Two-phase reactors applied to the removal of substituted phenols: comparison between liquid-liquid and liquid-solid systems.

In this paper, a comparison is provided between liquid-liquid and liquid-solid partitioning systems applied to the removal of high concentrations of 4-nitrophenol. The target compound is a typical representative of substituted phenols found in many industrial effluents while the biomass was a mixed culture operating as a conventional Sequencing Batch Reactor and acclimatized to 4-nitrophenol as the sole carbon source. Both two-phase systems showed enhanced performance relative to the conventional single phase bioreactor and may be suitable for industrial application. The best results were obtained with the polymer Hytrel which is characterized by higher partition capability in comparison to the immiscible liquid solvent (2-undecanone) and to the polymer Tone™. A model of the two systems was formulated and applied to evaluate the relative magnitudes of the reaction, mass transfer and diffusion characteristic times. Kinetic parameters for the Haldane equation, diffusivity and mass transfer coefficients have been evaluated by data fitting of batch tests for liquid-liquid and liquid-solid two phase systems. Finally, preliminary results showed the feasibility of polymer regeneration to facilitate polymer reuse by an extended contact time with the biomass.

Operation of a two-phase partitioning bioreactor for the oxidation of anthracene by the enzyme manganese peroxidase.

A study was conducted to determine the potential of a two-phase partitioning bioreactor (TPPB) for the treatment of a poorly soluble compound, anthracene, by the enzyme manganese peroxidase (MnP) from the fungus Bjerkandera sp. BOS55. Silicone oil was used as the immiscible solvent, which contained anthracene at high concentrations. The optimization of the oxidation process was conducted taking into account the factors which may directly affect the MnP catalytic cycle (the concentration of H(2)O(2) and malonic acid) and those that affect the mass transfer of anthracene between the organic and the aqueous phase (solvent and agitation speed). The main objective was carried out in terms of improved efficiency, i.e., maximizing the anthracene oxidized per unit of enzyme used. The TPPB reached nearly complete oxidation of anthracene at a conversion rate of 1.8mgl(-1)h(-1) in 56h, which suggests the application of enzymatic TPPBs for the removal of poorly soluble compounds.

Phosphonium ionic liquids for degradation of phenol in a two-phase partitioning bioreactor.

Six ionic liquids (ILs), which are organic salts that are liquid at room temperature, were tested for their biocompatibility with three xenobiotic-degrading bacteria, Pseudomonas putida, Achromobacter xylosoxidans, and Sphingomonas aromaticivorans. Of the 18 pairings, seven were found to demonstrate biocompatibility, with one IL (trihexyl(tetradecyl)phosphonium bis(trifluoromethylsulfonyl) amide) being biocompatible with all three organisms. This IL was then used in a two-phase partitioning bioreactor (TPPB), consisting of 1 l aqueous phase loaded with 1,580 mg phenol and 0.25 l IL, inoculated with the phenol degrader P. putida. This initially toxic aqueous level of phenol was substantially reduced by phenol partitioning into the IL phase, allowing the cells to utilize the reduced phenol concentration. The partitioning of phenol from the IL to the aqueous phase was driven by cellular demand and thermodynamic equilibrium. All of the phenol was consumed at a rate comparable to that of previously used organic-aqueous TPPB systems, demonstrating the first successful use of an IL with a cell-based system. A quantitative (31)P NMR spectroscopic assay for estimating the log P values of ILs is under development.

The treatment of gaseous benzene by two-phase partitioning bioreactors: a high performance alternative to the use of biofilters.

A 2-l (1-l working volume) two-phase partitioning bioreactor (TPPB) was used as an integrated scrubber/bioreactor in which the removal and destruction of benzene from a gas stream was achieved by the reactor's organic/aqueous liquid contents. The organic solvent used to trap benzene was n-hexadecane, and degradation of benzene was achieved in the aqueous phase using the bacterium Alcaligenes xylosoxidans Y234. A gas stream with a benzene concentration of 340 mg l(-1) at a flow rate of 0.414 l h(-1) was delivered to the system at a loading capacity of 140 g m(-3) h(-1), and an elimination capacity of 133 g m(-3 )h(-1) was achieved (the volume in this term is the total liquid volume of the TPPB). This elimination capacity is between 3 and 13 times greater than any benzene elimination achieved by biofiltration, a competing biological air treatment strategy. It was also determined that the evaluation of TPPB performance in terms of elimination capacity should include the cell mass present in the system, as this is a readily controllable quantity. A specific benzene utilization rate of 0.57 g benzene (g cells)(-1) h(-1) was experimentally determined in a bioreactor with a cell concentration that varied dynamically between 0.2 and 1 g l(-1). If it assumed that this specific benzene utilization rate (0.57 g g(-1) h(-1)) is independent of cell concentration, then a TPPB operated at high cell concentrations could potentially achieve elimination capacities several hundred times greater than those obtained with biofilters.

