Poly-B-hydroxybutyrate (PHB): A Biodegradable Polymer of Microbial Origin - Natural Polymers, Biopolymers, Biomaterials, and Their Composites, Blends, and IPNs - page 52

PhB BiosyNthesis
In the case of normal metabolism and cell growth, organisms convert glucose into
two pyruvate molecules via the glycolytic pathway. Acetyl coenzyme A is synthe-
sized from this pyruvate, which then enters the citric acid cycle, releasing energy in
the form of ATP, and GTP as well as NADH which then enters the electron transport
chain and donates its electrons to O 2 ,where the energy released is trapped in the form
of ATP.
The PHB synthesis is nothing but the conversion of acetyl-CoA to PHB as a mech-
anism for storing carbon. The P(3HB) biosynthesis, is the three step biosynthesis path-
way, mainly consists of three enzymatic reactions catalyzed by three distinct enzymes.
The enzymes are β-ketothiolase, acetoacetyl-CoA reductase and PHB synthase.
enzymes involved
b -ketothiolase
It catalyzes the first step in P(3HB) synthesis. It is a member of the family of enzymes
of thiolytic cleavage of substrate into acyl-CoA and acetyl-CoA. The two acetyl CoA
molecules are combined to form acetoacetyl-CoA catalyzed by 3-ketothiolase (acetyl-
CoA acetyl transferase)
It is divided into two groups; first group of thiolases fall into the enzyme Class
EC.2.3.1.16 and the second group, belonging to enzyme Class EC.2.3.1.9. The first
group is involved mainly in the degradation of fatty acids and is located in the cyto-
plasm of prokaryotes and in the mitochondria. The second type is considered to take
part in P(3HB) biosynthesis (Madison and Huisman, 1999).
In Ralstonia eutropha, two β-ketothiolases, enzyme A and enzyme B, have been
discovered to take part in biosynthesis of PHB. Enzyme A is a homo-tetramer of 44-kDa
subunits and enzyme B, a homo-tetramer of 46 kDa subunits. (Madison and Huisman,
1999). Slater et al. (1998) have shown that enzyme B is the primary catalyzer for the
P(3HB-3HV) formation. The enzymatic mechanism of β-ketothiolase consists of bio-
logical condensation of two acetyl-CoA-moieties with formation of carbon-carbon bond.
Acetoacetyl-CoA Reductase
It catalyzes second step in the P(3HB) biosynthetic pathway by stereo-selective re-
duction of acetoacetyl-CoA formed by β-ketothiolase to 3-hydroxybutyryl-CoA. Two
types of reductases are found in organisms (Haywood et al., 1988a, 1988b). First type
is a NADH dependant reductase, EC.1.1.1.35, while the second type, EC.1.1.1.36, is
a NADPH dependant. The former is a tetramer with identical subunits of 30 kDa. The
latter is a homo-tetramer of 25 kDa subunits. (Schembri et al., 1994).
P(3HB) Synthase
The last reaction in the polymer formation is catalyzed by the enzyme PHB synthase
which links D(-)-β-hydroxybutyryl moiety to an existing polyester molecule by an es-
ter bond. This key enzyme determines the type of PHB synthesized. The PHB synthase
is soluble only as long as no PHB synthesis and accumulation occurs in an organism.
It, however, becomes granule associated under storage conditions (Haywood, 1989).