Polymerization, enzyme

Production of all naturally occurring polymers in vivo is catalyzed by enzymes. Polymerizations catalyzed by an enzyme ( enzymatic polymerizations ) have received much attention as new methodology [6-11], since in recent years structural variation of synthetic targets on polymers has begun to develop highly selective polymerizations for the increasing demands in the production of various functional polymers in material science. So far, in vitro syntheses of not only biopolymers but also non-natural synthetic polymers through enzymatic catalysis have been achieved [6-11]. 

Fig. 12. Example of a proteolytic enzyme-polymeric substrate interaction. For explanation of symbols see text...

Because of the very high molar masses of enzymically polymerized lignin sulfonates, Sephacryl S-300 is used as the gel matrix. The fractionation range is 10 000 to 1 000 000 dalton. Even in this case proteins of known molar mass can be used as calibration standards (Figs. 7 and 8). 

Figure 7. Fractionation of enzymically polymerized lignin sulfonates through Sephacryl S-300. Eluent 0.5M NaCl, 0.1M Tris-HCl (pH 8).

Molded Dry Chemistry. In general, most enzymes are very fragile and sensitive to pH. solvent, and elevated lemperaiurts. The catalytic activity of most enzymes i> reduced dramatically ils the temperature is increased, Typi cal properties of diagnostic enzymes are given in Table 1. t he presence of ionic salts and other chemicals can considerably influence enzyme stability. To keep or sustain enzymatic activity, the redox centers must remain intact. The bulk of the enzyme, polymeric in composition, is an insulaior. thus. altering ii does not reduce the enzyme s catalytic activity, li... 

Another immobilization method was described by Maeda and coworkers [344], They developed a facile and inexpensive preparation method for the formation of an enzyme-polymeric membrane on the inner wall of the microchannel (PTFE) through cross-linking polymerization in a laminar flow. With this approach, a-chymotrypsin was immobilized successfully. The activity of the immobilized enzyme was tested using N-glutaryl-L-phenylalanine p-nitroanilide as substrate, and the reaction products were analyzed offline by HPLC. There was no significant difference in the hydrolysis efficiency compared to solution-phase batchwise reactions using the same enzyme/substrate molar ratio (Scheme 4.87). 

A problem that still has to be solved is the deficiency of water resistance. The sulfonate groups are so polar that the polymerizate is still water-soluble. As was mentioned before, water-soluble fractions of organosolv lignins became insoluble in water after enzymic polymerization (11) Therefore, we hoped that an addition of phenol-rich organosolv lignin would improve the water resistance However, as can be seen in the last line of Table I, this is not the case. 

Unfolding of globular proteins and subunits. Data on frozen storage of HMM, actin and sarcoplasmic enzymes have led us to propose that denaturation involves unfolding of the protein chain based on a decrease in enzymatic activity (myosin, HMM, and sarcoplasmic enzymes), polymerizing ability (actin) and filament forming properties (myosin) (82,99,113-116,122). 

Kobayashi S, Shimada J, Kashiwa K, Shoda S. Enzymic polymerization of a-D-maltosyl fluoride using a-amylase as catalyst a new approach for synthesis of maltooligosaccharides. Macromolecules 1992 25 3237-3241. 

Figure 2 Mode of action of the prototypical lantibiotic nisin. (a) The peptidoglycan precursor lipid II is composed of an N-acetylglucosamine-p-1,4-N-acetylmuramic acid disaccharide (GIcNAc-MurNAc) that is attached to a membrane anchor of 11 isoprene units via a pyrophosphate moiety. A pentapeptide is linked to the muramic acid. Transglycosylase and transpeptidase enzymes polymerize multiple lipid II molecules and crosslink their pentapeptide groups, respectively, to generate the peptidoglycan. (b) The NMR solution structure of the 1 1 complex of nisin and a lipid II derivative in DMSO (6). (c) The amino-terminus of nisin binds the pyrophosphate of lipid II, whereas the carboxy-terminus inserts into the bacterial membrane. Four lipid II and eight nisin molecules compose a stable pore, although the arrangement of the molecules within each pore is unknown (5).

