Penicillin acylase-catalyzed synthesis of beta-lactam antibiotics in highly condensed aqueous systems: beneficial impact of kinetic substrate supersaturation

Advantages of performing penicillin acylase-catalyzed synthesis of new penicillins and cephalosporins by enzymatic acyl transfer to the beta-lactam antibiotic nuclei in the supersaturated solutions of substrates have been demonstrated. It has been shown that the effective nucleophile reactivity of 6-aminopenicillanic (6-APA) and 7-aminodesacetoxycephalosporanic (7-ADCA) acids in their supersaturated solutions continue to grow proportionally to the nucleophile concentration. As a result, synthesis/hydrolysis ratio in the enzymatic synthesis can be significantly (up to three times) increased due to the nucleophile supersaturation. In the antibiotic nuclei conversion to the target antibiotic the remarkable improvement (up to 14%) has been gained. Methods of obtaining relatively stable supersaturated solutions of 6-APA, 7-ADCA, and D-p-hydroxyphenylglycine amide (D-HPGA) have been developed and syntheses of ampicillin, amoxicillin, and cephalexin starting from the supersaturated homogeneous solutions of substrates were performed. Higher synthetic efficiency and increased productivity of these reactions compared to the heterogeneous "aqueous solution-precipitate" systems were observed. The suggested approach seems to be an effective solution for the aqueous synthesis of the most widely requested beta-lactam antibiotics (i.e., amoxicillin, cephalexin, cephadroxil, cephaclor, etc.).     

One-pot synthesis in aqueous system for water-soluble chitosan-graft-poly(ethylene glycol) methyl ether

Chitosan is functionalized with poly(ethylene glycol) methyl ether (mPEG) at the amino and hydroxyl groups via a single step reaction in a homogeneous aqueous system. A chitosan aqueous solution obtained from the mixture of chitosan and hydroxybenzotriazole (HOBt) in water is a key factor in providing mild conditions to conjugate mPEG by using a carbodiimide conjugating agent. The reaction at ambient temperature for 24 h gives chitosan-g-mPEG with water solubility with mPEG content as high as 42%. This work demonstrates that a water-soluble chitosan-HOBt complex is an effective system for the preparation of chitosan derivatives via the aqueous system without the use of acids or organic solvents.     

The application of papain,ficin and clostripain in kinetically controlled peptide synthesis in frozen aqueous solutions

The capability of the cysteine proteases ficin, papain and clostripain to form peptide bonds in frozen aqueous solutions was investigated. Freezing the reaction mixture resulted in increased peptide yields in kinetically controlled coupling of Bz–Arg–OEt with various amino acid amides and dipeptides. Under these conditions, peptide yields increased up to 70% depending on the enzyme and the amino component used. Enzyme-catalysed peptide syntheses were carried out under optimized reaction conditions (temperature, amino component concentration and pH before freezing) using the condensation of Bz–Arg–OEt and H–Leu–NH₂ as a model reaction.     

Kinetically controlled acylation of 6-APA catalyzed by penicillin acylase from Streptomyces lavendulae: effect of reaction conditions in the enzymatic synthesis of penicillin V

Abstract

Enzymatic synthesis of penicillin V (penV) by acylation of 6-aminopenicillanic acid (6-APA) was carried out using methyl phenoxyacetate (MPOA) as activated acyl donor and soluble penicillin acylase from Streptomyces lavendulae (SlPVA) as biocatalyst. The effect of different reaction conditions on penV synthesis was investigated, such as enzyme concentration, pH, molar ratio of 6-APA to MPOA, as well as presence of DMSO as water-miscible co-solvent at different concentrations. Time-course profiles of all reactions followed the typical pattern of kinetically controlled synthesis (KCS) of β-lactam antibiotics: penV concentration reached a maximum (highest yield or Y_max) and then decreased gradually. Such maximum was higher at pH 7.0, observing that final penV concentration was abruptly reduced when basic pH values were employed in the reaction. Under the selected conditions (100?mM Tris/HCl buffer pH 7.0, 30?°C, 2.7% (v/v) DMSO, 20?mM MPOA, 0.3 UI/ml of SlPVA), Y_max was enhanced by increasing the substrate molar ratio (6-APA to MPOA) up to 5, reaching a maximum of 94.5% and a S/H value of 16.4 (ratio of synthetic activity to hydrolytic activity). As a consequence, the use of an excess of 6-APA as nucleophile has allowed us to obtain some of the highest Y_max and S/H values among those reported in literature for KCS of β-lactam antibiotics. Although many penicillin G acylases (PGAs) have been described in kinetically controlled acylations, SlPVA should be considered as a different enzyme in the biocatalytic tool-box for novel potential synthetic processes, mainly due to its different substrate specificity compared to PGAs.     

