Efficient biotransformation

Odoux E, Chauwin A, Brillouet JM (2003) Purification and characterization of vanilla bean (Vanillaplanifolia Andrews) / -glucosidase. J Agric Food Chem 51 3168-3173 Overhage J, Steinbuechel A and Priefert H (2003) Highly efficient biotransformation of euge-nol to ferulic acid and further conversion to vanillin in recombinant strains of Escherichia coli. Appl Environ Microbiol 69 6569-6576... 

In 1978 Givaudan patented a transformation of a-ionone [127] a fermentation of Botryodiplodia theobromae IFO 6469 with a-ionone (152) yielded a mixture of compounds (155 -157) with honeysuckle aroma, Fig. (31). The same fungi which efficiently biotransformed / -ionone also transformed a-ionone [128]. As observed for /1-ionone, a-ionone (152) transformations with Lasiodiplodia theobromae ATCC 28570 also suggested an oxygenase-type enzyme system to be responsible for the degradation of the molecule by loss of one C2-unit. 

Mucuna pruriens cell cultures are known to hydroxylate a variety of phenolic compounds (Pras, 1990). The solubility of the phenolic steroid, 1713-estradiol (Fig. 6.1), is only 12 pM in culture medium and no biotransformation products could be detected after administration to freely suspended cells, immobilized cells or partially purified Mucuna phenoloxidase. Complexation with (3-cyclodextrin dramatically enhanced the solubility of 17(3-estradiol. Alginate-entrapped cells, cell homogenates and the phenoloxidase were able to o-hydroxylate 17(3-estradiol when supplied as the cyclodextrin complex, the most efficient biotransformation being achieved with the isolated enz)mrie (Woerdenbag et at, 1990). 

Evidently, the use of the whole cell biocatalyst enables an efficient biotransformation with high substrate concentrations. 

However, determination and interpretation of explosive concentrations in organisms are faced with significant challenges, as explosives typically bioaccumulate at low whole body concentrations relative to environmental concentrations and are efficiently biotransformed and/or quickly eliminated following cessation of exposure (see Chapter 6). 

Efficient biotransformation of TNT has been reported for fish [3,7,9] and an aquatic invertebrate [6], most of which accumulated biotransformed products including tissue-bound molecules, at higher concentrations relative to the parent compound at steady state [6,7,9], The ability of aquatic animals to biotransform other explosives and related compounds is unknown. Recent studies examining the uptake from water and elimination kinetics of nitroaromatic and cyclic nitroamine explosives in aquatic invertebrates and fish [2,3,5-9] revealed that elimination of those compounds is very efficient, leading to steady-state levels within hours. 

Aquatic vascular plants and macroalgae can take up TNT dissolved in water very efficiently as indicated by removal rates determined for several species [11,12] some of which are promoted for use in phyto-treatment of explosives-contaminated water [11]. Less efficient removal of dissolved RDX was reported for wetland and aquatic plants [13,14], Efficient biotransformation and elimination mechanisms in aquatic vascular plants and macroalgae resulted in a lack of bioconcentration of TNT and its solvent-extractable transformation products [11,12], This chapter summarizes and discusses the bioconcentration, bioaccumulation, biotransformation, and toxicoki-netic processes of explosives in aquatic organisms. 

The nitro groups of TNT characteristically undergo biochemical reduction in living systems (see Chapter 2). Biotransformation of TNT to ADNTs during aqueous exposures occurred very quickly in T. tubifex (0.212 and 0.187 h 1 for 2-ADNT and 4-ADNT, respectively) [6], but was markedly slower (0.06 h 1 for ADNTs) in juvenile sheepshead minnows [3], Substantial biotransformation of TNT to ADNTs in fathead minnows exposed to TNT for periods as short as 10 min was observed [2], Efficient biotransformation of TNT to ADNTs was also reported for soil invertebrates... 

Fujii T, Fujii Y, Machida K, Ochiai A, Ito M (2009) Efficient biotransformations using Escherichia coli with tolC acrAB mutations expressing cytochrome P450 genes. Biosci Biotechnol Biochem 73 805-810... 

VAN Rensburg, P. and I.S. Pretorius. 2000. Enzymes in winemaking harnessing natural catalysts for efficient biotransformations irreview. S. Afr. J. Enol. Vitic. 21 52-73. 

Amino acid separations represent another specific application of the technology. Amino acids are important synthesis precursors - in particular for pharmaceuticals -such as, for example, D-phenylglycine or D-parahydroxyphenylglycine in the preparation of semisynthetic penicillins. They are also used for other chiral fine chemicals and for incorporation into modified biologically active peptides. Since the unnatural amino acids cannot be obtained by fermentation or from natural sources, they must be prepared by conventional synthesis followed by racemate resolution, by asymmetric synthesis, or by biotransformation of chiral or prochiral precursors. Thus, amino acids represent an important class of compounds that can benefit from more efficient separations technology. 

