Enzymatic synthesis Escherichia coli enzyme

Enzyme preparations from liver or microbial sources were reported to show rather high substrate specificity [76] for the natural phosphorylated acceptor d-(18) but, at much reduced reaction rates, offer a rather broad substrate tolerance for polar, short-chain aldehydes [77-79]. Simple aliphatic or aromatic aldehydes are not converted. Therefore, the aldolase from Escherichia coli has been mutated for improved acceptance of nonphosphorylated and enantiomeric substrates toward facilitated enzymatic syntheses ofboth d- and t-sugars [80,81]. High stereoselectivity of the wild-type enzyme has been utilized in the preparation of compounds (23) / (24) and in a two-step enzymatic synthesis of (22), the N-terminal amino acid portion of nikkomycin antibiotics (Figure 10.12) [82]. 

Compound 25 (Fig. 18.9), a prodrug of 9-P-D-arabinofuranosyl guanine (26), was developed for the potential treatment of leukemia. Compound 24 is poorly soluble in water and its synthesis by conventional techniques is difficult. An enzymatic demethoxylation process was developed using adenosine deaminase (Mahmoudian et al., 1999, 2001). Compound 25 was enzymatically prepared from 6-methoxyguanine (27) and ara-uracil (28) using uridine phosphorylase and purine nucleotide phosphorylase. Each protein was cloned and overexpressed in independent Escherichia coli strains. Fermentation conditions were optimized for production of both enzymes and a co-immobilized enzyme preparation was used in the biotransformation process at 200 g/L substrate input. Enzyme was recovered at the end of the reaction by filtration and reused in several cycles. A more water soluble 5 -acetate ester of compound 26 was subsequently prepared by an enzymatic acylation process using immobilized Candida antarctica lipase in 1,4-dioxane (100 g/L substrate) with vinyl acetate as the acyl donor (Krenitsky et al., 1992). 

Three immobilized enzyme or microbial cell systems currently used industrially in synthesis of chiral amino acids plus one presently under development are described. L-amino acids are produced by enzymatic hydrolysis of DL-acylamino acid with aminoacylase immobilized by ionic binding to DEAE-Sephadex. Escherichia coli cells immobilized by K-carrageenan crosslinked with glutaraldehyde and hexamethylenediamine are used to convert fumaric acid and cimmonia to L-aspartic acid and Brevibacterium flavum cells similarly immobilized are used to hydrate fumaric acid to L-malic acid. The decarboxylation of L-aspcirtic acid by immobilized Pseudomonas dacunhae to L-alanine is currently under investigation. 

Such universal distribution and distinct function does not mean that the enzymes could be readily studied everywhere. Due to their cell cycle dependence described later and rather poor activity in cell-free extracts ribonucleotide reductases can only be purified and characterized under carefully chosen conditions. Therefore the organisms named as enzyme source in this chapter are not too numerous. They include some obvious, biochemically well-known candidates for the study of DNA precursors like Escherichia coli, yeast, or calf thymus, while other, more remote ones (e.g., Brevibacterium ammoniagenes or the alga, Euglena gracilis) have been added because of some peculiarity of their DNA synthesis. A systematic view of enzymatic deoxyribonucleotide formation is assembled in Table 4. 

Kurochkina VB, Nys PS (2002) Kinetic and thermodynamic approach to design of processes for enzymatic synthesis of betalactams. Biocatal Biotransform 20(1) 35-41 Lee SB, Ryu DDY (1982) Reaction kinetics and mechanism of penicillin amidase a comparative study of computer simulation. Enzyme Microb Technol 4 35-38 Lin WJ, Kuo BY, Chou CP (2001) A biochemical engineering approach for enhancing production of recombinant penicillin acylase in Escherichia coli. Bioproc Biosys Eng 24 239-247 Lindsay JP, Clark DS, Dordick JS (2004) Combinatorial formulation of biocatalyst preparation for increased activity in organic solvents salt activation of penidllin amidase. Biotechnol Bioeng 85(5) 553-560... 

Schiirmann M, Schiirmann M, Sprenger GA (2002) Fructose 6-phosphate aldolase and 1-deoxy-D-xylulose 5-phosphate synthase from Escherichia coli as tools in enzymatic synthesis of 1-deoxysugars. J Mol Catal B Enzym 19 247-252 Shelton CM, Toone EJ (1995) Differential dye-ligand chromatography as a general purification protocol for 2-keto-3-deoxy-6-phosphogluconate aldolases. Tetrahed Asymm 6 207-211 Silvestri MG, Desantis G, Mitchell M et al. (2003) Asymmetric aldol reactions using aldolases. Top Stereochem 23 267-342... 

