Oxidoreductase biocatalysts

Alcohol oxidoreductases capable of oxidizing short chain polyols are useful biocatalysts in industrial production of chiral hydroxy esters, hydroxy adds, amino adds, and alcohols [83]. In a metagenomic study without enrichment, a total of 24 positive clones were obtained and tested for their substrate specifidty. To improve the detedion frequency, enrichment was performed using glycerol or 1,2-propanediol and further 24 positive clones were deteded in this study. 

Further research in improving the BDS activity of the biocatalysts was targeted towards the search of co-catalysts and co-factors to enhance overall desulfurization rates as well as promoters to enhance enzyme expression. This research resulted in identification of NADH and FMNH2 as co-factors essential for electron transfer and related oxidoreductase enzymes as co-catalysts as described in detail below. Additionally, other bacterial strains were also investigated as hosts and are reported below. 

A BDS method consisting in the incubation of a mixture formed by a fossil fuel and an aqueous phase containing a biocatalyst and a rate-enhancing amount of a protein having NADH FMN oxidoreductase activity or enzymatically active mutant thereof. The oxidoreductase has the amino acid sequence set forth in SEQ ID No. 2, described in the original document (see Ref. [57]). A separation stage is also claimed. 

In the field of bioremediation, oxidoreductases are considered to be excellent biocatalysts for environmentally friendly processes. Laccases and peroxidases are widely used to treat effluents from pulp/cotton mills, food/fruit processing plants and breweries [1, 2, 37]. Laccases, peroxidases and other oxygenases are also being studied for their abihty to degrade hazardous coal substances, especially the sulfur-containing components, and in the treatment of industrial waste and contaminated soil and water in the transformation of xenobiotics, polycycHc aromatic hydrocarbons and other pollutants (biodetoxification and biodecontamination)... 

This chapter covers some general aspects of the use of enzymes in aqueous and organic media. Although lipases are the most common biocatalysts in these processes [4], other hydrolytic enzymes such as esterases and nitrilases have also shown their utility in the manufacture of pharmaceuticals. In addition, some representative examples using oxidoreductases and lyases will be also discussed. 

Oxidoreductases are, after lipases, the second most-used kinds of biocatalysts in organic synthesis. Two main processes have been reported using this type of enzymes-bioreduction of carbonyl groups [39] and biohydroxylation of non-activated substrates [40]. However, in recent few years other processes such as deracemization of amines or alcohols [41] and enzymatic Baeyer-Villiger reactions of ketones and aldehydes [42] are being used with great utility in asymmetric synthesis. 

Biocatalysts based on hydrolases (E.C. class 3, Table 5.2) ate mostly used as (purified) enzymes since they are cofactor independent, since these preparations are commercially available and because a number of hydrolases can be applied in organic solvents. Oxidoreductases (E.C. class 1) however, are relatively complex enzymes, which require cofactors and frequently consist of more than one protein component. Thus, despite the fact that efficient cofactor regeneration systems for NADH based on formate dehydrogenase (FDH) have been developed (Bradshaw et al, 1992 Chenault Whitesides, 1987 Wandrey Bossow, 1986, chapter 10) and that also an NADPH dependent FDH has been isolated (Klyushnichenko, Tishkov Kula, 1997), these enzymes are still mostly used as whole-cell biocatalysts. 

In current research, oxidoreductases are second in the number of applications of enzymes in organic synthesis. The number of commercially available biocatalysts of this class has increased tremendously during the last few years and various screening kits for oxidation and reduction are sold. Many oxidoreductases are rather easy to handle, though, in contrast to hydrolases, they are dependent on cofactors [22]. 

Biocatalysts, mainly hydrolytic enzymes and oxidoreductases, have been used for organic reactions due to their excellent enantioselectivities and environmentally friendliness.1 Typical enzymatic reactions used for the organic synthesis are shown in Figure 1. Especially, hydrolytic enzymes for kinetic resolutions of racemates have been utilized widely because of their high stabilities, wide substrate specificities, lack of cofactor requirements and high availabilities. 

Optical resolution of racemic compounds by biocatalysts has been a useful method as shown in this review. For this purpose, two types of biocatalysts are mainly used hydrolytic enzymes and oxidoreductases. 

The oxidoreductase class of biocatalysts is one of the most common of all biological reactions, comprising dehydrogenases, oxidases, and reductases. All these enzymes act on substrates through the transfer of electrons with various co-factors or co-enzymes serving as acceptor molecules. Only a select group of reactions will be discussed because of space limitations, so the reader is referred to other texts for more in-depth discussions of other oxidation-reduction reactions.17 20 25-28... 

Chiral alcohols are some of the most important chiral building blocks for the production of pharmaceuticals. The creation of chiral alcohols through the asymmetric reduction of prochiral carbonyl compounds using biocatalysts, such as microbial cells and commercially available oxidoreductases, has been... 

Peroxidase (donor hydrogen-peroxide oxidoreductase, EC 1.11.1.7) is a prominent biocatalyst that polymerizes a wide range of aromatic compounds, generally through the following consecutive steps (Yamazaki and Yokota, 1973 Sakurada et al., 1990) ... 

Catalase (EC 1.11.1.6) is a heme containing oxidoreductase that acts on peroxides liberating oxygen and water. The enzyme is a very fast biocatalyst, i.e., Pichia pastoris catalase has a turnover number of 3 x 10 H2O2 s [199]. The enzyme consists of multiple subunits containing each heme as active site. 

A composite biomaterial formed by Pd metal, carbon-ceramic mixture and oxidoreductase enz3ones constitutes a new t3rpe of renewable smface biosensor with a controllable size reaction layer [198]. The carbon provides the electrical conductivity, the enzymes are used for biocatalyst process, metallic palladimn is used for electrocatalysis of biochemical reaction product and the porous silica provides a rigid skeleton. The hydrophobicity of this composite material allows only a limited section of the electrode to be wetted by the aqueous analyte, thus providing a controlled thickness reactive layer. Another biocomposite material containing enzyme-modifled boron-doped diamond was used in the development of biosensors for the determination of phenol derivatives [199], alcohol [200] and glucose [201]. 

The types of enzymes used by organic chemists vary widely and include such well-known biocatalysts as lipases, esterases, oxidoreductases, oxinitrilases, transferases and aldolases [4]. An example which illustrates the industrial application of a lipase concerns the kinetic resolution of a chiral epoxy ester used as the key intermediate in the synthesis of the calcium antagonist Diltiazem, a major therapeutic in the treatment of high blood pressure [6] (Fig. 1). In developing the industrial process for the production of this drug, many different lipases were screened, but only the bacterial lipase from Serratia marescens showed both a sufficiently high activity and enantioselectivity. The intermediate is produced industrially on a scale of 50 tons/year. 

The application of the biocatalysts in this overview is limited to these stable enzymes, which do not need cofactors, such as the various hydrolytic enzymes, some lyases, transferases and isomerases. In addition to these groups, oxidoreductases, which demand NAD or NADP as cofactors, some pyridoxyl-phosphate dependent lyases with simple systems for cofactor regeration and finally, various aldolases in combination with L-glycerol- phosphate oxidase and catalase are useable to some extent in cell-free form. 

Yeast contains a variety of enzymes, and in some cases use of a single purified enzyme is preferable. These are divided into oxidoreductases, transferases, hydrolases, lyases, isomerases, and lipases. Many of these are commercially available (but expensive). Purified reductases usually require expensive cofactors. In addition individual microbes can be used as biocatalysts. A general review of microbial asymmetric reductions is available. These reductions can be the opposite of those of yeast. 

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