Mol Biol Rep (2014) 41:2645–2656 DOI 10.1007/s11033-014-3123-8

Cloning and molecular modelling of pectin degrading glycosyl hydrolase of family 28 from soil metagenomic library T. A. Sathya • Ani Methew Jacob • Mahejibin Khan

Received: 10 March 2013 / Accepted: 11 January 2014 / Published online: 23 January 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract Western Ghats of India is recognized as one of the 12 mega diversity regions of the world and is the hot spot for unrevealed microbial diversity. To explore the diversity of polysaccharide degrading enzymes in that region, metagenomic library was constructed from forest soil of Southern Western Ghats region. Nine pectinolytic clones with the ability to degrade citrus pectin were isolated based on function based screening of the library. Sequence analysis of pg_4 clone containing revealed that it contained GH family 28 domain (pfam00295) belonging to polygalacturonase superfamily (PLN03003). Its amino acid sequence analysis showed 25–55 % identity to the other well-characterized polygalacturonases. Molecular modeling of pg_4 revealed that it comprised of three right handed-parallel b sheets, one anti-parallel b sheet and one a helix with three conserved catalytic residue D 2263, D 284-85 and H 312 at the C terminal end. The enzyme characterized was able to hydrolyze both apple and citrus pectin with Km values of 1.685 and 1.542 mg ml-1 and retained more that 80 % of activity at pH 5–9 and temperature 20–60 °C. Keywords Glycosyl hydrolase  Metagenomics  Polygalacturonase

T. A. Sathya  A. M. Jacob  M. Khan (&) Department of Food Microbiology, CSIR—Central Food Technological Research Institute, Mysore 570020, India e-mail: [email protected] T. A. Sathya  M. Khan Academy of Scientific & Innovative Research, New Delhi 110001, India

Introduction Polysaccharides, which are a- or b-linked chains of carbohydrate moieties, play essential roles in biological processes. Glycoside hydrolases (GHs) performing hydrolytic reactions and b-elimination due to lyases break these linkages. GHs are a prominent group of enzymes that hydrolyze the bond between a carbohydrate and another compound, such as a second carbohydrate, a protein, or a lipid. These enzymes perform hydrolysis of glycosidic bonds, which can lead to inversion or retention of the anomeric configuration, depending on the enzyme family. The enzymes allow an organism to degrade carbohydrate polymers completely into oligomers and eventually, monosaccharide units. GHs are the best characterized of all carbohydrate-active enzymes (CAZymes) and are categorized into at least 115 different families, as defined by sequence similarity [1]. The pectin-degrading enzymes, classified into the family 28 of GHs, are one of the most widely distributed enzymes in bacteria, fungi, yeast and plants. These enzymes have been used for decades in the food and wine-making industry for the processing of fruit juices. These enzymes are very important from an industrial perspective. For example, the conversion of plant biomass into its constituent simple sugars is a crucial step in the production of second generation biofuels [2, 3]. Other applications have also been envisaged for pectinases in the production of oligogalacturonides as functional food components from pectin cleavage [4–6]. These oligogalacturonides play an important role in the survival of health promoting gut microbial symbionts. Various applications and working conditions of enzymes require broad-spectrum enzymes which allow for a more versatile use in different applications. Due to the

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availability of molecular biology techniques and gene sequencing, the numbers of genes encoding putative pectinolytic enzymes have been increased. However, with only a few exceptions, characteristics of the enzymes found so far are not very appropriate from the point of view of their industrial applications. Therefore, there is still a great need to expand the current enzyme repertoire. Thus, further metagenomics-based search for novel pectinolytic enzymes from different sources, and with greater industrial applicability, is the need of the hour. The metagenomic DNA represents an enormous genetic and biological pool for mining of new genes, entire pathways and compounds with wide applications in industries. The Western Ghats, a region in Southern India, covered with native tropical wet evergreen forests [7, 8] is one of the hot spots of unrevealed microbial diversity in the world [9]. The relatively higher thermal gradients and widely varying elevations along this belt may be due to volcanism that brought in large nutrients and high biodiversity [10]. Although many studies have been conducted on the diversity of animals and plants of Western Ghats, microbial diversity has received only a less attention. Searching of less explored ecosystems like forest can lead to identification of novel enzymes with unique properties that may be useful in industry and biotechnology. Further, forest communities appeared to be better adapted to decompose recalcitrant carbon compounds. The present study was conducted to perform construction and functional screening of soil metagenomic library from Southern Western Ghats region of India for GHs family enzymes. One novel pectin degrading gene (pg_4) was identified and characterised from the constructed library. A three-dimensional structure of pg_4 was build on the basis of comparative homology and functional/active sites in the modeled structure were identified.

