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A J Daugulis

Publications and source records attributed to A J Daugulis.

At least 19 recordsLinked to original sources
PubMed2002

Identification and characterization of the AgmR regulator of Pseudomonas putida: role in alcohol utilization.

Two-phase partitioning bioreactors (TPPBs) comprise an aqueous phase containing all non-carbon nutrients necessary for microbial growth and a solvent phase containing high concentrations of inhibitory or toxic substrates that partition at sub-inhibitory levels to the aqueous phase in response to cellular demand. This work aimed at eliminating the growth of Pseudomonas putida ATCC 11172 on medium-chain-length (C8-C12) aliphatic alcohols, hence enabling their use as xenobiotic delivery solvents within two-phase partitioning bioreactors. Experiments resulted in the isolation of a mini-Tn5 mutant unable to utilize these alcohols. The mutation, which also eliminated growth on glycerol and ethanol, was identified to be within a homologue of the P aeruginosa agmR gene, which encodes a response regulator. Enzyme analysis of the agmR::Tn5Km mutant cell extracts revealed a 10-fold decrease in pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase activity. A knockout in a gene (exaA) encoding a PQQ-linked alcohol dehydrogenase slowed but did not eliminate growth on medium-chain-length alcohols or ethanol, suggesting metabolic redundancy within P. putida ATCC 11172. Analysis of P. putida KT2440 genome sequence data indicated the presence of two PQQ-linked alcohol dehydrogenase-encoding genes. The successful elimination of alcohol utilization in the agmR mutant indicates control by AgmR on multiple pathways and presents a useful strain for biotechnological applications requiring alcohol non-utilizing microbial catalysts.

Alcohol Dehydrogenase↗
PubMed2001

Development of a novel bioreactor system for treatment of gaseous benzene.

A novel, continuous bioreactor system combining a bubble column (absorption section) and a two-phase bioreactor (degradation section) has been designed to treat a gas stream containing benzene. The bubble column contained hexadecane as an absorbent for benzene, and was systemically chosen considering physical, biological, environmental, operational, and economic factors. This solvent has infinite solubility for benzene and very low volatility. After absorbing benzene in the bubble column, the hexadecane served as the organic phase of the two-phase partitioning bioreactor, transferring benzene into the aqueous phase where it was degraded by Alcaligenes xylosoxidans Y234. The hexadecane was then continuously recirculated back to the absorber section for the removal of additional benzene. All mass transfer and biodegradation characteristics in this system were investigated prior to operation of the integrated unit, and these included: the mass transfer rate of benzene in the absorption column; the mass transfer rate of benzene from the organic phase into the aqueous phase in the two-phase bioreactor; the stripping rate of benzene out of the two-phase bioreactor, etc. All of these parameters were incorporated into model equations, which were used to investigate the effects of operating conditions on the performance of the system. Finally, two experiments were conducted to show the feasibility of this system. Based on an aqueous bioreactor volume of 1 L, when the inlet gas flow and gaseous benzene concentration were 120 L/h and 4.2 mg/L, respectively, the benzene removal efficiency was 75% at steady state. This process is believed to be very practical for the treatment of high concentrations of gaseous pollutants, and represents an alternative to the use of biofilters.

Alcaligenes↗
PubMed2001

A rational approach to improving productivity in recombinant Pichia pastoris fermentation.

