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Anita Ruhal

Reg. no. 0909902

Deptt. of Bio and NanoTechnology

GJUS&T, Hisar

Under supervision

Prof. J.S Rana

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FRUIT MATURITY INDICATOR

Malic acid is an important indicator for fruit maturity (Barden et al.,

1997, Jayaprakasha et al., 1998, Kader 1999, Arif et al., 2002,Prodomodis et al., 2002, Shobha Jawaheer et al., 2003, Gokhan

Durmaz et al., 2010).

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MALIC ACID

Malic acid is a dicarboxylic acid with formula C4H6O5found in many

sour or tart-tasting foods.

The salts of malic acid, known as malates are an important

intermediary step in the citric acid cycle.

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1. Malic acid was originally isolated in an apple by the Swedish chemist Carl Wilhelm Scheele

(1785).

2. Fruit maturity is the most important factor that determines final fruit quality (Arif et al., 2000)

3. Life stages of fruits such as growth, maturation, senescence, color and antimicrobial activity

all depend on organic acids (Cano et al., 1994).

4. Organic acids are responsible for the taste of a fruits or vegetables along with balance of

sugar and acids.

5. Major organic acids in most fruits is L-malic acid (Arif et al., 2002, Davis et al., 1966).

6. Determination of L-malic acid concentration provides important information about the

ripening of fruits and vegetables.

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Malic acid

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Sugarconfectionary

Softdrink

Cider

Dental hygine

Wines

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MALIC ENZYME

Oxidative decarboxylation of malic acid is carried out by NADP-malate dehydrogenase

(also called malic enzyme) using NADP+

as a coenzyme (cofactor) to producepyruvate, CO2 and NADPH (Franke et al.,1995, Gajovic et al., 1997, J. Justin

Gooding 2000, Lupu et al., 2004, Rana et al., 2010, Doubnerova et al., 2011).

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REACTION MECHANISM

Malic enzyme catalyzes a reversible oxidative decarboxylation of L-

malate to give carbon dioxide and pyruvate in the concomitant reduction

of NAD(P)+ to NAD(P)H.

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The catalysis by malic enzymes

generally proceeds in three

steps dehydrogenation of

malate to produce oxaloacetate

(k1), decarboxylation of

oxaloacetate to produce

enolpyruvate (k2), and finally

tautomerization of enolpyruvate

to produce pyruvate (k3)

(Cleland 1999).

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SCREEN PRINTED MULTIWALLED CARBON

NANOTUBE ELECTRODE

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IMMOBILIZATION OF ENZYME ON SCREEN PRINTEDMULTIWALLED CARBON NANOTUBE ELECTRODE

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PREPARATION OF ENZYME ELECTRODE

All works performed on screen printed electrode on which MWCNT already

immobilized.

The fabricated screen printed electrode washes with milli Q water and then left it for

dry.

The working electrode of screen printed was treated with mixture of 0.2M [N-ethyle-N-

(3-dimethylaminopropyl)carbodimide] (EDC) and 0.2M NHS for 1h.

The electrode was washed with PBS Buffer (50mM NaH2PO4, 50mM Na2HPO4, 0.9%

NaCl) pH 7.4 and dried before immobilization of enzyme.

An enzymatic solution of Malic enzyme with an activity of 0.4 units in PBS buffer, pH

7.4 was prepared and coated surface of working electrode with 5 l solution. Left it for

1 h at room temperature.

The resulting electrode stored in the refrigerator at 40C.

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ROLE OF EDC AND NHS IN THE IMMOBILIZATION OF

ENZYMES ON TO C-MWCNT ELECTRODE

Firstly EDC converts free COOH groups of c- MWCNT into reactive intermediate,

which is susceptible to amine attacks. EDC catalyzes the formation of amide bonds

between COOH groups and NH2 groups by activating carboxyl to form an O-urea

derivative. This intermediate was unstable and random reactions were results in

undesired products.

After that NHS was often used to assist the carbodimide coupling in the presence of

EDC.

Finally the active ester intermediate further reacts with the NH2 groups on the

surface of enzymes to yield the final amide bond confirming the covalent

immobilization of enzymes on the surface of c-MWCNT through amide bond formation

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SCHEMATIC REPRESENTATION OF CHEMICAL REACTION INVOLVED IN

THE FABRICATION OF ENZYME/ SPMWCNT ELECTRODE.