Materials and methods Sample collection Soil samples from forest topsoil (5–10 cm) of the southern side of the Western Ghats were collected from the Sathyamangalam forest (11°380 2400 N, 77°130 3400 E), Erode District, Tamilnadu, India. They were stored at 4 °C until DNA extraction. Strains, plasmids and chemicals E. coli JM109 culture was grown at 37 °C on Luria–Bertani (LB) agar or in LB broth supplemented with 100 lg/ml ampicillin. Big easy linear kit from Lucigen Corporation, USA, was used to construct forest soil metagenomic

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library and pUC19 was used to subclone the pectinolytic gene. Metagenomic library construction and functional screening of clones DNA from soil sample was isolated and metagenomic library of 1–3 kb fragments constructed as described earlier [11]. Clones were screened for pectinolytic activity by growing on 1 % pectin containing plates. The activity was visualized as clear zones after the plates were flooded with 0.05 % ruthenium red solution for 1 h and washed with tap water. Bioinformatics analysis Clones were confirmed by plasmid isolation and restriction enzyme digestion. The positive clone was sequenced on gene analyzer 310, ABI prism, USA. Sequences were screened for vector contamination and quality trimmed. The nucleotide sequence of pg_4 deposited in the GenBank database under the accession number AFU91007. Open reading frames (ORFs) in sequence were identified using the ORF Finder, homology search and conserved domain analysis were performed with protein–protein BLAST provided by National Centre for Biotechnology Information NCBI. To classify pg_4, a phylogenetic tree was constructed on phylogene.fr server which uses maximum likelihood method for phylogenetic tree construction. Multiple sequence alignments were calculated using ClustalW [12] and exported by ESPript [13]. The threedimensional model of pg_4 was constructed by Geno3D [14] using Erwinia corotovora endopolygalacturonase (PDB id: 1bhe) as a template [15] and presented using chimera [16]. The qualities of the refined models were evaluated using PROCHECK [17], VERIFY3D [18], QMEAN [19] and ERRAT server [20]. The protein structure analysis (ProSA) [21] tool was employed in the refinement and validation of modelled structures. The root mean square deviation (RMSD) between the main chain atoms of the model and respective templates were calculated by structural superimpositions of predicted structures with their respective templates using iPBA web server [22]. Function prediction and identification of functional surface The 3d2GO server (http://www.sbg.bio.ic.ac.uk/phyre/ pfd/index.html) was used to predict functions of the validated models using sequence and structure in the reference of gene ontology (GO). CASTp server was used to identify their functional surfaces, which is taken as the surface pocket containing annotated binding site residues [23].

