Table 1. Substrate preparation were done by applying indicated amounts of ONPG by simple dilution. Stock solution of ONPG was 15mg/ml.Then, reaction mixture were prepared by adding Z buffer, Chloroform, 0.1 % SDS by respective manner. Na2CO3 was added to end the reaction.Each tube contained 800 μl cell lysate.
|Substrate Concentration||Distilled water||ONPG||Assay Reaction Mix|
|Z Buffer||Chloroform||0.1% SDS||Na2CO3|
|15mg/ml||0 μl||300 μl||500 μl||100μl||50μl||300μl|
|12mg/ml||60 μl||240 μl||500 μl||100μl||50μl||300μl|
|10mg/ml||100 μl||200 μl||500 μl||100μl||50μl||300μl|
|7.5 mg/ml||150 μl||150 μl||500 μl||100μl||50μl||300μl|
|5 mg/ml||200 μl||100 μl||500 μl||100μl||50μl||300μl|
|4 mg/ml||220 μl||80 μl||500 μl||100μl||50μl||300μl|
|3mg/ml||240 μl||60 μl||500 μl||100μl||50μl||300μl|
|2 mg/ml||260 μl||40 μl||500 μl||100μl||50μl||300μl|
|1 mg/ml||280 μl||20 μl||500 μl||100μl||50μl||300μl|
|0.5 mg/ml||290 μl||10 μl||500 μl||100μl||50μl||300μl|
Table2. Substrate concentration obtained from M1xV1=M2xV2 formula. Needed amounts of ONPG were indicated below.
|Substrate Concentration||Calculation for substrate||Needed ONPG|
Table3.OD measurements at 420 nm for each ONPG concentration were indicated below.
|ONPG concentration (mg/ml)||OD420|
Table 4. Shows Miller Unit calculations for enzyme velocity. 1000x(OD420) / TxVxOD600 that formula was applied.OD600
was obtained as 1.305.
Graph1 shows velocity versus substrate concentration of enzyme. Velocity’s unit was indicated as Miller unit.Expected logarithmic line added for visualized how accurate data were.Predicted vmax was seen as 120 MU. Km predicted as approximately 2 mg/ml.
Table 5. shows 1/Miller Units and 1/Substrate Concentration calculations.
|1/Miller Units||1/Substrate||Calculation for 1/miller unit||Calculation for 1/ Substrate|
Graph 2. Lineweaver-Burk Plot were plotted by 1/velocity versus 1/substrate concentration. 1/velocity unit is 1/MU and 1/Substrate unit is ml/mg.
|Km calculation||V max calculation|
|For y=0 -1/Km=-0.176 Km=5.68mg/ml||From slope Km/Vmax=0.0296 Vmax=191.9|
|5.68mg/mlx1g/1000mgx1mole/1/301.25×1000/1Lx1000ml/1 mole = 1.8mM||Vmax conversion : 1.8mM /(1.91 ) x1000 = 942 micromolar/min/mg|
In this laboratory enzyme kinetics were studied by investigating β galactosidase activity. Michaelis-Menten and Lineweaver Burk plots were drawed for indicate Km and Vmax values.
According from Michaelis-Menten graph, approximate Vmax was close to calculated Vmax from Lineweaver-Burk plot value. Expected value for Km was 0.12mM and Vmax was Expected 360μg/min/mg.(*7).However obtained Km value was greater than expected. Expected value was obtained when manganase were used as cofactor, maybe this affected the result.According from obtained result, enzyme needs more substrate to reaction occur. However, expected value shows taht less amount of substrate is needed for reaching maximum velocity. When calculated Vmax turn to related unit , Vmax seen as greater than expected value. Enzyme catalyze more faster than expected. When substrate amount not reach its expected value, velocity of an enzyme can not reach greater value. Because of their specific interactions between substrate and enzyme. Even though substrate can not increase the rate of reaction, to observe velocity enzyme and substrate must made complex.