Modification of Polyhydroxyalkanoates (PHAs) - Polyhydroxyalkanoate (PHA) Based Blends, Composites and Nanocomposites - page 166

monomer release. 128 A drop in the solution's pH was due to secretion of PHA
monomers by the bacteria. 12

d n 2 r 4 n g | 1

7.4.2 In Vitro Enzymatic Depolymerization
7.4.2.1 Extracellular PHA Depolymerase
Many PHA-degrading microorganisms have been isolated and their ability to
secrete extracellular PHA depolymerase could be exploited. The degraded
monomer can be extracted and used as a macro-initiator for further modi-
fication. 129 The extracellular PHA depolymerases for mcl-PHA have been
identified mainly from Gram-negative bacteria, predominantly Pseudomonas
species; Pseudomonas fluorescens GK13, actinomycete species; Streptomyces
roseplus SL3, and scl-PHA depolymerase from Alcaligenes faecalis T1. 130-132
This enzyme belongs to the family of serine hydrolases with lipase con-
sensus sequence Gly-X-Ser-X-Gly and it is strongly hydrophobic. 133 The
catalytic triad for PHA depolymerase consists of a serine residue that acts as
a nucleophile with aspartate (or glutamate) and histidine to stabilize it
(serine-histidine-aspartate). 134,135
Shirakura et al. 131 observed that PHB depolymerase acts as an endo-type
hydrolase as the enzyme only cleaves at the second ester linkage from the
hydroxy terminus of the trimer, tetramer, pentamer and higher oligomers as
shown in Figure 7.12. At the same time, the PHA depolymerase prefers PHA
with alkyl side chains rather than linear PHA. 135 According to Mukai et al., 136
PHA depolymerase enzyme is very selective towards chiral monomers, es-
pecially alkyl side chains such as 3HB rather than 4HB. The rate of enzymatic
hydrolysis is primarily dependent on the composition and length of the
PHA side chain. Longer side chains provide steric hindrance for the enzyme
to be adsorbed effectively on the polymer backbone chain as shown in
Figure 7.13. 137

.

H ∗

HO HB HB HB HB HB HB HB

H ∗

(a)

HO HB COOH

H ∗

HO HB HB COOH

(b)

H ∗

HO HB HB HB COOH

(c)

H ∗

(d)

HO HB HB HB HB COOH

Figure 7.12 Bond cleavage during initial action of poly(3-hydroxybutyrate) depoly-
merase on radioactive oligomers start from blue arrow. (a), (b), (c), (d)
are some products of hydrolysis with (a) degradation product not
undergoing further hydrolysis.