Enzyme-substrate reactions

As our reporter system, we use orthogonal hydrolase enzymes that react with specific substrates to give a chromogenic product. The color produced from the enzyme-substrate reaction gives the cue for the player to carry out the next logical move. The player adds a multi-substrate solution on to colonies on the agar mine-field. In case the player is certain of a colony to be a mine colony, another second specific substrate is added. The substrates react with the enzymes to give the color in couple of minutes and are visible by eye.

Our minesweeper is becoming better and better so keep on track for updates !

The picture to the left shows different colors produced by the hydrolase-substrate reactions. Each hydrolase was tested with the corresponding substrate in liquid cultures. The hydrolases are lacZ, nagZ , aes, gusA and phoA. Multiple substrates were tested for the hydrolases as well.

The following table lists each of the specific hydrolase-substrate reactions with the corresponding color produced from the reaction.

Hydrolase		Complementary substrate / IUPAC name		Visible color	Concentration[M]	Time(s)for colorimetric response
LacZ	Beta-Galactosidase	X-Gal	5-Bromo-4chloro-3-indolyl-beta-galactopyranoside	Blue
LacZ	Beta-Galactosidase	Green-beta-D-Gal	N-Methyl-3-indolyl-beta-D_galactopyranoside	Green
GusA	Beta-glucuronidase	Magenta glucuronide	6-chloro-3-indolyl-beta-D-glucuronide-cycloheylammonium salt	Red
PhoA	Alkaline phosphatase	pNPP	4-Nitrophenylphosphatedi(tris) salt	Yellow
Aes	Acetyl esterase	Magenta butyrate	5-bromo-6-chloro-3-indoxyl butyrate	Magenta
NagZ	Glycoside hydrolase	X-glucosaminide X-Glucnac	5-bromo-4-chloro-3-indolyl-N-acetyl-beta-D-glucosaminide	Blue

Beta-galactosidase(LacZ)

Chemical conversion



Colorimetric response in liquid culture

Colorimetric response on LB-Agar

Fig.5: X-Gal on LacZ expressing colony

Fig.6: Beta-green-X-gal on LacZ expressing colony

Alkaline phosphatase(PhoA)

Chemical conversion



Colorimetric response in liquid culture

Colorimetric response on LB-Agar

Acetyl esterase(Aes)

Chemical conversion



Colorimetric response in liquid culture

Colorimetric response on LB-Agar

Beta-glucuronidase(GusA)

Chemical conversion



Colorimetric response in liquid culture

Colorimetric response on LB-Agar

β-N-Acetylglucosaminidase(NagZ)

Chemical conversion



Colorimetric response in liquid culture

Colorimetric response on LB-Agar

Do the substrates and enzymes cross-react ?

Figure 6.2: Expected colorimetric response

An enzyme-substrate test matrix (Figure 6) was established to test each substrate against each enzyme. The results were as expected (Figure 6.2) and no cross reaction is visible. The NagZ-X - glucosaminide X-Glunac reveal some difficulties in the liquid culture as well as on the agar plate.

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