Incomplete mixing in large bioreactors — a study of its role in the fermentative production of streptokinase

The production of streptokinase in a batch fermentation has been analysed for the role of incomplete macromixing of the broth. The analysis is based on a kinetic model exhibiting inhibition by the substrate and a primary metabolite (lactic acid), and a mixing model comprising two continuous flow reactors (CFRs) with closed-loop recycle. The inoculum is introduced into one region (one CFR) and the mixing process determines its distribution, growth and reactivity. By varying the dilution rates of the CFRs, any degree of macromixing can be simulated. For dilution rates larger than 1.0 h^?1 almost complete macromixing is achieved, for which an analogy has been drawn with micromixing. Increasing the volume of the inoculated region relative to the noninoculated region improves the maximum attainable activity of streptokinase and shortens the time for this. In such a situation an imperfectly mixed bioreactor is superior to a perfectly mixed one, implying that good productivity requires a large inoculated region and incomplete macromixing. These inferences are supported by earlier studies of fluid mixing and relaxation times in bioreactors.     

An evaluation of a neural network for the start-up phase of a continuous recombinant fermentation subject to disturbances

Summary A radial basis neural network was applied to a process for glyceraldehyde-3-phosphate dehydrogenase produced by an Escherichia coli strain containing the plasmid pBR Eco gap. A neural network trained with a pure culture predicted the performance of a fermentation containing wild type cells and/or product in the inoculum better than in the reverse case; this is explained. In general, the network learnt the trends in the concentrations of plasmid-containing cells and the recombinant product more accurately than those of wild type cells and the substrate. This similarity with deterministic networks and the good predictability with some training vectors suggests that neural networks can be used to simulate the start-up phase of recombinant fermentations corrupted by disturbances.     

The effect of micro- and macromixing on enzyme reaction in a real CSTR

The effect of micromixing and macromixing on enzyme reaction of Michaelis-Menten type in a real continuously stirred tank reactor (CSTR) is considered. The effect of bypassing of a fraction of feed stream, dead space, initial enzyme concentration and Michaelis-Menten constant on substrate conversion is evaluated. Bypass reduces the substrate conversion significantly compared with other parameters in the case of micro and macromixing. Micromixing predicts higher substrate conversions compared with macromixing. The effect of micro and macromixing on substrate conversion is negligible at low and high conversions.List of Symbols C kmol/m³ concentration of reactant - ¯C kmol/m³ average concentration of reactant - C_A kmol/m³ exit concentration of reactant A - C_Aa kmol/m³ exit concentration of reactant A from active zone - C_AO kmol/m³ initial concentration of reactant A - C_EO kmol/m³ initial enzyme concentration - C_O kmol/m³ initial concentration of reactant - E(t) 1/s exit age distribution function - k 1/s reaction rate constant - M kmol/m³ Michaelis-Menten constant - r kmol/(m³s) rate of reaction - –r_A kmol/(m³s) rate of reaction with respect to A - t s time - v m³/s volumetric feed rate - v_a m³/s volumetric feed rate entering the active zone - v_b m³/s volumetric feed rate entering the bypass stream - V m³ total volume of the vessel - V_a m³ active volume of the vessel - V_d m³ volume of dead space - X_A conversion of A Greek Letters fraction of feed stream bypassing the vessel (v_b/v) - fraction of the total volume as dead space (V_d/V) - (t) 1/s Dirac delta function, an ideal pulse occurring at time t = 0 - s life expectancy of a molecule - 1/s intensity function or escape probability function - s space time or mean residence time     

A Method forin SituMitotic Spindle Binding Assay

TheXenopuscentrosome protein kinase pEg2, involved in spindle assembly, binds to microtubules polymerizedin vitro.We have developed a method to investigate the affinity of purified recombinant pEg2 protein for the cellular mitotic spindle. Briefly, cells grown on coverslips are fixed, permeabilized, and incubated with recombinant pEg2 protein. Localization of the protein is revealed by probing with a specific monoclonal antibody that recognizes recombinant but not endogenous pEg2. Using this method we show that recombinant pEg2 binds to microtubulesin vitro,while,in vivo,pEg2 localized only to the mitotic spindle and not the interphase microtubule network. We also demonstrate that the catalytic activity of pEg2 is not necessary for its binding ability. This technique can be used to analyze the binding of various tagged proteins to cellular mitotic spindle.     

