Identification and characterization of a novel D-amidase gene from Variovorax paradoxus and its expression in Escherichia coli.

The gene for the newly described D-amidase from Variovorax paradoxus (Krieg et al. 2002) was cloned and functionally expressed in Escherichia coli. Since native enzyme was available in minute amounts only, we determined the N-terminal sequence of the enzyme and utilized the Universal GenomeWalker Approach to make use of the common internal sequence of the amidase signature family. The high GC content of the gene made it necessary to employ an appropriate DNA polymerase in the amplification reactions. Thus, the sequence of the complete gene and the flanking regions was established. In independent experiments, the gene was then amplified from genomic DNA of V. paradoxus, expressed in E. coli, and characterized. The recombinant enzyme has a specific activity of 1.7 units/mg with racemic tert-leucine amide as substrate and is a homodimer of 49.6-kDa monomers.

Improving the carboligase activity of benzoylformate decarboxylase from Pseudomonas putida by a combination of directed evolution and site-directed mutagenesis.

Benzoylformate decarboxylase (BFD) from Pseudomonas putida was subjected to directed molecular evolution to generate mutants with increased carboligase activity which is a side reaction of the enzyme. After a single round of random mutagenesis mutants were isolated which exhibited a 5-fold increased carboligase activity in aqueous buffer compared to the wild-type enzyme with a high enantiomeric excess of the product (S)-2-hydroxy-1-phenyl-propanone. From the same library, mutants with enhanced carboligase activity in water-miscible organic solvents have been isolated. The selected mutants have been characterized by sequencing, revealing that all mutants carry a mutation at Leu476, which is close to the active site but does not directly interact with the active center. BFD-L476Q has a 5-fold higher carboligase activity than the wild-type enzyme. L476 was subjected to saturation mutagenesis yielding eight different mutants with up to 5-fold increased carboligase activity. Surprisingly, all L476 mutants catalyze the formation of 2-hydroxy-1-phenyl-propanone with significantly higher enantioselectivity than the wild-type enzyme although enantioselectivity was not a selection parameter. Leu476 potentially plays the role of a gatekeeper of the active site of BFD, possibly by controlling the release of the product. The biocatalyst could be significantly improved for its side reaction, the C-C bond formation and for application under conditions that are not optimized in nature.

Gene cloning, overexpression and biochemical characterization of the peptide amidase from Stenotrophomonas maltophilia.

The peptide amidase (Pam) from the gram-negative bacterium Stenotrophomonas maltophilia catalyzes predominantly the hydrolysis of the C-terminal amide bond in peptide amides. Its gene ( pam) was isolated by Southern hybridization using a DNA probe derived from the known N-terminal amino acid sequence. Pam is a member of the amidase signature family and was identified as a periplasmic protein by an N-terminal signal peptide found in the gene. The processed protein consists of 503 amino acids with a molecular mass of 53.5 kDa. The recombinant enzyme with a C-terminal His(6) tag has a monomeric structure and its isoelectric point is 6.3. The dipeptide amide L-Ala- L-Phe-NH(2) is hydrolyzed in the absence of cofactors to L-Ala- L-Phe-OH and ammonia with V(max)=194 U/mg and K(m) <0.5 mM. The natural function of Pam remains unclear. Chymostatin ( K(i)<0.3 microM) and Pefabloc SC ( K(i) not determined) were identified as inhibitors. When the gene was expressed in Escherichia coli on a 12-l scale, the specific activity in the crude extract was 60 U/mg, compared to 0.24 U/mg in S. maltophilia. In the expression system, Pam made up about 31% of the total soluble cell protein. From 75 g wet cells, 2.1 g of >95% pure enzyme was obtained, which corresponds to a total activity of 416,000 units.

The use of ion-selective electrodes for evaluating residence time distributions in expanded bed adsorption systems.

