Biotechnology Progress (BIOTECHNOL PROGR)

Publications in this journal

• Article: Optimal design of an enzymatic reactor for flow injection analysis.

  M Poch, J L Montesinos, M del Valle, J Alonso, A Araujo, J L Lima
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  ABSTRACT: A simulation procedure for the optimization of enzymatic reactors used in sandwich flow injection systems is evaluated. The system is modeled as a plug-flow reactor with axial dispersion. To calibrate it, dispersion coefficients can be evaluated using residence time distribution techniques; meanwhile, enzymatic kinetics must be determined for the system considered, according to the values of the substrate conversion attained. The model has been linked to an optimization routine based on the Powell algorithm. The proposed approach has been evaluated in a system performing simultaneous determinations of glucose and glycerol, considered the common carbon sources in a fermentation process.
  Biotechnology Progress 06/2013; 9(5):473-80.
• Article: Poly(vinyl alcohol) hydrogel as a biocompatible viscoelastic mimetic for articular cartilage.

  Colin Grant, Pete Twigg, Alex Egan, Alexandra Moody, Annie Smith, Donald Eagland, Nicholas Crowther, Steve Britland
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  ABSTRACT: The prevalence of suboptimal outcome for surgical interventions in the treatment of full-thickness articular cartilage damage suggests that there is scope for a materials-based strategy to deliver a more durable repair. Given that the superficial layer of articular cartilage creates and sustains the tribological function of synovial joints, it is logical that candidate materials should have surface viscoelastic properties that mimic native articular cartilage. The present paper describes force spectroscopy analysis by nano-indentation to measure the elastic modulus of the surface of a novel poly(vinyl alcohol) hydrogel with therapeutic potential as a joint implant. More than 1 order of magnitude decrease in the elastic modulus was detected after adsorption of a hyaluronic acid layer onto the hydrogel, bringing it very close to previously reported values for articular cartilage. Covalent derivatization of the hydrogel surface with fibronectin facilitated the adhesion and growth of cultured rat tibial condyle chondrocytes as evidenced morphologically and by the observance of metachromatic staining with toluidine blue dye. The present results indicate that hydrogel materials with potential therapeutic benefit for injured and diseased joints can be engineered with surfaces with biomechanical properties similar to those of native tissue and are accepted as such by their constituent cell type.
  Biotechnology Progress 09/2008; 22(5):1400-6.
• Article: Modeling transport processes in sterilization-in-place.

  P T Noble
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  ABSTRACT: SIP (sterilization-in-place) of equipment using saturated steam is limited by transport processes that restrict the distribution of sterilizing steam. The following are two crucial operations: the removal of air prior to sterilization, and the removal of condensate during the sterilization. Using simple model systems of pipes and tanks, characteristic operating parameters were examined and steady-state models were analyzed. The results were used to evaluate design aspects of SIP, including heat insulation, spacing of steam traps, sloping of lines, steam velocities and consumption, placement of temperature sensors, and scale factors in piping. A more reliable SIP design is achievable by insulating equipment, spacing steam traps to limit condensate buildup, providing an effective air removal operation, and providing reliable, high-quality steam.
  Biotechnology Progress 09/2008; 8(4):275-84.
• Article: Oxidative burst in suspension culture of Taxus cuspidata induced by a laminar shear stress in short-term.

  Rong-Bin Han, Ying-Jin Yuan
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  ABSTRACT: Generation of active oxidative species induced by shear stress in suspension cultures of Taxus cuspidata was investigated in a Couette-type shear reactor. It was found that T. cuspidata cells respond to a shear rate of 95 s(-)(1) with oxidative bursts. Their triphasic characteristics in 6 h were similar in both intracellular H(2)O(2) production and extracellular O(2)(-)( )(*) production. Additionally, inhibition studies with diphenylene iodonium and azide suggested that the key enzyme responsible for oxidative bursts under the shear rate of 95 s(-)(1) is primarily NADPH oxidase and the contribution of peroxidase for oxidative bursts was less. Investigation of the relationship between active oxidative species and defense responses induced by the shear stress indicated that the O(2)(-)( )(*) burst may account for the change of membrane permeability, and the H(2)O(2) burst plays an important role in inducing secondary metabolites such as the activation of phenylalanine ammonia lyase enzyme and phenolic accumulation. Furthermore, oxidative bursts elicited by the shear rate of 95 s(-)(1) were suppressed by treatment with suramin, nifedipine, and neomycin prior to the shear stress treatment, suggesting that G-protein, Ca(2+) channel, and phospholipase C are involved in the signal pathway for oxidative bursts induced by the shear stress. A model is proposed to explain the oxidative burst in cultured T. cuspidata cells challenged with the shear stress.
  Biotechnology Progress 09/2008; 20(2):507-13.
• Article: Effect of temperature and high pressure on the activity and mode of action of fungal pectin methyl esterase.

