Penicillin acylase-catalyzed synthesis of cefazolin in water–solvent mixtures: enhancement effect of ethyl acetate and carbon tetrachloride on the synthetic yield

The effects of organic solvents on the penicillin acylase-catalyzed, kinetically controlled synthesis of cefazolin have been examined in various water–solvent mixtures. In the presence of water-miscible solvents, the initial rate and maximum yield of cefazolin (CEZ) synthesis reaction were found to be reduced. The extent of inhibition was increased with increasing hydrophobicity of the solvent in the reaction mixtures. Enzymatic synthesis of cefazolin was also carried out in the water–solvent biphasic systems. Among the water-immiscible solvents tested, ethyl acetate (EtOAc) and carbon tetrachloride (CCl₄) were found to markedly improve the yield of cefazolin in the two-phase reaction system. Our study showed that the enhancement effect of EtOAc and CCl₄ on the synthetic yield was mainly caused by a reduction of the hydrolysis of acyl donor and product in the two-phase system rather than extraction of the product into the solvent phase.     

Application of analytical and preparative high-speed counter-current chromatography for the separation of Z-ligustilide from a crude extract of Angelica sinensis

Z-Ligustilide was separated and purified from the traditional Chinese medicinal plant Angelica sinensis by high-speed counter-current chromatography (HSCCC). Analytical HSCCC was first used for the systematic selection of the two-phase solvent system. Preparative HSCCC separation was performed with a two-phase solvent system composed of petroleum ether (60-90 degrees C)-ethanol-water at an optimum volume ratio of 10:17:10 (v/v). A total of 38 mg Z-ligustilide at 98.8% purity was obtained in one step from 200 mg crude extract as determined by HPLC analysis. The structure of the target compound was identified by electron impact ionisation mass spectrometry.     

高速逆流色谱技术在生物大分子分离纯化中的应用

高速逆流色谱是一种连续液-液色谱技术,具有无固相载体、样品无需严格预处理等优点。近10年来,在设备结构和溶剂体系等方面进行了大量的研究开发,已推广应用于生物技术、医药、天然产物、环境监测、食品等领域。为适应生物大分子和活性细胞的分离,采用条件温和的双水相体系,研究开发相应的高速逆流色谱设备已成为热点。针对双水相体系的特点,已经开发出了多种具有较高固定相保留率的新型高速逆流色谱设备,通过优化实验条件,成功地进行了多种蛋白质的分离纯化。本对该领域的最新进展进行了综述与评价。     

低水有机介质中的酶催化

酶不仅能在水溶液里催化化学反应,而且能在有机介质中显示催化活性．其中低水溶剂体系对有机合成最为有利．文章就低水溶剂体系中影响酶催化的三要素（水、溶剂和载体）以及酶在该体系表现出来的一些特殊性质进行了讨论,并列举了低水溶剂体系中的酶催化在有机合成,化学分析,和高分子化学等方面的应用．     

Functional selection of phage displayed peptides for facilitated design of fusion tags improving aqueous two-phase partitioning of recombinant proteins.

Aqueous two-phase systems allow for the unequal distribution of proteins and other molecules in water-rich solutions containing phase separating polymers or surfactants. One approach to improve the partitioning properties of recombinant proteins is to produce the proteins as fused to certain peptide tags. However, the rational design of such tags has proven difficult since it involves a compromise between multivariate parameters such as partitioning properties, solvent accessibility and production/secretion efficiency. In this work, a novel approach for the identification of suitable peptide tag extensions has been investigated. Using the principles of selection, rather than design, peptide sequences contributing to an improved partitioning have been identified using phage display technology. A 40 million member phagemid library of random nona-peptides, displayed as fusion to the major coat protein pVIII of the filamentous phage M13, was employed in the selection of top-phase partitioning phage particles in a PEG/sodium phosphate system. After multiple cycles of selection by partitioning, peptides with high frequencies of both tyrosine and proline residues were found to be over represented in selected clones. The identified peptide sequences, or derivatives thereof, were subsequently individually analyzed for their partitioning behavior as displayed on phage, as free synthetic peptides and as genetically fused to a recombinant model target protein. The results showed that novel peptide sequences capable of enhancing top-phase partitioning without interfering with protein production and secretion indeed could be identified for the aqueous two-phase system investigated.     

