Investigating the Enzymatic Activity During Paste Processing

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Investigating the Enzymatic Activity During Paste
Processing
ESRA CAPANOGLU Food Engineering Department
Istanbul Technical University
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Background
Wounding effect Enzyme activity
?...
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Literature-Wounding Effect
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Enzymes for Flavonol Synthesis

• Flavonol synthase

• Glucosyl transferase-rhamnosyl transferase

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3-Glucosyl Transferase (3-GT)

• Optimization of extraction procedure
• Setting up enzyme assay HPLC method
• Processing steps of paste samples
• Tomatoes of different developmental stages
• Tobacco (control)

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Establishment of the 3-GT Assay

• Assay conditions
• Optimum pH
• Buffer composition
• Bicine
• Tris-HCl
• MES
• CAPS
• Incubation conditions
• 30ºC
• 1 hour

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Establishment of the 3-GT Assay

• Detection of the product (HPLC)

• SAMPLE

substrate
product

• CONTROL
• (-UDP GLUCOSE)

• CONTROL
• (-ENZYME EXTRACT)

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3-GT Assay Results for Paste Processing Steps
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3-GT Assay for Paste Processing Steps
Enzyme activity doubles during breaking!..
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3-GT-Fruit vs Breaker
360 nm
Tomato
?
?
Quercetin
Isoquercitrin
Breaker
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UPLC-MS Data
360 nm
Control (-UDP Glucose)
Tomato
Breaker
Finisher seedskin
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(No Transcript)
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360 nm
Quercetin-3-O-glucoside
Quercetin-7-O-glucoside
Quercetin
Quercetin-4-O-glucoside
m/z 301
m/z 301 463
m/z 301 463
m/z 301 463
?M- ? gt
Aglycon
Aglycon Glc
Aglycon Glc
Aglycon Glc
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3-GT Assay Results for Developmental Stages
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3-GT Assay for Developmental Stages of Tomatoes

• Green
• Turning 1
• Turning 2
• Pink
• Red
• Dark red

Flesh
Skin
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Flavonoid Analysis-Developmental Stages of
Tomatoes
Skin part 360 nm
Chlorogenic acid
rutin apioside
rutin
naringenin chalcone
Green
Turning 1
Turning 2
Pink
Red
Dark red
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FLESH
SKIN
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PAL Assay
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PAL (Phenylalanine ammonia lyase)
PAL
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PAL (Phenylalanine ammonia lyase)
Kinetic reaction 290 nm 41 scans
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PAL Assay- Developmental Stages
FLESH
3895 ukat/mg

SKIN
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(No Transcript)
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Correlations
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Protein Purification
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Control vs Tomato
Tomato
360 nm
isoquercitrin
Tobacco
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Protein purification
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Purification steps
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FPLC (280 nm) Gel Filtration
MW 70 kDa
Pool active fractions
2D- Gel
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2D-Gel
70 kDa
30 kDa
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Conclusion Future work

• The increase in the rutin content is consistent
  with the enzyme activity for rutin biosynthesis.
• High PAL activity in the skin part gtmore
  substrate available
• Initial protocols for 3-GT extraction, assay and
  purification are composed
• Further characterization of 3-GT in tomatoes

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People involved
Hans-Peter Mock Andrea Matros Silke
Peterek
Esra Capanoglu Dilek Boyacioglu Artemis Karaali
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• Thank you