Isolation,purification, and study of properties of recombinant hepsin from <Emphasis Type="Italic">Escherichia coli</Emphasis>

A recombinant hepsin-producing strain of Escherichia coli was obtained and the conditions for hepsin expression in a bacterial system were optimized. To study the physicochemical properties of the enzyme, a procedure for purification of active recombinant hepsin using metal-chelate affinity chromatography and ion-exchange chromatography was developed. The interaction of recombinant hepsin with various peptide substrates is characterized. The dose-dependent inhibition of the recombinant hepsin enzyme activity by anthralin in vitro and an increase in the hepsin enzymatic activity in the presence of resveratrol were revealed.     

Mixing state of xanthan gum solution in an aerated and agitated fermentor—Effects of impeller size on volumes of mixing regions and circulation time distribution

The effects of the impeller diameter and width on the volumes of the micromixing and macromixing regions, and on the circulation time distribution were investigated at various agitation speeds to formulate the relationships of them in emperical equations. A fermentor was a 10-l capacity, which was equipped with a turbine impeller with six flat balades and aerated at 1 vvm. It was found that the volumes of the micromixing and macromixing regions depended on the tip speed of the impeller, ND, and the discharging performance of the impeller, ND²W, respectively, in the xabthan gum solution with concentrations of 0.9, 1.8, 2.7, and 3.9%. Empirical equations were derived to estimate the volume of each mixing region from the impeller diameter, D, impeller width, W, agitation speed, N, and consistency coefficient of the xanthan gum solution. On the other hand, the circulation time distribution could be estimated empirically from only the impeller diameter and agitation speed, regardless of variation in the impeller width and consistency coefficient of the xanthan gum solution tested.     

Mechanistic analysis of xanthan gum production in a stirred tank

The overall effect of agitation on xanthan gum production by Xanthomonas campestris ATCC13951 in a stirred vessel was mechanistically analyzed considering local variation of the specific production rate due to variation of shear stress in the vessel. The whole liquid volume in a fermentor was roughly divided into three regions; the micromixing region around the impeller with high shear stress, the macromixing region dominated by a circulating flow and the stagnant region. The value of the shear rate was first ascertained by experiments in order to obtain a picture of shear rate variation in a radial direction from the impeller, and the equivalence between the volumes of the high shear stress region and micromixing region was confirmed. The shear stress obtained using a correlation between the shear rate at the impeller tip and Reynolds number of Wichterle et al. was used as a representative of the shear stress in the micromixing region, and the shear stress estimated by use of an empirical correlation between the average shear rate in a fermentor and agitation speed derived by Metzner et al. was adopted as a representative of the shear stress in the macromixing region. The information about the circulation time distribution was also used to take into account oxygen deficiency during circulation of liquid elements in the macromixing region, considering that oxygen from the gas phase was supplied mainly in the high shear region. The calculated values of xanthan gum concentrations which were obtained by the proposed simulation method agreed well with the experimental data in the time course of xanthan gum production at various agitation speeds. Experimental results of the relationship between the overall specific production rate and ND (N, agitation speed, and D, impeller diameter) was also verified by the proposed method.     

Effects of E. coli chaperones on the solubility of human receptors in an in vitro expression system