  Thomas Duvetter, Ilse Fraeye, Daniel N Sila, Isabel Verlent, Chantal Smout, Elke Clynen, Liliane Schoofs, Henk Schols, Marc Hendrickx, Ann Van Loey
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  ABSTRACT: Pectin was de-esterified with purified recombinant Aspergillus aculeatus pectin methyl esterase (PME) during isothermal-isobaric treatments. By measuring the release of methanol as a function of treatment time, the rate of enzymatic pectin conversion was determined. Elevated temperature and pressure were found to stimulate PME activity. The highest rate of PME-catalyzed pectin de-esterification was obtained when combining pressures in the range 200-300 MPa with temperatures in the range 50-55 degrees C. The mode of pectin de-esterification was investigated by characterizing the pectin reaction products by enzymatic fingerprinting. No significant effect of increasing pressure (300 MPa) and/or temperature (50 degrees C) on the mode of pectin conversion was detected.
  Biotechnology Progress 09/2008; 22(5):1313-20.
• Article: Cloning of the thaumatin I cDNA and characterization of recombinant thaumatin I secreted by Pichia pastoris.

  Nobuyuki Ide, Ryosuke Kaneko, Ritsuko Wada, Alka Mehta, Shinobu Tamaki, Tomoko Tsuruta, Yuki Fujita, Tetsuya Masuda, Naofumi Kitabatake
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  ABSTRACT: Thaumatin is a sweet-tasting protein comprising a mixture of some variants. The major variants are thaumatins I and II. Although the amino acid sequence of thaumatin I was known and the nucleotide sequence of cDNA of thaumatin II was elucidated, the nucleotide sequence of thaumatin I has been controversial. We have cloned two thaumatin cDNAs from the fruit of Thaumatococcus daniellii Benth. One is the same nucleotide sequence as that of thaumatin II already reported, and the other is a novel nucleotide sequence. The amino acid sequence deduced from the novel cDNA was the same amino acid sequence as that of thaumatin I, the only exception being the residue at position 113 (Asp instead of Asn), indicating that the novel thaumatin cDNA is that for thaumatin I. This thaumatin I cDNA was transformed into Pichia pastoris X-33, and the recombinant thaumatin I expressed was purified and characterized. The threshold value of sweetness of the recombinant thaumatin I was the same as that of the plant thaumatin I, although several unexpected amino acid residues were attached to the N-terminal of the recombinant thaumatin I. These indicate that the N-terminal portion of thaumatin is not critical for the elicitation of sweetness.
  Biotechnology Progress 09/2008; 23(5):1023-30.
• Article: Carbon material and bioenergetic balances of xylitol production from corncobs by Debaryomyces hansenii.