As previously pointed out, considerable data have been accumulated from experiments in vivo indicating that D-xylose in plants originates from D-glucose by a series of known enzymic reactions. It was assumed that, after this pentose is formed, it is enzymically polymerized to D-xylan. 

Abequose residues in S. typhimurium occur as branches of O side-chain, and are not found in its main chain (see Fig. 1). Indeed, the enzymic polymerization readily occurs in the absence of CDP-abequose, hence, it was thought possible that abequose residues are added after the synthesis of the main chain of O side-chain polysaccharide is completed. However, this hypothesis appears unlikely in view of the following results. (I) Under certain experimental conditions, the incorporation of D-mannose, L-rhamnose, and D-galactose is strongly stimulated by the simultaneous presence of CDP-abe-quose. (2) By skilful manipulation of the conditions of reaction. 

We focus on the enzymic polymerization of nucleotides in a reversed micellar system utilizing the liquid/solid interface. 

IV. STUDY OF ENZYMIC POLYMERIZATION OF NUCLEOTIDES IN A REVERSED MICELLAR SYSTEM AS A DEVELOPMENT OF BIOPOLYMER SYNTHESIS UTILIZING THE LIQUID-SOLID INTERFACE... 

Oparin and coworkers [125,126] have studied the enzymic polymerization of ADP by polynucleotide phosphorylase (PNPase) and Mg ions in coacervates in an attempt to construct primitive forms of precellular structures. Walde et al. [127] have investigated this enzymic ADP polymerization in AOT reversed micellar solutions instead of coacervates. The PNPase-catalyzed synthesis of poly(A) (polyadenylic acid) in the AOT reversed micelles was carried out by mixing two reversed micellar solutions, one containing ADP and the other containing the enzyme. 

This enzymic polymerization needed a high concentration of ions (10 mM in water pools). The reaction usually proceeded at a low concentration of (1.56 mM)... 

Another subject is concerned with biopolymer synthesis utilizing the liquid/solid interface in a reversed micellar system. The enzymic polymerization of ADP in AOT reversed micellar solution containing a Mg ions resulted in the precipitation of poly(A) together with the PNPase. Further polymerization could proceed by the enzyme in the precipitate by feeding ADP through the dynamic AOT monolayer on a glass surface. This is concluded to be a kind of solid polymerization in a reversed micellar solution. This process of polymerization provides a simple isolation of both the product and enzyme the maintenance of the enzyme activity for a long time and a novel solid polymerization on the oil/solid interface. This polymerization at the interfaces in the reversed micellar solution could be applied to other biopolymer syntheses. 

Protein-polymeric membrane in a microchannel is prepared by using a concentric laminar flow (Fig. 43) [267]. Crosslinking condensation of a crosslinked enzyme aggregate (CLEA) [268] with aldehyde groups, which react with amino groups of the enzyme, in a concentric laminar flow results in the formation of a cylindrical enzyme-polymerized membrane on the inner wall of the microtube. The use of this technology for membrane formation in a microchannel can be extended to a broad range of functional proteins. 

We mentioned that enzymes polymerize DNA and RNA according to the base sequence information. These enzymes are themselves proteins, and DNA and RNA make proteins. It means that the genetic material and its product (the protein) are like a chicken and an egg. This is a poetically wonderful symbiosis ... 

The key enzyme for PHA biosynthesis is the PHA synthase. This enzyme polymerizes (/ )-3-hydroxyacyl-CoA thioester monomers into polyester with the release of Co A. 

In Figure 5, a plot of percent monomer conversion as a function of time is presented. The monomer conversion increased steadily with time. For example, after 1.5 h the conversion was 19% and reached 74% after 9 h. The monomer conversion kept increasing beyond 50% because the enzyme polymerized both enantiomers in the racemic monomer mixture although the (. -enantiomer was polymerized in preference to the (R)-enantiomer. 

Miyazaki and Maeda accomplished immobilization of acylase by the formation of an enzyme-polymeric membrane on the inner wall of the microreactor [172]. The same group used a microreactor system connected to a microextractor, which allowed liquid-liquid microextraction in a flow stream, as shown in Scheme 7.44. Using this microreaction system, optical resolution of racemic acetylphenylalanine was achieved to give D-acelyl phenylalanine with high optical purity [173]. 

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