Separation of cephalosporin C and desacetyl cephalosporin C by high speed counter-current chromatography in aqueous two-phase systems

Summary In the recovery of cephalosporin C (CPC) from fermentation broth, the separation of desacetyl cephalosporin C (DAC) is a major concern. Multistage extraction in aqueous two-phase systems, mainly PEG/ammonium sulfate systems, proved to be promising. In preparative scale operation, high speed counter-current chromatography (HSCCC) with eccentric columns was used with aqueous two-phase systems to obtain baseline resolution of CPC and DAC. Solvents (e.g. 5% acetone) or neutral salts (e.g. 1.45% KSCN) added into aqueous two-phase systems enhanced the separation efficiency. Operation parameters of HSCCC such as rotational speed and mobile phase flow rate can affect the retention of the stationary phase and HETP.     

Genetic engineering approach to reduce undesirable by-products in cephalosporin C fermentation

Deacetoxycephalosporin C (DAOC) is produced by Acremonium chrysogenum as an intermediate compound in the cephalosporin C biosynthetic pathway, and is present in small quantities in cephalosporin C fermentation broth. This compound forms an undesirable impurity, 7-aminodeacetoxycephalosporanic acid (7-ADCA), when the cephalosporin C is converted chemically or enzymatically to 7-aminocephalosporanic acid (7-ACA). In the cephalosporin C biosynthetic pathway of A. chrysogenum, the bifunctional expandase/hydroxylase enzyme catalyzes the conversion of penicillin N to DAOC and subsequently deacetylcephalosporin C (DAC). By genetically engineering strains for increased copy number of the expandase/hydroxylase gene, we were able to reduce the level of DAOC present in the fermentation broth to 50% of the control. CHEF gel electrophoresis and Southern analysis of DNA from two of the transformants revealed that one copy of the transforming plasmid had integrated into chromosome VIII (ie a heterologous site from the host expandase/hydroxylase gene situated on chromosome II). Northern analysis indicated that the amount of transcribed expandase/hydroxylase mRNA in one of the transformants is increased approximately two-fold over that in the untransformed host. Received 5 January 1998/ Accepted in revised form 29 May 1998     

Two-step immobilized enzyme conversion of cephalosporin C to 7-aminocephalosporanic acid

The first large-scale production of 7-aminocephalosporanic acid (7ACA) from cephalosporin C (CPC) using a wholly enzymatic synthesis method is reported here. We produced 7ACA from CPC in as high a molar yield as 85% using the immobilized enzymes D-amino acid oxidase (D-AOD) and glutaryl-7-ACA acylase (GL-acylase). In the first reactor, CPC is converted to keto-adipyl-7-aminocephalosporanic acid (keto-7ACA) using an immobilized D-AOD isolated from a yeast, Trigonopsis variabilis. The keto-7ACA is then spontaneously converted to glutaryl-7-aminocephalosporanic acid (GL-7ACA) via a chemical reaction with hydrogen peroxide. The hydrogen peroxide is also a product of the D-AOD reaction. Near quantitative conversion of the keto-7ACA to GL-7ACA was observed. The second reactor converts GL-7ACA to 7ACA using an immobilized GL-acylase, which was isolated from a reconbinant Escherichia coli. The final 7ACA crystalline product is a high quality product. The reactions are conducted under very mild aqueous conditions: pH 8.0 and 20 degrees to 25 degrees C. The production of desacetyl side products is minimal. This process is currently being implemented on an industrial scale to produce 7ACA. (c) 1995 John Wiley & Sons, Inc.     

ATPS applied to extraction of small molecules - polycetides - and simultaneous clarification of culture media with filamentous microorganisms

Aqueous two-phase systems (ATPS) were applied for extraction of small molecules (polycetides) - retamycin, an anthracyclin, and two red pigments, rubropunctamin and monascorubramin - from the whole culture media of Streptomyces olindensis and Monascus purpureus. ATPS allows, in one step, the separation of the small hydrophobic molecules in the PEG rich phase, from the filamentous microorganisms, which remains in the salt phase. Through experimental designs, the main variables and their levels were defined, as follows: for retamycin extraction, PEG 6000 (10%, w/w), phosphate at 20% (w/w) and pH 6.0 led to the higher partition coefficient, K(r) = 8.2, and yield = 91.3%; for red pigments, the statistical analysis indicate PEG 6000 (20%, w/w) and phosphate at 15% (w/w), for a high partition coefficient, (K(pig) = 113 and 150).     

