Protein Expression and Purification 87 (2013) 11–16

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Protein Expression and Purification

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Purification and physicochemical properties of polygalacturonase fromAspergillus niger MTCC 3323

Shashi Kant, Anuja Vohra, Reena Gupta ⇑Department of Biotechnology, Himachal Pradesh University, Summer Hill, Shimla 171005, India

a r t i c l e i n f o

Article history:Received 21 July 2012and in revised form 22 September 2012Available online 13 October 2012

Keywords:PolygalacturonasePurificationGel filtration chromatographySDS and NATIVE PAGEClarification

1046-5928/$ - see front matter � 2012 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.pep.2012.09.014

⇑ Corresponding author. Fax: +91 177 2831948.E-mail address: reenagupta_2001@yahoo.com (R.

1 Abbreviations used: PG, polygalacturonase; GRAS,PDA, Potato Dextrose Agar; PGA, polygalacturonic acid;TSS, total soluble solids.

a b s t r a c t

Polygalacturonases are the pectinolytic enzymes that catalyze the hydrolytic cleavage of the polygalac-turonic acid chain. In the present study, polygalacturonase from Aspergillus niger (MTCC 3323) was puri-fied. The enzyme precipitated with 60% ethanol resulted in 1.68-fold purification. The enzyme waspurified to 6.52-fold by Sephacryl S-200 gel-filtration chromatography. On SDS–PAGE analysis, enzymewas found to be a heterodimer of 34 and 69 kDa subunit. Homogeneity of the enzyme was checked byNATIVE-PAGE and its molecular weight was found to be 106 kDa. The purified enzyme showed maximumactivity in the presence of polygalacturonic acid at temperature of 45 �C, pH of 4.8, reaction time of15 min. The enzyme was stable within the pH range of 4.0–5.5 for 1 h. At 4 �C it retained 50% activity after108 h but at room temperature it lost its 50% activity after 3 h. The addition of Mn2+, K+, Zn2+, Ca2+ andAl3+ inhibited the enzyme activity; it increased in the presence of Mg2+ and Cu2+ ions. Enzyme activitywas increased on increasing the substrate concentration from 0.1% to 0.5%. The Km and Vmax values ofthe enzyme were found to be 0.083 mg/ml and 18.21 lmol/ml/min. The enzyme was used for guava juiceextraction and clarification. The recovery of juice of enzymatically treated pulp increased from 6% to 23%.Addition of purified enzyme increased the %T650 from 2.5 to 20.4 and �Brix from 1.9 to 4.8. The pH of theenzyme treated juice decreased from 4.5 to 3.02.

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Introduction

Pectinases are a heterogeneous group of related enzymes thathydrolyze the pectic substances, present mostly in plants. Theyare of prime importance for plants as they help in cell wall exten-sion [1] and softening of some plant tissues during maturation andstorage. Pectinases are among the most important industrial en-zymes [2]. Based on mode of action and preferred substrate theseenzymes are classified into esterases, eliminative depolymerases(lyases) and hydrolytic depolymerases (polygalacturonases, PGs)1.PGs are the pectinolytic enzymes that degrade polygalacturonanpresent in the cell walls of plants by hydrolysis of the glycosidicbonds that link galacturonic acid residues. Endo-PGs (E.C. 3.2.1.15)bring about random hydrolysis of the polymer, whereas exo-PGs(E.C. 3.2.1.67) act sequentially from the non-reducing end [3]. PGsare widely distributed, among fungi, bacteria and many types ofyeast and also found in higher plants and some plant parasitic nem-atodes [4].

The enzyme preparations used in the food industry are mostlyof fungal origin because fungi are potent producers of pectic en-

ll rights reserved.

Gupta).Generally Regarded As Safe;TPP, three-phase partitioning;

zymes and the optimum pH of fungal enzymes is very close tothe pH of many fruit juices, which range from pH 3.0 to 5.5 [5].Aspergillus niger is the most commonly used fungal species forindustrial production of pectinolytic enzymes [6]. A. niger pectinas-es are most widely used in industries because this strain possessesGRAS (Generally Regarded As Safe) status so that metabolites pro-duced by this strain can be safely used.

