purification and characterization of pectinase …

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Niharika et al. World Journal of Pharmacy and Pharmaceutical Sciences

PURIFICATION AND CHARACTERIZATION OF PECTINASE

(PECTIN METHYL ESTERASE) FROM APPLE POMACE

Niharika Sood1*, Abhishek Mathur

2

1*

Shri Venkateshwara University, Gajraula (U.P), India.

2Institute of Transgene Life Sciences, Dehradun (U.K), India.

ABSTRACT

Pectinases have extensive applications in extraction, clarification, and

cloud stabilization of fruit juices, in degumming and retting of natural

fibers. In the present study, Pectinases/ Pectin Methyl Esterase (PME)

was purified from apple pomace by consecutive steps of enzyme

extraction and further precipitated by saturated concentration of

ammonium sulphate. The results confirm that the enzyme can activated

at 45 to 55 °C and work well at a pH of 4.5 to 5.5. The enzyme was

further purified and characterized by HPLC. The purified pectinase

showed the retention time, 1.812 minutes with reference to standard

pectinases which showed the retention time, 1.790 minutes. Purified

pectinases were further utilized to observe the degradation of pectin in

pectin-agar plate. The zone of degradation of pectin by pectinases was observed by

overlaying 1% Iodine solution prepared in 95% ethyl alcohol. Purified pectinases degrade the

pectin in pectin-agar plates in comparison to control. The enzyme purified was loaded on

agarose gel and further stained with ruthenium red and coomassie brilliant blue separately.

The enzyme purified showed molecular weight 43 kDa as compared to standard marker. The

enzyme purified was qualitatively assessed for pectin degradation in fruits. It was observed

that in case of test, orange slice incubated with pectinases/PME, the pectin degrades and

converts the orange slice into juicy lump in comparison to control, in which orange slice was

treated with distilled water but not with enzyme. The juice/extract was purified and viscosity

was determined. It was found that enzyme treated sample (test) was more viscous in

comparison to control. The protein content in pectinases was determined by Bradford

method. It was found that total protein content per gram of apple was 1.7 g/apple sample. The

antimicrobial activity of purified pectinases was determined by well diffusion method. The

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 2.786

Volume 4, Issue 1, 749-764. Research Article ISSN 2278 – 4357

Article Received on

21 Oct 2014,

Revised on 15 Nov 2014,

Accepted on 08 Dec 2014

*Correspondence for

Author

Niharika Sood

Shri Venkateshwara

University, Gajraula (U.P),

India.


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results confirmed the strong antimicrobial potential against all the pathogens studied. The

results were found to be more surprising when the purified enzyme showed the maximum

activity against MRSA amongst all the pathogens studied.

KEYWORDS: Pectin, apple pomace, Pectin Methyl Esterase (PME)/Pectinase, protein

content, electrophoresis, HPLC, antimicrobial activity.

INTRODUCTION

Pectin is the complex polysaccharide found in fruits and vegetables and helps in ripening.

The highest concentration of pectin is found in the middle lamella of cell wall, with a gradual

decrease as one passes through the primary wall toward the plasma membrane. Apple is

particularly rich in pectin, the name applied to any one of a group of white, amorphous

complex carbohydrates with a high molecular weight. Researchers have found that raw

apples are the richest fruit sources in pectin. The characteristics structure of pectin is a linear

chain of a (β-1-4)-linked D-galacturonic acid that forms the protein-backbone, a

homogalacturonan. The highest concentrations of pectin are found in the middle lamella of

cell wall, with a gradual decrease as one passes through the primary wall toward the plasma

membrane. [1]

Pectin esterase catalyzes the deesterification of methyl ester linkages of

galacturonan backbone of pectic substances to release acidic pectins and methanol. [2]

The

resulting pectin is then acted upon by PG and PL. [3]

Production, biochemical

characterization, and applications of PNL have been reviewed extensively. [4]

Among all

pectinases, PNLs are of particular interest because these degrade pectin polymers directly by

a b-elimination mechanism that results in the formation of 4,5-unsaturated

oligogalacturonides, while other pectinases act sequentially to degrade the pectin molecule

completely. Pectin esterases are found in plants, plant pathogenic bacteria, and fungi [5]

while

PGs are widely distributed among fungi, bacteria, and many yeasts. [6]

Numerous studies on

fungal pectolytic enzymes have been carried out and several fungal PNL genes have been

isolated and characterized from Aspergillus niger, A. oryzae and Glomerella cingulata.

(ramie, hemp, flax, bast), maceration of plant tissues, isolation of protoplasts, and

saccharification of biomass.[7-10]

Antimicrobial and antioxidant activities of the pectin

extracted from apple and pomace were determined. [11]

In the present study, PME (Pectinases)

was purified and characterized from apple pomace. The enzyme was assayed for its

biochemical and physical properties. The enzyme was evaluated for its antimicrobial profile

against pathogens and drug resistant strains.