The implementation of efficient technologies for the production of recombinant mammalian membrane receptors is an outstanding challenge in understanding receptor-ligand actions and the development of therapeutic antibodies. In order to improve the solubility of recombinant extracellular domains of human membrane receptors expressed in Escherichia coli, proteins were synthesized by an E. coli in vitro translation system supplemented with bacterial molecular chaperones, such as GroEL-GroES (GroEL/ES), Trigger factor (TF), a DnaK-DnaJ-GrpE chaperone system (DnaKJE), and/or a heat shock protein Hsp100, ClpB. The following three proteins that are prone to aggregation were examined: the extracellular domain (ECD) or the second immunoglobulin-like domain (IgII) of the human neurotrophin receptor TrkC (TrkC-ECD and TrkC-IgII), and the C-type lectin carbohydrate recognition domain of the human asialoglycoprotein receptor (ASGPR HI CRD). The cooperative chaperone system including GroEL/ES, DnaKJE and ClpB had a marked effect on the solubility of TrkC-ECD and TrkC-IgII, and the GroEL/ES-DnaKJE-TF chaperone system was more effective for TrkC-IgII. The GroEL/ES-DnaKJE-TF chaperone network increased the yield of soluble ASGPR HI CRD. The present findings demonstrate that E. coli molecular chaperones are useful in improving the yield of soluble recombinant extracellular domains of human membrane receptors in an E. coli expression system.     

Metabolic approaches for the optimisation of recombinant fermentation processes

The aim of this work was the establishment of a novel method to determine the metabolic load on host-cell metabolism resulting from recombinant protein production in Escherichia coli. This tool can be used to develop strategies to optimise recombinant fermentation processes through adjustment of recombinant-protein expression to the biosynthetic capacity of the host-cell. The signal molecule of the stringent-response network, guanosine tetraphosphate (ppGpp), and its precursor nucleotides were selected for the estimation of the metabolic load relating to recombinant-protein production. An improved analytical method for the quantification of nucleotides by ion-pair, high-performance liquid chromatography was established. The host-cell response upon overexpression of recombinant protein in fed-batch fermentations was investigated using the production of human superoxide dismutase (rhSOD) as a model system. E. coli strains with different recombinant systems (the T7 and pKK promoter system) exerting different loads on host-cell metabolism were analysed with regard to intracellular nucleotide concentration, rate of product formation and plasmid copy number. Received: 30 April 1999 / Received revision: 26 July 1999 / Accepted: 1 August 1999     

Fusion with maltose-binding protein (MBP) affects neither RNA N-glycosidase activity nor immunogenicity of karasurin-A, a ribosome-inactivating protein from Trichosanthes kirilowii var. japonica

A genomic clone encoding mature karasurin-A (KRNA), a ribosome-inactivating protein from Trichosanthes kirilowii var. japonica, was efficiently expressed in E. coli using an expression cassette vector pMAL-c2. The resultant recombinant KRNA fused with maltose-binding protein (MBP) was recovered from the soluble fraction of the bacterial cells and purified to near homogeneity after one round of the affinity chromatography. Neither the karasurin precursor retaining both N- and C-terminal peptides, nor the protein with the N-terminal peptide was successufully produced even as a MBP-fusion. The protein with its C-terminal peptide was over-produced but was recovered in an insoluble fraction. Both the recombinant MBP-KRNA fusion protein and recombinant KRNA with MBP removed were as active as the native KRNA from root tubers. The immunogenicity of the recombinant KRNA was also unaffected by fusion with MBP.     

Bioengineering problems connected with the use of conventional and unconventional raw materials in fermentation

Some bioengineering problems connected with the use of conventional and unconventional raw materials in fermentation research and industry are reviewed. They include the effect of the physical state of different substrates (solid, liquid, gaseous) and considerations of physico-chemical processes, especially the identification of limiting steps. A new concept of classification of fermentors with respect to the macromixing properties is suggested and its applicability for different substrates is considered. Presented at the seminar “Raw materials in the fermentation industry”, organized by the Czechoslovak Society for Microbiology, Nové Město na Moravě, April 28–30, 1976.     

High-level expression, purification and characterization of recombinant Aspergillus oryzae alkaline protease in Pichia pastoris

The alkaline protease gene from Aspergillus oryzae was cloned, and then it was successfully expressed in the heterologous Pichia pastoris GS115 with native signal peptide or α-factor secretion signal peptide. The yield of the recombinant alkaline protease with native signal peptide was about 1.5-fold higher than that with α-factor secretion signal peptide, and the maximum yield of the recombinant alkaline protease was 513 mg/L, which was higher than other researches. The recombinant alkaline protease was purified by ammonium sulfate precipitation, ion exchange chromatography and gel filtration chromatography. The purified recombinant alkaline protease showed on SDS–PAGE as a single band with an apparent molecular weight of 34 kDa. The recombinant alkaline protease was identical to native alkaline protease from A. oryzae with regard to molecular weight, optimum temperature for activity, optimum pH for activity, stability to pH, and similar sensitivity to various metal ions and protease inhibitors. The native enzyme retained 61.18% of its original activity after being incubated at 50 °C for 10 min, however, the recombinant enzyme retained 56.22% of its original activity with same disposal. The work demonstrates that alkaline protease gene from A. oryzae can be expressed largely in P. pastoris without affecting its enzyme properties and the recombinant alkaline protease could be widely used in various industrial applications.     

