PHA depolymerase

Practically nothing is known about how PHA synthases and intracellular PHA depolymerases, which are both bound to the granules, interact and whether or... 

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. 

PHA depolymerases, 20 253, 256 pH adjusting agents, 12 61-62 Phage cloning, DNA isolation for,... 

The enzymatic hydrolysis of PHB by PHA depolymerase from Acidovo-rax sp. TP4 produced 3HB monomer as a major product, while mainly 3HB... 

Extracellular PHA depolymerases are ubiquitous in the environment (Tokiwa and Calabia 2004). The earliest discovery of PHA-degrading bacteria belongs to... 

Many extracellular PHA depolymerases have been pirtilied from elifferent microorganisms anel/or characterized. The purified PHA depolymerases... 

A nirmber of extracellular bacterial PHA depolymerase genes have been cloned and analyzed. 

Enz57matic hydrolysis experiments using linear and cyclic oligomers of 3HB show that the PHA depolymerases have endo-hydrolase activity in addition to exo-hydrolase activity. In addition, the results demonstrated that the... 

The enzymatic degradation of water-insoluble P(3HB) material by water-soluble PHA depolymerase is a heterogeneous reaction, involving two steps, namely, adsorption and hydrolysis the first step is adsorption of the enzyme on the surface of P(3HB) material by the binding domain of the enzyme, and the second step is hydrolysis of polymer chains by the active site of the enzyme. 

Kasu) et investigated the kinetits and mechanism of surface hydrolysis of P(3HB) with PHA depolymerase from Raktonia pickettii T1 at different reaction temperature and pH. The rate constant of enzymatic hydrolysis increased with a rise in temperature, while the adsorption equilibrium constant decreased. The activation energy of hydrolysis by the catalytic domain calculated from the results obtained was found to be 82kfmor. ... 

Figure 17 A schematic model of enzymatic hydrolysis of P(3HB) single crystals by PHA depolymerase consisting of binding and catalytic domains. Reproduced with permission from Iwata, T. Doi, Y. Kasuya, K. Inoue, Y. Macromolecules l, 30, 833. ° Copyright 1997 American Chemical Society.

The adsorption of PHA depolymerase to P(3HB) single crystals has been investigated using immunogold labeling techniques.It has been found that PHA depolymerase adsorbed homogeneously on the surfaces of single crystals without site specificity. PHA depolymerases are inactive toward rubbery amorphous polyesters such as native amorphous PHA... 

Since the PHA depolymerases have ndo-hydrolase activity, as mentioned above, the random copolymers with 3HB unit as a major constituent are degradable by PHA depolymerase even though enzymatically inactive monomeric units are introduced. The effects of chemical structure of second monomer units and copolymer compositions on the rate of enzymatic erosion have been examined through the enzymatic degradation of solution-cast films of random copolymers of 3HB with various HA units in the presence of PHA depolymerase. " The enzymatic degradation of solution-cast films of these PHA copolymers was performed in an aqueous solution of purified PHA depolymerase from R. pickettii T1 at 37 °C. The rate of enzymatic erosion of solution-cast PHA films increased markedly with an increase in the fraction of second monomer units up to 10-20 mol.% to reach a maximum value followed by a decrease in the erosion rate (Figure 18). The highest rates of enzymatic erosion were 5-10 times that of the P(3HB) homopolymer film. 

Figure 18 Relation between the rate of enzymatic hydrolysis by PHA depolymerase from Ralstonia pickettii and the fraction of second monomer unit for random copolymers of 3HB with different HA units, (a) P(3HB-co-3HHx) (o) P(3HB-co-3HP) and (a) P(3HB-co-4HB). Reprinted from Abe, H. Doi, Y. int. J. Bioi. Macromoi. 1999, 25,185. Copyright (1999), with permission from Elsevier.

Iwata and coworkers ° ° also investigated enzyme adsorption to copolymer single crystals, and reported that PHA depolymerase adsorbed randomly on the surfaces of single crystals. However, the concentrations of adsorbed enzjune on the surface of copolymer single crystals are lower than that for P(3HB) homopolymer single crystals. PHA depolymerase molecules cannot bind tighdy to the irregular surface of copolymer crystals since the copolymer chains with second monomer units have loose loop folds. 

Figure 19 A molecular surface representation of the PHA depolymerase from Penicillium funiculosum cemmQ on the mouth of the crevice. The positions of solvent-exposed hydrophobic residues (purple), as well as polar (green) and catalytic triad (cyan) residues, are indicated. A model of the 3HB trimer bound in the crevice is shown as a yellow stick model. Reproduced with permission from Hisano, T. Kasuya, K. Tezuka, Y. etal. The Crystal Structure of Polyhydroxybutyrate Depolymerase from Penicillium funiculosum Provides Insights into the Recognition and Degradation of Biopolyesters, J. Mol. Biol. 2006, 356, 993, Copyright (2006), with permission from Elsevier.

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