In principle, numerous reports have detailed the possibility to modify an enzyme to carry out a different type of reaction than that of its attributed function, and the possibility to modify the cofactor of the enzyme has been well explored [8,10]. Recently, the possibility to directly observe reactions, normally not catalyzed by an enzyme when choosing a modified substrate, has been reported under the concept of catalytic promiscuity [9], a phenomenon that is believed to be involved in the appearance of new enzyme functions during the course of evolution [23]. A recent example of catalytic promiscuity of possible interest for novel biotransformations concerns the discovery that mutation of the nucleophilic serine residue in the active site of Candida antarctica lipase B produces a mutant (SerlOSAla) capable of efficiently catalyzing the Michael addition of acetyl acetone to methyl vinyl ketone [24]. The oxyanion hole is believed to be complex and activate the carbonyl group of the electrophile, while the histidine nucleophile takes care of generating the acetyl acetonate anion by deprotonation of the carbon (Figure 3.5). 

Such isolated enzyme approaches for deracemization have a clear disadvantage in that they require two operational manipulations with an intermediate recovery step. A one-pot strategy is offered by employing whole-cell biotransformations with strains containing set(s) of complementary dehydrogenases operating in both biooxidative and bioreductive modes. Trace amounts of the intermediate ketone species can be isolated in several cases. In order to lead to an efficient deracemization... 

The most important removal pathways of PhACs during wastewater treatment are biotransformation/biodegradation and abiotic removal by adsorption to the sludge. The efficiency of their removal at WWTP depends on their physico-chemical properties, especially hydrophobicity and biodegradability, and process operating parameters (i.e., HRT, SRT, and temperature). For certain NSAIDs (e.g., ibuprofen, acetaminophen), high removals (>90%) are consistently reported in literature... 

Some of the industrial biocatalysts are nitrile hydralase (Nitto Chemicals), which has a productivity of 50 g acrylamide per litre per hour penicillin G amidase (Smith Kline Beechem and others), which has a productivity of 1 - 2 tonnes 6-APA per kg of the immobilized enzyme glucose isomerase (Novo Nordisk, etc.), which has a productivity of 20 tonnes of high fmctose syrup per kg of immobilized enzyme (Cheetham, 1998). Wandrey et al. (2000) have given an account of industrial biocatalysis past, present, and future. It appears that more than 100 different biotransformations are carried out in industry. In the case of isolated enzymes the cost of enzyme is expected to drop due to an efficient production with genetically engineered microorganisms or higher cells. Rozzell (1999) has discussed myths and realities... 

The volumetric ratio of the two liquid phases (j6 = Forg/ Faq) can affect the efficiency of substrate conversion in biphasic media. The biocatalyst stability and the reaction equilibrium shift are dependent on the volume ratio of the two phases [29]. In our previous work [37], we studied the importance of the nonpolar phase in a biphasic system (octane-buffer pH 9) by varying the volume of solvent. The ratio /I = 2/10 has been the most appropriate for an improvement of the yield of the two-enzyme (lipase-lipoxygenase) system. We found that a larger volume of organic phase decreases the total yield of conversion. Nevertheless, Antonini et al. [61] affirmed that changes in the ratios of phases in water-organic two-phase system have little effect upon biotransformation rate. 

Based on reactions they catalyze, enzymes can be broadly classified into six major categories (Table 1.1) [1], It was estimated that about 60% of biotransformations currently rely on the use of hydrolases, followed by 20% of oxidoreductases [2]. On the other hand, some of the C—C bond-forming and oxygenation enzymes catalyze reactions with very high reaction efficiency and very low waste generation, underlining the potential of emerging enzymes. 

These first examples illustrate the importance of a sufficient separation of products and byproducts, whereas membranes are one possibility in pharmaceutical production to obtain this aim. Therefore, they are one key tool to obtaining better quality products and environmentally friendly processes. For a more detailed article about the state of the art of membranes in biotechnology, see Rios et al. [27]. At the same time, it can be seen that stoichiometric cofactor need is no longer a limitation for industrial biotransformations, since they can be overcome with efficient recyclization methods. 

Kaup, B., Bringer-Meyer, S. and Sahm, H. (2004) Metabolic engineering of Escherichia coir, construction of an efficient biocatalyst for D-mannitol formation in a whole-cell biotransformation. Applied Microbiology and Biotechnology, 64 (3), 333-339. 

Degradation percentages of several UV filters and some of their metabolites in a solid-phase treatment of sewage sludge by T. versicolor are shown in [80]. As treatments were performed in sterile conditions, the near 100% removal efficiencies of those compounds can be attributed to fungal biotransformation. However, experiments in synthetic liquid media with the fungus in the form of pellets are preferable for the in-depth study of each contaminant. 

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