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Subcloning of pg_4 gene Plasmid was isolated from RB4 clone by alkaline lysis method and amplified using primer PGLF 50 AAAGGATC CCGGCTTGTTTAGCGCATCGG30 and PGLR 50 AAA GGATCCCGCTACCGATCGACATGCCA30 (containing BamHI restriction site). The 50-ll reactions included 19 PCR buffer (Hi Media, India), 0.4 mM dNTPs, and 0.25 U Taq polymerase (Hi Media, India). PCR was carried out under the following conditions: 95 °C for 5 min, followed by 94 °C for 1 min, 58 °C for 1 min and 72 °C for 1 min. The final extension step was at 72 °C for 5 min. PCR products were visualized on an agarose gel, purified and ligated to BamHI digested and dephosphorylated pUC19 vectors. The ligation mixtures were transformed into E. coli JM109. The transformants were spread onto LB plates supplemented with 100 lg/ml ampicillin and 1 % pectin and grown at 37 °C for 24 h followed by the overlaying of plates with ruthenium red dye and scored for clear halo zone around the colonies. Clones were further confirmed by colony PCR. Purification of enzyme RB4 clone containing the pectinase gene was grown for 24 h in a medium containing 0.5 % pectin and the cell-free supernatant was fractionated with ammonium sulfate. 40–70 % fractionated precipitate was dissolved in the minimum amount of phosphate buffer saline (pH 7.6), dialyzed against PBS overnight and subjected to gel filtration in a Sephadex G-150 column. 1.5 ml fractions obtained after the void volume (20 ml) were assayed for activity. The purity of the active fractions were analyzed on 12.5 % SDS-PAGE. Enzyme characterization The pectinase activity was determined by measuring the amount of reducing sugars liberated from pectin using 3,5dinitrosalicylic acid (DNS) reagent [24]. Briefly, 0.5 ml of the enzyme was mixed with 0.5 ml of the pectin (1 % w/v) and incubated for 5 min at room temperature. After adding 1 ml DNS, the mixture was incubated at 60 °C for 15 min. The mixture was finally diluted to 5 ml with deionized water. Absorbance was read at 540 nm using UV-spectrophotometer. One unit of enzyme activity (U) was defined as 1 lmol reducing groups released per min. Temperature and pH optima were determined by measuring enzyme activity at different temperatures (20–70 °C) and pH (2–9), respectively. After temperature and pH optimization, pH stability of the enzyme was determined by incubation of the enzyme in 0.1 M sodium acetate buffer of varying pH (2–9) for 1, 3 and 6 h and the residual

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activity were measured. For the thermostability assay, the enzyme was incubated at temperatures ranging from 20 to 70 °C in 0.1 M sodium acetate buffer for 1, 3, 6 and 12 h and the residual activity was measured. Samples without any treatment were used as control. The effect of metal ions on enzyme activity was determined by pre-incubation of purified enzyme with 1 and 10 mM of metal ion at room temperature for 60 min. The metal ions used were Cacl2, MgCl2, ZnCl2, HgCl2, MnSO4 and FeCl3. The enzyme activity without any metal was used as a control (100 % of relative activity) and the residual activity was measured. Results and discussion In this study, nearly 2,000 colonies of the 1–3 kb metagenomic library constructed from the forest soil samples of the Southern Western ghat region of India were screened. Nine colonies selected on the basis of hydrolysis zone and the pectinase positive plasmids were sequenced. The clone designated as RB4 that contained 2,750 base sequence, designated as pg_4, showed strong pectinolytic activity. The ORF that encoded 430 amino acids was identified as polygalacturonase (PG). The ProtPram tool was used to calculate the theoretical parameters of the protein and the deduced molecular mass calculated as 45.0 kDa, based on ORF, corobotated the molecular mass deduced by SDS PAGE . Phylogenetic analysis and three dimensional computational modeling of pg_4 In order to classify pg_4, a phylogenetic tree was constructed on phylogene.fr server [25]. Since it uses maximum likelihood method for phylogenetic tree construction, which is commonly recognized as the most accurate approach (along with Bayesian) in molecular phylogenetics. Comparison of amino acid sequence of pg_4 with the non-redundant sequence database deposited in the NCBI showed 52 % identity with PG form Pseudomonas syringae (Gen Bank: EGH65102.1), 51 % with peh-1 gene product from Xanthomonas axonopodis pv. citri (GenBank: NP_641014.1), 50 % with Erwinia amylovora (GenBank: YP_003531536.1), 49 % with Catenulispora acidiphila (YP_003114374.1), and 45 % with Pectobacterium (Gen Bank: AAA57139.1), respectively (Fig. 1). Among the known 3D structure pg_4 showed 44, 23 and 21 % identity with E. corotovora (PDB id: 1bhe), Yersinia enterocolitica (PDB id: 2uve) and Thermotoga maritima (PDB id: 2jbr), respectively. Multiple sequence alignment with known PG revealed that pg_4 contained four highly conserved regions, the first segment, Asn 261-Thr 262-Asp 263, the second segment, Gly 283-Asp 284-Asp 285, the third