Protein sorption and recovery by hydrogels using principles of aqueous two-phase extraction

Use of the thermodynamic principles of aqueous two-phase extraction (ATPE) to drive protein into a crosslinked gel is developed as a protein isolation and separation technique, and as a protein loading technique for drug delivery applications. A PEG/dextran gel system was chosen as a model system because PEG/dextran systems are widely used in aqueous two-phase extraction and dextran gels (Sephadex(R)) are common chromatographic media. The effects of polymer concentrations and molecular weights, salts, and pH on the partitioning of ovalbumin matched ATPE heuristics and data trends. Gel partition coefficients (Cgel/Csolution) increased with increasing PEG molecular weight and concentration and decreasing dextran concentration (increased gel swelling). The addition of PEG to the buffer solution yielded partition coefficients more than an order of magnitude greater than those obtained in systems with buffer alone, or added salt. A combined salt/PEG system yielded an additional order of magnitude increase. For example, when ovalbumin solution (2.3 mg/mL) was equilibrated with Sephadex(R) G-50 at pH 6.75, the partition coefficients were 0.13 in buffer, 0.11 in buffer with 0.22M KI, 2.3 in 12 wt% PEG-10,000 and 32.0 in 12 wt% PEG-10, 000 with 0.22M KI. The effect of anions and cations as well as ionic strength and pH on the partitioning of ovalbumin also matched ATPE heuristics. Using the heuristics established above, partition coefficients as high as 80 for bovine serum albumin and protein recoveries over 90% were achieved. In addition, the wide range of partition coefficients that were obtained for different proteins suggests the potential of the technique for separating proteins. Also, ovalbumin sorption capacities in dextran were as high as 450 mg/g dry polymer, and the sorption isotherms were linear over a broad protein concentration range.     

头孢菌素C生物合成调控研究进展北大核心CSCD

头孢菌素C由丝状真菌顶头孢霉产生,属于β-内酰胺类抗生素。其经改造后的7-氨基头孢烷酸是头孢类抗生素的重要中间体。头孢类抗生素在国内外抗生素市场中占有巨大的份额,是临床上的主要抗感染药物。随着分子生物学的发展,头孢菌素C的生物合成途径已基本阐明。为提高头孢菌素C的产量和降低生产成本,越来越多的研究者开始关注其较为精细、复杂的调控机制。本文重点对头孢菌素C生物合成及其调控机制的最新进展进行了简述,希望为今后头孢菌素C生产菌株的菌种改造和传统产业的升级换代提供一定的借鉴。     

Metal affinity extraction of human hemoglobin in an aqueous polyethylene glycol-sodium sulfate two-phase system

Summary We have developed a protein extraction technique which uses metal affinity ligands in PEG/salt aqueous two-phase systems. Cu(II)IDA-PEG will partition proteins according to their surface histidine contents in two-phase systems formed from sodium sulfate and polyethylene glycol. The nearly complete separation of human hemoglobin and human serum albumin in a single stage is presented as a demonstration of the effectiveness of metal affinity extraction in PEG/salt systems.     

