Oxygen transfer effects on recombinant benzaldehyde lyase production

Abstract Oxygen transfer (OT) effects on benzaldehyde lyase (BAL) production by recombinant Escherichia coli carrying pUC18::bal gene were investigated on a defined medium with glucose and (NH 4 ) 2 HPO 4 as the sole carbon and nitrogen sources respectively, in 3.0 dm 3 bioreactor systems at six different conditions with the parameters, air inlet rate of Q O / V R = 0.5 vvm, and agitation rates of N = 250 , 375, 500, 625, 750  min - 1 and at Q O / V R = 0.7 vvm , N = 750 min - 1 . Cell concentration ( C X ), BAL volumetric and specific activities, organic acid concentrations, yield values, maintenance coefficients, oxygen transfer coefficients ( K L a ) and oxygen uptake rates (OUR) were determined throughout the bioprocess. Increase in the oxygen transfer rate increased the cell formation up to 0.5 vvm, 500  min - 1 ; however, further increase inhibited cell formation. The highest BAL volumetric activity was obtained at 0.5 vvm, 500  min - 1 as 860 U cm - 3 . K L a , and enhancement factor E ( K L a / K L a o ) increased with the cultivation time and agitation rate, their values varied between 0.008–0.046 s - 1 and 1.00–3.07, respectively. OUR first increased, and then decreased with the cultivation time at all OT conditions, the highest OUR was observed at 0.5 vvm, 500 min - 1 as 3.6 mmol m - 3  s - 1 at t = 8 h ; while the highest specific OUR was obtained at 0.5 vvm, 250 min - 1 as 0.74 kg  kg - 1  h - 1 at t = 1 h . With the increase in Q O / V R and/or N, Damkohler number ( Da ) , that is the OT limitation, decreased. The effectiveness factor ( η ) values were close to 1.0 at medium and high OT conditions at the beginning of the processes as cells consumed oxygen with such a high rate that maximum possible oxygen utilization rate (oxygen demand) value was approached; thereafter, η decreased with the increase in cultivation time and Da, and the decrease rate was higher at lower agitation rates due to the lower K L a . Oxygen consumption for maintenance ( m O ) was the highest at 0.5 vvm, 500  min - 1 and the value was 0.20 kg kg - 1  h - 1 .

[1]  Enrique Galindo,et al.  Effects of dissolved oxygen tension on the production of recombinant penicillin acylase in Escherichia coli , 2003 .

[2]  R. Vicuña,et al.  Cloning and sequencing of the gene encoding benzaldehyde lyase from Pseudomonas fluorescens biovar I. , 1994, Gene.

[3]  P. Çalık,et al.  Metabolic flux analysis for serine alkaline protease fermentation by Bacillus licheniformis in a defined medium: Effects of the oxygen transfer rate , 1999 .

[4]  S. J. Parulekar,et al.  Expression of β‐lactamase by recombinant Escherichia coli strains containing plasmids of different sizes—effects of pH, phosphate, and dissolved oxygen , 1989, Biotechnology and bioengineering.

[5]  G. L. Miller Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar , 1959 .

[6]  J. Nielsen,et al.  Bioreaction Engineering Principles , 1994, Springer US.

[7]  P. Hagander,et al.  Avoiding acetate accumulation in Escherichia coli cultures using feedback control of glucose feeding. , 2001, Biotechnology and bioengineering.

[8]  Determination methods of the volumetric oxygen transfer coefficient kLa in bioreactors , 1990 .

[9]  Michael Müller,et al.  Benzaldehyde lyase-catalyzed enantioselective carboligation of aromatic aldehydes with mono- and dimethoxy acetaldehyde. , 2003, Organic letters.

[10]  P. Çalık,et al.  Oxygen transfer effects in serine alkaline protease fermentation by Bacillus licheniformis: use of citric acid as the carbon source , 1998 .

[11]  S. Enfors,et al.  Oxygen enriched air supply in Escherichia coli processes : production of biomass and recombinant human growth hormone , 2002 .

[12]  Michael Müller,et al.  Enantioselective Synthesis of α‐Hydroxy Ketones via Benzaldehyde Lyase‐Catalyzed C−C Bond Formation Reaction , 2002 .

[13]  S. Bhattacharya,et al.  Effects of dissolved oxygen and oxygen mass transfer on overexpression of target gene in recombinant E. coli , 1997 .

[14]  R Vicuña,et al.  Benzaldehyde lyase, a novel thiamine PPi-requiring enzyme, from Pseudomonas fluorescens biovar I , 1989, Journal of bacteriology.

[15]  Michael J Cooney,et al.  Tracking the acetate threshold using DO‐transient control during medium and high cell density cultivation of recombinant Escherichia coli in complex media , 2003, Biotechnology and bioengineering.

[16]  Michael Müller,et al.  Enantioselective synthesis of hydroxy ketones through cleavage and formation of acyloin linkage. Enzymatic kinetic resolution via C-C bond cleavage , 2001 .

[17]  P. Çalık,et al.  Oxygen-transfer strategy and its regulation effects in serine alkaline protease production by Bacillus licheniformis. , 2000, Biotechnology and bioengineering.