Continuous cell partitioning using an aqueous two-phase flow system in microfluidic devices

We present a novel microfluidic system in which an aqueous two-phase laminar flow is stably formed, and the continuous partitioning of relatively large cells can be performed, eliminating the influence of gravity. In this study, plant cell aggregates whose diameters were 37-96 microm were used as model particles. We first performed cell partitioning using a simple straight microchannel having two inlets and two outlets and examined the effects of the flow rate and the phase width on partitioning efficiency. Second, by using a microchannel with a pinched segment, the partitioning efficiency was successfully improved. This microscale aqueous two-phase flow system can further be incorporated into micro total analysis systems (microTAS) or lab-on-a-chip technology, owing to its simplicity, applicability, and biocompatibility.     

Advantages of using non-isothermal bioreactors for the enzymatic synthesis of antibiotics: the penicillin G acylase as enzyme model

A new hydrophobic and catalytic membrane was prepared by immobilizing Penicillin G acylase (PGA, EC.3.5.1.11) from E. coli on a nylon membrane, chemically grafted with butylmethacrylate (BMA). Hexamethylenediamine (HMDA) and glutaraldehyde (Glu) were used as a spacer and coupling agent, respectively. PGA was used for the enzymatic synthesis of cephalexin, using D(-)-phenylglycine methyl ester (PGME) and 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) as substrates. Several factors affecting this reaction, such as pH, temperature, and concentrations of substrates were investigated. The results indicated good enzyme-binding efficiency of the pre-treated membrane, and an increased stability of the immobilized PGA towards pH and temperature. Calculation of the activation energies showed that cephalexin production by the immobilized biocatalyst was limited by diffusion, resulting in a decrease of enzyme activity and substrate affinity. Temperature gradients were employed as a way to reduce the effects of diffusion limitation. Cephalexin was found to linearly increase with the applied temperature gradient. A temperature difference of about 3 degrees C across the catalytic membrane resulted into a cephalexin synthesis increase of 100% with a 50% reduction of the production times. The advantage of using non-isothermal bioreactors in biotechnological processes, including pharmaceutical applications, is also discussed.     

A novel method for repetitive peptide synthesis in solution without isolation of intermediates.

A novel method was developed for the large-scale manufacture of peptides in solution, called DioRaSSP-Diosynth Rapid Solution Synthesis of Peptides. This method combines the advantages of the homogeneous character of classical solution-phase synthesis with the universal character and the amenability to automation inherent to the solid-phase approach. The process consists of repetitive cycles of coupling and deprotection in a permanent organic phase and is further characterized by the fact that intermediates are not isolated. Couplings are mediated by water-soluble carbodiimide. Several types of function may be applied for temporary amino protection depending on the sequence of the actual peptide, including Z, Fmoc, Msc and Nsc. Formate is the preferred hydrogen donor during hydrogenolysis of the Z function, while 1,8-diazabicyclo[5.4.0]undec-7-ene is used to deprotect Fmoc, Msc and Nsc. Morpholine is added during the deprotection of Msc and Nsc to scavenge the arising alkenes. Processes according to this highly efficient synthesis method are easy to scale up and yield products of reproducible high purity, which is guaranteed by a new quenching method for residual activated compounds, applying an anion-forming amine such as a beta-alanine ester. This ester should display a lability similar to that of the temporary amino-protecting function, allowing simultaneous deprotection of the growing peptide and the quenched compound. The DioRaSSP approach assures the completely quantitative removal of deprotected quenched compounds before the coupling step of the next cycle of the synthesis by basic aqueous (that is active) extraction, while the growing peptide remains anchored in the organic phase due to the presence of hydrophobic protecting functions.     

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.     

Dissociation and isolation of chromatin proteins in salt solutions by an aqueous two-phase system

An aqueous two-phase system containing 7% Dextran T 500-5% polyethylene glycol (PEG) 6000 has been adopted for rapid selective stepwise extractions of high-mobility-group proteins and histones from both isolated chromatin and intact nuclei of calf thymus. After the dissociated proteins in the PEG phase were precipitated with 20% trichloroacetic acid at 4 degrees C, proteins were recovered from this phase by solubilization of PEG with acidified acetone at room temperature. This method allows preparation of nuclei depleted of histone H1.     