有机溶剂对HPLC测定头孢菌素C含量的影响

考察了10种水溶性有机溶剂对HPLC测定头孢菌素C含量的影响,采用C_(18)色谱柱,流动相为磷酸二氢钠-甲醇(90:10),波长254 nm。发现不同的有机溶剂有不同程度的影响,其中乙醇的影响最大;乙醇浓度越高,峰面积就越小。将含有乙醇的头孢菌素C溶液稀释到一定程度,能够有效消除乙醇的影响。稀释法具有较好的精密度、重复性和回收率,为在工业生产中准确测定头孢菌素C的含量提供了依据。     

Production of cephalosporin C,and its intermediates,by raised-titre strains of Acremonium chrysogenum

Three different strains of Acremonium chrysogenum have been grown under identical fermentation conditions and their profiles with respect to cephalosporin C and its intermediates were compared. Clear differences were found between the strains; one notably accumulated a large pool of penicillin N, showing a reduced ability to convert this antibiotic to the later intermediates in the pathway, deacetoxycephalosporin C, deacetylcephalosporin C and cephalosporin C.     

Cybernetic modeling of the cephalosporin C fermentation process by Cephalosporium acremonium

A cybernetic mathematical model has been developed to describe the production of cephalosporin C. In developing the model, diauxic behavior of substrate consumption, morphological differentiation of cells, and catabolite repression of cephalosporin C production by the preferred substrate, glucose, were considered. The proposed model was tested on the experimental data from the literature and could adequately describe the morphological differentiation of cells, the sequential utilization of carbon sources and the production of cephalosporin C. It could be a useful tool to optimize the production of cephalosporin C by Cephalosporium acremonium in batch, fed-batch or continuous operations.     

Undefined xylose media extracted from biorefinery waste for enhanced and eco-friendly production of cephalosporin C by Acremonium chrysogenum M35

Xylose-rich undefined broth, extracted from the dilute acid pretreatment wastes of barley straw, serves as resourceful media for Acremonium chrysogenum M35 culture and production of cephalosporin C (CPC). Concentrating the extract with proper reprocessing enables to prepare various concentrations of xylose broth (2%–8%). The undefined xylose media were prepared for CPC production from A. chrysogenum M35 by the addition of other nutrients. Cell growth and CPC production were the most effective at 6% xylose and additional 2% glycerol, with maximum CPC production of 9.07 g/L after 6 days, which is higher production than that in defined media prepared with laboratory-level nutrients and reagents. Investigation of autotrophic and reverse trans-sulfuration pathways for cysteine synthesis, a limited element of three precursors for CPC synthesis, supports the enhanced CPC production in undefined media. Abundance of xylose ensures a maintained NADPH concentration required for sulfate reduction and synthesis of amino sulfide such as cysteine. Cystathionine-γ-lyase activity profiling indicated more efficient biosynthesis in undefined media than in other cultures use glycerol and glucose, and the biosynthesis pathway of CPC production by the cephalosporin gene cluster (i.e. pcbC and cefG genes) was investigated. The process using undefined xylose media was designed, and process simulation program confirmed our results.     

AQUEOUS TWO-PHASE EXTRACTION FOR THE PURIFICATION OF ALKALINE AGARASES FROM CULTURE EXTRACTS OF Pseudomonas aeruginosa AG LSL-11

The agarases were purified for the first time an using aqueous two-phase system (ATPS) consisting of polyethylene glycol (PEG) and phosphate salt. The three extracellular, alkaline agarases produced by Pseudomonas aeruginosa AG LSL-11 were efficiently extracted into the top PEG-rich layer. The influencing factors on the partition of agarases—molecular weight of the PEG, system pH, system temperature, and NaCl concentration—were investigated. All the factors were found to have a significant effect on the partition of agarases except NaCl. The optimal ATPS parameters for the partitioning and purification of agarases were found to be 12% PEG 600 and 11.9% (w/w) phosphate salt at pH 8.0 and 4°C. All three agarases were concentrated in the top PEG phase with 6.19-fold purity and 71.21% recovery. The ATPS was found to be more convenient and economical than the conventional ion-exchange chromatography (IEC) method for extraction of three agarases and could be significantly employed for the purification of agarases from fermentation broth.     

