly higher than thoseobtained with nitrogen (39%) and magnesium (22.5%) limitationsat the same dilution rate. Phosphorus limitation at 0.09 h-1 alsorendered a high value (40.8%).

High lipid values were obtained with magnesium limitation inthe range of 0.05 to 0.11 h-1, reaching 30.4% lipid at 0.05 h-1, signif-

S352 Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576

icantly higher than those obtained with nitrogen (22.9%), carbon(19.4%) and phosphorus (23%) limitations.

The highest lipid/protein ratio was 0.89 obtained under magne-sium limitation at 0.09 h-1. While the amount of lipid is not as high(24.1%) the protein does not significantly diminish its value (27%).

In conclusion, it is possible to manipulate cell composition inorder to obtain high lipid to protein ratio.

doi:10.1016/j.jbiotec.2010.09.394

[P-I.29]

Recombinant Protein Purification Using Complementary Pep-tides as Affinity Tags

María C. Martínez Ceron, Alexandra M. Targovnik, Nicolás Urtasun,Osvaldo Cascone ∗, María V. Miranda, Silvia A. Camperi

University of Buenos Aires, ArgentinaKeywords: Recombinant protein; Purification; Peptides; Affinitytags

Affinity tags have become highly popular tools for purifyingrecombinant proteins from crude extracts. Besides, short peptidesare excellent ligands for affinity chromatography, as they are notlikely to cause an immune response in case of leakage into theproduct, they are more stable than antibodies to elution and clean-ing conditions and they usually have very acceptable selectivity.Hydropathically complementary peptides designed de novo showenough selectivity to be used successfully as peptide ligands forprotein purification from crude extracts. Recognition specificityand selectivity in the interaction between the complementarypeptide pair LSAAIIIPFLLH and KNYPKKKMEKRF have been demon-strated by other authors.

In this work we designed a recombinant protein purificationmethod using a peptide affinity tag that binds to a peptide-bindingpartner immobilised on a chromatographic matrix using the greenfluorescent protein (GFP) expressed in E. coli as the model.

The peptide LSAAIIIPFLLH was synthesised by solid phase usingthe Fmoc chemistry and immobilised in NHS-agarose. The GFP wasexpressed either with a fusion tag KNYPKKKMEKRF on the carboxyterminus or without a fusion tag.

After cell disruption, the extract was directly applied to aLSAAIIIPFLLH-agarose column using 20 mM sodium phosphatebuffer, pH 7.0, for the adsorption step. The GFP fused with the pep-tide tag KNYPKKKMEKRF was adsorbed and then eluted specificallywith 1 M Tris. The yield was 98% and the purification factor 4.6. Onthe other hand, GFP without tag pass through without interactingwith the peptide-agarose column.

The method designed can be applied for the purification of otherrecombinant proteins.

doi:10.1016/j.jbiotec.2010.09.395

[P-I.30]

Effect of additives on freeze-drying and storage of Yarrowialipolytica lipase

F. Darvishi 1,∗, J. Destain 2, I. Nahvi 1, P. Thonart 2, H. Zarkesh-Esfahani 1

1 University of Isfahan, Iran, Islamic Republic of2 CWBI, University of Liège, BelgiumKeywords: Yarrowia lipolytica lipase; Freeze-drying; Additives;Storage

The extracellular lipase of Yarrowia lipolytica is an enzyme thatpresents numerous potentialities for biotechnological applications,particularly in the food, pharmacology and chemical industries.This work describes the development and storage of powdersobtained from supernatants containing Y. lipolytica lipase by freeze-drying.

Lipase was produced by Yarrowia lipolytica U6 mutant strainin 20 L bioreactor. After centrifugation, non-concentrated culturesupernatant samples (211,460 U/L) to frezze drying were supple-mented with different concentration (0.5-1%) of maltodextrin andglycerol as additives. Effect of additives, temperature and storagetime on lipase powders were determined by lipase activity.

Maltodextrin gave the best protection of lipase during dehydra-tion treatment and its freeze-drying yield (73-76%) is better thanglycerol. Lipase powder with glycerol resembled glue and there-fore glycerol is not good for lipase freeze-drying. Powders producedfrom enzyme were stored for 20 weeks at 4 or 20 ◦C without lossof activity. All the powders obtained by freeze-drying were stableduring storage.

For the first time, maltodextrin and glycerol used as additivesto freeze-drying of Y. lipolytica lipase. In all cases, additives used todehydrate and stabilize lipase allowed for longer periods of stor-age. The formulation carried out with maltodextrin was better thanglycerol for lipase downstream processing. The present study dealswith stability of lipase powder during storage in order to use it inseveral applications. This study suggests that the use of additivesand freeze-drying should therefore be considered as an efficientmethod of enzyme preservation.

doi:10.1016/j.jbiotec.2010.09.396

[P-I.31]

Nucleotide Dependency in Thermal Protection Activities ofChaperonins from Hyperthermophilic Archaeum Aeropyrumpernix K1

Soo-Wan Nam 1,2,∗, Jeong-Hwan Kim 1

1 Department of Biomaterial Control (BK21 Program), Dong-Eui Uni-versity, Korea, Republic of2 Department of Biotechnology and Bioengineering, Dong-Eui Univer-sity, Korea, Republic ofKeywords: Chaperonin; Aeropyrum pernix; Thermal Protection;Nucleotide Dependency

In the present study, nucleotide dependency in thermal pro-tection activities of Aeropyrum pernix chaperonins (ApCpnA andApCpnB) was estimated using citrate synthase (CS) from porcineheart, alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae,and malate dehydrogenase (MDH) from Thermus flavus as foreignproteins. The molecular chaperonins, ApCpnA and ApCpnB, wereoverexpressed in E. coli and purified by heat treatment at 80◦C for20 min and HiTrap Q anion-exchange chromatography. The addi-