Disha NEET Physics Guide for classes 11 and 12.pdf
Alliinase importance and estimation method.pptx
1. Alliinase estimation
An enzyme found in garlic and other species of the
genus Allium, which when brought into contact with
the amino acid converts it to the strong-smelling
compound allicin.
Babanjeet
L-2021-H-85-D
PhD Vegetable Science
2. Importance
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• Alliinase is a glycoprotein with an estimated carbohydrate content of 4.6 % in onion and 5.5% in garlic (Nock
and Mazelis 1987).
• Responsible for catalyzing chemical reactions that produce the volatile chemicals that give these foods their
flavors, odors, and tear-inducing properties.
• Alliinases are part of the plant's defense against herbivores.
• When the plant is damaged by a feeding animal, the alliinase is released to catalyze the production of the
pungent chemicals.
3. Alliinase-catalysed
conversion of alliin
into allicin
Elimination of 2-propenesulfenic acid from the amino acid unit, with a amino
acrylic acid as a by-product
Condensation of two Sulfenic acid molecules.
5. Case study 1. Physical
Characterization of Alliinase:
The Flavor Generating
Enzyme In Onions
Protein Purification
and sequencing
Molecular Mass
Determination by
Gel filtration FPLC
In Vitro Translation
Clark et al 1997
6. Purification Of alliinase from onion bulbs
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Fraction Total protein (mg) Total activity
(nkat)
Specific activity
(nkat/mg)
Purification (fold) Recovery (%)
Crude extract 180 14217 78.98 1 100
1.33M-3.25M
(NH4)2SO4
206.7 26076 126.15 1.60 183
Sephacryl S200 138 17200 124.64 1.58 121
Con A-Sepharose 10.73 7300 680.34 8.61 51
Phenyl-Sepharose 6.50 6083 935.90 11.85 43
CM-Sepharose 3.33 2487 746.70 9.45 17
7. Analysis of purified alliinase by CM-
Sepharose CL6B
Gel filtration FPLC (at 280nm)
SDS-PAGE and silver staining
NH4 terminal sequence
Panel (A) is shoulder peak, and Panel (B) is main peak of alliinase activity and Panel (C)
is deglycosylated alliinase of main peak.
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8. iN vITROtRANSLATIONoF
aLLIINASE sEPARATEDbY 7-17%6M
uREASDS-PAGE
Staining by fluorography (A) and Coomassie Blue (B)
Lane 1: C14 labelled molecular weight markers
Lane 2: In vitro translated alliinase
Lane 3: Deglycosylated alliinase
Lane 4: Biorad molecular weight markers
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Clark et al 1997
9. Case study 2. Extraction and Purification
Alliin standard
Bovine serum
albumin
Ehyl acetate
Pyridoxine-5-
phosphate
Sodium pyruvate
Methanol and
EDTA
Garlic
Onion bulbs
Stored at temp 40
C.
Microwave oven
Spectrophotometer
Vortex
Ultrasonic bath
HPLC system
connected with UV-
VIS detector
Rotary evaporator
Calorimetric
method
Optimisation of
ultrasound
parameters
Quantification of
allicin by HPLC
Chemicals Plant
material
Equipment Principle
Wang et al 2010
11. Assay of alliinase activity!
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Procedure
• A sample of 0.05 mL was added to 1 mL of 60 mmol L−1
sodium phosphate buffer.
• Incubated at 25 ◦ C for 5 min and after that add 1.5 mL of 100
g kg−1 trichloroacetic acid.
• After centrifugation remove the precipitated protein, the
supernatant is assayed for pyruvate concentration.
• Add 0.5 mL volume of 1 g kg−1 2,4-dinitrophenylhydrazine to
the supernatant and incubated at 25◦ C for 5 min.
• Add 5 mL of 0.5 mol L−1 NaOH and incubated at 25◦ C for
10 min.
Absorbance at 520 nm
One unit of alliinase activity was defined as the amount of
enzyme releasing 1 nmol pyruvate min−1
Wang et al 2010
12. CONCLUSIO
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Alliinase is an important enzyme for plant's defense
mechanism.
Molecular analysis of alliinase cDNA indicated that
two genes and thus two protein subunits were expressed
in onion bulb tissue (Clark et al 1997).
The employed ultrasound increased the activity of the
purified alliinase by about 42.8%, did not affect the
enzyme’s temperature optimum and improved its
thermal stability (Wang et al 2010).