Acidophilic pectinases are used in apple juice preparation andclarification, to facilitate pressing and juice extraction. Moreover,pectic enzymes are used to reduce haze or gelling of grape juicein wine manufacture and to enhance the quality of cider applevarieties that are bitter, sweet, or sour [7,8]. The alkaline pectin-ases also have various industrial applications, such as wastewa-ter treatment, paper manufacturing, oil extraction, coffee and teafermentation, processing and degumming of many plant fibers[8]. Although pectinases are used in industrial processes in crudeform, their purification and knowledge of the biochemical char-acteristics of these enzymes are essential for an understandingof their structure and mechanisms of action and thermostability.The present study reports the purification, characterisation andapplication of polygalacturonase (PG) from A. niger MTCC 3323.Earlier, immobilization of polygalacturonase from same organismwas carried out in our laboratory to study its application in ap-ple juice clarification [9].

12 S. Kant et al. / Protein Expression and Purification 87 (2013) 11–16

Material and methods

Material

The polygalacturonase producing fungus A. niger was isolatedfrom rotten banana peel and the organism was identified at Micro-bial Type Culture Collection, IMTECH, Chandigarh. All the chemi-cals used in the present investigation were either procured fromSigma Aldrich (USA) or Himedia (Mumbai, India) and were of highpurity analytical grade. Polygalacturonic acid from Citrus Fruit(Sigma) was used as substrate and D-Galacturonic acid was usedas a standard.

Methods

Maintenance of A. niger and polygalacturonase production

The fungal culture was maintained on Potato Dextrose Agar(PDA) slants and incubated at 30 �C for 5 days. The heavily sporu-lated slants were stored at 4 �C. Subculturing was done after every45 days. The spores were harvested by adding 2 ml sterile salinecontaining 0.01% Tween 20 to a sporulated slant. About 2 � 105

spores were found to be suitable for better production of enzyme.Production medium consisting of KCl (0.05%), MgSO4�7H2O (0.1%),Na2HPO4 (0.1%), KH2PO4 (0.1%), Yeast extract (0.1%), Caseinhydrolysate (0.1%) and Citrus Pectin (1%) was used. The pH of theproduction medium was adjusted to 3.5 with 0.1 N HCl. Themedium was autoclaved at 15 psi for 20 min. 25 ml of sterileproduction medium was taken in 250 ml conical flasks. About2 � 105 spores were inoculated in the medium and were incubatedin an Orbitek Rotary Shaker at 30 �C at 150 rpm. After 72 h, 12.5 mlof 2X production medium was fed to the growing culture. Theflasks were again incubated at 30 �C. After 96 h, the culture washarvested and filtered through Whatman No. 1 filter paper. Thefiltered, cell free broth was used as crude enzyme preparation.

Enzyme assay

The PG activity was assayed by estimating the amount of reduc-ing sugar released under assay conditions. The reducing sugar wasquantified by a standard colorimetric method [10] and [11]. Thesubstrate used for assay was 0.5% polygalacturonic acid (PGA) thatis, 0.5 g of PGA in 100 ml of 0.05 M citric acid-sodium citrate buffer,pH 4.8. The assay mixture consisted of 980 ll of freshly preparedsubstrate and 20 ll of enzyme was incubated at 45 �C for 15 min.The reaction was stopped by keeping the reaction mixture in boil-ing water bath for 3 min. To the reaction mixture, 500 ll of alkalinecopper tartrate reagent was added and tubes were again kept inboiling water bath for 20 min. The tubes were cooled and 500 llof arsenomolybdate reagent was added and thoroughly mixed.The absorbance was recorded at 620 nm (using Lab India spec-trometer) against a blank consisting of 1 ml citrate buffer, 500 llalkaline copper tartrate reagent, and 500 ll arsenomolybdate re-agent. The amount of galacturonic acid released was calculatedfrom standard curve of galacturonic acid. One unit of enzyme activ-ity was defined as the amount of enzyme required to release onemicromole of galacturonic acid per ml per minute under standardassay conditions.