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MATERIALS AND METHODS

All the chemicals and reagents used in the experiments were procured from C.D.H and

Ranchem. Glass wares used were of Borosil. The media and broth used for microbial culture

was procured from Hi-Media Ltd., Mumbai.

Collection of Sample

The apple fruit samples were collected in sterilized conditions from local gardens/retail

market in sterilized polythene bags and were stored at 4 0

C in a refrigerator until use.

Extraction and Purification of Pectinases (Pectin methyl Esterase)

The enzyme was produced by using the optimized conditions and strain, Aspergillus niger

was inoculated in apple pomace in solid state fermentation (SSF). For SSF, 800 g of

moistened apple pomace was supplemented with optimum concentration of best nitrogen

source. The flask was incubated for 48-72 h. After incubation, the whole content of the flask

was separately and enzyme was extracted with 0.25 M NaCl at 40 ml/100 ml of medium. The

extract was filtered through muslin cloth and was centrifuged at 10,000 rpm for 30 minutes to

obtain the clear extract of the enzyme. [12]

Pectin esterase in the crude extract was precipitated

between 20-80% saturation of ammonium sulphate. The active fractions were pooled out and

were stored at 40C. The partially purified enzyme preparation was used for characterization as

well as for evaluation of its activity and other related procedures. Further optimum pH and

temperature was determined for proper functioning of the enzyme.

Characterization of Pectinases (Pectin Methyl Esterase) via Chromatographic and

Spectroscopic Techniques

Characterization of pectinases (pectin methyl esterase) purified from apple pomace was

carried out by spectrophotometric methods as described. [13]

Purification and characterization

of Pectinases was done by using a combination of different techniques including HPLC and

FT-IR.

High-performance Liquid Chromatography (HPLC)

HPLC analysis was performed in Roorkee Research and Analytical Laboratory Pvt. Ltd.,

Roorkee (Uttarakhand), India using a Shimadzo LC- 2010 HPLC system (Kyoto, Japan),

equipped with a Shimadzo LC 2010 UV-VIS detector with a thermostated flow cell and a

selectable two wavelengths of 190 - 370 nm or 371–600nm. The detector signal was

recorded on a Shimadzo LC2010 integrator. The column used was a Chiral Column block


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heating-type Shim-pack VP-ODS (4.6 mm interior diameter × 150 mm long) with a particle

size of 5 μm. Mobile phase was used containing 50 % acetonitrile along with 50 % Phosphate

buffer was used at a flow rate of 3.0 ml/min, column temperature 25°C. Injection volume was

40 µl and detection was carried out at specific wavelength having maximum absorbance as

calculated by UV absorption spectra at maximum wavelength.

Fourier Transform Infrared (FTIR) Studies

The IR spectrum of purified enzyme was recorded in Roorkee Research and Analytical

Laboratory Pvt. Ltd., Roorkee (Uttarakhand), India using a computerized FTIR spectrometer

(Perkin Co., Germany) in the range of 4400–400 cm-1

by the KBr pellet technique.

SDS-PAGE Analysis and Zymography of Pectinases

The protein profile and the presence of enzyme were confirmed by SDS-PAGE analysis. [14]

The SDS-PAGE was performed in 13 % w/v gels and samples were heated for 10 minutes at

450C in the sample buffer before loading in the wells. After electrophoresis, gels was soaked

in 2.5%w/v Triton-X 100 for 30 minutes, washed in 100 mM Glycine buffer pH, 10.0, 1.5

mM CaCl2 for 30 minutes and overlaid with 1% agarose gel containing 0.1%

polygalacturonic acid in the same buffer as above. After 30 minutes of incubation at 450C,

gel was stained with 0.05% w/v ruthenium red (Sigma) for 10 minutes and was washed with

water until PME bands became visible. Further pectinases bands were also observed by

staining gel with Coomassie brilliant blue. [14]

Qualitative Assays of Pectinases Enzyme

(a) For ripening of raw fruits and pectin degradation

The efficiency of extra cellular activity of pectinases enzyme was determined as per the

method prescribed. [15]

Fine orange slices of 20g weight were introduced in two beakers. In

one beaker, 5-10 ml of partially purified pectinase was added and in the other beaker,

distilled water was introduced in same quantity. This set up (after stirring the contents well

enough) was placed inside the boiling water bath at 400C for 30 minutes. With the help of

filter paper, the juicy watery part was filtered from the beakers.

(b) For Degradation of Pectin in Pectate-Agar Plate

The enzyme assay of pectinases was performed using Pectate agar plates. [16]

The agar plates

were prepared using 0.5% pectin with 1.5% agar. A 10 mm diameter well was punched

aseptically with the help of sterile borer. The wells were filled with different concentrations


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of the pectinases enzyme sample will be incubated at 370C for overnight. The substrate

utilization zone was observed by overlaying 1% Iodine solution prepared in 95% ethyl

alcohol and was kept for 10-15 minutes for the appearance of zones.

