http://dx.doi.org/10.4314/tjpr.v11i6.6
Abstract
Purpose: To improve the biosynthesis of L-arginine
by overexpressing homologous NAD kinase (ppnk) in
Corynebacterium crenatum SYPA5-5 and to study its impact
in presence of high (HOS) and low oxygen supply (LOS).
Methods: A recombinant plasmid
(pJC1-tac-ppnK) harboring homologous NAD kinase (ppnk)
was constructed in a shuttle vector pJC1 and transferred
in L-arginine producing strain Corynebacterium crenatum
SYPA5-5. Furthermore, fermentation was performed by
shake flask method with consecutive determination of
cell growth and glucose concentration. NAD+
kinase activity was studied by stop method and NADP(H)
concentrations were determined by spectrophotometric
enzymatic cycling method. To check the biosynthesis of
amino acids, HPLC method was used to determine
extracellular amino acid concentrations.
Results: In HOS condition, NAD+
kinase activity increased by 116 %, while intracellular
concentrations of NADP+ and NADPH increased
by 7.3 and 36.8 %, respectively. Whereas, in LOS
condition , NAD+ kinase activity increased
49 % , with intracellular 14.67 and 15 % increases in
NADP+ and NADPH respectively. More
importantly, recombinant strain could produce 26.47 and
11.36 g/L L-arginine in HOS and LOS respectively, which
is higher than control strain value of 24.29 and 7.58
g/L respectively.
Conclusion: These results suggest that altering
the concentration of co-enzymes by NAD kinase in
Corynebacterium crenatum is an effective way to increase
NADP+ with concurrent production of NADPH for
further enhanced L-arginine biosynthesis in
Corynebacterium crenatum in both conditions of high and
low oxygen supply.
Keywords: NAD kinase, PpnK, L-arginine,
Corynebacterium crenatum