Enzyme catalysed reactions in water - Organic solvent two-phase systems

Enzyme-catalysed transformations carried out in two-phase systems consisting of water and a water-immiscible organic solvent are discussed. The systems appear advantageous when substrates poorly soluble in water, such as steroids, are used. The methodology also makes possible the use of hydrolytic enzymes for the synthesis of ester bonds. Several applications of such two-phase systems are illustrated. The criteria that must be taken into consideration for selecting the most suitable organic solvents and the operational conditions are discussed.     

Glycosidases in organic solvents: I. Alkyl-β-glucoside synthesis in a water-organic two-phase system

A low-water organic solvent two-phase system suitable for glycosylation of hydrophobic substrates is described. Almond β-glucosidase adsorbed on polymeric supports has been shown to catalyse alkyl-β-glucoside synthesis via a transferase reaction or through direct condensation of the glucosidic bond. High concentrations of glucosyl donors were present in the aqueous phase, while water-immiscible primary alcohols, which form the organic phase, served as acceptors of glucose. Reaction yield appeared to be thermodynamically controlled. The influence of various support materials, glucosyl donors, and glucosyl acceptors on reaction rate and product yield was investigated.     

Ionic liquids for two-phase systems and their application for purification,extraction and biocatalysis

The development of biotechnological processes using novel two-phase systems based on molten salts known as ionic liquids (ILs) got into the focus of interest. Many new approaches for the beneficial application of the interesting solvent have been published over the last years. ILs bring beneficial properties compared to organic solvents like nonflammability and nonvolatility. There are two possible ways to use the ILs: first, the hydrophobic ones as a substitute for organic solvents in pure two-phase systems with water and second, the hydrophilic ones in aqueous two-phase systems (ATPS). To effectively utilise IL-based two-phase systems or IL-based ATPS in biotechnology, extensive experimental work is required to gain the optimal system parameters to ensure selective extraction of the product of interest. This review will focus on the most actual findings dealing with the basic driving forces for the target extraction in IL-based ATPS as well as presenting some selected examples for the beneficial application of ILs as a substitute for organic solvents. Besides the research focusing on IL-based two-phase systems, the “green aspect” of ILs, due to their negligible vapour pressure, is widely discussed. We will present the newest results concerning ecotoxicity of ILs to get an overview of the state of the art concerning ILs and their utilisation in novel two-phase systems in biotechnology.     

Quantitative techniques for the comparison of solid supports

The host of solid supports available to the synthetic chemist adds yet another level of complexity to solid-phase synthesis. Although the selection of the optimal solid support for a specific synthetic transformation is still empirically driven, significant progress has been made in the development of quantitative techniques to compare solid supports, providing new insight into the microenvironment created by the interaction of the solid support with solvent.     

Biodegradation of 4-nitrophenol in a two-phase sequencing batch reactor: concept demonstration,kinetics and modelling

The objectives of this work were to demonstrate the potential of a two-phase sequencing batch reactor in degrading xenobiotics and to evaluate the kinetic parameters leading to a mathematical model of the system. 4-Nitrophenol (4NP), a typical representative of substituted phenols, was selected as the target xenobiotic; this compound has never been remediated in a two-phase bioreactor before. Partition tests were conducted to determine the most appropriate partitioning solvent, and among the three investigated solvents (1-undecanol, 2-undecanone and oleyl alcohol), 2-undecanone was chosen because of its favourable partition coefficient and its negligible emulsion-forming tendencies. Moreover, the selected solvent showed satisfactory biocompatibility characteristics with respect to the biomass, with only minor effects on the intrinsic microbial kinetics. Kinetic tests were then performed in a sequencing batch reactor (2-l volume) operated in both conventional one- and two-phase configurations, with the two-phase system showing a significant improvement in the process kinetics in terms of reduced inhibition and increased maximum removal rate. The obtained kinetic parameters suggest that the two-phase sequencing batch system may find full-scale application, as the maximum removal rate k _max (~3 mg 4NP mgVSS⁻¹ day⁻¹) is of the same order of magnitude of heterotrophic bacteria operating in wastewater treatment plants.     

