Partition and substrate concentration effect in the enzymatic synthesis of cephalexin in aqueous two-phase systems

The kinetically controlled synthesis of cephalexin in aqueous two-phase systems was studied, using immobilized penicillin acylase, 7-amino 3-desacetoxycephalosporanic acid as nucleophile and phenylglycine methyl ester as acyl donor. The organic phases used were 80% (v/v) polyethyleneglycol 400 and 600 and the aqueous phase was 2.5 M (NH₄)₂SO₄. 7-amino 3-desacetoxycephalosporanic acid and cephalexin partition coefficients were determined at pH 7.4 and 7.8, at 14 °C and 20 °C. Highest partition coefficient for cephalexin was obtained for polyethyleneglycol 400–(NH₄)₂SO₄ at pH 7.4 and 20 °C, while the lowest partition coefficient for 7-amino desacetoxycephalosporanic acid was obtained in the same system at pH 7.8 and 14 °C. No significant effect of pH was observed on conversion yield and productivity of cephalexin synthesis; however, higher values were obtained with polyethyleneglycol 400 as organic phase. Higher conversion yields with both biphasic systems were obtained at the lowest temperature, where product hydrolysis was lower; volumetric productivity was higher for the fully aqueous medium (control), being higher at 20 °C. All parameters of synthesis were improved at higher substrates concentrations, obtaining conversion yields of 78.2% and 65.4%, with 60 mM 7-amino desacetoxycephalosporanic acid for the polyethyleneglycol 400–(NH₄)₂SO₄ system and the control, respectively.     

Purification of recombinant cutinase by extraction in an aqueous two-phase system facilitated by a fatty acid substrate

Purification of recombinant wild-type cutinase from the culture supernatant of Saccharomyces cerevisiae by extraction in aqueous two-phase system was investigated. The partition of the enzyme in a polyethylene glycol (PEG)-potassium phosphate system to the top phase was increased with lower molecular weight PEG. Enzyme partition in a 20% PEG/15% phosphate two-phase system was studied in the presence of detergents, fatty acids, and alcohols, respectively. Addition of 0.5% (w/w) butyrate increased the partition coefficient from 17 to 135 and the purification factor from 10 to 23. The effect of butyrate was also confirmed by using the countercurrent mode of extraction. Recovery of cutinase from the top phase was achieved by a secondary extraction into a new salt phase at a lower pH or a lower temperature. A specific interaction of butyrate to the active site of the enzyme was demonstrated by fluorescence spectroscopy. Size exclusion chromatography showed the cutinase-butyrate complex to be over two times the size of the free enzyme.     

Enhancement of phase separation using a drop coalescer in an aqueous two-phase system

The effect of a drop coalescer on phase separation in a PEG/salt aqueous two-phase system (ATPS) in the absence and presence of protein has been investigated. Raschig rings of ceramic, PTFE and glass were used as a drop coalescer in order to separate the mixture into two phases. Among the three materials PTFE is the most effective in coalescing the dispersed drops, with the throughput with PTFE being twice that without the coalescer. Random packing gives good results for phase separation. Two types of fiber mesh coated with PTFE were also used as drop coalescers, one in a spirally folded form and the other in a three-dimensional lattice-form. Throughput in the PEG/salt system with the three-dimensional lattice-form is 1.2 times as high as that with the spirally folded form. Throughput with the coalescer formed by compiling PTFE Raschig rings and fiber mesh in lattice form is 1.6 and 1.2 times as high as the case of separate use of the fiber mesh and the PTFE Raschig rings, respectively. The hydrophobic surface of PTFE in the compiled coalescer has no significant effect on the recovery fraction of the protein in ATPS.     

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.     