A Mut(S) Pichia pastoris strain that had been genetically modified to produce and secrete sea raven antifreeze protein was used as a model system to demonstrate the implementation of a rational, model-based approach to improve process productivity. A set of glycerol/methanol mixed-feed continuous stirred-tank reactor (CSTR) experiments was performed at the 5-L scale to characterize the relationship between the specific growth rate and the cell yield on methanol, the specific methanol consumption rate, the specific recombinant protein formation rate, and the productivity based on secreted protein levels. The range of dilution rates studied was 0. 01 to 0.10 h(-1), and the residual methanol concentration was kept constant at approximately 2 g/L (below the inhibitory level). With the assumption that the cell yield on glycerol was constant, the cell yield on methanol increased from approximately 0.5 to 1.5 over the range studied. A maximum specific methanol consumption rate of 20 mg/g. h was achieved at a dilution rate of 0.06 h(-1). The specific product formation rate and the volumetric productivity based on product continued to increase over the range of dilution rates studied, and the maximum values were 0.06 mg/g. h and 1.7 mg/L. h, respectively. Therefore, no evidence of repression by glycerol was observed over this range, and operating at the highest dilution rate studied maximized productivity. Fed-batch mass balance equations, based on Monod-type kinetics and parameters derived from data collected during the CSTR work, were then used to predict cell growth and recombinant protein production and to develop an exponential feeding strategy using two carbon sources. Two exponential fed-batch fermentations were conducted according to the predicted feeding strategy at specific growth rates of 0.03 h(-1) and 0.07 h(-1) to verify the accuracy of the model. Cell growth was accurately predicted in both fed-batch runs; however, the model underestimated recombinant product concentration. The overall volumetric productivity of both runs was approximately 2.2 mg/L. h, representing a tenfold increase in the productivity compared with a heuristic feeding strategy.

Biotechnology↗
PubMed2001

Low-temperature increases the yield of biologically active herring antifreeze protein in Pichia pastoris.

Antifreeze proteins and antifreeze glycoproteins are structurally diverse molecules that share a common property in binding to ice crystals and inhibiting ice crystal growth. Type II fish antifreeze protein of Atlantic herring (Clupea harengus harengus) is unique in its requirement of Ca(2+) for antifreeze activity. In this study, we utilized the secretion vector pGAPZalpha A to express recombinant herring antifreeze protein (WT) and a fusion protein with a C-terminal six-histidine tag (WT-6H) in yeast Pichia pastoris wild-type strain X-33 or protease-deficient strain SMD1168H. Both recombinant proteins were secreted into the culture medium and properly folded and functioned as the native herring antifreeze protein. Furthermore, our studies demonstrated that expression at a lower temperature increased the yield of the recombinant protein dramatically, which might be due to the enhanced protein folding pathway, as well as increased cell viability at lower temperature. These data suggested that P. pastoris is a useful system for the production of soluble and biologically active herring antifreeze protein required for structural and functional studies.

Animals↗
PubMed2001

Two-phase partitioning bioreactors: a new technology platform for destroying xenobiotics.

Toxic organic compounds (xenobiotics) pose serious environmental and health risks worldwide. Biological treatment of these materials is severely constrained by their toxic and inhibitory nature and great care is required with respect to the rate at which they are provided to cells. The use of a second, distinct, organic phase in a bioreactor has been shown to provide a virtually foolproof means of feeding substrate to cells because this process concept relies only on thermodynamic equilibrium and the cells' own rate of metabolism. This technology can be applied to stockpiled xenobiotics as well as contamination of air, water and soil environments.

Biodegradation, Environmental↗
PubMed2000

Dynamic modeling and optimal fed-batch feeding strategies for a two-phase partitioning bioreactor.

A dynamic model for the degradation of phenol in a two-phase partitioning bioreactor has been developed based on mechanistic balances around the bioreactor. The key process characteristics including substrate transfer between the organic and aqueous phases, substrate inhibition, oxygen limitation, and cell entrainment were incorporated into the model. The model predictions were validated against existing experimental data obtained for a 2-L bioreactor, and good correlation was observed for the time frames of the simulations, as well as for trends in cell and substrate concentrations. Optimal fed-batch, phenol feeding strategies were then developed based on two approaches: (1) maximization of phenol consumption in a fixed time interval and (2) consumption of a fixed amount of phenol in minimal time. The optimal feeding policies, determined using the Iterative Dynamic Programming algorithm, provided substantial improvements in the amount of phenol consumed when compared to a typical experimental heuristic approach. For example, 45.73 g of phenol was predicted to be consumed in 50 h (not including lag phase) using the optimal feeding profile compared to 10.26 g of phenol consumed in the simulated experimental approach. Oxygen limitation was predicted to be a recurring operational challenge in the partitioning bioreactor, and had a strong impact on the optimization results.

Algorithms↗
PubMed2000

Structure-function relationships in spruce budworm antifreeze protein revealed by isoform diversity.