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Fig. 1 Phylogenetic analysis of pg_4 and relatives, based on conserved sequence motifs of bacterial pectinolytic enzymes. Amino acid sequences of polygalacturonase enzymes were obtained from

NCBI and the tree was created at phylogene.fr server. Scale bar at bottom indicates number of amino acid substitutions per site

segment Gly 311-His 312-Gly 313, and the fourth segment Arg 342-Ile 343-Lys 344 (Fig. 2). Pfam showed that pg_4 contained GHs family 28 domain (pfam00295) and belonged to probable polygalacturonase superfamily (PLN03003). On the basis of sequence comparisons carried out by means of homology and hydrophobic cluster analysis, pg_4 was classified into the GH family 28 that included polygalacturonase (EC: 3.2.1.15) as well as rhamnogalacturonase A (RGase A). Comparative modeling of protein is considered as one of the most accurate methods for three-dimensional structurefunction prediction. The pairwise sequence alignment of target pg_4 and the template 1bhe was refined using ClustalW and the alignment is shown in Fig. 3. pg_4 computational 3D model built using Geno 3D server, the structure refined and verified at verify3D, ERRAT and QMEAN server (Table 1) and the stereochemistry, quality and

accuracy of the predicted model were evaluated using Ramachandran plot in PROCHECK. The refined model showed 90 % residues in most favoured regions, additional allowed regions (8.1 %) and generously allowed regions (1.9 %). Absence of residues from disallowed regions supported its high geometric quality (Fig. 4a). Energy profiles of the model obtained using ProSA score (Fig. 4b) revealed a Z-score value of the model that lay between -5.0 to -7.5, the model accuracy as it measured the total energy of the structures. ProSA score for modeled pg_4 calculated was -5.82 within the permissible range of native conformational structures. Further, the overall quality factor and compatibility of an atomic model (3D) with amino acid sequence (1D) and the packing quality of each residue for the modeled protein was assessed 0.81 by Verify3D, residues with a score over 0.2 considered reliable. Thus Verify3D results confirmed the model was

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Fig. 2 Multiple amino acid sequence alignment of pg_4. Sequence alignment was performed using ClustalW version 2.0 and exported to ESPript programs. Conserved sequences are indicated by box, and similar sequences are indicated by colored background. (Color figure online)

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Fig. 2 continued

reliable and of good quality. Predicted secondary structure of the pg_4 with secondary structural elements, a-helices, b-strands and loops, are shown in Fig. 4c. The modeled structure and its template were superimposed on their backbone atoms, to investigate how well both the structures matched. RMSD values of the backbone atoms was calculated 0.72, which supports that the generated model was reasonably good and quite similar to the template (Fig. 5). The results of 3d2GO server showed that modeled pg_4 protein predominantly associated with different cellular process, i.e. cell wall macromolecules catabolic process, pectin catabolic process, hydrolase activity, hydrolyzing Oglycosyl compounds, hydrolase activity acting on glycosyl bond with a confidence value [0.75. Homology model of pg_4 revealed that the threedimensional structure of both pg_4 and 1bhe comprised of right handed-parallel b sheets. The major deviation between the two structures observed was, pg_4 consisted of three right handed-parallel b sheets and one additional antiparallel b sheet and one small a-helix 1bhe folded into a three right-handed parallel b-helical structures comprising 10 complete turns. However, the overall shape of pg_4 was the same as that of 1bhe. Similar type of structures have also been reported in the crystal structure of thermophilic bacterium, T. maritima. It was observed that the residues 434–447 in the C-terminal coil