The suitability of ion-selective electrodes (ISE) for the determination of residence time distribution (RTD) in turbid, cell-containing fluids was examined. The electrodes were found to give reproducible signals in biomass-containing feedstock with up to 20% wet weight of solids. The enhanced feedstock compatibility of IES, when compared to other tracer sensing devices, allows the study of expanded bed system hydrodynamics under relevant operating conditions. Within the linear range of the corresponding ISE-tracer pair, both examined ISE (Li(+)- or Br(-)-selective) showed to be insensitive against the range of flow rate and pH normally employed during expanded bed adsorption (EBA) of proteins. Analyzing the RTD obtained after a perfect ion tracer pulse in terms of the PDE model (PDE, axially dispersed plug-flow exchanging mass with stagnant zones) gave a quantitative description of the underlying hydrodynamic situation during EBA processing. These data provided a powerful tool to make predictions on the adsorptive global process performance with a defined feedstock type and composition. The link between the hydrodynamic events during feedstock application and the actual process performance was shown when applying intact yeast cell suspensions at different biomass content (up to 7.5% wet weight) and buffer conductivity (5-12 mS) onto an EBA column filled with the adsorbent Streamline Q XL as fluidized phase. On the basis of our experimental results, a guideline for the successful application of the ISE/RTD method to EBA process design is presented.

The hydrophobins HFBI and HFBII from Trichoderma reesei showing efficient interactions with nonionic surfactants in aqueous two-phase systems.

Fungal hydrophobins are a group of surface active, self-assembling proteins. The filamentous fungus Trichoderma reesei produces two (class II) hydrophobins, HFBI and HFBII. We have studied how these water-soluble hydrophobins behave in two-phase systems using a series of nonionic surfactants with different characteristics. It was found that both hydrophobins, but especially HFBI, had a very high affinity for the surfactants. The highest partitioning coefficient, over 2500, was observed for HFBI with C(11)EO(2). Reducing the disulfides in the protein resulted in a complete loss of affinity for the surfactant, which demonstrates that the interaction is dependent on the disulfide-stabilized conformation. The hydrophobins could be efficiently extracted back from the surfactant phase by addition of alcohols such as isobutanol. Effects of the type of surfactant, temperature, pH, and ionic strength were investigated. The use of this method for purifying the proteins from crude fungal culture supernatants is demonstrated and implications of the protein-polymer interaction are discussed.

Isolation of a recombinant formate dehydrogenase by pseudo-affinity expanded bed adsorption.

Formate dehydrogenase (FDH) is an enzyme of industrial interest, which is recombinantly expressed as an intracellular protein in Escherichia coli. In order to establish an efficient and reliable purification protocol, an expanded bed adsorption (EBA) process was developed, starting from the crude bacterial homogenate. EBA process design was performed with the goal of finding operating conditions which, on one hand, allow efficient adsorption of the target protein and which, on the other hand, support the formation of a perfectly classified fluidised bed (expanded bed) in the crude feed solution. A pseudo-affinity ligand (Procion Red HE3B) was used to bind the FDH with high selectivity and reasonable capacity (maximum equilibrium capacity of 30 U/ml). Additionally, a simplified modelling approach, involving small packed beds for generation of process parameters, was employed for defining the operating conditions during sample application. In combination with extended elution studies, a process was set up, which could be scaled up to 7.5 l of adsorbent volume yielding a total amount of 100,000 U of 94% pure FDH per run. On this scale, 19 l of a benzonase-treated E. coli homogenate of 15% wet-weight (pH 7.5, 9 mS/cm conductivity) were loaded to the pseudo-affinity adsorbent (0.25 m sed. bed height, 5 x 10(-4) m/s fluid velocity). After a series of two wash steps, a particle-free eluate pool was obtained with 85% yield of FDH. This excellently demonstrates the suitability of expanded bed adsorption for efficient isolation of proteins by combining solid-liquid separation with adsorptive purification in a single unit operation.

Human chymotrypsinogen B production from Pichia pastoris by integrated development of fermentation and downstream processing. Part 2. Protein recovery.