双水相生物催化技术的研究进展

双水相生物催化是一种高效且易于放大的生物催化技术,可以有效解决传统生物催化过程中产物浓度低、产物和副产物的抑制、以及生物催化剂难以回收等缺点。介绍了该技术的操作工艺及设备研究及其在抗生素、激素、肽类和有机化舍物酶法合成中的应用研究现状,展望了该技术的可能发展方向。     

Production of pectinases by Polyporus squamosus in aqueous two-phase system

The ability of Polyporus squamosus to grow and produce pectinases in an aqueous two-phase medium composed of polyethylene glycol and crude dextran is reported. Fungal growth was restricted to the bottom phase leaving the top phase cell free. Amounts of produced biomass and endo and exo-pectinase activities were superior or equal to those obtained in homogeneous medium. The partition coefficient for the endo-pectinase was 1.52 followed by a top phase yield of 70.86%. Although the phase system composition favours partition of a greater part of exo-pectinase activity to the bottom phase (K(exo) was 0.6 and yield in top phase 48.56%) the partitioned activity in the top phase was equal to that produced in homogeneous cultivation.     

Enhanced microbial corrosion of stainless steel by Acidithiobacillus ferrooxidans through the manipulation of substrate oxidation and overexpression of rus

Acidithiobacillus ferrooxidans cells can oxidize iron and sulfur and are key members of the microbial biomining communities that are exploited in the large-scale bioleaching of metal sulfide ores. Some minerals are recalcitrant to bioleaching due to the presence of other inhibitory materials in the ore bodies. Additives are intentionally included in processed metals to reduce environmental impacts and microbially influenced corrosion. We have previously reported a new aerobic corrosion mechanism where A. ferrooxidans cells combined with pyrite and chloride can oxidize low-grade stainless steel (SS304) with a thiosulfate-mediated mechanism. Here we explore process conditions and genetic engineering of the cells that enable corrosion of a higher grade steel (SS316). The addition of elemental sulfur and an increase in the cell loading resulted in a 74% increase in the corrosion of SS316 as compared to the initial sulfur- and cell-free control experiments containing only pyrite. The overexpression of the endogenous rus gene, which is involved in the cellular iron oxidation pathway, led to a further 85% increase in the corrosion of the steel in addition to the improvements made by changes to the process conditions. Thus, the modification of the culturing conditions and the use of rus-overexpressing cells led to a more than threefold increase in the corrosion of SS316 stainless steel, such that 15% of the metal coupons was dissolved in just 2 weeks. This study demonstrates how the engineering of cells and the optimization of their cultivation conditions can be used to discover conditions that lead to the corrosion of a complex metal target.     

Bovine serum albumin partitioning in an aqueous two-phase system: Effect of pH and sodium chloride concentration

The partitioning of bovine serum albumin (BSA) in a polyethylene glycol 3350 (8% w/w)–dextran 37 500 (6% w/w)–0.05 M phosphate aqueous two-phase was investigated at different pHs, at varying concentrations of sodium chloride at 20°C. The effect of NaCl concentration on the partition coefficient of BSA was studied for the PEG–dx systems with initial pH values of 4.2, 5.0, 7.0, 9.0, and 9.8. The NaCl concentrations in the phase systems with constant pH value were 0.06, 0.1, 0.2, 0.3, and 0.34 M. It was observed that the BSA partition coefficient decreased at concentrations smaller than 0.2 M NaCl and increased at concentrations greater than 0.2 M NaCl for all systems with initial pHs of 4.2, 5.0, 7.0, 9.0, and 9.8. It was also seen that the partition coefficient of BSA decreased as the pH of the aqueous two-phase systems increased at any NaCl salt concentration studied.     

Partitioning of yeast RNA in aqueous two-phase system

Summary The partition behaviour of RNA extracted from yeast was examined using three types of aqueous two-phase systems: potassium phosphate—polyethyleneglycol (PEG) system, ammonium sulphate—PEG system and dextran sulphate—PEG—NaCl system. The molecular weight of PEG showed rather remarkable effects of RNA partition than did the electrolyte. High molecular weight RNA was concentrated at the interface in these systems. This suggests the possibility of its simple concentration or extraction.     

Development of linear irreversible thermodynamic model for oxidation reduction potential in environmental microbial system

Nernst equation has been directly used to formulate the oxidation reduction potential (ORP) of reversible thermodynamic conditions but applied to irreversible conditions after several assumptions and/or modifications. However, the assumptions are sometimes inappropriate in the quantification of ORP in nonequilibrium system. We propose a linear nonequilibrium thermodynamic model, called microbial related reduction and oxidation reaction (MIRROR Model No. 1) for the interpretation of ORP in biological process. The ORP was related to the affinities of catabolism and anabolism. The energy expenditure of catabolism and anabolism was directly proportional to overpotential (eta), straight coefficient of electrode (L(EE)), and degree of coupling between catabolism and ORP electrode, respectively. Finally, the limitations of MIRROR Model No. 1 were discussed for expanding the applicability of the model.     

