New Trends in Polymers - Progress in Organic and Physical Chemistry: Structures and Mechanisms - page 191

enzymatic degradation of PHB are affected by many factors as monomer composition,
molecular weight and degree of crystallinity [39].
At the next step it is necessary to observe enzymatic degradation of PHB under
the conditions that modeled the animal tissues and body À uids containing nonspeci¿ c
esterases. In vitro degradation of PHB ¿ lms in the presence of various lipases as non-
speci¿ c esterases was carried out in buffered solutions containing lipases [41, 42], in
digestive juices (for example, pancreatin) [11], biological media (serum, blood etc.)
[18] and crude tissue extracts containing a mixture of enzymes [19] to examine the
mechanism of nonspeci¿ c enzymatic degradation process. It was noted that a Ser. His.
Asp triad constitutes the active center of the catalytic domain of both PHB depolymer-
ase [43] and lipases [44]. The serine is part of the pentapeptide Gly X1-Ser-X2-Gly,
which has been located in all known PHB depolymerases as well as in lipases, ester-
ases and serine proteases [43].
On the one hand, it was shown that PHB was not degraded for 100 days with a
quantity of lipases isolated from different bacteria and fungi [41, 42]. On the other
hand, the progressive PHB degradation by lipases was shown [24, 25, 34, 35]. The
PHB enzymatic biodegradation was studied also in biological media: it was shown
that with pancreatin addition no additional mass loss of PHB was observed in compar-
ison with simple hydrolysis [11], the PHB degradation process in serum and blood was
demonstrated to be similar to hydrolysis process in buffered solution [24, 25], whereas
progressive mass loss of PHB sutures was observed in serum and blood: 16 and 25%,
respectively, after 180 days incubation [18], crude extracts from liver, muscle, kidney,
heart, and brain showed the activity to degrade the PHB: from 2 to 18% mass loss of
PHB microspheres after 17 hr incubation at pH 7.5 and 9.5 [19]. The degradation rate
in solution with pancreatin addition, obtained from the decrease in M w of pure PHB,
was accelerated about threefold: 34% decrease in M w after incubation for 84 days in
pancreatin (10 mg/ml in Sorensen buffer) versus . 11% decrease in Mw after incubation
in phosphate buffer [11].The same data was obtained for PHB biodegradation in buff-
ered solutions with porcine lipase addition: 72% decrease in M w of PHB (450 kDa)
after incubation for 84 days with lipase (20 U/mg, 10 mg/ml in Tris buffer) versus .
39% decrease in Mw after incubation in phosphate buffer [24, 25]. This observation
is in contrast with enzymatic degradation by PHB depolymerases which was reported
to proceed on the surface of the polymer ¿ lm with an almost unchanged molecular
weight [20, 21]. It has been proposed that for depolymerases the relative size of the en-
zyme compared with the void space in solvent cast ¿ lms is the limiting factor for diffu-
sion into the polymer matrix [Jesudason J. J. et al., 1993] whereas lipases can penetrate
into the polymer matrix through pores in PHB ¿ lm [34, 35]. It was shown that lipase
(0.1 g/l in buffer) treatment for 24 hr caused signi¿ cant morphological change in PHB
¿ lm surface: transferring from native PHB ¿ lm with many pores ranging from 1 to 5
—m in size into a pore free surface without producing a quantity of hydroxyl groups on
the ¿ lm surface. It was supposed that the pores had a fairly large surface exposed to
lipase, thus it was degraded more easily (Figure 2) [34, 35]. It indicates also that lipase
can partially penetrate into pores of PHB ¿ lm but the enzymatic degradation proceeds
mainly on the surface of the coarse polymer ¿ lm achievable for lipase. Two additional
effects reported for depolymerases could be of importance. It was concluded that seg-