Substrate specificity, conversion

Most nitrile bioconversions published have been conducted in aqueous media and consequently few data are available on the effect of solvents on enzymatic nitrile hydrolysis. Such studies seem highly justified in order to investigate the effects of different solvents or co-solvents on substrate specificity, conversion rate, stereoselectivity, and catalyst half-life. 

Glucose oxidase is specific for P-D-glucose and where a-D-glucose is the available substrate, prior conversion from the a to the P form is required. 

The substrate specificity of ACE is low. ACE cleaves a variety of pairs of amino acids from the carboxy-terminal part of several peptide substrates. The conversion of ANGI to ANGII and the degradation of bradykinin to inactive fragments are considered the most important functions of ACE. Both peptides have profound impact on the cardiovascular system and beyond. ACE is thus an important target for ACE inhibitors. These compounds are frequently and efficiently used in the treatment of hypertension and cardiac failure. 

The 4-methoxybenzoate monooxygenase from Pseudomonas putida shows low substrate specificity. Although it introduces only a single atom of oxygen into 3-hydroxy- and 4-hydroxybenzoate, it accomplishes the conversion of 4-vinylbenzoate into the corresponding side-chain diol (Wende et al. 1989). 

Chang and co-workers isolated strain Nocardia sp. CYKS2 from a dyeing industry wastewater using DBT as the sole sulfur source [27]. This strain also desulfurized DBT to the same product 2-HBP however, it had broader substrate specificity and was reported to desulfurize thiophenes, sulfides, and disulfides (Table 3) in addition to DBT. However, it did not desulfurize trithiane, thianthrene and 4,4 -thiodiphenol. The desulfurization experiments were conducted in batch with the rate reported as 0.279 mg-sulfur/L dispersion/h for DBT conversion. 

In addition to desulfurization activity, several other parameters are important in selecting the right biocatalyst for a commercial BDS application. These include solvent tolerance, substrate specificity, complete conversion to a desulfurized product (as opposed to initial consumption/removal of a sulfur substrate), catalyst stability, biosurfactant production, cell growth rate (for biocatalyst production), impact of final desulfurized oil product on separation, biocatalyst separation from oil phase (for recycle), and finally, ability to regenerate the biocatalyst. Very few studies have addressed these issues and their impact on a process in detail [155,160], even though these seem to be very important from a commercialization point of view. While parameters such as activity in solvent or oil phase and substrate specificity have been studied for biocatalysts, these have not been used as screening criteria for identifying better biocatalysts. 

The investigation of the aminotransferase activity of apple ACS carried out by Feng et al reveals that it is able to reductively aminate PLP to PMP by transamination of some L-amino acids to their corresponding a-keto acids. The enzyme has shown substrate specificity with the preference of Ala > Arg > Phe > Asp. The addition of excess pyruvate causes a conversion of the PMP form of the enzyme back to the PLP form. The quite unstable PMP form of ACS can generate apoenzyme, which captures PLP to restore its physiologically active form. 

In addition to 9—12, several useful chiral carbonyl compounds have been obtained from the diols obtained by yeast treatment of the corresponding a-hydroxyketones. As a part of a study (2) on the substrate specificity of the multienzymic conversion shown in Eq. 2, a serie of racemic a-hydroxyketones has been prepared and submitted to the yeast treatment. The reduction process is stereospecific, but depending upon... 

A potential versatile route into a-amino acids and their derivatives is via a combination of (i) nitrile hydratase/amidase-mediated conversion of substituted malo-nonitriles to the corresponding amide/acid followed by (ii) stereospecific Hofmann rearrangement of the amide group to the corresponding amine. Using a series of a,a-disubstituted malononitriles 14, cyanocarboxamides 15 and bis-carboxamides 16, the substrate specificity of the nitrile hydratase and amidase from Rhodococcus rhodochrous IF015564 was initially examined (Scheme 2.7). The amidase hydrolyzed the diamide 16 to produce (R)-17 with 95% conversion and 98%e.e. Amide 17 was then chemically converted to a precursor of (S)-a-methyldopa. It was found... 

Recent developments on research into a bacterial C-F bond forming enzyme are reviewed. The fluorinase enzyme was isolated from Streptomyces cattleya in 2002 and shown to catalyse the conversion of fluoride ion and S-adenosyl-L-methionine (SAM) to 5 -fluoro-5 -deoxyadenosine (5 -FDA) and L-methionine. Subsequently, the enzyme has been the subject of cloning, crystallisation, mechanism and substrate specificity studies. This review summarises the current status of this research. 