Fusion protein of Vitreoscilla hemoglobin with D-amino acid oxidase enhances activity and stability of biocatalyst in the bioconversion process of cephalosporin C

In this study we constructed an artificial flavohemoprotein by fusing Vitreoscilla hemoglobin (VHb) with D-amino acid oxidase (DAO) of Rhodotorula gracilis to determine whether bacterial hemoglobin can be used as an oxygen donor to immobilized flavoenzyme. This chimeric enzyme significantly enhanced DAO activity and stability in the bioconversion process of cephalosporin C. In a 200-mL bioreactor, the catalytic efficiency of immobilized VHb-DAO against cephalosporin C was 12.5-fold higher than that of immobilized DAO, and the operational stability of the immobilized VHb-DAO was approximately threefold better than that of the immobilized DAO. In the scaled-up bioprocess with a 5-L bioreactor, immobilized VHb-DAO (2500 U/L) resulted in 99% bioconversion of 120 mM cephalosporin C within 60 min at an oxygen flow rate of 0.2 (v/v) x min. Ninety percent of the initial activity of immobilized VHb-DAO could be maintained at up to 50 cycles of the enzymatic reaction without exogenous addition of H(2)O(2) and flavin adenine dinucleotide (FAD). The purity of the final product, glutaryl-7-aminocephalosporanic acid, was confirmed to be 99.77% by high-performance liquid chromatography (HPLC) analysis. Relative specificity of immobilized VHb-DAO on D-alpha-aminoadipic acid, a precursor in cephalosporin C biosynthesis, increased twofold, compared with that of immobilized DAO, suggesting that conformational modification of the VHb-DAO fusion protein may be altered in favor of cephalosporin C.     

Extraction of tetracycline from fermentation broth using aqueous two-phase systems composed of polyethylene glycol and cholinium-based salts

Aiming at developing not only cheaper but also biocompatible and sustainable extraction and purification processes for antibiotics, in this work it was evaluated the ability of aqueous two-phase systems (ATPS) composed of polyethylene glycol (PEG) and cholinium-based salts to extract tetracycline from the fermented broth of Streptomyces aureofaciens. Conventional polymer/salt and salt/salt ATPS were also studied for comparison purposes. The novel systems here proposed are able to extract tetracycline directly from the fermentation broth with extraction efficiencies higher than 80%. A tailored extraction ability of these systems can also be achieved, with preferential extractions either for the polymer- or salt-rich phases, and which further depend on the cholinium-based salt employed. The gathered results support the applicability of biocompatible ATPS in the extraction of antibiotics from complex matrices and can be envisaged as valuable platforms to be applied at the industrial level by pharmaceutical companies.     

Optimization of bovine serum albumin sorption and recovery by hydrogels

Aqueous two-phase systems are composed of aqueous solutions of either two water-soluble polymers, usually polyethylene glycol (PEG) and dextran (Dx), or a polymer and a salt, usually PEG and phosphate or sulfate. Partitioning of proteins in such systems provides a powerful method for separating and purifying mixtures of biomolecules by extraction. If one of the phase forming polymers is a crosslinked gel, then the solution-controlled gel sorption may be considered as a modification of aqueous two-phase extraction. Since PEG/dextran systems are widely used in aqueous two-phase extraction and dextran gels (Sephadex) are common chromatographic media, we choose a PEG/dextran gel system as a model system in this study. The partitioning behavior of pure bovine serum albumin (BSA) in PEG/dextran gel systems is investigated to see the effects of variations in PEG and NaCl concentrations on the partition coefficient K. By making use of the Box-Wilson experimental design, K is shown to be maximized at 9.8 (%, w/w) PEG and 0.2 M NaCl concentrations, respectively, as 182.     

Specific cephalosporin C production of Acremonium chrysogenum is independent of the culture density

Specific cephalosporin C production of Acremonium chrysogenum grown on a glucose-based minimal medium using conventional batch and dialysis membrane reactor systems was independent of the cell density in the range of 0.4 to 40 g biomass l^–1.     