Efficient Itaconic acid production via protein–protein scaffold introduction between GltA,AcnA, and CadA in recombinant Escherichia coli

Itaconic acid, which is a promising organic acid in synthetic polymers and some base-material production, has been produced by Aspergillus terreus fermentation at a high cost. The recombinant Escherichia coli that contained the cadA gene from A. terreus can produce itaconic acid but with low yield. By introducing the protein–protein scaffold between citrate synthesis, aconitase, and cis-aconitase decarboxylase, 5.7 g/L of itaconic acid was produced, which is 3.8-fold higher than that obtained with the strain without scaffold. The optimum pH and temperature for itaconic acid production were 8.5 and 30°C, respectively. When the competing metabolic network was inactivated by knock-out mutation, the itaconic acid concentration further increased, to 6.57 g/L.     

Cloning of a superoxide dismutase gene from Listeria ivanovii by functional complementation in Escherichia coli and characterization of the gene product.

Summary A gene encoding superoxide dismutase (EC 1.15.1.1., SOD) was isolated from a plasmid library of chromosomal DNA from Listeria ivanovii by functional complementation of an SOD-negative Escherichia coli host. The nucleotide sequence of the cloned gene was determined and contained an open reading frame which codes for a protein of 202 amino acid residues (calculated molecular weight 22 755 Da including the amino-terminal methionine residue). Comparison of the deduced amino acid sequence of L. ivanovii SOD with previously reported SOD amino acid sequences revealed considerable homologies with Fe- and Mn-dependent SODs. Enzymatic analyses using cell lysates and the purified recombinant enzyme indicated that this SOD is manganese-dependent. The recombinant SOD accounted for up to 30% of the total soluble protein in recombinant E. coli and protected sodA sodB mutants against the toxic effects of paraquat. Subunits of the recombinant Listeria SOD and of both E. coli SODS formed enzymatically active hybrids in vivo.Some of our preliminary observations have been published as a conference report of SOD V (Jerusalem, 1989) in Free Rad Res Commun (1991) 12–13:371     

Characterization of an OmpA-like outer membrane protein of the acidophilic iron-oxidizing bacterium, <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis>

An OmpA family protein (FopA) previously reported as one of the major outer membrane proteins of an acidophilic iron-oxidizing bacterium Acidithiobacillus ferrooxidans was characterized with emphasis on the modification by heat and the interaction with peptidoglycan. A 30-kDa band corresponding to the FopA protein was detected in outer membrane proteins extracted at 75°C or heated to 100°C for 10 min prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). However, the band was not detected in outer membrane proteins extracted at ≤40°C and without boiling prior to electrophoresis. By Western blot analysis using the polyclonal antibody against the recombinant FopA, FopA was detected as bands with apparent molecular masses of 30 and 90 kDa, suggesting that FopA existed as an oligomeric form in the outer membrane of A. ferrooxidans. Although the fopA gene with a sequence encoding the signal peptide was successfully expressed in the outer membrane of Escherichia coli, the recombinant FopA existed as a monomer in the outer membrane of E. coli. FopA was detected in peptidoglycan-associated proteins from A. ferrooxidans. The recombinant FopA also showed the peptidoglycan-binding activity.     

