The enzyme activity against p-NPS w

The enzyme activity against p-NPS was measured at different
temperatures at pH 8.0 (Fig. 2). The enzyme when stored at 4
◦
C
for 7–14 days did not show any loss of activity and thus, was quite
stable. Activity gradually increased in the temperature range from
25 to 45
◦
C but optimum at 35
◦
C. The enzyme activity reduced to
almost half of the optimum at 15
◦
C and 55
◦
C. The enzyme activity was completely absent at temperature≥65
◦
C probably due to
the thermal denaturation of enzyme.KmandVmaxof the enzyme
was determined by linear regression, plotting the inverse enzyme
activity against the inverse of substrate concentration. The apparentKmandVmaxwere estimated to be 10 mM and 20 mM min−1
respectively at pH 8.0 and 35
◦
C.
The effect of additives on sulfohydrolase activity was examined
in the presence of various metal ions, chelators (5 mM) and organic
solvents (5%) and is shown inTable 2. Among the metals ions Ca
2+
and Mg
2+
ions enhanced the enzymatic activity by 21% and 40%
respectively, while Cu
2+
reduced the activity by 21% from the optimum values. The metal ions Hg2+
and Pb
2+
completely inhibited the
activity while no appreciable change in the activity was observed
in the presence of Na
+
and K
+
. The serine protease inhibitor (PMSF)
and metal chelator EDTA significantly reduced the enzyme activity
by 100% and 40% respectively. Most of the organic solvents used in
this study inhibited the enzyme activity by 10–55%, however, the
presence of CHCl3did not affect the enzyme activity.
3.2. Desulfation and physical properties of agar
The purified sulfohydrolase was allowed to react with agar
sample under optimum experimental conditions. A series of
enzyme concentrations (10, 25, 50 and 100 U) were used to
optimize the enzyme concentration for agar desulfation. A gradual increase in sulfate release and 3, 6-AG together with the
increase in viscosity of agar solution was observed when subjected to 10–50 U of enzyme while no appreciable change in
these parameters observed when enzyme concentration was >50 U
(Table 3). Therefore, the concentration of 50 U was chosen as optimum for subsequent experiment. A recovery of almost 90% was
observed in the agar subjected to enzymatic treatment. The physical properties like gel strength, gelling and melting temperature
Table 3
Optimization of enzyme concentration for the removal of sulfate from agar.
Enzyme (U) Sulfate (%) 3,6-AG (w/w) (%)
0 2.82±0.19 18±.5
10 2.51±0.096 19±1.0
25 2.05±0.05 23±1
50 1.11±0.046 30±2
100 1.1±0.076 30±1.5
and viscosity improved significantly in the enzyme treated agar
(Table 4).
The ICP-AES findings further showed that sulfate content
decreased remarkably from 2.81% (control) to 1.11% (enzyme
treated agar) that corresponded to reduction of about 60% (Table 4).
The release of sulfate was positively correlated with the amount
of 3,6-AG units that increased from 18% (control) to 30% (enzyme
treated agar). The capability of sulfohydrolase to increase the viscosity of agar solution was also measured and estimated to increase
by two-fold with values 9.67 cp in (control) and 18 cp (enzyme
treated agar). The sulfohydrolase treatment resulted in a noticeable decrease in gelling and melting temperature with 31
◦
C and
82
◦
C respectively and was quite low as compared with that of control agar with 38
◦
C and 90
◦
C. The enzymatic desulfation improved
the gel strength as it enhanced its value from 190 g/cm
2
in control to 470 g/cm
2
in enzyme treated agar and contributed to almost
2.5-fold increase.
3.3. Scanning electron microscopy
The scanning electron micrographs (SEM) of the xerogels
obtained for control agar and enzyme treated agar showed different
types of network morphologies (Fig. 3). The xerogels of control agar
showed fibrous structures of almost 215–650 nm width (Fig. 3A).
The enzyme treated agar xerogel exhibited much blunt structure
persisted with helixes of approximately 2500–3000 nm in width
together with strong cross linking (Fig. 3B).
3.4. FT-IR spectra
The quantitative FT-IR spectra of both control agar and enzyme
treated agar are shown in Fig. 4. The area of the band at
1250 cm
−1
which represents the total sulfate content get reduced
in the enzyme treated sample while the band in the range of
850–868 cm
−1
was completely absent in the enzyme treated sample with a significant increase in the band at 930 cm
−1
.
4. Discussion
The enzyme sulfohydrolase which catalyzes the formation of
3,6-AG ring froml-Gal-6-sulfate by removing sulfate ester moiety in porphyran has been first reported byRees (1961a,b). This
has opened new opportunities for researchers to improve the
gel strength of sulfated galactans phenomenally. Subsequently,
a few studies describing the conversion of-carrageenan into
-carrageenan using protein fraction from C. crispus(Wong &
Craigie, 1978) Gigartina stellata(Lawson & Rees, 1970) and -carrageenan to-carrageenan with Calliblepharis jubataextracts