The purification of human chymotrypsinogen B (hCTRB) after expression and secretion by the yeast Pichia pastoris is described based on two different approaches using integrated initial recovery. Extraction employing aqueous two-phase systems (ATPS) from poly(ethylene glycol) and sodium sulfate allows direct processing of cell containing yeast suspensions of 50% wet weight. The target protein is obtained partially purified in the top phase while cells and cell debris are partitioned to the bottom phase of the system. hCTRB is further purified by adsorption from the top phase to the cation exchanger SP Sepharose Big Beads and elution in a salt step. The single step isolation of hCTRB is possible by expanded bed adsorption (EBA) using a fluidized cation exchanger (Streamline SP XL). A design strategy is shown taking both target protein binding and stable fluidization of the stationary phase in cell containing suspensions into consideration. For the example of hCTRB isolation from cell containing P. pastoris suspensions, a successful use of this strategy is demonstrated. Both initial recovery strategies deliver a product that can be further purified and formulated by ultrafiltration/diafiltration followed by lyophilization, resulting in a homogeneous product. Scale-up to 30-90 L of culture suspension was shown for both methods, resulting in a product of similar quality. Comparing both strategies reveals that the two-step ATPS route is better suited for high cell density cultures, while the single step EBA method is preferred for cultures of moderate cell density. This is due to the fact that application of EBA is restricted to suspensions of 10-12.5% wet weight cell concentration, thus necessitating dilution of the original broth prior to sample application. The data presented show that integrated recovery operations are a valuable alternative to traditional processing for systems that are problematic during initial solid-liquid separation.

Studies on peptide amidase-catalysed C-terminal peptide amidation in organic media with respect to its substrate specificity.

Peptide amidase-catalysed amidations of the C- terminal carboxylic group of peptides were studied using model substrates of a large series of N(alpha)-protected di-, tri-, tetra- and penta-peptides in the presence of NH4HCO3 as the ammonium source. The maximal yields of amide syntheses were achieved in a medium consisting of acetonitrile with 20-25 vol% of dimethylformamide and 3 vol% of water. Under these conditions, the substrate specificity of the enzyme was more restricted in the synthetic reaction than was found for the amide hydrolysis. Elongation of the peptide chain had a negative effect on enzymic amidation. Thus the direct amidation of N(alpha)-t-butoxycarbonyl-protected Leu-enkephalin resulted in a low yield of protected enkephalin amide.

Screening, overexpression and characterization of an N-acylamino acid racemase from Amycolatopsis orientalis subsp. lurida.

Thirty-one different actinomycete strains were used in a genetic screening using PCR and Southern hybridization methods to detect N-acetylamino acid racemases (AAR) in order to obtain enzymes with different properties. Cloning and sequencing of a 2.5 kb EcoRI DNA fragment from Amycolatopsis orientalis subsp. lurida revealed the coding gene of an N-acetylamino acid racemase, which had identities to the aar gene of Amycolatopsis sp. TS-1-60 [Tokuyama and Hatano (1995) Appl Microbiol Biotechnol 42:884-889] of 86% at the level of DNA, and 90% at the level of amino acids. The heterologous overexpression in Escherichia coli resulted in a specific activity of about 0.2 U/mg of this racemase. A two-step purification with heat treatment followed by anion-exchange chromatography led to almost homogeneous enzyme. The optimum pH of the enzyme was 8.0 and it was stable at 50 degrees C for 30 min. The relative molecular mass of the native enzyme and the subunit was calculated to be 300 kDa and 40 kDa by gel filtration and SDS-PAGE, respectively. The isoelectric point (pI) of the AAR was 4.4. It catalyzed the racemization of optically active N-acetylamino acids such as N-acetyl-L- or -D-methionine and N-acetyl-L-phenylalanine. Further characterization of the racemase demonstrated a requirement for divalent metal ions (Co2+, Mn2+, Mg2+) for activity and inhibition by EDTA and p-hydroxymercuribenzoic acid. AAR is sensitive to substrate inhibition at concentrations exceeding 200 mM.