The spruce budworm, Choristoneura fumiferana, produces antifreeze protein (AFP) to assist in the protection of the overwintering larval stage. AFPs are thought to lower the freezing point of the hemolymph, noncolligatively, by interaction with the surface of ice crystals. Previously, we had identified a cDNA encoding a 9-kDa AFP with 10-30 times the thermal hysteresis activity, on a molar basis, than that shown by fish AFPs. To identify important residues for ice interaction and to investigate the basis for the hyperactivity of the insect AFPs, six new spruce budworm AFP cDNA isoforms were isolated and sequenced. They differ in amino-acid identity as much as 36% from the originally characterized AFP and can be divided into three classes according to the length of their 3' untranslated regions (UTRs). The new isoforms have at least five putative 'Thr-X-Thr' ice-binding motifs and three of the new isoforms encode larger, 12-kDa proteins. These appear to be a result of a 30 amino-acid insertion bearing two additional ice-binding motifs spaced 15 residues apart. Molecular modeling, based on the NMR structure of a short isoform, suggests that the insertion folds into two additional beta-helix loops with their Thr-X-Thr motifs in perfect alignment with the others. The first Thr of the motifs are often substituted by Val, Ile or Arg and a recombinantly expressed isoform with both Val and Arg substitutions, showed wild-type thermal hysteresis activity. The analysis of these AFP isoforms suggests therefore that specific substitutions at the first Thr in the ice binding motif can be tolerated, and have no discernible effect on activity, but the second Thr appears to be conserved. The second Thr is thus likely important for the dynamics of initial ice contact and interaction by these hyperactive antifreezes.

Amino Acid Motifs↗
PubMed1999

Benzene/toluene/p-xylene degradation. Part I. Solvent selection and toluene degradation in a two-phase partitioning bioreactor.

A two-phase organic/aqueous reactor configuration was developed for use in the biodegradation of benzene, toluene and p-xylene, and tested with toluene. An immiscible organic phase was systematically selected on the basis of predicted and experimentally determined properties, such as high boiling points, low solubilities in the aqueous phase, good phase stability, biocompatibility, and good predicted partition coefficients for benzene, toluene and p-xylene. An industrial grade of oleyl alcohol was ultimately selected for use in the two-phase partitioning bioreactor. In order to examine the behavior of the system, a single-component fermentation of toluene was conducted with Pseudomonas sp. ATCC 55595. A 0.5-1 sample of Adol 85 NF was loaded with 10.4 g toluene, which partitioned into the cell containing 1 l aqueous medium at a concentration of approximately 50 mg/l. In consuming the toluene to completion, the organisms were able to achieve a volumetric degradation rate of 0.115 g l-1 h-1. This system is self-regulating with respect to toluene delivery to the aqueous phase, and requires only feedback control of temperature and pH.

Benzene↗
PubMed1999

Benzene/toluene/p-xylene degradation. Part II. Effect of substrate interactions and feeding strategies in toluene/benzene and toluene/p-xylene fermentations in a partitioning bioreactor.

A two-phase aqueous/organic partitioning bioreactor scheme was used to degrade mixtures of toluene and benzene, and toluene and p-xylene, using simultaneous and sequential feeding strategies. The aqueous phase of the partitioning bioreactor contained Pseudomonas sp. ATCC 55595, an organism able to degrade benzene, toluene and p-xylene simultaneously. An industrial grade of oleyl alcohol served as the organic phase. In each experiment, the organic phase of the bioreactor was loaded with 10.15 g toluene, and either 2.0 g benzene or 2.1 g p-xylene. The resulting aqueous phase concentrations were 50 mg/l, 25 mg/l and 8 mg/l toluene, benzene and p-xylene respectively. The simultaneous fermentation of benzene and toluene consumed these compounds at volumetric rates of 0.024 g l-1 h-1 and 0.067 g l-1 h-1, respectively. The simultaneous fermentation of toluene and p-xylene consumed these xenobiotics at volumetric rates of 0.066 g l-1 h-1 and 0.018 g l-1 h-1, respectively. A sequential feeding strategy was employed in which toluene was added initially, but the benzene or p-xylene aliquot was added only after the cells had consumed half of the initial toluene concentration. This strategy was shown to improve overall degradation rates, and to reduce the stress on the microorganisms. In the sequential fermentation of benzene and toluene, the volumetric degradation rates were 0.056 g l-1 h-1 and 0.079 g l-1 h-1, respectively. In the toluene/p-xylene sequential fermentation, the initial toluene load was consumed before the p-xylene aliquot was consumed. After 12 h in which no p-xylene degradation was observed, a 4.0-g toluene aliquot was added, and p-xylene degradation resumed. Excluding that 12-h period, the microbes consumed toluene and p-xylene at volumetric rates of 0.074 g l-1 h-1 and 0.025 g l-1 h-1, respectively. Oxygen limitation occurred in all fermentations during the rapid growth phase.