EDC

NHS

NH2

COOH COOH

Working electrode(c- MWCNT) EDC modified c-MWCNT electrode

O

C

O

C

O

O

NHS modified c-MWCNT electrode

C

O

C

O NH

NH

Enzyme/c- MWCNT electrode

Screen printed c-MWCNT electrode

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SURFACE CHARACTERIZATION OF ENZYME

ELECTRODE(ENZYME/SPMWCNT) BY SEM

SPMWCNT electrode Enzyme/SPMWCNT electrode.

MWCNT Enzyme on MWCNT

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FTIR SPECTROSCOPY

Peak at 2358 cm-1 associated with O-H

stretch from strongly hydrogen bonded

COOH. Increased strength of signal at 1166

cm-1may be associated with C-O stretching

in same functionalities. Peak at 1566 cm1

can be associated with the stretching of

carbon nanotubes backbone. Chemical

treatment with the acid mixture introduces

additional peaks in the spectra. Peak at 3014

cm-1 shows the O-H stretching.

An FTIR spectrum of immobilized enzymewith peaks at 3026, 1636cm-1 and 1188 cm-

corresponds to C-H stretching; N-H was

bending and C-N stretching, respectively.

Medium intensity peaks of N-H occur at

3497.

FTIR Spectra obtained for (A) SPMWCNT electrode

(B) Enzyme/SPMWCNT electrode.

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CV FOR DIFFERENT CONCENTRATION OF MALIC ACID

To evaluate the catalytic activity of

Enzyme/SPMWCNT, the modified electrode

was characterized by a cyclic voltammogram

in the presence of different concentration of

Malic acid (0.03mM to 2.00 mM) at the

potential range from -0.7V to -0.1V. The

maximum response was observed at -0.34 V

and hence subsequent studies were carried

out at this stage. The CV was measured in a

microcell containing 1.0 l NADP (4mM) and

1.0 l of different concentration of malic acid

in 48 l of PBS, pH 7.4 on

enzyme/SPMWCNT. CV peaks increase with

increasing concentration of malic acid due to

increased oxidation of malic acid

CV of amperometric response studies as a function of

Enzyme/SPMWCNT in malic acid concentration from

(0.03 to 2.00mM) in PBS, pH 7.4.

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DPV FOR DIFFERENT CONCENTRATION OF MALIC ACID

Differential pulse

Mode = Potentiostatic

Current range = 100 A

Initial potential = -0.6

End potential = -0.1

Interval time = 5 sec

Scan rate =80 mV/sec

DPV of amperometric response studies as a function

of Enzyme/SPMWCNT in malic acid concentration

from (0.03 to 2.00mM) in PBS, pH 7.4.

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EFFECT OF SUBSTRATE CONCENTRATION ON BIOSENSOR (KM)

To study the effect of substrate

concentration on biosensor, the

concentration of malic acid was varied

from 0.03 mM to 2 mM in PBS, pH 7.4.

A hyperbolic relationship was found

between malic acid concentrations

versus current. The standard

calibration curve of the sensor

response at different concentration of

malic acid showed that the sensor

response was linear from 0 to 0.25 mM

malic acid (Fig inset) which is lower

than 0.01 to 0.4mM (Doubnerov and

Ryslava 2011), 0.1 to 1mM

(Jayapraksha and Sakariah 1998),

0.028 to 0.7mM (Arif et al. 2002).

Effect of substrate concentration study (hyperbolic

curve) and calibration plot (inset) of current (A)

responses at different malic acid concentrations (mM)

by malate biosensor based on Enzyme/SPMWCNTelectrode

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Lineweaver-Burk plot for effect of malic acid

concentration on response of malate biosensor (Km)

based on NADP-specific malate dehydrogenase

Km value for malic acid as

calculated from Lineweaver-

Burke plot was 0.19 mM which is

lower than 0.6 mM reported

earlier (Siebert et al. 1979).

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OPTIMIZATION OF MALATE BIOSENSOR

Various kinetic properties of immobilized enzymes onto c-MWCNT/SPC electrode

were studied such as effect of pH, incubation temperature, effect of substrate

concentration, effect of co-factor concentration to optimize the working conditions of

enzyme electrode.

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EFFECT OF pH

The effect of pH on electrochemical

response of enzyme electrode was

studied in the pH range 5.0 to 9.0.

Highest current responses were

obtained between pH 7.0 to 8.0. At

below pH 7.0 and above pH 8.0, the

response of malate biosensor

decreases sharply. Therefore, pH 7.4

was used throughout the experiment.