Bromelain purification through unconventional aqueous two-phase system (PEG/ammonium sulphate)

This paper focuses on the feasibility of unconventional aqueous two-phase systems for bromelain purification from pineapple processing waste. The main difference in comparison with conventional systems is the integration of the liquid–liquid extraction technique with fractional precipitation, which can decrease the protein content with no loss of biological activity by removing of unwanted molecules. The analysis of the results was based on the response surface methodology and revealed that the use of the desirability optimisation methodology (DOM) was necessary to achieve higher purification factor values and greater bromelain recovery. The use of DOM yielded an 11.80-fold purification factor and 66.38 % biological activity recovery using poly(ethylene glycol) (PEG) with a molar mass of 4,000, 10.86 % PEG concentration (m/m) and 36.21 % saturation of ammonium sulphate.     

Free polymeric bioligands in aqueous two-phase affinity extractions of microbial xylanases and pullulanase

Two reversibly soluble-insoluble polymers (viz. Eudragit S-100 and alginate) were used as free macroaffinity bioligands in polyethylene glycol (PEG)/salt two-phase systems for separation of enzymes. Incorporation of Eudragit S-100 and alginate in the PEG phase led to considerable selectivity in separation of microbial xylanases and pullulanase, respectively. Xylanase from Aspergillus niger was recovered 93% with 56-fold purification, whereas the enzyme from Trichoderma reesei and Bacillus amyloliquefaciens was obtained with 93% activity recovery (31-fold purification) and 90% activity recovery (32-fold purification), respectively. From Bacillus acidopullulyticus pullulanase, 85% enzyme activity recovery with 44-fold purification was obtained. The approach described here shows the potential of developing into a general approach for use of reversibly soluble-insoluble macroaffinity ligand in two-phase affinity extraction.     

Characterisation of anaerobic mixed cultures by partition in an aqueous two-phase system

A method to characterise a dynamic microbial consortium is described. By exploiting differences in surface properties between different cells and between cells of different physiological status, it was possible to develop a partition pattern for a mixed culture under different conditions. The separation method used was partition in aqueous two-phase systems and when using a counter current extraction process one could clearly differentiate the partition profile between resting, active and overloaded biomethanation cultures.     

Purification of acetohydroxy acid synthase by separation in an aqueous two-phase system

Extraction in a polyethylene glycol (PEG)–phosphate aqueous two-phase system was considered as a primary step in purification of the acetohydroxy acid synthase III large catalytic subunit from an E. coli extract. Extraction optimization was achieved by varying the system parameters. Two systems with the following weight compositions were chosen for purification: PEG-2000 (16%)–phosphate (6%) and PEG-4000 (14%)–phosphate (5.5%)–KCl (8%), both at pH 7.0 and 1 mg total protein per 1 g system. Significant purification was achieved by a single extraction step with 70% recovery of the enzyme. After an additional ion-exchange chromatography step, pure enzyme was obtained in a 50% overall yield.     

Production of alkaline protease by Bacillus licheniformis in an aqueous two-phase system

Alkaline protease production by Bacillus licheniformis was studied in an aqueous two-phase system composed of 5% (w/w) polyethylene glycol 6000 (PEG 6000) and 5% (w/w) dextran T500. The top phase was continuous and rich in PEG while the bottom phase was dispersed and rich in dextran. The cells were retained in the bottom phase and at the interface. The two-phase system produced less enzyme in total amount than the control in the early phase, but after 50 h the enzyme produced in the control system decreased while the aqueous two-phase system continued its production and finally the total enzyme activity reached 1.3 times that of the control culture. In order to improve the productivity of protease, repeated batch cultivation were successfully carried out four times by optimizing the top phas composition of freshly added media, which resulted in 13.8, 35.9, 27.8 and 34.7 units ml⁻¹ h⁻¹ of protease based on the amounts of replaced top phases, respectively.     