Determination of protein concentration

The protein concentration was determined using Bovine SerumAlbumin as standard by the method of Lowry et al. [12].

Purification by gel-filtration (Sephacryl S-200) chromatography

PG was purified by ethanol precipitation (60%) and Gel-Filtra-tion (Sephacryl S-200) chromatography. The crude enzyme wasprecipitated by adding 60% of chilled ethanol and allowed to pre-cipitate overnight at 4 �C. The solution was centrifuged at15,000g for 15 min at 4 �C. The pellet obtained was dissolved inminimum volume of assay buffer (sodium-citrate buffer), pH 4.8.The ethanol-precipitated enzyme was centrifuged; supernatantwas taken and filtered through 0.45 l filter. This filtered enzymewas loaded to the column. All eluted fractions of 2.0 ml volumewere collected and measured at 280 nm for protein and assayedfor enzyme activity. The fractions showing maximum polygalactu-ronase activity were pooled for further studies. The relative molec-ular weight of the purified enzyme was estimated by SDS–PAGE(12%) and NATIVE-PAGE (8%) according to the method of Laemmli[13] using a Mini GEL electrophoresis system (Bangalore GeneiPvt., Ltd., Bangalore, India). Proteins were stained with coomassiebrilliant blue R-250. Molecular mass of purified PG was estimatedusing a standard ‘‘protein marker’’ of known molecular mass 14.3–97.4 kDa for SDS–PAGE and 66–669 kDa for NATIVE PAGE.

Characterization of polygalacturonase

Optimization of temperature, pH and incubation time of enzymeThe optimum temperature and pH of the enzyme were deter-

mined by measuring the PG activity at various temperatures rang-ing from 30 to 60 �C and at different pH (3.0, 3.6, 4.2, 4.8, 5.0, 5.5and 6.0) in 0.05 M sodium-citrate buffer (pH 4.8) with PGA as sub-strate. Optimum reaction time was determined by incubating thereactants for different time intervals (5, 10, 15, 20, 25, 30 and35 min) and performing the assay for PG activity at optimized tem-perature and pH.

pH stability of enzyme

In order to determine the pH stability, the enzyme was preincu-bated in 0.05 mM sodium-citrate buffer of different pH (3.5, 4.0,4.5, 4.8, 5.0, 5.5, 6.0 and 6.5) at 4 �C for 1 h and then assayed forpolygalacturonase activity in 0.05 M sodium-citrate buffer (pH4.8) with PGA as substrate.

Stability profile of enzyme at different temperatures

The enzyme PG was incubated at 4, 25, 45 �C in sodium-citratebuffer (0.05 M, pH 4.8). At 4 �C its activity was determined from time0 h till 120 h with an interval of 12 h. At 25 �C its activity was deter-mined from 0 h till 6 h with an interval of 1 h and at 45 �C its activitywas assayed from 0 h till 1.5 h with an interval of 15 min. Enzymeactivity was used to determine the stability profile.

Effect of metal ions on the enzyme activity

The assay was performed in the presence of various metal ionsat a final concentration of 1 mM in 50 mM sodium-citrate bufferpH 4.8. The reaction mixture in which metal ion was not addedwas used as control.

Determination of substrate specificity and substrate concentration

To determine the substrate specificity, different substrates i.e.polygalacturonic acid, citrus pectin, potato dextrose agar, applepectin and amylopectin 0.5% each in 0.05 M sodium-citrate bufferwere used and to determine the optimum substrate concentration,different concentrations of substrate 0.1%, 0.2%, 0.3%, 0.4%, 0.5%,0.6% and 0.7% were used.

Table 1Purification of polygalacturonase produced extracellularly by Aspergillus niger.