E.S. and Kornberg, (1958) A. Enzymatic synthesis of deoxyribonucleic acid. I. Preparation of substrates and partial purification of an enzyme from Escherichia coli. / Bid. Chem., 233, 163-170. 

Uridine diphosphate glucose (UDP-Glc) serves as a glucosyl donor in many enzymatic glycosylation processes. A multiple enzyme, one-pot, biocatalytic system was developed for the synthesis of UDP-Glc from low cost raw materials maltodextrin and uridine triphosphate. Three enzymes needed for the synthesis of UDP-Glc (maltodextrin phosphorylase, glucose-l-phosphate thymidyly-transferase, and pyrophosphatase) were expressed in Escherichia coli and then immobilized individually on amino-functionalized magnetic nanoparticles. The conditions for biocatalysis were optimized and the immobilized multiple-enzyme biocatalyst could be easily recovered and reused up to five times in repeated syntheses of UDP-Glc. After a simple purification, approximately 630 mg of crystallized UDP-Glc was obtained from 1 L of reaction mixture, with a moderate yield of around 50% (UTP conversion) at very low cost. ... 

In this chapter, the focus is on in vitro enzyme catalysis for vinyl polymerization. To the best of our knowledge, prior to the work of Derango et al. (1992) there is a single short report showing the formation of low molecular weight vinyl polymers when studied in a suspension of Escherichia coli in the presence of methyl methacrylate [15,16]. Unhke polyaromatics, vinyl polymerization offers better control of polymer characteristics, as has been demonstrated with ternary systems (enzyme, oxidant, and initiator such as b-diketone). The number of different vinyl monomer chemistries investigated for susceptibility toward enzymatic polymerization (1-12) is fewer than reported aromatics, as is the extent of literature covering these types of syntheses. In addition, the discovery of multienzymatic approaches for the synthesis of antioxidant-functionalized vinyl polymers provides new impetus for the use of enzymatic methods related to vinyl polymers. 

Of the enzymes listed in Table 10.1, Upases are the woikhorses. The employment of Upases in non-aqueous media is an estabUshed art, with over 25 years of research serving as a foundation. Lipases are abundant and relatively inexpensive enzymes that require no co-factors and are easily immobilized. Lipases from several thermophiUc organisms have been isolated, cloned, and mass produced via recombinant DNA technology in common vectors such as Escherichia coli. Some of the examples in Table 10.1 are surfactants formed from enzymatic hydrolysis of oleochemical feedstocks, such as MAG formed from lipase-catalyzed hydrolysis of TAG, and lysophospholipids via hydrolysis by Upases or phosphoUpase A. Ui the foUowing sections some specific examples from the literature are given of enzyme-catalyzed synthesis of bio-based surfactants. Other examples not described, such as the oxidation of fatty alcohols to aldehydes (OrUch et al., 2000) and the covalent attachment of fatty alcohols and bio-based diethyl carbonate (Banno et al., 2007, 2010 Matsumura 2002 Lee et al., 2010) are covered in the references provided. 

Hausmann, R. Synthesis of an S-adenosylmethionine-cleaving enzyme in T3-infected Escherichia coli and its disturbance by coinfection with enzymatically incompetent bacteriophage. J. Virol. 1, 57-63 (1967). 

Taking into account that enzymatic methods basically mimic in vitro the natural pathways of oligosaccharide biosynthesis, a reasonable short-cut is the production of the oligosaccharide in a living recombinant organism e.g. Escherichia coli). Unfortunately this task is not simple to accomplish, because of the characteristics of the biosynthetic machinery involved in the synthesis of the desired products. Production in E. coli of active eukaryotic GTs in suitable amounts is not always possible or requires eonsiderable effort for reasons that are not well understood. This problem can be overcome by use of homologous enzymes from prokaryotic organisms. 

Larsson, a. Enzymatic synthesis of deoxyribonucleotides. III. Reduction of purine ribonucleotides with an enzyme system from Escherichia coli B. J. Biol. Chem. 238, 3414 (1963). 

Ribonucleotide reductase, an allosteric enzyme from Escherichia coli, converts ribonucleotide diphosphates to the corresponding deoxyribo-nucleotides, and therefore provides the necessary precursors for DNA synthesis (Reichard, 1967). During purification ribonucleotide reductase separates into two nonidentical subunits, proteins B1 and B2, each enzymatically inactive (Brown et a/., 1969a). The active enzyme is formed in the presence of magnesium ions and consists of a 1 1 complex of the two subunits (Thelander, 1973). Proton subunit B2 (mol. wt. 78,000), which participates in the formation of the catalytic site (Thelander et al, 1976), contains nonheme iron (Brown et al, 1969b) and an ESR-detectable organic... 

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