Enzymes: depolymerase

Each fruit has specific quantities and ratio of pectin, hemicelluloses and cellulose. These polysaccharides are important concerning enzymes activities required to produce juices and concentrates. Moreover, even if molecular weight and methylation degree of the pectin are specific for each fruit, during the fruit maturation, endogenous pectinases depolymerases and esterase are changing the pectin characteristics This broad variability of raw material makes difficult the standardisation of fruits processing. 

R. Verger, in Hydrolases et Depolymerases. Enzymes d Interet Industriel Gauthier-Villars, Paris, 1985, pp. 313-329. 

Synthesis of PHAs in plants can not only be used directly in biotechnology for the creation of novel crop plants, but can also be a utilized as a unique novel tool in the basic studies of plant biochemistry. PHA synthesized in plants acts as a terminal carbon sink, since plants do not have enzymes, such as PHA depolymerases [68], required for degradation of the polymer. The quantity and composition of PHA can thus be used to monitor the quantity and quality of the carbon flux to different pathways. 

Polyesters, such as microbially produced poly[(P)-3-hydroxybutyric acid] [poly(3HB)], other poly[(P)-hydroxyalkanoic acids] [poly(HA)] and related biosynthetic or chemosynthetic polyesters are a class of polymers that have potential applications as thermoplastic elastomers. In contrast to poly(ethylene) and similar polymers with saturated, non-functionalized carbon backbones, poly(HA) can be biodegraded to water, methane, and/or carbon dioxide. This review provides an overview of the microbiology, biochemistry and molecular biology of poly(HA) biodegradation. In particular, the properties of extracellular and intracellular poly(HA) hydrolyzing enzymes [poly(HA) depolymerases] are described. 

Most poly(HA) depolymerases are inhibited by reducing agents, e.g., dithio-erythritol (DTT), which indicates the presence of essential disulfide bonds, and by serine hydrolase inhibitors such as diisopropyl-fluoryl phosphate (DFP) or acylsulfonyl derivates. The latter compounds covalently bind to the active site serine of serine hydrolases and irreversibly inhibit enzyme activity [48]. 

Poly(HASCL) depolymerases are able to bind to poly(3HB)-granules. This ability is specific because poly(3HB) depolymerases do not bind to chitin or to (crystalline) cellulose [56,57]. The poly(3HB)-binding ability is lost in truncated proteins which lack the C-terminal domain of about 60 amino acids, and these modified enzymes do not hydrolyze poly(3HB). However, the catalytic domain is unaffected since the activity with water-soluble oligomers of 3-hy-droxybutyrate or with artificial water-soluble substrates such as p-nitrophenyl-esters is unaffected [55, 56, 58, 59]. Obviously, the C-terminal domain of poly(3HB) depolymerases is responsible and sufficient for poly(3HB)-binding [poly(3HB)-binding domain]. These results are in agreement ... 

The poly(3HO) depolymerase differs from poly(HASCL) depolymerases in several of its biochemical properties it is specific for poly(HAMCL) and for artificial esters such as p-nitrophenylacyl esters with six or more carbon atoms in the fatty acid moiety. Poly(3HB) and other poly(HASCL) are not hydrolyzed. The enzyme is not inhibited by dithioerythritol or by EDTA and therefore apparently does not contain essential disulfide bonds. It is also not dependent on Ca2+ or other divalent cations. 

Although there is evidence that all poly(HA) depolymerases cleave the polyesters by the same mechanism (catalytic triad), the poly(3HO) depolymerase differs considerably from poly(HASCL) depolymerases in terms of primary sequence and polymer-binding. This might be due to different approaches of these enzymes to get access to the polymers reflecting the distinctive physicochemical properties of poly(HASCL) and poly(HMCLA) rather than coevolution. 

