Introduction

• Cadaverine can be found in polymers, such as polyamides and

chelating agents
• polyamines -bio-based alternatives to conventional petroleum-based
polyamines.
• synthesized by the decarboxylation of lysine
• Low pH , E. coli produces cadaverine to increase the extracellular pH,
which helps the microorganism to survive
• lysine decarboxylase is encoded by cadA

• Enzyme immobilization reduces the cost of enzymes and increases enzyme

stability
• in situ immobilization of enzyme is better option in terms of cost and
reusability

• In situ enzyme immobilization to polyhydroxyalkanoate (PHA)biopolymers

• This method eliminates the need for additional enzyme purification and
immobilization processes. In situ immobilization increased enzyme stability
• PhaP1 from Ralstonia eutropha was fused to CadA to produce fusion protein.

• The fusion protein is immobilised on intracellular P(3HB) granules in E.coli which was produced by engineering a
heterologous metabolic pathway of R.eutropha into E. coli.

Lysine Decarboxylase

Phasin (PhaP 1)
 Fusion protein production

phaP1 gene is PCR colony using

Protein
inserted into Transformed in to T7 primers and
Protein expression quantification
plasmid containing DH5α competent transform into
using SDS page using Bradford
cadA gene at the C- cell BL21 (DE3) for
assay
terminal expression
 Lysine decarboxylase activity

specific activity of lysine

decarboxylase was Incubate at 37◦C for 5 min The specific activity was
measured by calculating and reaction is stopped at calculated based on the
0.2 mM PLP was added to
the amount of cadaverine 95◦C for 5 min. The protein concentration and
start the
produced in 100 mM mixture is diluted 1:10 for the amount of cadaverine
lysine–HCl and 100 mM analysis. produced.
(pH 6) acetate buffer
Enzyme purification and characterization
cadA-Phap1 –
Free cadA
PH3 complex

Supernatant after
Pellet after
sonication is
sonication is
purified with NI-
washed with PBS
NTA beads

Tested for pH stability ,

thermal stability and
enzyme reusablity
• CadA (82.1 kDa) expression level was
reduced after the introduction of P3HB
• co-expression of the P(3HB) synthesis
operon affects the production of lysine
decarboxylase
Specific Activity of Lysine Carboxylase enzyme
• crude cell extract of all strains, with the
exhibited lysine decarboxylase activity.
• Introduction of the P(3HB) synthesis
operon reduced the activity by ∼38%.
• soluble fraction of all strains exhibited
specific lysine decarboxylase activity
• insoluble fraction of only the strains
expressing phasin-fused CadA exhibited
activity
• highest specific lysine decarboxylase
activity is due to the presence of purified
phasin-fused CadA by associating with
P(3HB) granules
• immobilization efficiency of the phasin-
fused CadA was calculated to be 72.5%.
• insoluble fraction of the BCADPcell extract,
which contains phasin-fused CadA without
any intra-cellular P(3HB), also showed
lysine decarboxylase activity
Effect of the fusion cadA-phasin on cell
growth and P3HB production
• expression of phasin-fused CadA
increased P(3HB) production

• BCADP19 produced 51% more

P(3HB)

• CadA-fused phasin stabilize P(3HB)

granules by forming a layer on the
P(3HB) surface and therefore
increase the accumulation of P(3HB).
pH stability of the Fusion protein

• CadA–P(3HB) maintained activity up to

a pH of 8, whereas free CadAactivity
decreased with increasing pH.

• Immobilization did not pre-vent the loss

of activity above pH 9
Thermal Stability

• CadA–P(3HB) complex showed lower

activity at temperaturesof 4–80◦C
• Immobilization increased the thermal stability of
CadA. Immobilized CadA had a half-life of 70 h
and retained 91.8% of activity after 48 h of
incubation at 50◦C, but free CadA retained only
26.5% of activity after 20 h
• CadA–P(3HB) complex in five sequential 1-h
reactions resulted in conversion of 75–80% of
lysine into cadaverine.

• The CadA–P(3HB) complex retained its

conversion ability after five 1-h reaction
cycles, whereas the activity (Unit/mgprotein)
of free CadA was reduced to 43% of the initial
value.
Conclusion