Question

Which of the following is expected to have the highest yield of ONP in the enzyme kinetics experiment? 5.00 ml of PO4,0.50 ml of ONPG, 0.50 ml of lactase = 6 ml 1 2.00 ml of PO4, 4.0 ml of ONPG, 0.00 ml of lactase = 6 ml 5.50 ml of PO4, 0.00 ml of ONPG,0.50 ml of lactase - 6 ml 5.25 ml of PO4, 0.25 ml of ONPG, 0.50 ml of lactase 6 ml

Answer 1

Media and Reagents used :

Sodium phosphate buffer, 1 M, pH 7.0

O-Nitrophenyl-β-D-galactopyranoside (ONPG), 0.75 M

Physiologic saline

Toulene

The main thing from which we conclude the answer is in procedure:

Bacteria grown in medium containing lactose (to induce the production of the galactosidase enzyme), such as Kligler iron agar (KIA) or Triple sugar Iron (TSI) agar, produces optimal results in ONPG Test.

Procedure:

Note: β-galactosidase enzyme (inducible enzyme) is made ONLY in the presence of the lactose substrate

A loopful of bacterial growth is emulsified in 0.05mL of physiologic saline to produce a heavy suspension

One drop of toluene is added to the suspension and vigorously mixed for a few seconds to release the enzyme for bacterial cells.

****An equal quantity of buffered ONPG solution is added to the suspension.**** equal to bacterial growth suspension producing lactase. This means ONPG=lactase

So, 1st is correct.

The mixture is placed in a 37°C water bath.

Both enzyme and substrate Or reactant if equal then all substrate attached to the active site of the enzyme and form product in high yield.