Purification steps Total activity (U) Total protein (mg) Specific activity (U/mg) Fold purification Yield (%)

Crude 4284 514.5 8.32 1 100Ethanol precipitation (60%) 264.74 18.9 14 1.68 6.17Sephacryl S-200 column chromatography 215.04 3.96 54.3 6.52 5.01

1 2 3

20.1 kDa

29 kDa

43 kDa

66 kDa

97.4 kDa

34 kDa

69 kDa

Fig. 1a. SDS–PAGE of purified polygalacturonase from Aspergillus niger showing 69and 34 kDa subunit size LANE 1: Bangalore Genei Protein Molecular Weight Marker(medium range), Phosphorylase b – 97.4, Bovine serum albumin – 66.0, Ovalbumin– 43.0, Carbonic anhydrase – 29.0, Soyabean trypsin inhibitor – 20.0, Lysozyme –14.3. LANE 2: Ethanol precipitated enzyme LANE 3: Concentrated fraction showingheterodimer from S-200 column.

1 2 3 4

66 kDa

440 kDa

232 kDa

140 kDa

106 kDa

669 kDa

Fig. 1b. Native PAGE of purified polygalacturonase from Aspergillus niger showingmolecular weight of 106 kDa LANE 1: Amersham Biosciences UK Limited Marker,Thyroglobulin – 669, Ferritin – 440, Catalase – 232, Lactate dehydrogenase – 140,Albumin – 66. LANE 2: Purified polygalacturonase from Sephacryl S-200 Column.The amount of protein loaded was 0.3 mg/ml. LANE 3 and 4: Lanes 3 and 4 areduplicates. Each contains purified polygalacturonase from Sephacryl S-200 Column.The amount of protein loaded was 0.9 mg/ml.

S. Kant et al. / Protein Expression and Purification 87 (2013) 11–16 13

Determination of Km and Vmax values

Km and Vmax values of the enzyme were determined by measur-ing the reaction velocity at different concentrations of the sub-strate (PGA) 0.1–0.7% (w/v). The reciprocal of the reactionvelocity (1/V) was plotted against the reciprocal of the substrate

concentration (1/[S]) to determine the Km and Vmax values by theLineweaver–Burke plot.

Effect of thiols and phenolics

The assay was performed in the presence of various thiols andphenolic acids at a final concentration of 1 mM in 50 mM so-dium-citrate buffer, pH 4.8 by pre-incubating the enzyme for5 min at room temperature before starting the reaction with PGA.

Application of purified enzyme in guava juice extraction andclarification

Extraction of juice: Guava fruits were purchased from the localmarket and stored at 4 �C until used. The juice was extracted fromdried fruits using a lab mixer for 2–3 min until a homogeneousfruit pulp was obtained. The different amount of purified enzyme(8.96, 17.92, 26.88, 35.84 and 44.80 U) was added to pulp of guavataken in different flasks. The control without enzyme was keptalong with other samples at 45 �C for 4 h of incubation for extrac-tion of juice. Each treated pulp was then filtered in muslin clothand juice obtained was analyzed for juice yield.

Juice clarification: The different amount of purified enzyme(8.96, 17.92, 26.88, 35.84 and 44.80 U) was added to extractedjuice (15 ml each) and kept overnight at room temperature. Trea-ted juices were analyzed for change in pH, �Brix and %T650.

Results and discussion

Purification of polygalacturonase

Crude enzyme was saturated with 60% of chilled ethanol whichindicated 1.68-fold purification of the enzyme at the precipitationstage (Table 1). During the studies on partial purification of anextracellular pectin lyase from a strain of A. niger, Yadav and Sha-stri [14] obtained a fold purification of 1.3% with 60% ethanol pre-cipitation. In an earlier study, the fold purification ofpolygalacturonase from Mucor circinelloides was found to be 2.12with 60% ethanol precipitation [15].

The enzyme was purified about 6.52-fold with an increase inspecific activity to 54.3 U/mg giving a yield of 5.01% (Table 1). Inanother study 6.1-fold purification of polygalacturonase from A. ni-ger U-86 has been reported using Sephadex G-75 column [16]. Exo-polygalacturonase enzyme produced by Aspergillus sojae ATCC20235 was purified using three-phase partitioning (TPP), anemerging bio-separation technique where a single step as com-pared to the classical multi-step purification was used and the en-zyme was purified to 6.7-fold [17].