  Beatriz Rivas, Paolo Torre, José Manuel Domínguez, Patrizia Perego, Attilio Converti, Juan Carlos Parajó
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  ABSTRACT: The effect of oxygenation on xylitol production by the yeast Debaryomyces hansenii has been investigated in this work using the liquors from corncob hydrolysis as the fermentation medium. The concentrations of consumed substrates (glucose, xylose, arabinose, acetate and oxygen) and formed products (xylitol, arabitol, ethanol, biomass and carbon dioxide) have been used, together with those previously obtained varying the hydrolysis technique, the level of adaptation of the microorganism, the sterilization procedure and the initial substrate and biomass concentrations, in carbon material balances to evaluate the percentages of xylose consumed by the yeast for the reduction to xylitol, alcohol fermentation, respiration and cell growth. The highest xylitol concentration (71 g/L) and volumetric productivity (1.5 g/L.h) were obtained semiaerobically using detoxified hydrolyzate produced by autohydrolysis-posthydrolysis, at starting levels of xylose (S(0)) and biomass (X(0)) of about 100 g/L and 12 g(DM)/L, respectively. No less than 80% xylose was addressed to xylitol production under these conditions. The experimental data collected in this work at variable oxygen levels allowed estimating a P/O ratio of 1.16 mol(ATP)/mol(O). The overall ATP requirements for biomass production and maintenance demonstrated to remarkably increase with X(0) and for S(0) >or= 130 g/L and to reach minimum values (1.9-2.1 mol(ATP)/C-mol(DM)) just under semiaerobic conditions favoring xylitol accumulation.
  Biotechnology Progress 09/2008; 19(3):706-13.
• Article: Modeling and advanced control of recombinant Zymomonas mobilis fed-batch fermentation.

  David B Hodge, M Nazmul Karim
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  ABSTRACT: This work presents the development of an unstructured kinetic model incorporating the differing degrees of product, substrate, and pH inhibition on the kinetic rates of ethanol fermentation by recombinant Zymomonas mobilis CP4:pZB5 for growth on two substrates. Product inhibition was observed to start affecting the specific growth rate at an ethanol concentration of 20 g/L and the specific productivity at about 35-40 g/L. Specific growth rate was also shown to be more sensitive to inhibition by lowered pH as well. A model for the inhibition of two competing substrates' cellular uptake via membrane transport is proposed. Inhibition functions and model parameters were determined by fitting experimental data to the model. The model was utilized in a nonlinear model predictive control (NMPC) algorithm to control the product concentration during fed-batch fermentation to offset the inhibitory effects of product inhibition. Using the optimal feeding policy determined online, the volumetric productivity of ethanol was improved 16.6% relative to the equivalent batch operation when the final ethanol concentration was reached.
  Biotechnology Progress 09/2008; 18(3):572-9.
• Article: Characterization of an ethanol-inducible promoter system in Catharanthus roseus hairy roots.

  Christie A M Peebles, Susan I Gibson, Jacqueline V Shanks, Ka-Yiu San
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  ABSTRACT: Efforts to engineer Catharanthus roseus hairy roots to produce commercially significant amounts of valuable compounds, such as the terpenoid indole alkaloids vinblastine and vincristine, require the development of tools to study the effects of overexpressing key metabolic and regulatory genes. The use of inducible promoters allows researchers to control the timing and level of expression of genes of interest. In addition, use of inducible promoters allows researchers to use a single transgenic line as both the control and experimental line, minimizing the problems associated with clonal variation. We have previously characterized the use of a glucocorticoid-inducible promoter system to study the effects of gene overexpression within the terpenoid indole alkaloid pathway on metabolite production. Here the feasibility of using an ethanol-inducible promoter within C. roseus hairy roots is reported. This ethanol-inducible promoter is highly sensitive to ethanol concentration with a concentration of 0.005% ethanol causing a 6-fold increase in CAT reporter activity after 24 h of induction. The ethanol-inducible CAT activity increased 24-fold over a 72-h induction period with 0.5% ethanol.
  Biotechnology Progress 09/2008; 23(5):1258-60.
• Article: Purification and characterization of an anti-apoptotic protein isolated from Lonomia obliqua hemolymph.

  Alvaro P B Souza, Cristina C Peixoto, Luis Maranga, Ana V Carvalhal, Roberto H P Moraes, Rita M Z Mendonça, Carlos A Pereira, Manuel J T Carrondo, Ronaldo Z Mendonça
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  ABSTRACT: Previously it was reported that supplementation of insect cell culture with Lonomia obliqua hemolymph could extend culture longevity (Maranga et al. Biotechnol. Prog. 2003, 19, 58-63). In this work the anti-apoptotic properties of this hemolymph in Spodoptera frugiperda (Sf-9) cell culture were investigated. The presence or absence of apoptotic cells was characterized by light microscopy, flow cytometry, and agarose gel electrophoresis. Hemolymph was fractionated by several ion exchange and gel filtration chromatographic steps for identification of the compounds responsible for this effect. Fractions exhibiting a potent anti-apoptotic effect were isolated and tested in cell culture. A protein of about 51 kDa was identified, isolated, and tested for apoptosis inhibition. Addition of this purified protein to Sf-9 cultures was able to prevent apoptosis induced by nutrient depletion as well as by potent apoptosis chemical inducers such as Actinomycin D. This work confirms that the enhanced culture longevity obtained by supplementation with L. obliqua hemolymph is due to the presence of potent anti-apoptotic factors.
  Biotechnology Progress 09/2008; 21(1):99-105.
• Article: Asymmetric reduction of o-chloroacetophenone with Candida pseudotropicalis 104.