Benzene↗
PubMed1998

The solution structure of type II antifreeze protein reveals a new member of the lectin family.

A recombinant form of the sea raven type II antifreeze protein (SRAFP) has been produced using the Pichia pastoris expression system. The antifreeze activity of recombinant SRAFP is indistinguishable from that of the wild-type protein. The global fold of SRAFP has been determined by two-dimensional 1H homonuclear and three-dimensional 1H-¿15N¿ heteronuclear NMR spectroscopy using 785 NOE distance restraints and 47 angular restraints. The molecule folds into one globular domain that consists of two helices and nine beta-strands in two beta-sheets. The structure confirms the proposed existence of five disulfide bonds. The global fold of SRAFP is homologous to C-type lectins and pancreatic stone proteins, even though the sequence identity is only approximately 20%.

Amino Acid Sequence↗
PubMed1997

Characterization and optimization of a two-phase partitioning bioreactor for the biodegradation of phenol.

A two-phase partitioning bioreactor containing Pseudomonas putida ATCC 11172 was used to degrade high concentrations of phenol in batch and fed-batch mode. The 2-1 (nominal volume) partitioning bioreactor employs a 1-1 cell-containing aqueous phase, and a 500-ml immiscible and biocompatible second organic phase (2-undecanone), which partitions the toxic substrate into the aqueous phase at a rate based on the metabolic activity of the microorganisms. Using this reactor configuration, operated in batch mode, 10-g phenol was degraded to completion within 84-h. The system was, however, oxygen-limited during the rapid growth phase of the fermentation. A second experiment, using enriched air to prevent oxygen limitation, resulted in the complete degradation of 10-g phenol within 72-h. The use of a sequential feeding strategy, in which a 10-g phenol load was added in sequential 5-g aliquots, resulted in a significant reduction in the lag phase, from 36-h to 12-h, and the consumption of 10-g phenol in 60 h. Finally, fed-batch fermentation was used to attempt to determine the ultimate capacity of the system to degrade phenol. The organic phase was loaded with 10-g phenol, the microorganisms were allowed to consume this aliquot almost to completion, and a second 10-g aliquot was then added. The organic phase was spiked in this manner a total of four times, resulting in the degradation of 46.55-g phenol within 12 days. The system was also monitored for nutrient depletion, and a nutrient-feeding schedule was formulated, in response to the mass of phenol consumed.

Aerobiosis↗
PubMed1997

Biosynthetic production of type II fish antifreeze protein: fermentation by Pichia pastoris.

Sea raven type II antifreeze protein (SRAFP) is one of three different fish antifreeze proteins isolated to date. These proteins are known to bind to the surface of ice and inhibit its growth. To solve the three-dimensional structure of SRAFP, study its ice-binding mechanism, and as a basis for engineering these molecules, an efficient system for its biosynthetic production was developed. Several different expression systems have been tested including baculovirus, Escherichia coli and yeast. The latter, using the methylotrophic organism Pichia pastoris as the host, was the most productive. In shake-flask cultures the levels of SRAFP secreted from Pichia were up to 5 mg/l. The recombinant protein has an identical activity to SRAFP from sea raven serum. In order to increase yields further, four different strategies were tested in 10-l fermentation vessels, including: (1) optimization of pH and dissolved oxygen, (2) mixed feeding of methanol and glycerol with Mut(s) clones, (3) supplementation of amino acid building blocks, and (4) methanol feeding with Mut+ clones. The mixed-feeding/Mut(s) strategy proved to be the most efficient with SRAFP yields reaching 30 mg/l.