On SDS–PAGE analysis, enzyme was found to be heterodimer of69 and 34 kDa subunits (Fig. 1a). The Native PAGE gave a singleband indicating that enzyme was purified to homogeneity. Themolecular weight of the purified enzyme was estimated to be106 kDa using Gel doc analysis (Fig. 1b). Three multiple forms ofpolygalacturonase (PG) namely PGI, PGII and PGIII were isolated,purified and characterized from ripe mango (Mangifera indica cv.Dashehari) fruit. Native molecular weights of PGI, PGII and PGIIIwere found to be 120, 105 and 65 kDa respectively. On SDS–PAGE

Fig. 2a. Effect of temperature on activity of polygalacturonase from Aspergillus nigerdepicting optimum activity of 17.52 at a temperature of 45 �C.

Fig. 2b. Effect of pH on activity of polygalacturonase from Aspergillus niger showingoptimum activity of 17.95 at pH of 4.8.

Fig. 2c. Effect of incubation time on activity of polygalacturonase from Aspergillusniger at maximum pH and temperature depicting maximum activity of 17.89 at15 min time of incubation.

Table 2pH stability of polygalacturonase from Aspergillus niger.

pH Enzyme activity (U/ml) Relative activity (%)

3.5 9.21 51.454.0 11.71 65.414.5 14.4 80.444.8 17.9 1005.0 13.19 73.685.5 11.25 62.846.0 8.79 49.106.5 5.79 32.34

⁄Enzyme was preincubated at different pH values at 4 �C for 1 h.

18.15 17.8916.18

15.4414.34

13.812.15

11.5710.44

9.318.2

0

2

4

6

8

10

12

14

16

18

20

0 12 24 36 48 60 72 84 96 108 120

Enz

yme

acti

vity

(U/m

l)

Time (h)

Fig. 3a. Stability of polygalacturonase from Aspergillus niger at 4 �C. The enzymewas fairly stable at 4 �C up to 108 h, as it retained more than 50% of its activity andafter 48 h it retained about 79% of its activity.

14 S. Kant et al. / Protein Expression and Purification 87 (2013) 11–16

analysis, PGI was found to be a homodimer of subunit size 60 kDaeach while those of PGII and PGIII were found to be heterodimersof 70, 35 and 38, 27 kDa subunit size each, respectively [18]. Exo-enzymes secreted by some microorganisms had varied molecularweight polygalacturonases e.g. from Paecilomyces variotii(39.4 kDa) [19], Streptomyces erumpens (63 kDa) [20], Sclerotiniasclerotiorum (82 and 56 kDa) [21].

Characterization of polygalacturonase

The purified PG exhibited optimum activity at a temperature of45 �C as depicted in Fig. 2a. Earlier similar results were obtained forPG from same organism in our laboratory [10]. In the previousstudy PGs produced from M. circinelloides showed optimum

temperature of 42 �C [15]. The optimum pH of the purified enzymewas found to be 4.8 as shown in Fig. 2b. The PG activity was foundto be near optimum pH at pH of range 3.6–5.5. In a previous studysame pH was observed to be optimum for PG from same organismin our laboratory [10]. The results are very close to those reportedfor PGs from T. harzianum [22], T. reesei [23]. The PG activity atoptimum temperature and pH was found to be maximum at15 min time of incubation (Fig. 2c). The enzyme was stable withinpH range of 4.0 to 5.5 for 1 h (Table 2). PGs produced from fungusAspergillus niveus showed pH stability between 3.0 and 5.0 [24].