  Qing Xie, Jianping Wu, Gang Xu, Lirong Yang
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  ABSTRACT: The asymmetric reduction of o-chloroacetophenone 1 with Candida pseudotropicalis 104 produced the corresponding (S)-1-(2-chloro-phenyl)-ethanol 2 with the enantiomeric excess (ee >99%) without addition of any cosolvent. The cell could tolerate high ketone 1 concentration of 233.8 mmol/L (i.e., 36 g/L) with considerable reduction activity in this method. The product 2 concentration achieved 38.9 and 58.4 mmol/L with cells of 40 and 60 g(DCW) (dry cell weight)/L, respectively, in 24 h. The optimum reaction time, the effect of substrate concentration, cosubstrate type and concentration, and cell concentration in the reaction were investigated in this paper.
  Biotechnology Progress 09/2008; 22(5):1301-4.
• Article: Screening of novel excipients for improving the stability of retroviral and adenoviral vectors.

  Pedro E Cruz, Ana Carina Silva, António Roldão, Marlene Carmo, Manuel J T Carrondo, Paula M Alves
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  ABSTRACT: In the past decade there has been an increase in the application of viral vectors in the laboratory and clinical trials of human gene therapy, retroviral and adenoviral vectors among the most used. However, the limited stability of these vectors creates problems in the design of experiments, transport, and storage. Vectors stored at -80 degrees C must be quickly shipped on dry ice, which is somewhat cumbersome. Alternatively, viral vectors can be preserved in a lyophilized form. However, loss of viral activity during lyophilization can also be a serious problem. In this report we identify novel candidate formulations containing new compatible solutes, ectoin, hydroxyectoin, and firoin, that allow better stability of retroviral and adenoviral vectors during storage. For retroviral vectors, the maximum stabilization for long-term storage was achieved through lyophilization followed by storage at -20 degrees C using a formulation of Tris buffer pH 7.2 containing firoin (0.5 M), a half-life of 340 days being obtained. Adenoviral vectors storage at -80 degrees C in solution using Tris buffer pH 8.0 with firoin was the best method for long-term storage, with a half-life exceeding 1 year.
  Biotechnology Progress 09/2008; 22(2):568-76.
• Article: Carbon mass balance evaluation of cellulase production on soluble and insoluble substrates.

  Juan Carlos Sáez, Daniel J Schell, Arun Tholudur, Jody Farmer, Jenny Hamilton, José A Colucci, James D McMillan
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  ABSTRACT: A methodology is described and applied for performing carbon mass balances across cellulase enzyme production processes using both soluble sugar and insoluble cellulose substrates. The fungus Trichoderma reesei was grown on either glucose, lactose, or cellulose in aerobic batch mode, and the evolution of the main carbonaceous components (cell mass, cellulose, soluble protein, adsorbed protein, sugars, and carbon dioxide) was followed. A variety of analytical techniques were utilized to measure these components, including (i) gravimetric analysis, (ii) near-infrared spectroscopy, (iii) bicinchoninic acid based soluble protein measurement, (iv) gas mass spectrometry and flow rate, (v) CHNS/O elemental analyses, and (vi) high-performance liquid chromatography. The combined set of measurements allowed carbon mass balances across the cellulase production process to be assessed to determine the consistency of the underlying kinetic data. Results demonstrate the capability to determine the levels and distribution of all major carbonaceous components during the cellulase production process on both soluble and insoluble substrates. Average carbon mass balance closures were near 100% during early stages (<72 h) of the cultivations using glucose, lactose, or cellulose as the substrates, but carbon mass closures trended high later in the cultivation. Analysis of carbon allocation results suggests that an error in the gas mass flow rate measurement was the primary cause for carbon mass balance closures to exceed 110% late in the process.
  Biotechnology Progress 09/2008; 18(6):1400-7.
• Article: Comparison of fluidized bed and ultrasonic cell-retention systems for high cell density mammalian cell culture.