Toxicity of homologous series of organic solvents for the gram-positive bacteria Arthrobacter and Nocardia Sp. and the gram-negative bacteria Acinetobacter and Pseudomonas Sp

The toxicity of homologous series of organic solvents has been investigated for the gram-positive bacteria, Arthrobacter sp. and Nocardia sp., and the gram-negative bacteria, Acinetobacter sp. and Pseudomonas sp. The hydrophobicity of the solvent, expressed by its logP(octanol), proves to be a good measure for the toxicity of solvents in a two-phase system. The transition from toxic to nontoxic solvents occurs between logP(octanol) 3 and 5 and depends on the homologous series. No correlation has been found between the hydrophobicity of the substituent on the alkyl backbone of the solvent and the location of the transition point in toxicity. The logP(octanol), above which all solvents are nontoxic, is used to express the solvent tolerance of the bacteria. In general, the solvent tolerance of gram-negative bacteria is found to be slightly higher than that of gram-positive bacteria, but this does not hold for all homologous series of organic solvents investigated.Because the toxicity effects of organic solvents in a two-phase system can be ascribed to molecular as well as phase toxicity effects, molecular toxicity effects were investigated separately in a one-phase system with subsaturating amounts of organic solvent. The solvent concentration in the aqueous phase, at which 50% of the metabolic activity of the bacteria is lost, is used to express solvent toxicity. This concentration is found to be similar for the gram-positive Arthrobacter and the gram-negative Acinetobacter. Assuming the critical membrane concentration theory (G. J. Osborne et al. Enzyme Microb. Technol. 1990, 12: 281-291) to be valid, it can be concluded that differences in solvent tolerance between these two bacteria, cannot be ascribed to differences in response to molecular toxicity. Prediction of the toxicity of any solvent, using the critical membrane theory, appears to be possible in the case of alkanols or alkyl acetates. However, prediction of the toxicity of ethers appears to be impossible. (c) 1993 John Wiley & Sons, Inc.     

The potential of cloud point system as a novel two-phase partitioning system for biotransformation

Although the extractive biotransformation in two-phase partitioning systems have been studied extensively, such as the water–organic solvent two-phase system, the aqueous two-phase system, the reverse micelle system, and the room temperature ionic liquid, etc., this has not yet resulted in a widespread industrial application. Based on the discussion of the main obstacles, an exploitation of a cloud point system, which has already been applied in a separation field known as a cloud point extraction, as a novel two-phase partitioning system for biotransformation, is reviewed by analysis of some topical examples. At the end of the review, the process control and downstream processing in the application of the novel two-phase partitioning system for biotransformation are also briefly discussed.     

Strategies for enzymatic esterification in organic solvents: comparison of microaqueous, biphasic, and micellar systems.

The kinetics of butyl butyrate synthesis by a lipase from Mucor miehei in different types of organic media were investigated. The three systems studied were a microaqueous medium containing enzyme in suspension in hexane, a water-hexane two-phase system, and reverse micelles. The synthesis of butyl butyrate was possible in all cases because of a favorable partition of the ester into the organic solvent. A sufficient stirring rate was necessary to achieve good reaction rates in the case of the liquid-liquid biphasic medium. The effect of water content was different according to the type of system used. The dependence of reaction rate and of conversion yield on enzyme and substrate concentrations was also investigated. From an applied point of view, the best performances were obtained with either microaqueous or liquid-liquid two-phase systems. The use of reverse micelles can be advocated only in particular conditions, such as low enzyme concentration, compatible with the specific constraints it involves.     

A facile enzymatic process for the preparation of (s)-Naproxen ester prodrug in organic solvents

A facile enzymatic synthesis process has been developed to directly prepare the 4-morpholinoethyl ester prodrug of (s)-Naproxen from racemic Naproxen using lipases as the biocatalysts in the organic solvent. With the careful selection of lipase (Lipase MY) and reaction medium (cylohexane), a high enantiomeric ratio of 136 for the enzyme was obtained. A Ping-Pong Bi Bi mechanism with competitive inhibition by the alcohol was illustrated from the variation of initial rates with substrate concentrations.     

An enzymatic method for radiolabeling vertebrate vitellogenin

Partition in aqueous two-phase systems should have great potentialities for studies of biological material. However, the general use of the method has probably been hindered by the difficulties in finding the composition of two-phase systems with desired qualities. Experimental design has proved to be of immense value for optimization in many different areas and should also be useful in selecting the best possible two-phase system. Therefore the simplex method has been applied to the search for the composition of an aqueous two-phase system in which the partition behavior of filamentous actin and human spectrin differ as much as possible.     