Substrate specificity studies have revealed that the fluorinase can catalyse the conversion of 2 -deoxyadenosine substrates 16 and 17 to 2 d-SAM 18 (Scheme 5). 

Further detailed study of the substrate specificity of yeast squalene synthetase has been reported (see Vol. 7, p. 130). The enzyme is very sensitive to changes in substrate. For example, 10,11-dihydrofarnesyl pyrophosphate was converted into 2,3,22,23-tetrahydrosqualene with only 60% of the efficiency of farnesyl pyrophosphate whereas 6,7-dihydro- and 6,7,10,11-tetrahydro-farnesyl pyrophosphates were not metabolized. The first of the two binding sites has a greater preference for farnesyl pyrophosphate and this accounts for the formation of the unsymmetrical squalene product when mixtures of farnesyl pyrophosphate and a modified substrate are used. The importance of the methyl groups, especially that at C-3, for binding is emphasized by the low efficiency of conversion of 3-desmethylfarnesyl, , -3-methylundeca-2,6-dien-l-yl (1), and E,E-7-desmethylfarnesyl pyrophosphates. The prenylated cyclobutanones (2) and (3)... 

Figure 5.6. The production of NPs using matrix pathways was predicted by Jones and Firn because of the opportunity to produce and retain chemical diversity efficiently. In this diagrammatic scheme, three enzymes (ei, e2 and es) have access to one substrate. The upper panel shows that if each of the enzymes has a strict substrate specificity, a linear pathway producing three new chemicals would be expected, ffowever, if the three enzymes have a broad substrate specificity then the order of conversion can vary and a matrix pathway will result. Now three enzymes will produce 11 novel substances. Furthermore, such matrix pathways are more robust to the loss of any one enzyme activity (see Figure 5.4).

Substrate specificity site The binding of nucleoside triphosphates to an additional allosteric site (known as the substrate specificity site) on the enzyme regulates substrate specificity, causing an increase in the conversion of different species of ribonucleotides to deoxyribonucleotides as they are required for DNA synthesis. 

A characteristic of the liver P4so enzymes is their almost total lack of substrate specificity, distinguishing them from the adrenal gland enzymes which are much more specific (B-74MI11003). In addition to hydroxylation of hydrocarbons, which involves the conversion of C—H bonds to C—OH bonds and C=C bonds to epoxide rings, a multitude of other types of reaction are catalyzed. These include iV-oxidation, 5-oxidation, N-, S- and O-dealkylation, peroxidation, deamination, desulfuration and dehalogenation, as well as... 

As de novo synthesis has been proven unsuccessful in most cases, biotransformation of added precursors has been studied extensively. There is evidence that plant cell cultures retain an ability to transform specifically exogenous substrates administered to the cultured cells. Therefore, plant cell cultures are considered to be useful for transforming cheap and plentiful substances into rare and expensive compounds by using the cell culture as a bioreactor. For instance, cofactor dependent specific conversions of terpenoids in suspension cultures of aromatic plants often proceed with high yields and negligible amounts of byproducts. In Fig. (1), three examples of biotransformations of terpenes by plant cell cultures are shown (after [6]). 

The first data confirming this oxidoreductive epimerization were obtained by measuring the enzyme activity in protein extracts from a tomato cell suspension [24], It could be demonstrated, using a very sensitive fluorimetric detection method, that two different enzymes were involved in this subpathway. No enzyme activity could be detected with 24-ep/-teasterone as substrate and NAD+ or NADP+ as electronacceptors. But using the proposed intermediate 3-dehydro-24-epi-teasterone as substrate, enzymatic conversion to 24-epi-teasterone was measured in a microsomal fraction of tomato cell cultures (Fig. (9)). 3-dehydro-24-epi-teasterone-reductase showed a specific activity of 361 fkat/mg protein with NADPH as the only accepted electrondonor. 

The key enzymes involved in these conversions are transaldolase and transketolase. The two enzymes are similar in their substrate specificities. Both require a ketose as a donor and an aldose as an acceptor. The steric requirements at positions C-1 through C-4 are the same as the requirements of aldolase in the glycolytic pathway, except that aldolase requires phosphorylation at C-1, and both transaldolase and transketolase require a free hydroxyl group at C-1. 

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