Production of phosphomonoesterases by Nicotiana tabacum 1507 in an aqueous two-phase system

Studies were conducted on Nicotiana tabacum 1507 cultivation in an aqueous two-phase system (PD(5)) formed by adding 4% PEG (MW 20,000) and 7.5% dextran (MW 70,000) to the medium. The time course of growth and changes in the phase volumes of the PD(5) system, as well as the biosynthesis, secretion, and partitioning of phosphomonoesterases during 11 days of cultivation, were followed. In comparison with N. tabacum 1507 cultivation as a free suspension, on day 8 of cultivation in the PD(5) system the yields of acid and alkaline extracellular phosphomonoesterases were 18 and 10 times higher, respectively. Partitioning took place mainly in the bottom phase with specific activity being 4.5 and 3.5 times higher, respectively. (c) 1996 John Wiley & Sons, Inc.     

Steroid bioconversion in a novel aqueous two-phase system

A novel aqueous two-phase system, based on polyethyleneglycol (PEG) and monosodium glutamate, was tested for the 1-dehydrogenation of hydrocortisone-based substrates. This system led to higher substrate solubilities and biocatalyst/steroid separation levels when compared with alternative systems. The addition of short-chain monohydric alcohols resulted in higher solubilities and more favourable partition coefficients for the tested substrates. Bioconversion activities in PEG/glutamate systems with 2,5% (v/v) methanol were comparable to those measured in monophasic buffer-methanol medium.     

Chemoenzymatic synthesis of CMP-sialic acid derivatives by a one-pot two-enzyme system: comparison of substrate flexibility of three microbial CMP-sialic acid synthetases

Three C terminal His₆-tagged recombinant microbial CMP–sialic acid synthetases [EC 2.7.7.43] cloned from Neisseria meningitidis group B, Streptococcus agalactiae serotype V, and Escherichia coli K1, respectively, were evaluated for their ability in the synthesis of CMP–sialic acid derivatives in a one-pot two-enzyme system. In this system, N-acetylmannosamine or mannose analogs were condensed with pyruvate, catalyzed by a recombinant sialic acid aldolase [EC 4.1.3.3] cloned from E. coli K12 to provide sialic acid analogs as substrates for the CMP–sialic acid synthetases. The substrate flexibility and the reaction efficiency of the three recombinant CMP–sialic acid synthetases were compared, first by qualitative screening using thin layer chromatography, and then by quantitative analysis using high performance liquid chromatography. The N. meningitidis synthetase was shown to have the highest expression level, the most flexible substrate specificity, and the highest catalytic efficiency among the three synthetases. Finally, eight sugar nucleotides, including cytidine 5′-monophosphate N-acetylneuraminic acid (CMP–Neu5Ac) and its derivatives with substitutions at carbon-5, carbon-8, or carbon-9 of Neu5Ac, were synthesized in a preparative (100–200 mg) scale from their 5- or 6-carbon sugar precursors using the N. meningitidis synthetase and the aldolase.     

Effects of temperature and phosphorous concentration on microbial sulfate reduction by Desulfovibrio desulfuricans

The effects of temperature and phosphorous concentration on the rate and the extent of microbial sulfate reduction with lactate as carbon and energy source were investigated for Desulfovibrio desulfuricans. The continuous culture experiments (chemostat) were conducted at pH 7.0 from 12 to 48 degrees C. The maximum specific growth rate (mu(max)) was relatively constant in the range 25 degrees C-43 degrees C and dramatically decreased outside this temperature range. The half-saturation coefficient was minimum at 25 degrees C. Cell yield was highest in the optimum temperature range (35 degrees C-43 degrees C) for growth. Maintenance energy requirements for D. desulfuricans were not significant. Two moles of lactate is consumed for every mole of sulfate reduced, and this stoichiometric ratio is not temperature dependent. Steady state rate and stoichiometric coefficients accurately predicted transient behavior during temperature shifts. The extent of extracellular polymeric substance (EPS) is related to the concentration of phosphorous in the medium. EPS production rate increased with decreased phosphorous loading rate. Failure to discriminate between cell and EPS formation by D. desulfuricans leads to significant overestimates of the cell yield. The limiting C:P ratio for D. desulfuricans was in the range of 400:1 to 800:1.