Biocatalysis whole cell

Previously, we have shown that functional secretion of OPH molecules into the periplasmic space induced about 2.8-fold higher specific whole cell OPH activity [10]. From the detail reaction kinetic studies in this work, we showed that this periplasmic space-secretion strategy provided much improved bioconversion capability and efficiency ( 1.8-fold) for Paraoxon as a model organophosphate compound. From these results, we confirmed that Tat-driven periplasmic secretion of OPH can be successfully employed to develop a whole cell biocatalysis system with notable enhanced bioconversion efficiency and capability for environmental toxic organophosphates. 

Xie, X. and Tang, Y. (2007) Efficient synthesis of Simvastatin by use of whole-cell biocatalysis. Applied and Environmental Microbiology, 73, 2054-2060. 

An interesting example of the application of recombinant whole-cell biocatalysis is the conversion of 2-hydroxybiphenyl (2-phenylphenol) to 2,3-dihydroxybiphenyl... 

Many reported biotransformations are initially only demonstrated on a very small scale, the substrates or products may be subject to competing reactions if other enzymes are present (this can be a serious issue in whole-cell biocatalysis), or the desired enzyme is insufficiently active or produced in low levels. For many biotransformations a little care and attention is needed in the growth of the microbe to achieve the desired results. Production of a specific enzyme from a microbe can often be increased by growing the cells in the presence of a very small concentration (typically micromolar) of an inducer. The inducer could be a natural enzyme substrate, a substrate mimic or a molecule which is in some way associated with a substrate s availability or role in metabolism. This process is called induction and represents a genetic switch which cells use to respond... 

A strain of Pseudomonas aeruginosa has been recently described, which shows the opposite enantioselectivity, converting racemic arylaminonitriles efficientiy into the D-amino acids. Again, whole-cell biocatalysis worked well, the cells being entrapped in alginate beads. It is unclear whether this biotransformation involves an amide intermediate. 

Setti, L., Lanzarini, G. and Pifferi, P.G. (1997) Whole cell biocatalysis for an oil AQsa fanz2Aon rocQSS. Fuel Processing Technology, 52, 145-153. 

P. Nikolova and O. P. Ward, Whole cell biocatalysis in nonconventional media,... 

In an influential early investigation, correlation of biocatalytic activity data of aerobic and anaerobic whole-cell biocatalysis with solvent properties resulted in the strongest correlation for the partition coefficient log P, whereas both Hildebrand s solubility parameter 6 and the dielectric constant e showed either a weak correlation with activity data or none at all (Laane, 1985,1987) (Figure 12.2). 

Combining whole-cell biocatalysis and radical polymerization, researchers at Imperial Chemical Industries (ICI) published a chemoenzymatic route to high-molecular-weight poly(phenylene) [86], This polymer is used in the fibers and coatings industry. However, since it is practically insoluble, the challenge was to make a soluble polymer precursor that could first be coated or spun, and only then converted to poly(phenylene). The ICI process starts from benzene, which is oxidized by Pseudomonas putida cells to cyclohexa-3,5-diene-l,2-diol (see Figure 5.17). The... 

Whole cell biocatalysis is a productive and practical style of conducting biocatalytic reactions. Such reactions, as implied by the term, are done with structurally intact cells. Usually, viable respiring cells are used but not exclusively. There are many reasons why a whole-cell reaction might be preferred to a... 

Enzyme Based Biocatalysis vs. Whole-Cell Biocatalysis... 

The whole-cell biocatalysis approach is typically used when a specific biotransformation requires multiple enzymes or when it is difficult to isolate the enzyme. A whole-cell system has an advantage over isolated enzymes in that it is not necessary to recycle the cofactors (nonprotein components involved in enzyme catalysis). In addition, it can carry out selective synthesis using cheap and abundant raw materials such as cornstarches. However, whole-cell systems require expensive equipment and tedious work-up because of large volumes, and have low productivity. More importantly, uncontrolled metabolic processes may result in undesirable side reactions during cell growth. The accumulation of these undesirable products as well as desirable products may be toxic to the cell, and these products can be difficult to separate from the rest of the cell culture. Another drawback to whole-cell systems is that the cell membrane may act as a mass transport barrier between the substrates and the enzymes. 

