Intracellular Degradation of PHA

The major role of PHAs is of a beneficial reserve material, the ability to store large quantities of reduced carbon without significant effects on the osmotic pressure of the cell. As discussed earlier, the proteins attached to PHA granules include PHA polymerase/synthase, PHA depolymerase, structural proteins known as phasins and some other proteins with unknown functions. PHA depolymerases present inside the cells are known as intracellular PHA depolymerases (i-PHA depolymerases). i-PHA depolymerase is an enzyme responsible for degradation/mobilization/hydrolysis of PHA for the survival of the cell in absence of an exogenous carbon source.There are many studies on intracellular mobilization of PHB in PHB-accumulating organisms. 

1 Diversity of Microbial Strains Studied for Intracellular PHA Degradation 

Intracellular degradation of PHA by depolymerases has not been studied in as much depth as compared to extracellular PHA degradation. C. necator, an organism able to accumulate PHB, was the organism of research to study the intracellular mobilization. This is mediated by a cyclic metabolic route from 

Intracellular PHB Depolymerase of Cupriavidus necator. Note that C. necator was previously known as Ralstonia eutropha, Wautersia eutropha, Alcaligenes eutropha and Hydrogenomonas eutropha. iPHB depolymerases of PHAscl-accumulating bacteria were not described before the year 2000 but since then not less than seven putative iPHB depolymerases and two 3-hydroxybutyrate oligomer hydrolases have been postulated for C. 

Intracellular PHB Depolymerase of Rhodospirillum rubrum. R. rubrum possesses a putative intracellular PHB depolymerase system consisting of a soluble PHB depolymerase, a heat-stable inactivator and a 3-hydroxybutyrate dimer hydrolase. It consists of one polypeptide of around 35 kDa and has a pH optima of 9 and temperature optima of 55 °C. The purified enzyme was inactive with dPHB. It does not show lipase, protease or esterase activity with p-nitrophenyl fatty acid esters. It is highly substrate specific.  

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Intracellular degradation of PHAs in the PHA-producing organisms is a physiological process, and this activity can potentially influence the quality and yield of PHAs to be isolated... 

Biodegradation of PHAs can thus be viewed as two major aspects (1) extracellular degradation of PHA and (2) intracellular degradation of PHAs. 

Intracellular degradation occurs via hydrolytic cleavage [7] and 3-ketothiolase is the enzyme that plays a key role in the production and degradation of PHA. The presence of a limited carbon source in the medium stimulates the process of degradation. In these conditions, the level of acetyl-coenzyme A (CoA) increases and prevents the inhibition of 3-ketothiolase by acetoacetyl-CoA. This allows the release of acetyl-CoA from P(3HB) and inhibits the condensation of acetyl-CoA to acetoacetyl-CoA [8]. PhaZ enzymes are secreted in order to break down the polymer into hydroxyacids which are then ntilised by the microorganism as a carbon source for growth [9]. Once... 

Abstract Polyhydroxyalkanoates (PHAs) are energy- and intracellular carbon-storage compounds that can be mobilized and used when carbon is a limiting resource. Intracellular accumulation of PHA enhances the survival of several bacterial species under environmental stress conditions imposed in water and soil, such as UV irradiation, salinity, thermal and oxidative sttess, desiccation, and osmotic shock. The ability to endure these sttesses is linked to a cascade of events concomitant with PHA degradation and the expression of genes involved in protection against... 

Biodegradability is one of the most attractive properties of PHAs. Degradation of PHAs occurs extraceUularly as well as intracellularly. 

Degradation of PHA is important for two practical reasons it affects the yield and quality of PHA during commercial production, and it affects the disposal of consumer goods manufactured from PHA. Degradation of PHA can occur as a nonbiological process or as the result of extracellular and intracellular activities of PHA-degrading enzymes. This topic has been reviewed by Dawes and Senior (1973), Anderson and Dawes (1990), Hocking and Marchessault (1994), Steinbuchel (1996), and Jendrossek et al. (1996). 

The key concept consists of PHA biosynthesis and subsequent depolymerization of produced PHA in cells therefore, it canbe called an in vivo PHA depolymerization process (15). RHAs produced by the depolymerization of PHAs can be excreted into the medium when the further intracellular metabolism of RHAs is inhibited. Because all the PHA-producing bacteria also possess PHA degradation pathways, in vivo depolymerization of PHA can be achieved by natural PHA-producing bacteria when the proper conditions are met. 

Limitation of the carbon source during the production phase can lead to the degradation of the intracellular polymer due to PHA... 

Due to the differences between the physical structures of the intracellular native granules and extracellular denatured PHA, intracellular PhaZ is unable to hydrolyse extracellular PHA, and extracellular PhaZ cannot hydrolyse intracellular PHA [14]. The enzymatic degradation of P(3HB) is known as a heterogeneous reaction because the polyhydroxybutyrate (PHB) depolymerase is water-soluble, whereas the P(3HB) polymer is water-insoluble. Therefore, the enzymatic degradation of P(3HB) involves two steps, namely adsorption and hydrolysis. During adsorption, the enzyme is attached to the surface of P(3HB) via the binding domain of the polymer. This is followed by hydrolysis of the polymer chain at the active site of the enzyme [5]. 

The PHA degradation pathway as described in most bacteria studied begins with the depolymerization of PHA to D-3-hydroxybutyrate monomers by PHA depolymerase (encoded by phaZ). Extracellular and intracellular PHA degradation have been described (Jendrossek and Handrick 2002) for utilization of PHAs present in the environment or accumulated in PHA granules, respectively (Tanio et al. 1982 Saegusa et al. 2001 Jendrossek and Handrick 2002 Pbtter and Steinbiichel 2005). 

PHA is accumulated intracellularly in Gram-negative bacterial strains and normally its recovery after the fermentation include several steps - briefly these are (i) the separation of cells from the fermentation broth by centrifugation (ii) after that, the bacterial cells are pre-treated by heat, freeze dried, or salted, before extraction to avoid polymer degradation (iii) the PHA is therefore extracted, normally by using chlorinated solvents or other methods such as enzymatic digestion or mechanical cell disruption and (iv) PHA purification. The process of PHA recoveiy is shown in Figure 2.9. 

Intracellular degradation In this mechanism, PHAs are hydrolysed by intraceUular depolymerases also known as i-depolymerases. These enzymes are secreted by the PHA-accumulating organism itself. An important factor that differentiates the extracellular and intracellular degradation is the physical form of PHA. PHAs exist as granules... 

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