Ionic enzymatic transformation

The hydrophobic ionic liquid [BMIMJPFg has been consistently shown to provide the desired conformational flexibility to enzymes without drastically altering their catalytically active conformations. Therefore, this ionic liquid has been widely studied as a suitable medium for enzymatic transformations. 

When the water-miscible ionic liquid [MMIM][MeS04] was used as a neat medium for the enzymatic transformations, however, poorer performance was observed. For the kinetic resolution of mc-l-phenylethanol by transesterification with vinyl acetate with a set of different lipases dispersed in the pure ionic liquid, it was found that [MMIM][MeS04] was among the poorest media for the enzymes (291). It has been recognized that some water-miscible ionic liquids in the pure form are denaturants (27), but, when they are used in the presence of excess water, their tendency to... 

Reactions proceed faster and more smoothly when the reactants are dissolved, because of diffusion. Although reactions in the solid state are known [1] they are often condensations in which a molecule of water is formed and reaction takes place in a thin film of water at the boundary of the two solid surfaces. Other examples include the formation of a liquid product from two solids, e.g. dimethylimidazolium chloride reacts with aluminum chloride to produce the ionic liquid, dimethylimidazolium tetrachloroaluminate [2]. It is worth noting, however, that not all of the reactant(s) have to be dissolved and reactions can often be readily performed with suspensions. Indeed, so-called sol-id-to-solid conversions, whereby a reactant is suspended in a solvent and the product precipitates, replacing the reactant, have become popular in enzymatic transformations [3]. In some cases, the solvent may be an excess of one of the reactants. In this case the reaction is often referred to as a solvolysis, or, when the reactant is water, hydrolysis. 

Scheme 25 Enzymatic transformation using ionic liquids for immobilization of the enzyme and supercritical CO2 as fluid...

Fig. 13. The effect of the minimum ionic strength, / , on the pH-rate profile for a typical enzymatic reaction. Two types of curves are generated Type I, bell shaped type II, monotonically decreasing, depending on the pH of the experiment. Graphing the pH behavior as a function of ionic strength (a and b show the transformation) and applying the / cut-off (c), it can be seen that, if experimental pH at is lower than the pH optimum, a type I curve is obtained. If the experimental pH is greater, a type II is obtained.

In biphasic reactors or two-phase partitioning bioreactors (TPPB), the substrate is located mostly in the immiscible phase and diffuses to the aqueous phase. The enzyme catalyzes conversion of the substrate at the interface and/or in the aqueous phase. The product/s of the reaction then may partition to the organic phase. The system is self-regulated, as the substrate delivery to the aqueous phase is only directed by the partitioning ratio between the two phases and the enzymatic reaction rate [53]. The use of ionic liquid/supercritical carbon dioxide for enzyme-catalyzed transformation is gaining attention [69]. 

Nearly any type of cell (prokaryotic or eukaryotic) can be transformed by the technique of electroporation. Protoplasts are first prepared by enzymatic or chemical disruption of the host-cell membrane polysaccharides. Next, the recombinant vector is introduced to the protoplast suspension residing in a very low ionic strength buffer (or distilled water). This DNA-protoplast suspension is then subjected to one or several 250-V pulses delivered from a cathode and anode placed directly into the solution. This applied voltage gradient will cause a certain population of the cells (—1010 per... 

Ionic liquids were initially developed as solvents for electrochemical applications. The electrochemical window of clean ionic liquids can be huge/11 allowing for a wide range of redox reactions/2,31 It has further been demonstrated that they are also suitable solvents for enzymatic oxidations14 71 but both topics are beyond the scope of this book. Only transformations that involve the metal-catalysed addition of oxygen to unsaturated carbon bonds as well as the oxidation of alcohols, aldehydes and ketones to their corresponding ketones, carboxylic acids and esters shall be discussed in this chapter. 

Quantification of microbial PHA using GC method is rapid, sensitive, reproducible, and requires only small amount of samples (5-10 mg) for the analysis. Other techniques of analysis such as IR spectrometry at 5.75 A (Juttner et al. 1975), two-dimensional fluorescence spectroscopy, flow cytometry (Degelau et al. 1995) HPLC (Karr et al. 1983), ionic chromatography, and enzymatic determination (Hesselmann et al. 1999) were also desalbed. For online determination of PHA content in recombinant E. coli system, Fourier transform mid-infrared spectrometry (FTIR) and microcalorimetric technique (Ruan et al. 2007 Jarute et al. 2004) were also reported. For precise composition determination and structural elucidation of PHA, a variety of nuclear magnetic resonance (NMR) spectroscopy techniques have also been applied and the most commonly used are proton ( H) and carbon-13 ( C) NMR (Doi et al. 1986 Jacob et al. 1986). 

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