Characteristics of protein partitioning in an aqueous micellar-gel system

Partitioning of proteins has been studied experimentally in a system combining a gel-bead phase and a nonionic micellar phase. The micellar phase consists of cylindrically shaped micelles, which are completely excluded from the gel-bead phase. Partitioning of single-component protein solutions (myoglobin, ovalbumin, and BSA) is determined by excluded-volume interactions in the micellar phase, and as a result the proteins prefer the gel-bead phase to the micellar phase. The protein concentration inside the gel beads increases with an increase in volume fraction of the micelles and increases with an increase in the size of the proteins. The protein partition coefficients obtained for a binary mixture of myoglobin and bovine serum albumin (BSA) show the same protein concentration dependence as the single-component protein partition coefficients.     

Pulsed column reactor as a novel tool for continuous enzymatic synthesis of peptide in an aqueous/organic biphasic medium

We propose a novel effective method for a continuous peptide synthesis in an aqueous/organic biphasic medium using a pulsed column reactor. N-Formyl-l-aspartyl-l-phenylalanine methyl ester was enzymatically synthesized continuously. With this extractive method using a pulsed column reactor, we can synthesize peptides with a stable performance even if a peptide (or a peptide-amino acid complex) is precipitated due to its high hydrophobicity.     

Construction of an efficient Escherichia coli cell‐free system for in vitro expression of several kinds of proteins

Cell‐free protein synthesis systems have offered several advantages over traditional cell‐based expression methods. In this study, the effects of extract preparation and an energy‐regenerating system on protein synthesis were investigated in an Escherichia coli cell‐free system. The results indicated that the expression level of enhanced green fluorescent protein (eGFP) with the S12 extract was higher than that with the S30 extract. Among four adenosine triphosphate‐regenerating sources, the cAMP/CP/CK system (including cAMP, creatine phosphate, and creatine kinase) proved to be the most efficient one to support high‐level expression of eGFP. Further studies showed that this established cell‐free system could be successfully used to produce one model protein (eGFP), two human proteins (AK2 and coenzyme synthase) and two membrane proteins (subunit b of F₁F₀ adenosine triphosphate synthase and aquaporin Z). This outcome will be helpful to develop the highly efficient cell‐free technology for the production of various proteins with different bio‐origins.     

Protein prenylation in an insect cell-free protein synthesis system and identification of products by mass spectrometry

To evaluate the ability of an insect cell-free protein synthesis system to carry out proper protein prenylation, several CAIX (X indicates any C-terminal amino acid) sequences were introduced into the C-terminus of truncated human gelsolin (tGelsolin). Tryptic digests of these mutant proteins were analyzed by MALDI-TOF MS and MALDI-quadrupole-IT-TOF MS. The results indicated that the insect cell-free protein synthesis system possesses both farnesyltransferase (FTase) and geranylgeranyltransferase (GGTase) I, as is the case of the rabbit reticulocyte lysate system. The C-terminal amino acid sequence requirements for protein prenylation in this system showed high similarity to those observed in rat prenyltransferases. In the case of rhoC, which is a natural geranylgeranylated protein, it was found that it could serve as a substrate for both prenyltransferases in the presence of either farnesyl or geranylgeranyl pyrophosphate, whereas geranylgeranylation was only observed when both prenyl pyrophosphates were added to the in vitro translation reaction mixture. Thus, a combination of the cell-free protein synthesis system with MS is an effective strategy to analyze protein prenylation.     

Continuous extraction of β-d-galactosidase from Escherichia coli in an aqueous two-phase system: effects of biomass concentration on partitioning and mass transfer

High concentrations of Escherichia coli disintegrate move the binodial of a poly(ethylene glycol) (PEG) 4000/potassium phosphate aqueous two-phase system towards lower concentrations. It has also been shown that the yield and purification factor of β-d-galactosidase (β-d-galactoside galactohydrolase, EC 3.2.1.23) in the PEG phase was gradually improved by moving the experimental system to a composition closer to the binodial. The mass transfer rates of cell debris, total protein, β-d-galactosidase and DNA have been studied and were found to be fast enough to reach equilibrium between the phases after 1.9 s of mixing in a static mixer with 24 mixing elements. A continuous extraction process for β-d-galactosidase from E. coli has been designed on the basis of these studies with a mean residence time of 6.3 min from the disintegrator inlet to the β-d-galactosidase containing PEG-phase outlet of the centrifuge. This PEG phase contained 83.5% of the total β-d-galactosidase with a purification factor of 13.6, and only 2.8% of the total protease activity of the disintegrate. All cell debris and almost all DNA were confined to the potassium phosphate phase.     