The enzyme was fairly stable at 4 �C up to 108 h, as it retainedmore than 50% of its activity (Fig. 3a). After 48 h it retained about79% of its activity and after 120 h enzyme had residual activity of45%. At 25 �C the enzyme was found to be fairly stable 2 h only;it retained about 88% of its activity after 1 h and about 68% of itsactivity after 2 h and after 6 h it lost 72% of its activity (Fig. 3b).At 45 �C the relative activity after 30 min of incubation was ob-served to be 45.23%, i.e., it lost more than half of its activity(Fig. 3c). A further increase in incubation time dramatically re-duced the enzyme activity with only 6.8% of its activity remainingafter 75 min of incubation. PG produced from Mucor flavus hasbeen reported to be stable at 40 �C for 4 h [25].

Among various metal ions, Mn2+, K1+, Zn2+, Ca2+, Fe2+ and Al3+

inhibited the enzyme activity, while activity increased in the pres-ence of Mg2+ and Cu2+ ions (Table 3). But in case of Hg2+the enzymeactivity decreased to greater extent. The effects of different metalcations at the concentration of 1 mM on P. ostreatus PGs assay sys-tem were studied by Rashad et al., [26]. The effectiveness of metalcations as inhibitors for PG was in the order of Ba2+ < Co2+ < Zn2+ <Ni2+ < Ca2+ < Cu2+ < Mg2+ < Hg2+ < Fe2+ with 6.5%, 15.3%, 26.5%,29.4%, 33.0%, 48.2%, 56.3%, 90.4% and 95.82% inhibition, respec-tively. Among different substrates studied the PGs showed maxi-mum activity towards polygalacturonic acid, followed by apple

Table 4Substrate specificity of polygalacturonase from Aspergillus niger.

Substrate (0.5%) Enzyme activity (U/ml)

Poly-galacturonic acid 18.19Citrus pectin 6.4Apple pectin 7.1Potato dextrose agar 2.29Amylopectin 5.31

Fig. 4. Km and Vmax of polygalacturonase from Aspergillus niger. The Km and Vmax

values of the enzyme were found to be 0.083 mg/ml and 18.21 lmol/ml/min.

Fig. 3b. Stability of polygalacturonase from Aspergillus niger at 25 �C. The enzymewas found to be fairly stable at 25 �C up to 2 h only as it retained about 88% of itsactivity after 1 h and about 68% of its activity after 2 h.

Fig. 3c. Stability of polygalacturonase from Aspergillus niger at 45 �C. The relativeactivity after 30 min of incubation at 45 �C was observed to be 45.23%, i.e., it lostmore than half of its activity.

Table 3Effect of metal ions on activity of polygalacturonase from Aspergillus niger.

Metal ion (1 mM) Enzyme activity (U/ml) Relative activity (%)

Mn2+ 12.21 67.49Ca2+ 13.21 73.02Mg2+ 20.18 111.55K1+ 10.1 55.83Cu2+ 18.89 104.42Fe2+ 9.8 54.14Hg2+ 6.2 34.27Zn2+ 7.8 43.11Al3+ 13.2 72.96Control (no metal ion) 18.09 100

S. Kant et al. / Protein Expression and Purification 87 (2013) 11–16 15

pectin and citrus pectin. PGs showed minimum activity towardspotato dextrose agar (Table 4). The polygalacturonase from M. cir-cinelloides ITCC 6025 [15] showed maximum activity with PGA(0.1% w/v), but it decreased with all other substrates.

Enzyme showed increase in reaction spped, on increasing thesubstrate concentration from 0.1% to 0.5%, on increasing the sub-strate concentration further there was no increase in activity; itmight be due to saturation condition of enzyme. The Km of PG fromA. niger was 0.083 mg/ml and Vmax 18.21 lmol of galacturonic acid/ml/min (Fig. 4). In previous study, the Michaelis constant (Km)value of the pure P. ostreatus PG was 1.33 mg/ml using pectin asa substrate while its Vmax was 28.6 lmol/ml/min [26]. All the thiols

tested had inhibitory effect on activity of enzyme (Table 5). OnlyL-cystine showed enzyme activity of 12.34 U in comparison to18.11 U of control. Mercuric chloride was the strongest inhibitorof enzyme at followed by ascorbic acid. The effect of phenolics asinhibitor for polygalacturonase was in the order of Cinnamicacid > Ferulic acid > p-Coumaric acid > Salicylic acid > Chlorgenicacid. Previously, comparable results have been obtained for theeffect of thiols and phenolics [15,27].