Whole-cell based biocatalysis utilizes an entire microorganism for the production of the desired product. One of the oldest examples for industrial applications of whole-cell biocatalysis is the production of acetic acid from ethanol with an immobilized Acetobacter strain, which was developed nearly 200 yr ago. The key advantage of whole-cell biocatalysis is the ability to use cheap and abundant raw materials and catalyze multistep reactions. Recent advances in metabolic engineering have brought a renaissance to whole-cell biocatalysis. In the following sections, two novel industrial processes that utilize whole-cell biocatalysis are discussed with emphasis on the important role played by metabolic engineering. 

Experiments were also conducted to assess the impact of compressed N2, ethane, and propane on product selectivity of whole cell biocatalysis (45). Pressurized incubations, with and without a compressed solvent headspace, lead to an increase in the ratio of ethanol to acetate produced by the organism (Fig. 1). These results are consistent with increased dissolution of H2, a product gas which affects the pathways of acetate production (50), in the fermentation media with increasing pressure. Lactate formation was also decreased in the presence of compressed and liquid solvents (but not nitro-... 

Berberich JA. Whole cell biocatalysis in the presence of supercritical and compressed solvents. PhD dissertation. Lexington University of Kentucky, 2001. 

Leon R, Fernandes P, Pinheiro HM, Cabral JMS. Whole-cell biocatalysis in organic media. Enzyme Microb Tech 1998 23 483-500. 

Whole cell biocatalysis has been exploited for thousands of years. Historically biotechnology was manifested in skills such as the manufacture of wines, beer, cheese etc., where the techniques were well worked out and reproducible, while the biochemical mechanism was not understood. 

For cost reasons, if ever possible, whole-cell biocatalysis is used to perform biotransformations. This is possible when the following criteria are met (i) no diffusion limitations for substrate(s) and/or product(s) and (ii) no side or follow-up reactions due to the presence of other cellular enzymes. If these conditions are not fulfilled, the use of isolated enzymes - or in special cases of permeabilized cells - is indicated. 

An important technical issue is the large-scale applicability of co-factor-dependent enzymatic systems. It is generally accepted that, e.g., NADH-requiring oxidoreductases can easily be used in whole-cell biocatalysis such as baker s yeast-mediated reductions, where the cofactor recycling step is simultaneously performed within the intact cell, driven by the reduction equivalents introduced via the external carbon and energy source (glucose). 

The application of aqueous / supercritical biphasic media is not restricted to metal complex catalysis but has proven effective also for enzymatic and whole-cell biocatalysis [36]. In general, water plays an important role in coimection with biocatalysis. If water is completely absent, enzymes are often not catalytically active under supercritical conditions [37]. In the literature many examples of biocatalysis with supercritical fluids containing various amounts of water are known and a detailed account of this field is outside the scope of the present discussion. One example to highlight the use of a true biphasic system is the carboxylation of pyrrole... 

Apart from the usually low activity and sometimes insufficient selectivity of P450s towards steroids (which can be improved by means of protein engineering), the low solubility of steroid compounds in water (1-100 pM [334]) represents a challenging problem for the establishment of whole-cell biocatalysis. Consequently, several promising reaction-engineering techruques that were applied for biotransformations of other hydrophobic compoimds have also been tested with steroid substrates. Among these are (1) biphasic reaction setups with an organic phase, which serves as substrate reservoir, (2) surfactant-... 

Park JB (2007) Oxygenase-based whole-cell biocatalysis in organic synthesis. J Microbiol Biotechnol 17 379-392... 

Yoshida, A., Hama, S., Tamadani, N., Fukuda, H., Kondo, A. (2012). Improved performance of a packed-bed reactor for biodiesel production through whole-cell biocatalysis employing a high-lipase-expression system. Biochemical Engineering Journal, 63, 76—80. 

Enantioselective catalysis (and kinetic resolution) where the expensive chiral auxiliary is used in substoichiometric amounts attached to an active catalytic entity. The catalysts can be man-made (chemical catalysis), enzymes or whole cells (biocatalysis). In many respects, both chemical and biocatalysts have similar problems when used on an industrial scale (for chemical catalysts, see below). In biocatalysis, the often high effort for finding and developing an efficient biocatalyst, especially when the starting material is not a very close analog to the natural substrate, and the product isolation from an often rather dilute aqueous solution can be problematic. 

Weuster-Botz D (2007) Process intensification of whole-cell biocatalysis with ionic liquids. Chem Rec 7 334-340... 

Werner SR, Morgan JA (2009) Expression of a Dianthus flavonoid glucosyltransferase in Saccharomyces cerevisiae for whole-cell biocatalysis. J Biotechnol 142 233-241. doi 10.1016/j.jbiotec.2009.05.008... 

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