C-Phycocyanin (C-PC)
is a natural blue colored protein of Spirulina platensis. Several methods based on chemical,
physical, and enzymatic processes have been reported for the extraction
of C-PC. However, most of the processes are either costly and/or time-consuming
and/or produce C-PC with less purity. In view of this, a very simple
and effective method was developed to extract C-PC with high purity
and without involving any energy-consuming process from S.
platensis. Wet biomass of S. platensis was
harvested and incubated in an optimized 2-(N-morpholino)ethanesulfonic
acid buffer with no supplement of any other compounds under dark,
anaerobic, and still conditions. These conditions facilitated the
leaching out of C-PC from the cells. The purity ratio of the blue
color C-PC supernatant obtained was enhanced by precipitation and
dialysis. The purity ratio of C-PC produced was 0.644 and increased
to 1.345 upon simple dialysis of the crude C-PC. The purity of C-PC
was 6.17 upon purifying through column chromatography. Hence, a simple
process was developed for producing C-PC of much higher purity than
therapeutic grade (>4). Furthermore, the molecular regulation of
the
extracellular release of C-PC from S. platensis cells
was monitored through relative expression levels of α and β
subunits of C-PC genes during specific buffer vis-à-vis water
as a control. Expression levels of genes encoding α and β
subunits of C-PC were found to be upregulated in the case of the 2-(N-morpholino)ethanesulfonic acid buffer treated algal cells
compared to water treated algal cells. The higher level expression
of these genes documented the higher and specific production of C-PC
by the S. platensis upon 2-(N-morpholino)ethanesulfonic
acid buffer treatment.
The intrinsic role of lytic polysaccharide monooxygenases
(LPMOs)
for oxidative cleavage of a wide range of polysaccharides like cellulose,
starch, chitin, and xyloglucan has been studied extensively. Lignin
and its compounds are known to act as electron donors for activation
of LPMOs. However, the role of LPMOs in the cleavage of lignin compounds
and their complete kinetic analysis has not been well understood.
Here, an in-depth kinetic characterization was conducted to study
the depolymerization of the phenolic β-O-4 lignin compound,
guaicyl glycerol β-guaicyl ether (GGE), by LPMO. Ultrahigh-pressure
liquid chromatography, thin-layer chromatography, and NMR analysis
of the oxidized reaction mixture confirmed the formation of guaiacol
from selective cleavage of GGE at β-O-4 linkage by LPMO. Michaelis–Menten
and Lineweaver Burk plot kinetics of LPMOs revealed a K
m of 3.566 ± 1.002 mM and a catalytic efficiency
(k
cat/K
m)
of 43 × 103 M–1 s–1 for GGE. A complete catalytic mechanism involving O2 and
H2O2 was proposed and validated for the LPMO-driven
cleavage of GGE. The oxidation reactions involving GGE and LPMO in
the N2 atmosphere and competitive inhibition studies using
catalase confirmed the role of O2 and H2O2 in completing the catalytic cycle of LPMO. Molecular docking
and molecular dynamics simulation studies revealed the interaction
of amino residues around the copper active site of LPMO with GGE.
Per-residue interaction energy of amino acid depicted the maximum
interactive energy contribution by MET1 of LPMO. The presence of GGE
around the substrate-binding site further confirms the strong interaction
of LPMO and GGE. Hence, the interaction between GGE and LPMO confirmed
the role of LPMO in cleaving the β-O-4 linkage of lignin, thus
making the LPMO an attractive biocatalyst for use in enzymatic cocktails
for lignin valorization.
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