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of b-helix was antiparallel to the other strands and paired antiparallel with the C-terminal strand of a neighboring molecule. Apparently the residues shielded the hydrophobic interior of the b-helix from solvent and were responsible for strong intermolecular circular b-sheet formation. The N-terminal side of the b-helix was capped by an a-helix, a feature observed in most other b-helix folds [26]. Comparative analysis of amino acid sequences showed that the in 1bhe, conserved sequences Asn-Thr-Asp, GlyAsp-Asp and Gly-His-Gly and Arg-Ile-Lys were clustered on or before b-strands 5, 6, 7 and 8 of PB1 whereas in pg_4, Asn-Thr-Asp, Gly-Asp-Asp and Arg-Ile-Lys formed strands 6, 7 and 9, respectively. Gly-His-Gly forms turned before strand 8 of parallel b sheets. This clustering confirmed the functional conservation of all the four residues and suggested that the region on the surface of b-strands 6–9 of PB1 and the adjacent loops formed the catalytic site in pg_4. Markovic and Janecek [27], reported that all the members of GH family 28 contained its functionally important residues in the segments equivalent to the four conserved active site segments. However, some exceptions in bacterial and fungal species were also observed. Asn and Asp were strictly conserved while, Thr was replaced either by Gly or Ala in bacterial exo-PGs. In the fourth segment, Ile was replaced with Leu.

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Fig. 3 Sequence alignment of modeled pg_4 and the template E. corotovora polygalacturonase (PDB ID: 1bhe). The key conserved amino acids found in the active site are indicated in boxes

Table 1 Model validation scores of the pg_4 model with template Enzyme

Verify3D

ERRAT

ProSA (Z score)

QMEAN score

1bhe

0.79

87.02

-7.57

0.883 (Z-score 1.29)

pg_4

0.81

83.5

-5.82

0.808 (Z-score 0.97)

Prediction of functional surface and the binding site of pg_4 was carried out using CASTp server. For proteins and other molecules it provided identification and measurement of surface accessible pockets as well as interior inaccessible cavities. CASTp server measured the area and volume of each pocket and cavity, both in solvent accessible surface (SA, Richards’ surface) and in molecular surface (MS, Connolly’s surface). The ligand binding sites, usually in the largest pocket, defined energetic criteria. Amino acid residues that formed the large cavity in 1bhe and pg_4 are given in Table 2. Among all residues involved in the formation of largest pocket, Asp 263, Asp 284-85 and His 312 were crucial for catalytic activity. These residues were conserved in all the groups of family 28 of GH. Palanevelu

[28], studied the role of active site residues in the mechanism of catalysis of polygalacturonase and revealed that the NTD and RIK motifs play an important role in substrate binding and cleavage of glycosidic bond. Comparative analysis of amino acid sequences of the GH 28 family also revealed the presence one S–S bridge, Cys 26-Cys 65 in pg_4. The presence of cysteine residues in the crystal structure of PGs helped in its stabilization through an entropic effect, by decreasing the entropy of the protein’s unfolded state [15, 29, 30]. Enzyme characterization The specific activity of pg_4 after ammonium sulphate precipitation and gel filtration chromatography was 2599.84 U (Fig. 6). Since it corresponded to over 4.5 fold purity, stability studies were carried out to determine the structure and function of the enzyme. Optimum pH for the enzyme activity was 6.0. It retained over 75 % activity when incubated for 1 h at pH between 5.0 and 8.0 (Fig. 7). Even after 6 h incubation, it retained 38.7 and 25 % activity at pH 5 and 9.

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a c

b

Sec. struc:

Helices labelled H1, H2, ... and strands by their sheets A, B, ...