3.6 Quantitative Estimation of Protein Content in Pectinases for Enzymatic Activity

Pectinases enzyme was assayed by using TBA (0.01M)/ HCl (1N). One ml of crude enzyme

was added to 1.5ml of substrate (1.2% pectin in 0.2M Tris-HCl, pH 8.5) and was incubated at

350C for 1 hour. The reaction mixture was terminated by adding 1.5ml of 0.01M TBA and

0.7ml of 1N HCl. The contents were boiled in water bath for 20 minutes. Absorbance then

after was measured at 540nm in UV-VIS spectrophotometer. The total protein content will be

measured using Bradford method. One ml of enzyme sample was mixed with 5 ml of

Bradford reagent. BSA (100 µg/ml) was used as standard.

Determination of in Vitro Antimicrobial Activity of Pectinases Enzyme Purified

The pectinases enzyme as such (in dilutions) was used for determination of in vitro

antimicrobial activity.

Culture Media

The media used for antibacterial and antifungal test was Nutrient agar/broth and Sabouraud’s

dextrose agar/broth respectively procured from Hi media Pvt. Bombay, India.

Inoculum

The pure pathogenic bacteria cultures were separately inoculated into nutrient broth and

incubated at 37 0

C for 4 h and the suspension will be checked to provide approximately 105

CFU/ml. Similar procedure was done for fungal strains by inoculating in Sabouraud’s

dextrose broth for 6 h.

Microorganisms Used

Pure cultures of various pathogenic bacterial and fungal strains, E. coli NCIM 2065,

Lactobacillus plantarum NCIM 2083, Micrococcus luteus ATCC 9341, Salmonella abony

NCIM 2257, Candida albicans NCIM 3471, Aspergillus niger NCIM 1196 and Multidrug

resistant (MDR) strains of Staphylococcus aureus isolated from clinical specimens viz. pus

and blood of infected patients were procured with authentication for the study.


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Agar Well Diffusion Method

The agar well diffusion method was modified. [17]

Nutrient Agar medium (NAM) was used

for bacterial cultures. The culture medium was inoculated with the bacterial strains separately

suspended in nutrient broth. Sabouraud’s dextrose agar (SDA) was used for fungal cultures.

The culture medium was inoculated with the fungal strains separately suspended in

Sabouraud’s dextrose broth. A total of 8 mm diameter wells were punched into the agar and

filled with pectinases (100 µl) and solvent blank (N-saline) separately. Standard antibiotic

(Azithromycin/Erythromycin, concentration 1mg/ml) were used as positive control. The

bacterial plates were then incubated at 37oC for 18 h. The antibacterial activity was evaluated

by measuring the diameter of zone of inhibition observed. The same procedure was used for

determining antifungal activity but in this case standard antibiotic (Fucanazole, concentration

1 mg/ml) was used as positive control and fungal plates were incubated at 37oC for 72 h.

RESULTS

Purification and Characterization of PME (Pectinases)

Pectinases/ Pectin Methyl Esterase (PME) was purified from apple pomace by consecutive

steps of enzyme extraction and further precipitated by saturated concentration of ammonium

sulphate. The extraction process is shown in Image 1. The results also confirm that the

enzyme can activated at 45 to 55 °C and work well at a pH of 4.5 to 5.5. The enzyme was

further purified and characterized by HPLC. The purified pectinase showed the retention

time, 1.812 minutes with reference to standard pectinases which showed the retention time,

1.790 minutes. The HPLC chromatograms of purified pectinase and standard are recorded in

Figure 1 (a &b). Further FT-IR spectra were recorded of the purified pectinases. The FT-IR

spectrum of PME is shown in Figure 2.

SDS PAGE Analysis and Zymography of PME (Pectinases)

The enzyme purified was loaded on agarose gel and further stained with ruthenium red and

coomassie brilliant blue separately. The enzyme purified showed molecular weight 43 kDa as

compared to standard marker. The results are shown in Figure 3 and 4.

Qualitative Assays of Pectinases Enzyme

The enzyme purified was qualitatively assessed for pectin degradation in fruits. It was

observed that in case of Test, orange slice incubated with pectinases/PME, the pectin

degrades and converts the orange slice into juicy lump in comparison to Control, in which

orange slice was treated with distilled water but not with enzyme. The juice/extract was


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purified and viscosity was determined. It was found that enzyme treated sample (test) was

more viscous in comparison to control. The results are shown in Figure 5.

Purified pectinases were further utilized to observe the degradation of pectin in pectin-agar

plate. The zone of degradation of pectin by pectinases was observed by overlaying 1% Iodine

solution prepared in 95% ethyl alcohol. Purified pectinases degrade the pectin in pectin-agar

plates in comparison to control. The results are shown in Figure 6.