Parameters influencing protein extraction for whole broths in detergent based aqueous two-phase systems.

The parameters important for an optimisation of cloud point extraction in technical scale were investigated using a genetically engineered fusion protein derived from endoglucanase I expressed in Trichoderma reesei and the nonionic polyoxyethylene Agrimul NRE 1205. The key parameters are temperature, detergent concentration, and additional salts. These parameters are interdependent, thus there is an optimum in the partition coefficient with respect to detergent concentration and a maximum for the partition coefficient and the yield with respect to temperature. These results were confirmed for the detergent C12E5 to demonstrate that these optima are due to the nature of polyoxyethylenes. Cloud point extraction was found to be only slightly affected by pH. In the case studied extraction of whole broth is favourable for a high yield and partition coefficient, since fusion protein adhering to the cells can be solubilized. However some loss of detergent which remains in the fungal biomass was observed.

Physical properties of detergent-based aqueous two-phase systems.

The physical behavior of the binary phase systems of the non-ionic polyoxyethylene detergent Agrimul NRE 1205 and water was investigated. This technical detergent can be used for the large-scale recovery of biomolecules in detergent based aqueous two-phase systems. The phase diagram was determined. It shows significant and unexpected differences to highly purified detergents. Very similar to neat detergents the phase diagram can be influenced by auxiliary chemicals thus shifting the entire phase diagram in general to lower temperatures. This was demonstrated by lowering the cloud-point by various additions. The concentration factor, as an important parameter of a first capture step in purification was investigated and modeled. Auxiliary chemicals, temperature change and change in detergent concentration also influence the viscosity and density of the phases. These experimental data are shown. They can help to explain the separation behavior of proteins. In large-scale separations aqueous two-phase systems are separated using disc-stack centrifuges. It is demonstrated that this is not a feasible method for detergent-based aqueous two-phase extraction and the physical reason is presented.

Minimising biomass/adsorbent interactions in expanded bed adsorption processes: a methodological design approach.

Expanded bed adsorption (EBA) is an integrated technology for the primary recovery of proteins from crude feedstock. Interactions between solid matter in the feed suspension and fluidised adsorbent particles influence bed stability and therefore have a significant impact on protein adsorption in expanded beds. In order to design efficient and reliable EBA processes a strategy is needed, which allows to find operating conditions, where these adverse events do not take place. In this paper a methodological approach is presented, which allows systematic characterisation and minimisation of cell/adsorbent interactions with as little experimental effort as possible. Adsorption of BSA to the anion exchanger Streamline Q XL from a suspension containing S. cerevisiae cells was chosen as a model system with a strong affinity of the biomass towards the stationary phase. Finite bath biomass adsorption experiments were developed as an initial screening method to estimate a potential interference. The adhesiveness of S. cerevisiae to the anion exchanger could be reduced significantly by increasing the conductivity of the feedstock. A biomass pulse response method was used to find optimal operation conditions showing no cell/adsorbent interactions. A good correlation was found between the finite bath test and the pulse experiment for a variety of suspensions (intact yeast cells, E. coli homogenate and hybridoma cells) and adsorbents (Streamline Q XL, DEAE and SP), which allows to predict cell/adsorbent interactions in expanded beds just from finite bath adsorption tests. Under the optimised operating conditions obtained using the prior methods, the stability of the expanded bed was investigated during fluidisation in biomass containing feedstock (up to 15% yeast on wet weight basis) employing residence time distribution analysis and evaluation by an advanced model. Based on these studies threshold values were defined for the individual experiments, which have to be achieved in order to obtain an efficient EBA process. Breakthrough experiments were conducted to characterise the efficiency of BSA adsorption from S. cerevisiae suspensions in EBA mode under varying operating conditions. This allowed to correlate the stability of the expanded bed with its sorption efficiency and therefore could be used to verify the threshold values defined. The approach presented in this work provides a fast and simple way to minimise cell/adsorbent interactions and to define a window of operation for protein purification using EBA.