Biodegradation of polycyclic aromatic hydrocarbons in a two-phase partitioning bioreactor in the presence of a bioavailable solvent.

Mycobacterium PYR-1 was used in a two-phase partitioning bioreactor (TPPB) to degrade low and high molecular weight polycyclic aromatic hydrocarbons. TPPBs are characterized by a cell-containing aqueous phase, and an immiscible and biocompatible organic phase that partitions toxic substrates to the cells based on their metabolic demand and the thermodynamic equilibrium of the system. A bioavailable solvent, that is, a solvent usable as a carbon source, was used as the organic layer. Although bioavailable solvents are traditionally deemed unsuitable for use in TPPBs, bis(ethylhexyl) sebacate had superior chemical properties to other solvents examined and was cost-effective. In this system, 1 g of phenanthrene and 1 g of pyrene were completely degraded within 4 days, at rates of 168 mg l(-1) day(-1) and 138 mg l(-1 )day(-1), respectively, based on a 3-l aqueous volume. This is the highest pyrene degradation rate reported in the literature to date. Significant degradation of naphthalene and anthracene was also obtained. This work demonstrates that bioavailable solvents can be successfully used in TPPB systems, and may change the protocols commonly used to select solvents for TPPBs in the future.

Enhancement of a two-phase partitioning bioreactor system by modification of the microbial catalyst: demonstration of concept.

Application of two-phase partitioning bioreactors (TPPB) to the degradation of phenol and xenobiotics has been limited by the fact that many organic compounds that would otherwise be desirable delivery solvents can be utilized by the microorganisms employed. The ability to metabolize the solvent itself could interfere with xenobiotic degradation, limiting remediation efficiency, and hence represents a microbial characteristic incompatible with process goals. To avoid the issue of bioavailability, previous TPPB applications have relied on complex and often expensive delivery solvents or suboptimal catalyst-solvent pairings. In an effort to enhance TPPB activity and applicability, a genetically engineered derivative of Pseudomonas putida ATCC 11172 mutated in its ability to utilize medium-chain-length alcohols was generated (AVP2) and applied as the catalyst within a TPPB system with decanol as the delivery solvent. Kinetic analysis verified that the genetic alteration had not negatively affected phenol degradation. The volumetric productivity of AVP2 (0.48 g/L x h(-1)) was equivalent to that seen for wild-type ATCC 11172 (0.51 g/L x h(-1)), but a comparison of initial cell concentrations and yields revealed an improved phenol-degrading efficiency for the mutant under process conditions. Yield coefficients, cell dry weight, and viable count determinations all confirmed the stability of the modified phenotype. This work illustrates the possibilities for TPPB process enhancement through a careful combination of genetic modification and solvent selection.

Use of a two-phase partitioning bioreactor for degrading polycyclic aromatic hydrocarbons by a Sphingomonas sp.

A two-phase partitioning bioreactor (TPPB) utilizing the bacterium Sphingomonas aromaticivorans B0695 was used to degrade four low molecular weight (LMW) polycyclic aromatic hydrocarbons (PAHs). The TPPB concept is based on the use of a biocompatible, immiscible organic solvent in which high concentrations of recalcitrant substrates are dissolved. These substances partition into the cell-containing aqueous phase at rates determined by the metabolic activity of the cells. Experiments showed that the selected solvent, dodecane, could be successfully used in both solvent extraction experiments (to remove PAHs from soil) and in a TPPB application. Further testing demonstrated that solvent extraction from spiked soil was enhanced when a solvent combination (dodecane and ethanol) was used, and it was shown that the co-solvent did not significantly affect TPPB performance. The TPPB achieved complete biodegradation of naphthalene, phenanthrene, acenaphthene and anthracene at a volumetric consumption rate of 90 mg l(-1) h(-1) in approximately 30 h. Additionally, a total of 20.0 g of LMW PAHs (naphthalene and phenanthrene) were biodegraded at an overall volumetric rate of 98 mg l(-1) h(-1) in less than 75 h. Degradation rates achieved using the TPPB and S. aromaticivorans B0695 are much greater than any others previously reported for an ex situ PAH biodegradation system operating with a single species.