Disulphides:

disulphide bond

Fig. 4 Evaluation of modeled structure of pg_4. a Ramachandran plot for modeled pg_4, showing the residue in most favored regions (90 %), additional allowed regions (8.1 %) and generously allowed regions (1.9 %). b ProSA energy profile of pg_4, Z score for modeled

pg_4 calculated was -5.82. c Predicted secondary structure for the pg_4. The secondary structural elements a-helices, b-strands and loops are shown above the alignment

The enzyme also showed stability and activity at broad temperature range from 20 to 70 °C (Fig. 8). The enzyme activity increased with the increase in temperature up to 60 °C, and then decreased beyond that level. At 70 °C, enzyme retained 74.7 % activity. Fungal PGs are generally acidic and less thermostable compared to bacterial enzymes [31]. Alkaliphilic enzymes from bacterial strains like Bacillus and Streptomyces are known [32] and the hyperthermophilic bacterium T. maritima reportedly produced an enzyme with optimum activity at

80 °C but active at very narrow pH range [33]. The structure predicted of pg_4 suggested its bacterial origin. In recent years, broad-spectrum enzymes have attracted much attention in biotechnology that must act in different condition according to the process in which they are involved. Low temperatures active PGs are preferred for the production and retention of flavour and colour components in wine making. While in the extraction and clarification of fruit juice enzymes must be functional at

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Fig. 5 Three dimensional structure of pg_4 and comparison with 1bhe. a Ribbon diagram of the modeled pg_4, showing a-helices, bstrands and loops in blue, yellow and brown colour, respectively. b Superimposition of the template E. corotovora (PDB ID: 1bhe)

(blue) and the generated model of pg_4 (black). c Electrostatic potential distribution on the surface of the pg_4. d Cavity/active site in pg_4, shown in black in color. (Color figure online)

Table 2 The active sites and the amino acids involved in the formation of the large cavity in pg_4 and 1bhe with their surface areas and volumes predicted by CASTp server Enzyme

Area (sq. A°)

Volume (cubic A°)

Amino acid residues involved in cavity formation

1pg_4

985.5

2525.6

K123, S124, G125, A126, C127, G171, A171, K175, N178, Q179, V180, Q181, N181, S183, P184, D185, K188, V189, Q190, Y207, F208, F211, H213, I214, N261, D263, D266, I268, D284, D285, K290, Y292, K295, H312, S318, E319, R342, K344, D346, S348, N349, Y376, L377, T378, G379

1bhe

1133.7

3071.4

N82, D93, K94, N95, G96, K97, C99, D100, A101, T104, A138, A139, A141, K142, V143, K144, K145, L146, K147, Q148, N149, T150, P151, R152, Q156, N158, N174, F175, S180, D181, R199, N200, D202, D223, D224, K229, Y231, R234, H252, S257, E258, R280, K282, D284, S286, A287, V313, 314, E315, K316, K317, E318

higher temperature (between 45 and 95 °C). Since pg_4 is stable in the pH range of 3–9, and retained more than 75 % activity in the temperature range of 20–70 °C up to 1 h and *45 % activity after 3 h of incubation, it has wide applications in various industries.

Effect of metal ions on enzyme activity Influence of different metal ions such as magnesium chloride, zinc chloride, ferric chloride, mercuric chloride and manganese chloride were checked at a concentration of

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1

Zn2? had no effect on enzyme activity. Whereas with the increase in metal ion concentration to 10 mM, relative activity decreased to 400 in presence of Mn2? and other metal ions had no significant effect on enzyme activity (Table 3). These results confirm the absolute requirement of Mn2? as cofactor for pg_4. It has been found that the metal ion requirement of different PGs is different and various cations (Co2?, Mn2?, Zn2?, Fe2?, Hg2?, Ca2? or Ni2?) are used as cofactors [29, 32, 33].

2

97.4 67.0

Enzyme kinetics

43.0

29.0

20.0

Fig. 6 SDS-PAGE analysis of purified enzyme of pg_4. M marker, lane 1 crude protein, lane 2 purified protein

a

b

Relative enzyme activity

Relative enzyme activity(%)

1 and 10 mM. It appeared that all the metal ion at the conc. of 1 mM activate enzyme activity. However, the best cofactors for this enzyme was Mn2? with relative activities of 473 %, other metal ions such as Fe2?, Hg2? and Mg2? could activate enzyme activity moderately with relative activities 140, 60 and 158 % respectively and Ca2? and