Cloning and nucleotide sequence of a linear DNA plasmid from Xanthophyllomyces dendrorhous (Phaffia rhodozyma).

Extrachromosomal elements were found in a strain of X. dendrorhous, and were characterized as linear DNA forming two well defined groups, pPh1 with 3 high-copy-number molecules, pPh11 (6.9 kb), pPh12 (5.7), pPh13 (4.7), and pPh2 with 2 low-copy-number molecules, pPh21 (3.6 kb), pPh22 (3.0). A 4077 bp fragment from pPh13 was cloned in pUC18 (pDK1) and sequenced (accession no. AJ 278,424). Seven putative ORF and some possible regulator sequences were defined.

Fed-batch production of recombinant human calcitonin precursor fusion protein using Staphylococcus carnosus as an expression-secretion system.

A pH-auxostatic fed-batch process was developed for the secretory production of a fusion protein consisting of the pro-part of Staphylococcus hyicus lipase and two synthetic human calcitonin (hCT) precursor repeats under the control of a xylose-inducible promotor from Staphylococcus xylosus. Using glycerol as the energy source and pH-controlled addition of yeast extract resulted in the production of 2000 mg 1(-1) of the fusion protein (420 mg 1(-1) of the recombinant hCT precursor) within 14 h, reaching 45 g 1(-1) cell dry mass with Staphylococcus carnosus in a stirred-tank reactor. Product titer and space-time yield (30 mg calcitonin precursor 1(-1) h(-1)) were thus improved by a factor of 2, and 4.5, respectively, compared to Escherichia coli expression-secretion systems for the production of calcitonin precursors. Two hundred grams of the fusion protein was secreted by the recombinant S. carnosus on a 150-1 scale (scale-up factor of 50) with a minimum use of technical-grade yeast extract (40 mg fusion protein g(-1) yeast extract).

Investigating expression systems for the stable large-scale production of recombinant L-leucine-dehydrogenase from Bacillus cereus in Escherichia coli.

The established Escherichia coli expression vectors ptrc99a, pKK223-3, pPLlambda, pAsk75, pRA95, and pRA96, which differ in copy number, mode of induction, selection marker, and use of par sequences for stabilization, were investigated for the stable expression of recombinant L-leucine dehydrogenase from Bacillus cereus with a view to large-scale production. Best results were achieved with pIET98, a runaway-replication system derived from pRA96. Expression of L-leucine dehydrogenase was controlled by its constitutive B. cereus promoter and depended on host strain, cultivation temperature, induction time, and media composition. After cell cultivation at 30 degrees C and shifting to 41 degrees C to induce plasmid replication, E. coli BL21[pIET98] yielded 200 U LeuDH/mg protein, which corresponds to >50% of the soluble cell protein. Continuous cultivation in a semisynthetic high-cell-density medium verified structural and segregational stability over 100 generations in the absence of a selection pressure.

Investigation of mathematical methods for efficient optimisation of aqueous two-phase extraction.

Mathematical strategies were applied to optimise the extraction of recombinant leucine dehydrogenase from E. coli homogenates and endoglucanase 1 from culture filtrates of Trichoderma reesei in polyethylene glycol-phosphate systems. The goal was to test mathematical tools which could facilitate the optimisation procedure in aqueous two-phase systems. A modified simplex approach, the method of steepest ascent and a genetic algorithm were successfully applied and compared. The methods differ in the height of the optimum found, the number of experiments and the time required. The genetic algorithm proved to be an optimisation procedure which can be used well in aqueous two-phase systems. The simplex procedure has to be further improved.

Production of recombinant L-leucine dehydrogenase from Bacillus cereus in pilot scale using the runaway replication system E. coli[pIET98].