  Markus P Dürrschmid, Karlheinz Landauer, Gordana Simic, Helga Klug, Timo Keijzer, Felix Trampler, Arthur Oudshoorn, Martin Gröschl, Dethardt Müller, Otto Doblhoff-Dier
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  ABSTRACT: Economically viable biopharmaceutical production is to a high degree dependent on high product yields and stable fermentation systems that are easy to handle. In the current study we have compared two different fermentation systems for the production of recombinant protein from CHO cells. Both systems are fully scaleable and can be used for industrial high cell density bioprocesses. As a model cell line we have used a recombinant CHO cell line producing the enzyme arylsulfatase B (ASB). CHO cells were cultivated as adherent cell culture attached on Cytoline macroporous microcarrier (Amersham Biosciences, Sweden) using a Cytopilot Mini fluidized bed bioreactor (FBR, Vogelbusch-Amersham Biosciences, Austria) and as suspension culture using a stirred tank bioreactor equipped with a BioSep ultrasonic resonator based cell separation device (Applikon, The Netherlands). Both systems are equally well-suited for stable, long-term high cell density perfusion cell culture and provide industrial scalability and high yields. For products such as the recombinant ASB, high perfusion rates and therefore short product bioreactor residence times may be of additional benefit.
  Biotechnology Progress 09/2008; 19(3):1045-8.
• Article: Gelatin blends with alginate: gels for lipase immobilization and purification.

  Nitin W Fadnavis, Gurrala Sheelu, Bezavada Mani Kumar, Mahendra U Bhalerao, Ashlesha A Deshpande
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  ABSTRACT: Blends of natural polysaccharide sodium alginate (5%) with gelatin (3%) cross-linked with glutaraldehyde provide beads with excellent compressive strength (8 x 10(4) Pa) and regular structure on treatment with calcium chloride. Lipases from porcine pancreas, Pseudomonas cepacia, and Candida rugosa were immobilized in such a blend with excellent efficiency. The immobilized enzymes were stable and were reused several times without significant loss of enzyme activity both in aqueous and reverse micellar media. The beads were functionalized with succinic anhydride to obtain beads with extra carboxylic acid groups. These functionalized beads were then successfully used for 7.4-fold purification of crude porcine pancreatic lipase in a simple operation of protein binding at pH 5 and release at pH 8.5.
  Biotechnology Progress 09/2008; 19(2):557-64.
• Article: Effect of extracellular ph on matrix synthesis by chondrocytes in 3D agarose gel.

  Min-Hsien Wu, Jill P G Urban, Zhan Feng Cui, Zheng Cui, Xia Xu
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  ABSTRACT: In cartilage tissue engineering, the determination of the most appropriate cell/tissue culture conditions to maximize extracellular matrix synthesis is of major importance. The extracellular pH plays an important role in affecting energy metabolism and matrix synthesis by chondrocytes. In this study, chondrocytes were isolated from bovine articular cartilage, embedded in agarose gel, and cultured at varied pH levels (7.3-6.6). Rate of lactate production, total glycosaminoglycan (GAG) and collagen synthesis, as well as total cell numbers and cell viability were evaluated after culturing for up to 7 days. The results showed the rate of lactic acid production over the 7-day culture was significantly affected by extracellular pH; acidic pH markedly inhibited the production of lactate. Also, a biphasic response to extracellular pH in regard to total GAG synthesis was observed; the maximum synthesis was seen at pH 7.2. However, the collagen synthesis was not pH-dependent within the pH range explored. In addition, within the conditions studied, total cell numbers and cell viability were not significantly affected by extracellular pH. In conclusion, even minor changes in extracellular pH could markedly affect the metabolic activities and biosynthetic ability of chondrocytes. Consequently, the control of extracellular pH condition is crucially important for successful cartilage tissue engineering and for the study of chondrocyte physiology and functions.
  Biotechnology Progress 09/2008; 23(2):430-4.
• Article: Application of an antibody biochip for p53 detection and cancer diagnosis.