Application of the purified enzyme in juice extraction and clarification

While studying the effect of purified enzyme on guava juiceextraction it was found that increase in amount of enzyme in-creased the juice yield (Table 6). Addition of pectinases probablyhydrolysed the pectin into galacturonic acid that might have loos-ened the cell wall and released the sap thus, increased the juiceyield. In a recent study addition of pectinases at different concen-trations increased the juice recovery in all the fruits [28]. The addi-tion of enzyme increased the %T650 and �Brix (sugar content) but itdecreased the pH of the juice. The %T650 (clarification) and �Brix in-creased from 1.7 to 20.4 and 1.9–4.8, respectively. In a recentstudy, the apple juice treated with pectin methylesterase and com-mercial polygalacturonase showed the maximum clarification byincrease in %T650 from 1.7 to 5.6 [29]. Increase in �Brix might relateto greater degree of tissue breakdown, releasing more componentssuch as sugars. %T650 of treated juice increased because of removalof colloidal and suspended particles in the juice especially pectinpresent in the juice. The maximum juice clarification was observedwith 35.84 enzyme units. In a previous study on clarification ofapple juice using crude polygalacturonase from A. niger (sameenzyme as in present study) maximum increase in% Transmittance(85%) was observed at about 15 IU ml�1 with 0.01% gelatin [30] andoptimum temperature was found to be 45 �C [31]. In the present

Table 5Effect of thiols and phenolics on activity of polygalacturonase from Aspergillus niger.

Thiols (1 mM) Enzyme activity (U/ml) Relative activity (%)

L-Cystine 12.34 68.13

Ascorbic acid 4.72 26.06b-Mercapta ethanol 10.31 56.92Mercuric chloride 2.31 12.75Control 18.11 100

Phenolics (1 mM)Chlorogenic acid 10.21 56.37Cinnamic acid 6.21 34.29p-Coumeric acid 7.34 40.53Ferulic acid 7.29 40.25Salicylic acid 9.44 52.12Control 18.11 100

Table 6Effect of amount of enzyme on yield, pH, �Brix and %T650 of guava juice.

Enzyme units (U) Juice yield (ml) pH �Brix %T650

Control 15 4.5 1.9 1.78.96 16 3.24 2.8 2.517.92 16.5 3.19 3 5.826.88 17.2 3.14 3.4 7.935.84 17.9 3.11 4.2 14.644.80 18.5 3.05 4.8 20.4

16 S. Kant et al. / Protein Expression and Purification 87 (2013) 11–16

study, the pH of juice decreased from 4.5 to 3.05 with increase inamount of the enzyme (Table 6). In a similar study, addition of pec-tinases significantly increased the color, total soluble solids (TSS),titratable acidity and total sugar in enzymatically extracted juices.The pH and relative viscosity of extracted juices were decreased [28].

Conclusion

In the present study, polygalacturonase from A. niger (MTCC3323) was purified into 6.52-fold by Sephacryl S-200 gel-filtrationchromatography. On SDS–PAGE analysis, enzyme was found to be aheterodimer of 34 and 69 kDa subunit size. Homogeneity of the en-zyme was checked by NATIVE-PAGE and its molecular weight was106 kDa. An improved awareness of the properties of pectinases isimportant in commercialization of these enzymes in various fields.The quality of juice can be improved by appropriate treatmentwith polygalacturonase leading to the increased market potential,thereby, providing a significant boost to the economic state.

Acknowledgments

The financial support from Department of Biotechnology,Ministry of Science and Technology, Government of India, toDepartment of Biotechnology, Himachal Pradesh University,Shimla (India), is thankfully acknowledged. Financial assistancefrom UGC in the form of a Major Research Project is thankfullyacknowledged.

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