Quantitative Estimation of Protein Content in Pectinases

The protein content in pectinases was determined by Bradford method. It was found that total

protein content per gram of apple was 1.7 g/apple sample. The results are shown in Table 1

and Figure 7.

Antimicrobial Activity of Pectinases

The antimicrobial activity of purified pectinases was determined by well diffusion method.

The results confirmed the strong antimicrobial potential against all the pathogens studied.

The results were found to be more surprising when the purified enzyme showed the

maximum activity against MRSA amongst all the pathogens studied. The results are shown in

Table 2 and Figure 8 (a & b).

Image 1: Extraction of pectinase from apple pomace.


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Figure 1 (a): HPLC chromatogram of standard pectinases (PME)

Figure 1 (b): HPLC chromatogram of purified pectinases (PME)


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Figure 2: FT-IR spectra of purified pectinases (PME)

Figure 3: Electrophoretogram showing bands of PME stained with ruthenium red

showing molecular weight 43 kDa (MM, Marker; CE, Enzyme fraction before

ammonium sulphate precipitation; CM, Enzyme Fraction after ammonium sulphate

precipitation; Sdex 75, Final Gel chromatography fraction)


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Figure 4: Electrophoretogram showing single band of PME stained with Coomassie

brilliant blue showing molecular weight 43 kDa.

Figure 5: Conversion of orange juice by purified pectinases.


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Figure 6: Degradation of pectin by purified pectinases on pectin-agar plate.

Table 1: Estimation of protein content in pectinases by Bradford Method.

Figure 7: Protein content in purified pectinases.

S.No. BSA aliquots/Sample

(ml)

Phosphate buffer

(ml)

Bradford’s

reagent (ml)

Absorbance

(540 )

1. Blank 1.0 5 0.00

2. 0.2 0.8 5 1.27

3. 0.4 0.6 5 1.32

4. 0.6 0.4 5 1.42

5. 0.8 0.2 5 1.43

6. 1.0 ---- 5 1.47

7. Test; Purified enzyme

(1.0)

---- 5 1.43


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Table 2: Antimicrobial activities of purified pectinases.

Sample Diameter of zone of inhibition (mm)

Pathogens studied

E.coli S. abony M. luteus L. plantarum MRSA 35 MRSA 8 A. niger C.albicans

Purified

Pectinases/

PME

22 11 11 13 30 35 38 21

Erythromycin

(1 mg/ml) 27 28 32 33 30 NA NT NT

Fucanazole

(1 mg/ml) NT NT NT NT NT NT 24 12

*NA, No Activity; NT, Not Tested

Figure 8 (a): Graphical representation of antimicrobial spectrum of purified pectinases.


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Figure 8 (b): Antimicrobial spectrum of purified pectinases.

DISCUSSION

Previous studies already have already documented the enormous industrial significance of

enzyme pectinases/PME. Apple pomace is more susceptible to microbial decomposition and

thus acts as an ideal substrate for the production of pectinases. Pectinases constitute a unique

group of enzymes which catalyze the degradation of pectic polymers present in the plant cell


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walls. [18]

Pectinases are produced by many organisms such as bacteria, [19]

fungi [20]

and

yeasts. [21]

In the industrial sector, acidic pectinases are used in the extraction and clarification

of fruit juices, whereas alkalophilic pectinases are in immense use in the degumming of ramie

fibers, [22]

retting of wax, [23]

plant protoplast formation and treatment of effluents discharged

from fruit processing units. Pectinase enzyme which includes pectin methyl esterase and

depolymerizing enzymes find extensive application in fruit processing industries for

clarification of fruit juices and wines, in the manufacturing of pectin free starch, curing of

coffees, cocoa and tobacco, refinement of vegetable fibres and is used as an analytical tool

for the estimation of plant products. [24-26]

Pectinase is produced by several fungi but

Aspergillus is found to be the most prominent source. [19]

Therefore the study was undertaken

to reveal the various pharmacological properties of pectinase. Although the present studies

also adds a new chapter in the pharmacological importance and significance of enzyme,

pectinases/Pectin methyl esterase. The present investigation emphasized the role of apple and

other fruits for intake of pectinases which can be utilized as an effective strategy in treatment

of severe and non treatable microbial infections. Apart from this the purified pectinases has

molecular weight of 43 kDa which can be utilized as a standard protein marker in

determination of other unknown proteins and enzymes. The results also confirm that the

enzyme can activated at 45 to 55 °C and work well at a pH of 4.5 to 5.5. The enzyme can be

utilized in extraction of fruit juices without adding synthetic extractants in fruits. The work

thus can create a stepping stone in area of enzyme research. Further studies can be done in

order to investigate other pharmacological properties of the enzyme.

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