A method for the production of recombinant L-leucine dehydrogenase from Bacillus cereus in pilot scale is described employing the temperature induced runaway replication vector pIET98 and the Escherichia coli host strain BL21. Fed-batch cultivation using a semi-synthetic high-cell densitiy medium was adjusted in 5-L scale to yield a constant growth rate of 0,17 h(-1) and a final cell concentration of 27 g dry weight/L by exponentially increasing the nutrient supply. Runaway replication and thus, LeuDH expression was induced during the feeding phase by increasing the cultivation temperature to 41 degrees C yielding a specific enzyme activity of 110 U/mg, which corresponds to 30% of the soluble cell protein. The cultivation was terminated when the dissolved oxygen content fell below 10% saturation. The final volume activity was 600,000 U/L cultivation. No change in growth, cell density, or expression activity was observed scaling up the cultivation volume to 200 L. Thus, 120,000,000 units L-leucine dehydrogenase were obtained from one cultivation. The purification of L-leucine dehydrogenase to homogeneity was carried out by heat denaturation, liquid-liquid extraction, gel filtration, and anion-exchange chromatography to give pure enzyme in 65% yield. The integrity of the recombinant enzyme was tested measuring the molecular weight and determining the N-terminal amino acid sequence.

Aqueous two-phase systems containing urea: influence of protein structure on protein partitioning.

During recombinant E. coli fermentation with high-expression levels inclusion bodies are often formed. Aqueous two-phase systems have been successfully used in the presence of urea for the initial recovery step of inclusion bodies from E. coli. Basic studies of the complex interactions are lacking. For a systematic study of protein partitioning in the presence of urea we selected T4-lysozyme mutants with different thermal stability as a model. The stabilization of these variants by phase components was investigated measuring the fluorescence emission of tryptophan residues in the protein. Protein structure was stabilized at pH 7 in the order of S0(4)(2-) >> PEG = Dextran > H(2)O. The conformation of proteins was shown to have a strong influence on the partitioning in aqueous two-phase systems. Tryptophan and its homologuous di- and tripeptdides were partitioned in similar phase systems to normalize for contribution from hydrophobic interactions.

The influence of cell adsorbent interactions on protein adsorption in expanded beds.

Expanded bed adsorption (EBA) is a primary recovery operation allowing the adsorption of proteins directly from unclarified feedstock, e.g. culture suspensions, homogenates or crude extracts. Thus solid-liquid separation is combined with adsorptive purification in a single step. The concept of integration requires that the solid components of the feed solution are regarded as a part of the process, which influences stability, reproducibility, and overall performance. This aspect is investigated here at the example of the influence of presence and concentration of intact yeast cells (S. cerevisiae) on the adsorption of model proteins (hen egg white lysozyme and bovine serum albumin) to various stationary phases (cation and anion-exchange, hydrophobic interaction, immobilised metal affinity). The interaction of the cells with the adsorbents is determined qualitatively and quantitatively by a pulse response method as well as by a finite bath technique under different operating conditions. The consequence of these interactions for the stability of expanded beds in suspensions of varying cell concentration is measured by residence time distributions (RTDs) after tracer pulse injection (NaBr, LiCl). Analysis of the measured RTD by the PDE model allows the calculation of the fraction of perfectly fluidised bed (phi), a parameter which may be regarded as a critical quantity for the estimation of the quality of fluidisation of adsorbents in cell containing suspensions. The correlation between bed stability and performance is made by analysing the breakthrough of model proteins during adsorption from unclarified yeast culture broth. A clear relationship is found between the degree of cell/adsorbent interaction, bed stability in terms of the phi parameter, and the sorption efficiency. Only beds characterised by a phi value larger than 0.8 in the presence of cells will show a conserved performance compared to adsorption from cell free solutions. A drop in phi, which is due to interactions of the fluidised adsorbent particles with cells from the feed, will directly result in a reduced breakthrough efficiency. The data presented highlight the importance of including the potential interaction of solid feedstock components and the expanded adsorbents into the design of EBA processes, as the interrelation found here is a key factor for the overall performance of EBA as a truly integrated operation.