New Trends in Polymers - Progress in Organic and Physical Chemistry: Structures and Mechanisms - page 190

16.2.2 Enzymatic Degradation of PHB in vitro
The examination of enzymatic degradation of PHB in vitro is having the following im-
portant step for understanding of PHB operation in animal tissues and in environment.
The most studies observed degradation of PHB by depolymerases of its own bacterial
producers. The degradation of PHB in vitro by depolymerase was thoroughly exam-
ined and mechanism of enzymatic PHB degradation was perfectly clarified by Doi Y.
[20, 21]. At these early works it was shown that 68-85% and 58% mass loss of PHB
(Mw = 650-768 and 22 kDa, respectively) films (50-65 —m thick) occurred for 20 hr
under incubation at 37°C in phosphate solution (pH = 7.4) with depolymerase (1.5-3
—g/ml) isolated from A. faecalis . The rate (k e ) of enzymatic degradation of films from
PHB (M n = 768 and 22 kDa) was 0.17 and 0.15 mg/h, respectively. The thickness of
polymer films dropped from 65 to 22 —m (32% of initial thickness) during incubation.
The SEM examination showed that the surface of the PHB film after enzymatic degra-
dation was apparently blemished by the action of PHB depolymerase, while no change
was observed inside the film. Moreover, the molecular weight of PHB remains almost
unchanged after enzymatic hydrolysis: the M n of PHB decreased from 768 to 669 kDa
or unchanged (22 kDa) [20, 21].
The extensive literature data on enzymatic degradation of PHB by speci¿ c PHB
depolymerases were collected in detail in review of Sudesh K., Abe H., and Doi Y.
[39]. We would like to summarize some of the most important data. But at ¿ rst it is
necessary to note that PHB depolymerase is very speci¿ c enzyme and the hydrolysis
of polymer by depolymerase is a unique process. But in animal tissues and even in
environment the enzymatic degradation of PHB is occurred mainly by nonspeci¿ c
esterases [19, 40]. Thus, in the frameworks of this review, it is necessary to observe
the fundamental mechanisms of PHB enzymatic degradation.
The rate of enzymatic erosion of PHB by depolymerase is strongly dependent on
the concentration of the enzyme. The enzymatic degradation of solid PHB polymer is
heterogeneous reaction involving two steps, namely, adsorption and hydrolysis. The
¿ rst step is adsorption of the enzyme onto the surface of the PHB material by the bind-
ing domain of the PHB depolymerase, and the second step is hydrolysis of polyester
chains by the active site of the enzyme. The rate of enzymatic erosion for chemosyn-
thetic PHB samples containing both monomeric units of (R)- and (S)-3-hydrohybu-
tyrate is strongly dependent on both the stereo composition and on the tacticity of the
sample as well as on substrate speci¿ city of PHB depolymerase. The water soluble
product of random hydrolysis of PHB by enzyme shows a mixture of monomers and
oligomers of (R)-3-hydrohybutirate. The rate of enzymatic hydrolysis for melt-crys-
tallized PHB ¿ lms by PHB depolymerase decreased with an increase in the crystal-
linity of the PHB ¿ lm, while the rate of enzymatic degradation for PHB chains in an
amorphous state was approximately twenty times higher than the rate for PHB chains
in a crystalline state. It was suggested that the PHB depolymerase predominantly hy-
drolyzes polymer chains in the amorphous phase and then, subsequently, erodes the
crystalline phase. The surface of the PHB ¿ lm after enzymatic degradation was appar-
ently blemished by the action of PHB depolymerase, while no change was observed
inside the ¿ lm. Thus, depolymerase hydrolyses of the polyester chains in the surface
layer of the ¿ lm and polymer erosion proceeds in surface layers, while dissolution, the