N-Terminal protein modifications in an insect cell-free protein synthesis system and their identification by mass spectrometry

To evaluate the ability of an insect cell-free protein synthesis system to generate proper N-terminal cotranslational protein modifications such as removal of the initiating Met, N-acetylation, and N-myristoylation, several mutants were constructed using truncated human gelsolin (tGelsolin) as a model protein. Tryptic digests of these mutants were analyzed by MALDI-TOF MS and MALDI-quadrupole-IT-TOF MS. The wild-type tGelsolin, which is an N-myristoylated protein, was found to be N-myristoylated when myristoyl-CoA was added to the in vitro translation reaction mixture. N-myristoylation did not occur on the Gly-2 to Ala mutant, in which the N-myristoylation motif was disrupted, whereas this mutant was found to be N-acetylated after removal of the initiating Met. Analyses of Gly-2 to His and Leu-3 to Asp mutants revealed that the amino acids at positions 2 and 3 strongly affect the susceptibility of the nascent peptide chain to removal of the initiating Met and to N-acetylation, respectively. These results suggest that N-terminal modifications occurring in the insect cell-free protein synthesis system are quite similar to those observed in the mammalian protein synthesis system. Thus, a combination of the cell-free protein synthesis system with MS is an effective strategy to analyze protein modifications.     

Highly efficient detection of Cu2+ and B4O72− based on a recyclable asymmetric salamo‐based probe in aqueous medium

An asymmetric salamo‐based probe molecule ( H ₂ L ) was synthesized and characterized structurally. When DMF/H₂O (9:1) was used as the solvent, it was shown probe H ₂ L has high sensitivity to Cu²⁺. Using high‐resolution mass spectrometry and theoretical calculation, it was found that probe H ₂ L could form a more stable complex (1:1) with Cu²⁺, the minimum limit of detection (LOD) of H ₂ L for Cu²⁺ was calculated as 9.95 × 10^?8 M. In addition, probe H ₂ L could also be used to identify B₄O₇^2? under the same detection conditions and the minimum LOD of H ₂ L for B₄O₇^2? was calculated as 4.98 × 10^?7 M. At the same time, density functional theory theoretical calculation further proved the flexibility of probe H ₂ L . Through the action of EDTA, probe H ₂ L had a cyclic ability to recognize Cu²⁺, and showed a better response in the physiological pH range; probe H ₂ L had the characteristics of fast recognition speed and high efficiency. In addition, with probe H ₂ L test paper for Cu²⁺ and B₄O₇^2?, the effect was more obvious. Meanwhile, probe H ₂ L can be used to quantitatively detect Cu²⁺ in water samples.     

羧甲基茯苓多糖的水媒法制备及其免疫活性研究

羧甲基茯苓多糖carboxymethyl-pachymaran (CMP)具有良好的免疫调节活性,传统方法使用两步工序在醇相体系中制得,有机溶剂消耗大、成本高.本研究报道一种新制备方法,将提取茯苓多糖以及茯苓多糖与氯乙酸的醚化反应整合在一个碱液体系中完成.动物实验表明,新方法制备的CMP能提高环磷酰胺诱导的免疫低下小鼠...     

Technical improvement in the purification of enzymes from red algae using an aqueous two-phase partitioning system

The effective elimination of phycobiliproteins from crude enzyme preparation of the red alga Caloglossa continua (Okamura) King et Puttock (Ceramiales, Florideophyceae) was investigated in an aqueous two‐phase partitioning system (ATPS) by changing the concentrations of polyethylene glycol (PEG) and ammonium sulfate (AS). The phycobiliproteins shifted from the AS‐rich lower phase to the PEG‐rich upper phase in high PEG and AS concentrations. The best ATPS condition for the elimination of phycobiliproteins from the lower phase was obtained by the combination of 20% (weight/volume; w/v) PEG and 16% (w/v) AS. However, the recovery of aldolase and mannitol‐1‐phos‐phatase activities was significantly reduced. For purification of the enzymes, a combination of 15% (w/v) PEG and 16% (w/v) AS was the best ATPS condition, because a high specific activity and recovery of the enzymes were obtained. Under these conditions, 98% of the phycobiliproteins were removed from the lower phase. Therefore, the ATPS proved to be a very useful method as a first step in the purification of enzymes from red algae.