Cell disruption and determination of enzyme activity

1
Cell disruption and determination of enzyme
activity

• Abstract
• Materials and methods
• Laboratory Statics
• Experiment Analysis
• Active Calculation Layout

2
Abstract Two different methods of
cell disruption were studied in this work
mechanical and ultrasonical.
Mechanical method was used in my group to disrupt
the cell of E.coli, first, prepared the
incubation of E.coli K12 which got cell growth
enough and centrifuged it afterward , grinding it
with quartz in the mortar in the cold room with
1 ml of sodium phosphate with four times (0,25 ml
per time), grinding it again for 5 min and added
slowly 2,5 ml of buffer solution to get cell
disruption as much as possible. Transferred cell
disruption mixture to the bottle to be
centrifuged for 10 min at 2 c with 7000 rpm and
then transferred the supernatant to the bottle to
determinate the enzyme activity of the
intracellular. First, found the
suitable enzyme dilution from initial velocity
versus dilution series function which was as much
linear as possible. the values of Km- and Vm can
be determinated from the curve of Michalis-Menten
according to the initial velocity versus
substrate concentration and then according to it
to calculate the molar extinction coeffient to
get the enzyme activity. The values got here
were that Kmo.35mol/ml, Vm0.2053 dA/min, and
e7,76209ml/mol.cm. The enzyme activity of
intracellular was 16.00 ncat/ml.
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Cell disruption and determination of enzyme
activity

• Cell cultivation and relative centrifugal force
  formula RCF11,7rn2/106
• Cell disruption with mechanical method
• Determination of enzyme suitable dilution
• Determination of Km- and Vm- values
• Michaelis-Menten formula VVms/Kms
• Lineweaver-Burk formula1/V(1/Vm)Km/Vms
• Lambert_Beers formula Aelc which lwidth of
  cyvet(1 cm)
• Velocity for formation of reaction of reaction
  product VFV0Vtot/el
• The equation for enzyme activity AVF/Ea
• The formula for the final enzyme activityYxA

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Experimental Statics

• ONPG enzyme water
• 200 200 0
• 160 200 40
• 120 200 80
• 80 200
  120
• 40 200 160

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Relation between Substrate and Initial Velocity

• Substrate dA/min
• 248.00 0.205
• 198.40 0.201
• 148.80 0.195
• 99.20 0.174
• 49.60 0.133

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Relation between 1/Substrate and 1/Initial
Velocity

• 1/s 1/(dA/min)
• 403.23 4.87
• 504.03 4.96
• 672.04 5.12
• 1,008.06 5.72
• 2,016.13 7.47

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