  M Askari, J P Alarie, M Moreno-Bondi, T Vo-Dinh
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  ABSTRACT: Detection of the p53 tumor suppressor gene is important in early cancer diagnostics because alterations in the gene have been associated with carcinogenic manifestations in several tissue types in humans. We have developed an antibody-based detection instrument, the biochip, to detect the presence of the anti-p53 antibody in human serum. The design of this highly integrated detector system is based on miniaturized phototransistors having multiple optical sensing elements, amplifiers, discriminators, and logic circuitry on an IC board. The system utilizes laser excitation and fluorescence signals to detect complex formation between the p53 monoclonal antibody and the p53 antigen. Recognition antibodies are immobilized on a nylon membrane platform and incubated in solutions containing antigens labeled with Cy5, a fluorescent cyanine dye. Subsequently, this membrane is placed on the detection platform of the biochip and fluorescence signal is induced using a 632.8-nm He-Ne laser. Using this immuno-biochip, we have been able to detect binding of the p53 monoclonal antibody to the human p53 cancer protein in biological matrices. The performance of the integrated phototransistors and amplifier circuits of the biochip, previously evaluated through measurement of the signal output response for various concentrations of fluorescein-labeled molecules, have illustrated the linearity of the microchip necessary for quantitative analysis. The design of this biochip permits sensitive, selective and direct measurements of a variety of antigen-antibody formations at very low concentrations. Furthermore, the acquisitions of the qualitative and quantitative results are accomplished rapidly, in about 15 min. These features demonstrate the potential of this antibody-based biochip for simple, rapid and early biomedical diagnostics of cancer.
  Biotechnology Progress 09/2008; 17(3):543-52.
• Article: Enhanced hyaluronic acid production in Bacillus subtilis by coexpressing bacterial hemoglobin.

  Liang-Jung Chien, Cheng-Kang Lee
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  ABSTRACT: Bacillus subtilis strains that can produce hyaluronic acid (HA) were constructed by integrating the HA synthase gene (hasA) and the UDP-glucose dehydrogenase gene of group C Streptococcus (hasB) or of B. subtilis itself (tauD) into the amyE locus of the B. subtilis chromosome. All of the inserted genes were under the control of a strong constitutive vegII promoter of B. subtilis. Although HA production could be achieved by expressing hasA alone, coexpressing hasB or tauD with hasA could enhance HA production at least 2-fold. To replenish the energy consumed for HA biosynthesis, Vitreoscilla hemoglobin (VHb) was coexpressed with the HA-expressing genes. With the expression of VHb, not only the cell concentration was enhanced 25%, but also HA production was further increased by 100%. About 1.8 g/L of HA was obtained by the recombinant strain B. subtilis carrying VHb, hasA, and tauD genes in the expression cassette after 30 h cultivation.
  Biotechnology Progress 09/2008; 23(5):1017-22.
• Article: Use of dye affinity chromatography for the purification of Aerococcus viridans lactate oxidase.

  Sergio A Streitenberger, José A López-Más, Alvaro Sánchez-Ferrer, Francisco García-Carmona
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  ABSTRACT: Lactate oxidase was purified from Aerococcus viridans (A. viridans) by dye affinity chromatography and FPLC ion exchange chromatography. The lactate oxidase could be purified by comparatively simple procedures, the purification achieved from a crude extract of A. viridans was 41-fold with a specific activity of 143 units/(mg of protein). The purified enzyme was a L-lactate oxidase, which catalyses the conversion of L-lactate in the presence of molecular oxygen to pyruvate and H(2)O(2). This purified lactate oxidase showed an apparent molecular mass of 48,200 in SDS-PAGE and the native molecular weight, as estimated by FPLC gel filtration, was 187,300. This molecular weight indicates that lactate oxidase exists in tetrameric form after gel filtration. To differing degrees, all the triazine dyes tested were inhibitors of lactate oxidase, solutions of free triazine dyes showing an inhibition mechanism which was both time- and pH-dependent.
  Biotechnology Progress 09/2008; 18(3):657-9.