The effect of Nanopartical Doped water (Neowater®) on the activity and

stability of BglI and HindIII restriction enzymes

Millrod S. Guttman C.
Do-Coop Technologies, Israel

Abstract
There are many unresolved questions regarding the role of water in protein folding. Recent studies of water within and around proteins have made us acutely aware of
the critical role that water plays in protein structure and function. Neowater is an enabling technology that is based on breakthrough water-based nanotechnology.
Neowater builds upon the unique properties of nanoparticles to modify the physical properties of water molecules around them. My research aims to study the
influence of Nanoparticle Doped Water (Neowater) on BglI and HindIII restriction enzyme’s activity and heat stability and Whether Neowater can protect Restriction
Enzymes from heat inactivation and elevate its enzymatic activity in comparison to its resistance and activity in UPW. Experiments results showed that Replacement of
UPW by Neowater enhanced stability and resistance of HindIII and BglI restriction enzymes to thermal stress degradation.

Aims
study activity and heat resistance of restriction enzymes in NW in comparison to its activity and resistance in UPW

1.Transformation of Puc19 to DH5α Methods

2.Bacterial culture and harvest
3.Extract plasmid DNA from bacteria with Mini/maxi prep
4.Experiments of activity and heat resistance with restriction enzymes and Puc19
Activity experiments: final samples containing plasmid+ Enzyme were incubated at 37° for 1 hr
Heat resistance experiment: final samples were incubated in a thermal cycle and then were incubated at 37° for 1 hr
5.The results were obtained by loading the samples on a 1% agarose gel and photographing the gel following electrophoresis.

RESULTS
Experiment #1: Activity of BglI (NW Vs UPW) Experiment #4: Heat resistance of HindIII (temperature gradient)

M 1 M 1
Figure 1: serial dilution of BglI restriction enzyme were prepared in Neowater and UPW solutions . The
solutions were prepared at room temperature and then were incubated at 37° C for 1 hour. (M-ladder ; 1-uncut
plasmid, positive control ; the red circles point to the differences that were visible between UPW and NW). Experiment #5: Heat resistance of HindIII (80ºC- time gradient)

Experiment #2: Heat resistance of BglI (temperature gradient)

M 1
Figure 4 & 5: All samples were prepared with 10u (enzyme)/45 µl (UPW/NW). Tubes for Exp #4 were
incubated in a thermal cycler block in a gradient temperature ranging from 60ºC to 80C, 5C interval for a 15
min incubation and tubes from Exp #5 were incubated in a thermal cycler block in a time Gradient of 30 min .
Figure 2: All samples were prepared with a final concentration of 1u (enzyme)/36 µl (UPW/NW). Tubes were The rest of the tubes were incubated at 4ºC. buffer solution (NEB 2+plasmid (0.5µg/µl)) was added to all PCR
incubated in a thermal cycler block in a Heat gradient, (37°C for 15 min and 47°C for 10 min), the rest of the PCR tubes after the heat treatment. All PCR tubes were incubated for 1 hour at 37C. (M-ladder ; 1-uncut
tubes were stored at 4°C. buffer solution (NEB3+plasmid) was added to all PCR tubes after the heat treatment. plasmid, positive control ; the red circles point to the differences that were visible between UPW and NW).
All PCR tubes were incubated for 1 hour at 37°C. (M-ladder ; 1-uncut plasmid, positive control ; the red circles
point to the differences that were visible between UPW and NW)
Result summery:
Experiment #3: Heat resistance of BglI (50ºC- time gradient) Activity experiment  as seen in experiment #1, BglI activity
in the presence of Neowater at room temperature was favorable
at 0.03125u/50µl of enzyme concentration then the activity of
the same enzyme in the presence of UPW.
Heat resistance of BglI  as seen in experiment #3, BglI in
Neowater maintained its activity for 15 minutes at 50°C whereas
the same enzyme incubated in UPW under the same stress
M 1 conditions lost its activity after 5 minutes
Figure 3: All samples were prepared with 10u (enzyme)/45 µl (UPW/NW) . The tubes form each group were
Heat resistance of HindIII as seen in experiment # 5 HindIII in
incubated in a thermal cycler block (50°C) for a time gradient of 30 min. buffer solution (NEB 3+plasmid Neowater maintained its activity for 20 minutes at 80°C whereas
(0.5µg/µl)) was added to all PCR tubes after the heat treatment. All PCR tubes were incubated for 1 hour at
37°C. (M-ladder ; 1-uncut plasmid, positive control ; the red circles point to the differences that were visible
the same enzyme incubated in UPW under the same stress
between UPW and NW) conditions lost its activity after 5 minutes.

Conclusion
Replacement of UPW by Neowater® enhanced stability and resistance of HindIII and BglI restriction enzymes to thermal
stress inactivation