Aqueous two-phase extraction for the purification of alkaline agarases from culture extracts of Pseudomonas aeruginosa AG LSL-11

The agarases were purified for the first time an using aqueous two-phase system (ATPS) consisting of polyethylene glycol (PEG) and phosphate salt. The three extracellular, alkaline agarases produced by Pseudomonas aeruginosa AG LSL-11 were efficiently extracted into the top PEG-rich layer. The influencing factors on the partition of agarases--molecular weight of the PEG, system pH, system temperature, and NaCl concentration--were investigated. All the factors were found to have a significant effect on the partition of agarases except NaCl. The optimal ATPS parameters for the partitioning and purification of agarases were found to be 12% PEG 600 and 11.9% (w/w) phosphate salt at pH 8.0 and 4°C. All three agarases were concentrated in the top PEG phase with 6.19-fold purity and 71.21% recovery. The ATPS was found to be more convenient and economical than the conventional ion-exchange chromatography (IEC) method for extraction of three agarases and could be significantly employed for the purification of agarases from fermentation broth.     

Protein partitioning in aqueous two-phase systems composed of a pH-responsive copolymer and poly(ethylene glycol)

The effect of pH and salt concentration on the partitioning behavior of bovine serum albumin (BSA) and cytochrome c in an aqueous two-phase polymer system containing a novel pH-responsive copolymer that mimics the structure of proteins and poly(ethylene glycol) (PEG) was investigated. The two-phase system has low viscosity. Depending on pH and salt concentration, the cytochrome c was found to preferentially partition into the pH-responsive copolymer-rich (bottom) phase under all conditions of pH and salt concentrations considered in the study. This was caused by the attraction between the positively charged protein and negatively charged copolymer. BSA partitioning showed a more complex behavior and partitioned either to the PEG phase or copolymer phase depending on the pH and ionic strength. Extremely high partitioning levels (partition coefficient of 0.004) and very high separation ratios of the two proteins (up to 48) were recorded in the new systems. This was attributed to strong electrostatic interactions between the proteins and the charged copolymer.     

Studies on the isolation of penicillin acylase from Escherichia coli by aqueous two-phase partitioning

A method of enzyme release and aqueous two-phase extraction is described for the separation of penicillin acylase from Escherichia coli cells. Butyl acetate, 12% (v/v), treatment combined with freeze-thawing gives up to 70% enzyme release. For polyethylene glycol (PEG) + phosphate two-phase extraction systems the enzyme purity and yield were rather low. Modified PEG, including PEG-ampicillin, PEG-aniline, PEG-phosphate, and PEG-trimethylamine, were synthesized and used in aqueous two-phase systems; PEG-trimethylamine is the most satisfactory. A system containing 12% (w/w) PEG4000, 8% (w/w) of which is PEG-trimethylamine, with 0.7M potasium phosphate at pH 7.2, resulted in the enzyme selective partition being greatly enhanced by charge directed effects. Possible mechanisms for the separation process are discussed. (c) 1992 John Wiley & Sons, Inc.     

Extraction of protease from Aspergillus tamarii URM 4634 in aqueous two-phase system under continuous and discontinuous process

Abstract

Aqueous two-phase systems have been studied for almost a century to separate biomolecules in harmless conditions. Proteases produced by Aspergillus tamarii URM 4634 were extracted in polyethylene glycol (PEG)/phosphate aqueous two-phase system under discontinuous and continuous (perforated rotative discs column) process. On the discontinuous process, it was evaluated the effect of operational conditions (PEG molar mass and its concentration, phosphate concentration and pH) over the partition coefficient, activity yield and purification factor. Protease partitioned to PEG-phase with partition coefficients up to 55.73. The best process parameters were 17.5% of PEG, with molar mass 8000?g·mol^?1, 15% of phosphate salt at pH 6, with 113.15% of activity yield and purification factor of 2.62. Under continuous extraction, hold up data showed that 57.1% of the discontinuous phase was available for protein extraction. Further, separation achieved 90.0% of efficiency. The yields surpassed 100% in almost all runs, and the best purification factor was 1.84, with both flows of 2?mL·min^?1. Thus, the best operational conditions reached an activity yield of 95.3% and 90.0% of separation efficiency. Hence, aqueous two-phase system PEG/phosphate extraction is an efficient process for separation of proteases produced by Aspergillus tamarii URM 4634, under continuous extraction likewise under discontinuous process.