Gene cloning and enzyme structure modeling of the <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> N74 fructosyltransferase

The fructooligosaccharides (FOS) represent an important source of prebiotic compounds that are widely used as an ingredient in functional foods. Recently, the strain Aspergillus oryzae N74 was reported as a potential microorganism for the industrial production of FOS, due to its high yields of FOS production. In this work, we used a PCR-cloning strategy to clone the A. oryzae N74 ftase gene as a previous step for recombinant enzyme production. Ftase showed a 1630 bp size with a 99% similarity with other A. oryzae strains and between 1 to 68% identities with other Aspergillus strains. This gene encodes for a 525 amino acids protein with 99% similarity with other A. oryzae strains and between 11 to 69% similarities with other Aspergillus strains. Finally, an A. oryzae N74 FTase tertiary structure model was predicted base on its similarity with other glycoside hydrolase 32 family members. The active site was located inside the β-propeller domain and was formed for non-charged polar and charged amino acids. In summary, these results shows the high level of sequence conservation between A. oryzae strains and represent a first step towards the development of a FOS production industrial process using recombinant microorganism carrying the ftase gene from A. oryzae N74.     

Expression and purification of antimicrobial peptide buforin IIb in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A novel production method in Escherichia coli for an antimicrobial peptide of 21 amino acids, buforin IIb, which is a synthetic analog of buforin II, has been developed. The buforin IIb gene was cloned into the vector pET32a to construct an expression vector pET32a–buforin IIb. The fusion protein Trx-buforin IIb, purified by nickel nitrilo-triacetic acid (Ni-NTA) resin chromatography, was cleaved by hydroxylamine hydrochloride to release recombinant buforin IIb. Purification of recombinant buforin IIb was achieved by HPLC: about 3.1 mg/l active recombinant buforin IIb with purity >99% was obtained. The recombinant buforin IIb showed antimicrobial activities that were similar to the synthetic one.     

Production of a tumour‐targeting antibody with a human‐compatible glycosylation profile in N. benthamiana hairy root cultures

Hairy root (HR) cultures derived from Agrobacterium rhizogenes transformation of plant tissues are an advantageous biotechnological manufacturing platform due to the accumulation of recombinant proteins in an otherwise largely protein free culture medium. In this context, HRs descending from transgenic Nicotiana tabacum plants were successfully used for the production of several functional mAbs with plant‐type glycans. Here, we expressed the tumor‐targeting monoclonal antibody mAb H10 in HRs obtained either by infecting a transgenic N. tabacum line expressing H10 with A. rhizogenes or a glyco‐engineered N. benthamiana line (ΔXTFT) with recombinant A. rhizogenes carrying mAb H10 heavy and light chain cDNAs. Selected HR clones derived from both plants accumulated mAb H10 in the culture medium with similar yields (2–3 mg/L). N‐glycosylation profiles of antibodies purified from HR supernatant revealed the presence of plant‐typical complex structures for N. tabacum‐derived mAb H10 and of GnGn structures lacking xylose and fucose for the ΔXTFT‐derived counterpart. Both antibody glyco‐formats exhibited comparable antigen binding activities. Collectively, these data demonstrate that the co‐infection of ΔXTFT Nicotiana benthamiana with recombinant A. rhizogenes is an efficient procedure for the generation of stable HR cultures expressing the tumor‐targeting mAb H10 with a human‐compatible glycosylation profile, thus representing an important step towards the exploitation of root cultures for the production of ‘next generation’ human therapeutic antibodies.     

Metabolic flux analysis for optimizing the specific growth rate of recombinant <Emphasis Type="Italic">Aspergillus niger</Emphasis>

A comprehensive metabolic network comprising three intracellular compartments (cytoplasm, mitochondrion and peroxisome) was developed for Aspergillus niger. The metabolic flux network includes carbohydrate and amino acid metabolism in both anabolic and catabolic reactions. Linear programming was used for the optimization of the specific growth rates in combination with 37 measured input and output fluxes of the key metabolites to evaluate corresponding intracellular flux distributions throughout the batch fermentations. Logarithmic sensitivity analysis revealed that the addition of proline, alanine and glutamate benefited growth in defined media. The experimental observations and flux analysis showed that tyrosine was a potential candidate for biomass production improvement. Model predictions was verified by conducting batch and fed-batch fermentations and it was found that the addition of the four amino acids according to the predetermined schedule resulted in a 44 and 41% improvements in biomass and recombinant protein productions, respectively.