Expanded application of a two-phase partitioning bioreactor through strain development and new feeding strategies.

This research demonstrated the microbial treatment of concentrated phenol wastes using a two-phase partitioning bioreactor (TPPB). TPPBs are characterized by a cell-containing aqueous phase and an immiscible and biocompatible organic phase that partitions toxic substrates to the cells on the basis of their metabolic demand and the thermodynamic equilibrium of the system. Process limitations imposed by the capability of wild-type Pseudomonas putida ATCC 11172 to utilize long chain alcohols were addressed by strain modification (transposon mutagenesis) to eliminate this undesirable biochemical characteristic, enabling use of a range of previously bioavailable organics as delivery solvents. Degradation of phenol in a system with the modified strain as catalyst and industrial grade Adol 85 NF (primarily oleyl alcohol) as the solvent was demonstrated, with the system ultimately degrading 36 g of phenol within 38 h. Volumetric phenol consumption rates by wild type P. putida ATCC 11172 and the genetically modified derivative revealed equivalent phenol degrading capabilities (0.49 g/L x h vs 0.47 g/L x h respectively, in paired fermentations), with the latter presenting a more efficient remediation option due to decreased solvent losses arising from the modified strain's forced inability to consume the delivery solvent as a substrate. Two feeding strategies and system configurations were evaluated to expand practical applications of TPPB technology. The ability to operate with a lower solvent ratio over extended periods revealed potential for long-term application of TPPB to the treatment of large masses of phenol while minimizing solvent costs. Repeated recovery of 99% of phenol from concentrated phenol solutions and subsequent treatment within a TPPB scheme demonstrated applicability of the approach to the remediation of highly contaminated "effluents" as well as large masses of bulk phenol. Operation of the TPPB system in a dispersed manner, rather than as two distinct phases, resulted in volumetric consumption rates similar to those previously achieved only in systems operated with enriched air.

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.

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.

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.

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.

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.

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.

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.

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/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.

Simultaneous biodegradation of benzene, toluene, and p-xylene in a two-phase partitioning bioreactor: concept demonstration and practical application.

In this work, a mixture of benzene, toluene, and p-xylene was simultaneously biodegraded by Pseudomonas sp. ATCC 55595 in a two-phase partitioning bioreactor. This bioreactor consisted of a 1-L cell-containing aqueous medium phase and a 500-mL immiscible organic phase. The organic solvent systematically selected for use in the bioreactor was Adol 85 NF, an industrial-grade, biocompatible solvent. In the first of three experiments, the organic phase was loaded with 2.0 g of benzene, 10.15 g of toluene, and 2.1 g of p-xylene, which partitioned into the aqueous phase at concentrations of 25, 50, and 8 mg/L, respectively. The system ultimately biodegraded all of the substrates within 144 h. During the rapid growth phase of this fermentation, the cells were oxygen-limited. This fermentation was therefore repeated using an enriched air supply to remove the oxygen limitation. The use of enriched air ultimately reduced the length of the fermentation to 108 h, thereby improving the overall volumetric consumption rates. Finally, 500 mL of Adol were used to recover 2.0 g of benzene, 10.15 g of toluene, and 2.1 g of p-xylene from silica sand that was contaminated as part of a simulated soil "spill". The solvent washing procedure was able to recover greater than 99% of each compound from the contaminated soil. The Adol was then transferred to the two-phase bioreactor to permit biological treatment of the BTX contaminants. This process was repeated when the initial BTX load had been consumed almost to exhaustion, and the solvent was able to recover the contaminants at greater than 99% efficiency once again. The system was ultimately able to degrade 4.0 g of benzene, 20.2 g of toluene, and 4.2 g of p-xylene within 144 h. These results represent an unprecedented level of BTX degradation and illustrate a potential practical application for this novel biotechnology.

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%.

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.