Hydrolysis of end-labeled 3-hydroxybutyrate oligomers by purified A. faecalis T poly(3HB) depolymerase showed that the enzyme mainly cleaved the second and third ester linkage from the hydroxyl terminus [69]. However, since the enzyme also hydrolyzes cyclic oligomers, the A. faecalis depolymerase has endo-hydrolase activity in addition to exo-hydrolase activity [18, 70]. Results of... 

The first products of enzymatic hydrolysis of poly(3HB) by purified poly(3HB) depolymerases are a mixture of monomeric and/or oligomeric 3-hydroxybuty-rate esters. Some enzymes are able to hydrolyze oligomers and dimers to monomeric 3-hydroxybutyrate after prolonged time of hydrolysis in the presence of an excess of the appropriate depolymerase. These poly(3HB) depolymerases have high endogenous dimer-hydrolase activities (e.g., the poly(3HB) depolymerases of Comamonas strains, P. stutzeri, S. exfoliatus, and the depolymerases... 

The i-poly(3HB) depolymerase of R. rubrum is the only i-poly(3HB) depolymerase that has been purified [174]. The enzyme consists of one polypeptide of 30-32 kDa and has a pH and temperature optimum of pH 9 and 55 °C, respectively. A specific activity of 4 mmol released 3-hydroxybutyrate/min x mg protein was determined (at 45 °C). The purified enzyme was inactive with denatured poly(3HB) and had no lipase-, protease-, or esterase activity with p-nitro-phenyl fatty acid esters (2-8 carbon atoms). Native poly(3HO) granules were not hydrolyzed by i-poly(3HB) depolymerase, indicating a high substrate specificity similar to extracellular poly(3HB) depolymerases. Recently, the DNA sequence of the i-poly(3HB) depolymerase of R. eutropha was published (AB07612). Surprisingly, the DNA-deduced amino acid sequence (47.3 kDa) did not contain a lipase box fingerprint. A more detailed investigation of the structure and function of bacterial i-poly(HA) depolymerases will be necessary in future. 

In addition to the aforementioned quantitative methods, the activity of pectic depolymerases is often identified by the cup-plate method.113 Cups are cut out from solidified agar containing the substrate, and are filled with the enzyme solution. After elapse of a cer-... 

This principle has been utilized to assess intrinsic subsite binding energies for enzymes that have substrate binding subsites and exosites topologically distant from the reactive site (e.g., polysaccharide depolymerases and pro-teinases). 

This enzyme [EC 3.6.1.10] (also known as polyphosphate depolymerase, metaphosphatase, and polyphosphatase) catalyzes the hydrolysis of polyphosphate to yield oligo-phosphate products containing four or five phosphate residues. 

This enzyme [EC 3.2.1.15], also known as pectin depolymerase and pectinase, catalyzes the random hydrolysis of l,4-a-D-galactosiduronic linkages in pectate and other galacturonans. 

Although some depolymerases act processively in cleaving their polymeric substrates, others act by what can be described as multiple attack which results in nonselective scission or random scission. The analysis of cleavage products during the course of enzyme-catalyzed depolymerization can provide important clues about the nature of the reaction. With random scission, the rate of bond scission must be proportional to the total number of unbroken bonds present in the solution. Thomas measured the rate of base addition in a pH-Stat (a device with an automated feedback servomotor that expels ti-trant from a syringe to maintain pH) to follow the kinetics of DNA bond scission by DNase. The number of bonds cleaved was linear with time, and this was indicative of random scission. In other cases, one may apply the template challenge method to assess the processivity of nucleic acid polymerases. See Processivity... 

The macroscopic velocity is the sum of all microscopic velocities of an enzyme system which acts on a polymer at several different positions, thus yielding a number of different products from the same substrate. One good example is polysaccharide depolymerase ... 

The properties and action patterns of glycosidases and polysaccharide depolymerases have been reviewed,2 5 as also have the enzymes involved in biosynthetic pathways.6,7 An understanding of biosynthesis can provide insights into the structures of polysaccharides. Reviews concerning particular polysaccharides have appeared, and references to these will be given in relevant sections. 

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