Lipases,
which can be immobilized and reused for many reaction
cycles, are important enzymes with many industrial applications. A
key challenge in lipase immobilization for catalysis is to open the
lipase lid and maintain it in an open conformation in order to expose
its active site. Here we have designed “tailor-made”
graphene-based nanosupports for effective lipase (QLM) immobilization
through molecular engineering, which is in general a grand challenge
to control biophysicochemical interactions at the nano–bio
interface. It was observed that increasing hydrophobic surface increased
lipase activity due to opening of the helical lid present on lipase.
The molecular mechanism of lid opening revealed in molecular dynamics
simulations highlights the role of hydrophobic interactions at the
interface. We demonstrated that the open and active form of lipase
can be achieved and tuned with an optimized activity through chemical
reduction of graphene oxide. This research is a major step toward
designing nanomaterials as a platform for enhancing enzyme immobilization/activity.
Thermostable lipase produced by a genotypically identified extremophilic Bacillus subtilis NS 8 was purified 500-fold to homogeneity with a recovery of 16% by ultrafiltration, DEAE-Toyopearl 650M and Sephadex G-75 column. The purified enzyme showed a prominent single band with a molecular weight of 45 kDa. The optimum pH and temperature for activity of lipase were 7.0 and 60°C, respectively. The enzyme was stable in the pH range between 7.0 and 9.0 and temperature range between 40 and 70°C. It showed high stability with half-lives of 273.38 min at 60°C, 51.04 min at 70°C and 41.58 min at 80°C. The D-values at 60, 70 and 80°C were 788.70, 169.59 and 138.15 min, respectively. The enzyme's enthalpy, entropy and Gibb's free energy were in the range of 70.07-70.40 kJ mol(-1), -83.58 to -77.32 kJ mol(-1)K(-1) and 95.60-98.96 kJ mol(-1), respectively. Lipase activity was slightly enhanced when treated with Mg(2+) but there was no significant enhancement or inhibition of the activity with Ca(2+). However, other metal ions markedly inhibited its activity. Of all the natural vegetable oils tested, it had slightly higher hydrolytic activity on soybean oil compared to other oils. On TLC plate, the enzyme showed non-regioselective activity for triolein hydrolysis.
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