Multienzyme approach

At present, a few amino acids, however, single residue resolution with ETD/ECD (see Chapters 8 and 9) and/or multienzyme approaches (see Section 7.3 and Chapter 6) may be a reality soon [47, 48]... 

Table 2 Example of one-pot multienzyme approach for the s)mthesis of sialosides with versatile structural modification... 

A novel interesting approach is the use of multienzyme cocktails described by Scott et al. for the synthesis of precorrin-5159 starting from 8-amino levu-linic acid 158 (scheme 32).1871 For the process, a multienzyme cocktail of eight different enzymes including the ALA-dehydratase to form porphobilinogen as well as PBG deaminase and cosynthetase to give the tetracyclic uroporphyrinogen HI (10) was employed. 

A similar approach has been used to solve more complicated cases of two enzymes in one layer (Schulmeister and Scheller, 1985, p. Ill) and the multi-layer/multienzyme model (Schulmeister, 1987, p. 223). It is important to note that oxidases are one of the largest group of enzymes and therefore the improved sensors for substrates other than glucose can and have been developed according to this scheme. 

John Pedersen (Unizyme Laboratories) demonstrated how multienzyme systems can be used along with His affinity tags and subtractive IMAC to produce industrial enzymes in both packed or expanded bed adsorption systems. The efficiency of the approach was demonstrated with the produc-... 

In one type of study, the HPLC assay method has been applied to the question of assaying the activity of two enzymes that can use the same substrate but form different products. In another, the HPLC method was applied to the case of related two different enzymes that can use the same substrate and form the same product. It was also applied to the case a single enzyme that can use either of two substrates to form two different products. In a separate application, the so-called reconstitution approach, a multienzyme pathway is reconstructed by the addition of enzymes one after the other to form a multienzyme complex and to thereby reconstitute a naturally occurring multienzyme complex. And finally, this approach allows us to use the HPLC method to study an intact, naturally occurring multienzyme system. 

In a related approach, D,L-methionine can be efSdently deracemized to obtain the L-enantiomer using a multienzyme system consisting of D-amino acid oxidase, catalase, leucine dehydrogenase, and formate dehydrogenase. The a-keto acid 8 produced from the oxidation of the D-form is transformed into L-methionine 9 in the presence of ammonia, leucine dehydrogenase, and a stoichiometric amount of NADH. The NAD thus formed is recycled to NADH with ammonium formate and formate dehydrogenase [30] (Scheme 13.10). 

The linear approach described here is expandable to multienzyme electrodes as well as multilayer electrodes. At least for the stationary case, multilayer models of bienzyme electrodes may be easily treated, too. The whole system is readily adaptable to potentiometric electrodes (Carr and Bowers, 1980). It must be noted, however, that the superiority over purely numerical solution procedures decreases with increasing number of enzyme species and in the multilayer model. The advantage in calculation speed using the sum formulas described (e.g., in Section 2.5.2) amounts to about two orders of magnitude. With multilayer electrodes and formulas containing double and triple sums it is reduced to one order of magnitude. 

Such methods are likely to be more extensively used in the future to provide a highly focused combinatorial approach to generating molecular diversity. The multienzyme pathway responsible for converting simple linear unsaturated allylic alcohols to sterols, carotenoids, and terpenes is incompletely characterized, but offers excellent potential for genetic manipulation to provide directed bio-combinatorial chemistry (191,192). 

Most enzymes in biological cells function as complex enzyme systems. We have prepared artilicial cells that contain multienzyme systems with cofactor recycling." This approach can convert metabolic wastes such as urea and ammonia into essential amino acids such as leucine, isoleucine, and valine, which are required by the body." We have also prepared artificial cells containing hanoglo-bin with pseudoperoxidase activity and glucose oxidase to ranove bilirubin." " ... 

In an attempt to isolate a specific phosphatase for the carboxylase among multiple forms of the phosphatases in the cell, Krakower and Kim 61) purified acetyl-CoA carboxylase phosphatase by utilizing the multienzyme complex nature of acetyl-CoA carboxylase and its phosphatase. This approach assured the isolation of the specific phosphatase for the carboxylase from various other cytosolic phosphatases with broad substrate specificities 9,11,23,31,32,48,49,53,55,56,58,59, 63, 72, 73, 85, 114). 

Although Leloir-GTs are accepted as perfect candidates for the biocatalytic production of glycans their dependence on nucleotide sugars makes cost effective synthesis strategies more complex. One approach to overcome this drawback is the (re)generation of donor-substrates with multienzyme systems [24,... 

The total synthesis of sialosides by using the chemoenzymatic approach is as follows [74]. Sialic acid itself can be synthesized from ManNAc, mannose, or their derivatives by sialic acid aldolase enzyme through aldol condensation reaction. If ManNAc is chemically or enzymatically modified at C2, C4—C6 positions, sialic acid has structural modifications at C5, C7-C9 positions, respectively. The sialic acids are subsequently activated by a CMP-siahc acid synthetase to form a CMP-sialic acid, which is the donor used by sialyltransferases. Because CMP-sialic acid is tmstable, the CMP-Neu5Ac synthetase is valuable for the preparative enzymatic synthesis of sialosides. In the last steps, the CMP-sialic acid is transferred to galactose or GalNAc terminated glycosides by sialyltransferases to form structurally defined sialosides. Examples are that Chen and co-workers have recently developed a one-pot multienzyme system for the efficient synthesis of a-sialosides (Table 2) [12,76,79]. In this system, recombinant E. coli K-12 sialic acid aldolase catalyzed the synthesis of sialic acid precursors for... 

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