Amino Acids↗
PubMed1994

Integrated fermentation and recovery processes.

The application of integrated fermentation and product recovery continues to be improved for products such as ethanol, acetone and butanol. An extractive fermentation process for ethanol has now been scaled up and is approaching commercialization. Integrated fermentation process design is expanding into higher value materials, such as amino acids, plant cell products and antibodies. The general concept of selective partitioning of molecules between an aqueous and second (organic) phase has also been successfully applied to controlled substrate addition.

1-Butanol↗
PubMed1993

Importance of enzyme and solvent physical properties for the biocompatibility relationship of alpha-amino acid ester hydrolase.

The enzyme alpha-amino acid ester hydrolase was used with a variety of organic solvents to further explore the relationships between biocompatibility and solvent physical properties. Biocompatibility was shown to be system-specific; that is, the infection point of the characteristic log P trend (approximately 1.5) was specific to the enzyme employed, while the biocompatibility of solvents that were exceptions to the trend was dependent on the extent of agitation. In addition, phase toxic solvents all possessed a high interfacial tension with water. Finally, while the source of the enzyme may be significant, the extent of purification does not appear to be a factor in biocompatibility.

Biotransformation↗
PubMed1993

Polysaccharide microcapsules and macroporous beads for enhanced chromatographic separation.

One recent solution to the diffusion problem found in conventional chromatographic separation is the use of dual porosity beads in which large pores allow for convective flow and smaller pores allow for molecular diffusion. In our studies, dual porosity beads were prepared from carrageenan using an emulsion method. Effects of polymer type, polymer concentration, toluene content, gelling temperature, and stirring speed on the structure of porous beads were investigated. In an alternative approach, one of the interacting molecules in affinity chromatography, can be entrapped within semipermeable microcapsules. This has the advantage of increased adsorption capacities. Using blue dextran and albumin as a model system, alginate-chitosan capsules, containing blue dextran, were employed in the recovery of albumin from a saline solution.

Alginates↗
PubMed1992

Recombinant protein production in insect cell cultures infected with a temperature-sensitive baculovirus.

Spodoptera frugiperda (IPLB-SF-21) insect cells were grown in shake-flasks and infected with a temperature-sensitive baculovirus to express the gene of chloramphenicol acetyl transferase (CAT) in serum-free medium (SF-900) and two serum-supplemented media (IPL-41 and Grace's). In temperature-shift experiments (cell growth at 33 degrees C followed by virus replication at 27 degrees C 3-4 days later), virus and CAT production were much poorer in the serum-free medium than in serum-supplemented media, though cell growth was virtually the same in the different media tested. In all the three media, highest virus and CAT titers were obtained at the lowest MOI (multiplicity of infection 0.02). This result is contrary to that obtained in constant-temperature culture (27 degrees C for both cell growth and virus replication). Virus and CAT production was greatly improved when the entire culture was run at constant temperature. It appeared that infected cells were severely damaged at 33 degrees C (6 degrees C above the optimal 27 degrees C), resulting in little or no virus and protein production. As a result of these temperature-shift experiments, a larger-scale (14 1 air-lift bioreactor) serum-free culture of Sf-9 insect cells was conducted at constant temperature (27 degrees C) to produce recombinant protein (beta-galactosidase). A cell density as high as 1 x 10(7) cells.ml-1, and a beta-gal concentration of up to 104,000 unit.ml-1 were achieved.

Animals↗
PubMed1991

Integrated product formation and recovery.

As continues to be demonstrated, the in situ recovery of selected products froma bioreactor can have a significant positive impact on production. The strategies that are focused on here are: aqueous two-phase biocatalysis; non-aqueous biocatalysis; and membrane-enhanced biocatalysis. Additional fundamental understanding of molecular partitioning and biocatalytic activity in these environments will facilitate the rational selection of the components involved in these processing strategies.

Biotechnology↗