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.     

Enzymatic production of ceramide from sphingomyelin

Due to its major role in maintaining the water-retaining properties of the epidermis, ceramide is of great commercial potentials in cosmetic and pharmaceutical industries such as in hair and skin care products. Chemical synthesis of ceramide is a costly process, and developments of alternative cost-efficient production methods are of great interest. Present study was the first attempt to perform a systematic study on the production of ceramide through enzymatic hydrolysis of sphingomyelin. Sphingomyelin hydrolysis proved to be more efficient in two-phase (water:organic solvent) system than in one-phase (water-saturated organic solvent) system. Among the screened phospholipase C, the Clostridium perfringens enzyme had the highest sphingomyelin conversion rate, with very small temperature dependence. Addition of ethanol to the system markedly enhanced the rate of ceramide formation, and a mixture of ethylacetate:hexane (50:50) was the best organic solvent tested. Other factors such as (NH(4))(2)SO(4), NaCl and CaCl(2) were also tested but excluded for further consideration. On the basis of the initial experiments, the reaction system was optimized using response surface methodology including five factors (enzyme amount, water amount, ethanol amount, reaction time and the hexane ratio of organic solvent). Water content and enzyme amount was shown to have the most significant influence on the hydrolysis reaction in the fitted quadratic model. The efficiency of sphingomyelin hydrolysis was dramatically improved through system evaluation and optimization, with the optimal conditions at 75 min reaction time, 3 Uml(-1) enzyme amount, 6% water amount, 1.8% ethanol amount and 46% hexane in ethylacetate.     

An expedient synthesis of fluorescent labeled ceramide-1-phosphate analogues

A synthesis for fluorescent analogs of ceramide-1-phosphate bearing 9-anthrylvinyl or 4,4-difluoro-3a,4a-diaza-s-indacene-8-yl (Me₄-BODIPY) fluorophore at co-position of fatty acid residue was carried out. The key stage of the synthesis is hydrolysis of corresponding sphingomyelins catalyzed by phospholipase D from Streptomyces chromofuscus; the enzymatic yield has been raised to 50–70% by appliance of organic solvent in the incubation medium.     

Pseudomonas rhodesiae PF1: A New and Efficient Biocatalyst for Production of Isonovalal from α-pinene Oxide

A new bacterial strain, identified as Pseudomonas rhodesiae PF1 and deposited under the accession number CIP 107491, is presented. It is very active for the production of the acyclic compound Z-2-methyl-5-isopropyl-hexa-2,5-dien-1-al (isonovalal) from &#102 -pinene oxide. Enzyme synthesis is induced by culturing cells on &#102 -pinene; growing bacteria also have the ability to synthesize the epoxide derivative of &#102 -pinene. Isonovalal production was performed without aeration and with concentrated resting cells previously frozen at &#109 20°C, and subsequently thawed in a water-organic solvent, two-phase system. The organic layer was hexadecane, the volume ratio being 1:1. The best results achieved allowed recovery of c.a. 60 g/l organic solvent of isonovalal in 2.5 h operation, which is the most efficient process to date in the area of terpene biotransformations.     

Phenanthrene biodegradation by an algal-bacterial consortium in two-phase partitioning bioreactors

An algal-bacterial consortium formed by Chlorella sorokiniana and a phenanthrene-degrading Pseudomonas migulae strain was able to biodegrade 200-500 mg/l of phenanthrene dissolved in silicone oil or tetradecane under photosynthetic conditions and without any external supply of oxygen. Phenanthrene was only removed when provided in organic solvent, which confirms the potential of two-phase systems for toxicity reduction. Phenanthrene was degraded at highest rates when provided in silicone oil rather than in tetradecane since this solvent probably sequestered the PAH, reducing its mass transfer to the aqueous phase. The influence of phenanthrene concentration, amount of inoculum and light intensity on pollutant removal was also investigated and, under the best conditions, phenanthrene was degraded at 24.2 g m(-3).h(-1). In addition to being cost-effective and mitigating the release of greenhouse gases into the atmosphere, photosynthetic oxygenation was especially beneficial to the use of two-phase partitioning bioreactors since it prevented solvent emulsification and/or volatilization and evidence was found that the microalgae release biosurfactants that could further enhance phenanthrene degradation.