The response current in terms of Awas measured.

Effect of pH on current response of

Enzyme/c-MWCNT/SPC electrode

based malate biosensor

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EFFECT OF TEMPERATURE

Each enzyme exhibits its maximum activity at a

particular incubation temperature. In the present study,the effect of incubation temperature was studied on

the activity of malic enzyme immobilized onto c-

MWCNT/SPCE and SPMWCNT electrode. The

optimal temperature of enzyme electrode was studied

by measuring the current response at different

temperatures from 20 to 50 oC. It showed that the

current response of the biosensor increased with

increasing temperature and reached a maximum at

approximately 35 oC and then went down as the

temperature turned higher, hence 35oC was selected

as optimum temperature.Effect of incubation temperature on

current response of Enzyme/c-

MWCNT/SPC electrode based malate

biosensor

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EFFECT OF SUBSTRATE CONCENTRATIONS

Effect of substrate concentration study of current

(A) responses at different malic acid

concentrations (mM) by malate biosensor based

on Enzyme/c-MWCNT/SPCE

Lineweaver-Burk plot for effect of malic acid

concentration on response of malate biosensor (Km)

based on NADP-specific malate dehydrogenase

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EFFECT OF CO-FACTOR

CONCENTRATIONS

Differential pulse

Mode = Potentiostatic

Current range= 100A

Initial potential = -0.6

End potential = -0.1

Interval time = 5 sec

Scan rate = 80 mV/sec

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DETECTION OF MALIC ACID IN

FRUITSConcentration of Malic acid in fruit samples was

determined by biosensor with the help of

enzyme/SPMWCNT electrode.

Tomato was taken as the model fruit, along with

three variety of apple and two variety of watermelon

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Model fruit - Tomato

(A) Unripened tomato (B) Partially ripened tomato (C) Ripened tomato

(D) Over ripened tomato

A B

CD

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CYCLIC VOLTAMETRY OF TOMATO

CV staircase

Mode = Potentiostatic

Current range = 100A

Start potential= -0.1

End potential = 0.4

Upper vortex = 0.4

Lower vortex = -0.1

Stop potential = -0.1

Scan rate = 80 mV/sec

CV of amperometric response studies of

malic acid in tomato fruit in PBS, pH 7.4.

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DPV OF TOMATO

Differential pulse

Mode = Potentiostatic

Current range = 100 A

Initial potential = -0.4

End potential = 0.4

Interval time = 5 sec

Scan rate = 80 mV/sec

DPV of amperometric response studies of malic

acid in tomato fruit in PBS, pH 7.4.

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ELECTROCHEMICAL RESPONSE OF MALATE BIOSENSOR

Standard graph for estimation of malic acid concentration by

malate biosensor

Fruit Weight (g) Current (A) Malic acid

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g (g) ( )

Concen(mM)

Unripened Tomato 10 1.1 0.012

Partially ripened

Tomato

10 6 0.16

Ripened Tomato 10 6.75 0.23

Over ripened Tomato 10 6.06 0.17

Watermelon 10 0.8 0.01

Sugar baby

watermelon

10 0.1 0.001

Chinese apple 10 6.69 0.22

USA apple 10 6.33 0.19

Indian apple 10 7.4 1.3

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STORAGE STABILITY AND REUSABILITY OF ENZYME ELECTRODE

The long term storage stability of

enzyme /c-MWCNT/SPC electrode

was investigated by measuring

current response of the biosensor

after every 30 days under its storage

in PBS buffer at 40C over a period of

6 months.

It was revealed that current

response of the biosensor

maintained 82% of the initial current

response even after regular 100

uses over a period of 180 day. Effect of storage at 4 0C on the response of

malate biosensor

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CONCLUSIONS

From the above results (Shown in Table) we concluded that Malic acid concentration

increased from unripened stage of tomato to ripened stage but it decreased in over

ripened stage.. Malic acid is the fruit maturity indicator so it can be a viable tool for

estimation of maturity.

Malic acid biosensor has response time 2 minute and sensitivity is 0.001 mM.

Kmax value for malic acid as calculated from Lineweaver-Burke plot was 0.19 mM

which is lower than 0.6 mM reported earlier (Siebert et al. 1979).

Indian apple variety contain high level of malic acid concentration than other two

variety of apple (chinese and USA apple).

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