120 100 80 60 40 20 0 2

3

4

5

6

7

8

9

120

1hr

100

3hr

80

6hr

60 40 20 0 2

pH

Fig. 7 Effect of pH on pg_4 enzyme activity and stability. a Enzyme activity was measured at 60 °C in 0.1 M buffer, at various pH 2–9. Values are shown as percentage of maximal activity, defined as 100 %. b Effect of pH on stability of pg_4, the enzyme was incubated

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Citrus and apple pectin both have important applications in the food industry. Incubation of pg_4 with different concentrations (1–10 mg/ml) of apple and citrus pectin indicated that pectinase activity increased with the increase in the substrate concentration and reached its maximum at 5 mg/ml. Citrus and apple pectin have a similar sugar composition. Apple pectin contains more neutral sugars and less uronic acids than citrus pectin. However, the pg_4 activity on citrus pectin was higher than that on apple pectin. The Km and Vmax values of pectinase were 1.542 mg/ml and 310.4 U/ml and 1.685 mg/ml and 250.5 U/ml, respectively, for citrus and apple pectin. Km value indicates the affinity of substrate for the enzyme. The increasing activity with increase in substrate concentration may be attributed to the effective binding of the substrate to the active site, but further increase in substrate concentration above the optimal level will not produce any increase in the enzyme activity since no enzyme molecule will be available to react with the substrate. The present results are in agreement with reported Km values of various polygalacturonases. The Km of the reported polygalacturonases varies from 6.7 to 0.12 mg/ml [34–37].

3

4

5

6

7

8

9

pH

in buffer of varying pH from 2 to 9 for 1, 3 and 6 at room temperature. Residual enzyme activity was measured comparing with control (100 % of relative activity) at pH 6

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b Relative enzyme activity(%)

Relative enzyme activity(%)

a 120 100 80 60 40 20 0 20

30

40

50

60

120

1 hr

100

3hrs 80

6hrs 12hrs

60 40 20

70

0 20

Temperature

40

50

60

70

Temperature

Fig. 8 Effect of temperature on enzyme activity and stability. a The activity was measured by incubating the purified enzyme in the temperature range of 20–70 °C. b The stability of the enzyme to temperature was investigated by measuring the residual activity after

incubating the purified enzyme at 20–60 °C for 1, 3, 6 and 12 h. Residual activity was calculated comparing with control (100 % of relative activity) at 60 °C

Table 3 Effect of metal ions on enzyme activity Metal ions

30

Relative enzyme activity (%)

a

2.

1 mM

10 mM

Control

100 ± 3.1

100 ± 5.1

Cacl2

102 ± 3.9

101 ± 4.8

MgCl2

158 ± 2.9

99 ± 5.2

MnSO4

473 ± 8.1

400 ± 6.7

ZnCl2

113 ± 4.5

106 ± 3.9

HgCl2

160 ± 2.8

110 ± 4.1

FeCl3

140 ± 3.3

102 ± 3.8

a

Purified enzyme was incubated in the presence of 1 and 10 mM concentration of various metal ions at room temperature for 60 min. The remaining enzyme activity was measured and expressed as the mean of three determinations. The enzyme solution without any metal was used as a control (100 % of relative activity)

Conclusion Application of polygalacturonase in the perspective of food and pharmaceutical industries has been reviewed recently [38]. The pectin degrading enzyme identified and characterized from the soil metagenomic library belonging to GH 28 family appeared unique showing only 52 % sequence similarity with the known PGs. Importantly, it contained functionally important conserved residue, complete GH domain and was active at broad temperature and pH. Thus the study revealed that metagenes can be sourced for PGs with unique applications in biotechnological processes.

3.

4. 5.

6.

7.

8. 9.

10.

11.

12.

13.

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Cloning and molecular modelling of pectin degrading glycosyl hydrolase of family 28 from soil metagenomic library.

Western Ghats of India is recognized as one of the 12 mega diversity regions of the world and is the hot spot for unrevealed microbial diversity. To e...
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