A%20SIMPLE%20SOIL%20ASSAY%20TO%20DEMONSTRATE%20ENZYME%20REGULATION

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A SIMPLE SOIL ASSAY TO DEMONSTRATE ENZYME
REGULATION
CRAIG PHELPS Dept. of Environmental Sciences
Rutgers University
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Purpose To Demonstrate Soil Enzyme Activity from
an Environmental Perspective - Extracellular
enzymes for scavenging nutrients - Produced by
many different organisms - Measurable as a
collective Enzyme Activity - Dependent on -
Community composition - Community size -
Availability of substrates - Closely regulated
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NO3
NO3

PO4

• Phosphatase
• Extracellular or periplasmic enzyme
• Produced by almost all soil organisms
• Responsible for releasing soluble phosphate from
  organic molecules
• Can hydrolyze p-nitrophenol phosphate to form by
  p-nitrophenol
• Product is measured by absorbance at 460nm
• (Tabatabai and Bremner, 1969).

PO4
OH
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FIELD WORK
The lab starts with a field work class where the
students do water quality testing and collect
soil samples.
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• SOIL MICROCOSMS
• Set up four soil microcosms with different
  fertilizer treatments
• Treatment Fertilizer (15-30-15) Yeast Extract
  
• Organic 0.0 g 1.6 g
• Inorganic 0.2 g 0.0 g
• Combined 0.2 g 1.6 g
• Unamended 0.0 g 0.0 g
• Total phosphorus and nitrogen are comparable
• Procedure
• Cut out a circle of filter paper and use it to
  line the bottom of the flower pot.
• Use a glass beaker to measure out 200ml of soil.
• Weigh out the appropriate amount of fertilizer or
  yeast extract and mix it into the soil.
• Pour in the soil and fertilizer mix until it is
  1/2 inch from the top.
• Water the soil until it is all moist, then cover
  the top of the pot with parafilm.

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ENZYME ASSAY
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• Weigh out two 2-gram portions of each soil sample
  and pour them into labeled screw-cap tubes.

• Pipette 5ml of 0.5 M CaCl2 solution into each
  tube and shake well.
• Pipette 1ml of PNPP solution into one tube from
  each soil sample and 1ml of pH 10 buffer into the
  other tube to serve as a control.
• Incubate all tubes at 37?C for 1 hour

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• Centrifuge the screw-cap tubes in a clinical
  centrifuge for 5 min.
• Transfer 4ml of the supernatant into new tubes
  and re-centrifuge for 5 min.
• Transfer 3ml of the supernatant into clean test
  tubes.

• Read and record the absorbance for each of your
  samples at 460 nm.
• Calculate the concentrations of p-nitrophenol by
  comparing to a standard curve
• Calculate enzyme activity

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• RESULTS
• Under Ideal Conditions
• Soil fertilized with Yeast Extract shows an
  increase in activity over Un-amended soil
• Soil fertilized with 15-30-15 show a decrease in
  activity from Un-amended soil
• Soil fertilized with Combined sources show a
  decrease in activity compared to Yeast Extract
• Differences can happen based on the nutrient
  status of the soil
• - well fertilized soils will tend not to respond
  to treatment

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• CONCLUSIONS
• Demonstrates regulation of composite activity
  based on nutrient availability
• Easy to do in the teaching lab
• Large quantitative component
• Allows for hypothesis testing
• Flexible design
• - manipulate other environmental conditions
  (moisture, temp.,
• contamination, etc.)
• - test different soils