Thermostable and organic solvent-tolerant acid pectinase from Aspergillus terreus FP6: purification, characterization and evaluation of its phytopigment extraction potential

Abstract

The present study discusses the purification, characterization and application of pectinase from Aspergillus terreus FP6 in fruit pigment extraction. By the four-step purification involving precipitation, dialysis, ion-exchange chromatography, gel filtration chromatography, a 20.85-fold purification of the enzyme to homogeneity was achieved. The apparent molecular mass of the pectinase was 47 kDa, as found by sodium dodecyl sulphate–polyacrylamide gel electrophoresis. The optimum activity of the enzyme was recorded at pH 6.0 and 50 °C. The enzyme retained 80.3% and 79.1% residual activity, respectively at pH 6.0 and 50 °C for 90 min. The pectinase was best functional in the presence of toluene and retained its activity for 30 min. Cu²⁺ and Co²⁺ acted as enzyme activators, while Ca²⁺, β-mercaptoethanol, dimethyl sulfoxide and ethylenediaminetetraacetic acid proved to be the inhibitors. The K_m and V_max values of the pectinase with pectin as substrate were 0.002 mM and 27.39 U/mL, respectively thus indicating the high enzyme affinity towards the substrate. After 30-min treatment of the grape skin with the partially purified enzyme, microscopic observation revealed that a short time of the enzymatic treatment resulted in substantial loss of pigment and shrinkage of the grape skin cells thereby highlighting the high efficiency of the pectinase. The current study implies that the A. terreus FP6 pectinase may be applied as a bio-agent in the food and beverage industries and has the potential to replace harmful solvents by promoting a greener approach to extract plant pigments.

Introduction

Pectinases are the group of high molecular weight, negatively charged, acidic glycosidic macromolecular enzymes that break down pectin/polygalacturonic acid present in the middle lamellar region of plant tissues. Various forms of pectinases include polygalacturonase (EC 3.2.1.15), pectin lyase (EC: 4.2.2.10) and pectinesterase (EC: 3.1.1.11) (El Enshasy et al. 2018). Pectinases are among the important enzymes of the commercial sector, especially for the fruit juice and beverage industry. It also has the applications in pharmaceutical, textile, paper and pulp industries. Its usage is seen in the preparation of animal feeds and oil extraction (Kaur et al. 2021).

Commercially, filamentous fungi (prominently Aspergillus sp.) are preferred for pectinase production owing to certain characteristics like capacity for fermentation of large quantities of exoenzymes, feasibility of cultivation, and cost-effective production in large bioreactors (Radha et al. 2018). The search for an efficient pectinase from natural sources may involve investigating the pectinase producing capability of Aspergillus terreus due to several reasons. Under natural conditions, A. terreus is a prominent degrader of vegetative substances. Moreover, A. terreus is industrially utilized in the fermentation of primary metabolites (xylanase, itaconic acid, and cis-aconitic acid), and secondary metabolites (lovastatin) as it has the approval of the United States Food and Drug Administration to produce microbial metabolites (Sethi et al. 2016).

Purification and characterization of naturally synthesized enzymes enables the study of their physical and biological properties. Purification increases the enzyme efficiency and specificity, and characterization of the purified enzyme is essential to understand its optimum chemical and physical conditions for exhibiting maximum activity and in determining its application. Parameters like pH, temperature, substrate concentration, presence of solvents, activators and inhibitors greatly influence the catalytic site, thereby changes the enzyme function and stability. Thus, purified and well-characterized enzyme profile is always of industrial importance (Ahmed and Sohail 2019).

To the best of our knowledge, the present study is the first scientific literature elucidating the purification, characterization and application of A. terreus FP6 pectinase in extraction of commercially important phytopigment (anthocyanin) from grape skin. The present study will contribute to ease the short comings in terms of understanding the functional capabilities and utility of the enzyme.

Materials and methods

Chemicals and reagents

Pectin (≥ 85% esterified from citrus fruit) was purchased from Merck, Germany. Inorganic salts and solvents (analytical grade) were purchased from HiMedia, Mumbai, India.

Source of fungal culture

Aspergillus terreus FP6 (Genbank accession number MZ068227) was isolated from the compost soil and maintained at 4 °C as pure cultures on slants of potato dextrose agar for pectinase production.

Pectinase production, extraction and purification

The spore suspension of the isolate was prepared and the number of spores (1.0 × 10⁶ spores/mL) was counted using a Neubauer chamber (Leo Scientific, India). The spore suspension (1 mL) was inoculated in pectin broth (g/L: pectin, 5.0; yeast extract, 1.0; KH₂PO₄, 4.0; NaCl, 2.0; MgSO₄, 1.0; MnSO₄, 0.05; FeSO₄, 0.05; CaCl₂, 2.0; NH₄Cl, 2.0; pH 6.0 ± 0.2) and incubated at 28 ± 2 °C for 72 h, 150 rpm (Orbitek LT-orbital shaker, Scigenics Biotech Pvt. Ltd., India). After growth, the culture was centrifuged at 3864 × g, 4 °C, 15 min (Universal 32R, Hettich Lab Technology, Tuttlingen, Germany) and the supernatant was considered as the ‘crude pectinase’.

The crude enzyme (250 mL) was mixed with ammonium sulphate solution (70% saturation), precipitated and reconstituted with sodium phosphate buffer (10 mL, 100 mM, pH 7.4) (Mehmood et al. 2019). The reconstituted enzyme preparation was dialyzed overnight in Na₂HPO₄-citric acid buffer (0.02 M, pH5.2) and resuspended in the same buffer (20 mL, 0.02 M, pH 5.2). The dialyzed enzyme was further purified using a diethylaminoethyl‐cellulose (DEAE‐cellulose) column (1 × 30 cm, particle size 22–40 μm, bed volume 19.63 mL; Merck, Germany) equilibrated thrice with Na₂HPO₄-citric acid buffer (0.02 M, pH 5.2). The column was washed thrice with the same buffer. Elution was carried out in Na₂HPO₄-citric acid buffer (0.02 M, pH 5.2) with NaCl gradient (0–1 M, at 1 mL/min flow rate) (Yu and Xu 2018). The eluent from ion-exchange chromatography demonstrating the highest pectinase activity was applied to Sephadex G-100 column (1 × 30 cm, bed volume 19.63 mL; Merck, Germany). The column was equilibrated with Na₂HPO₄-citric acid buffer (0.04 M, pH 5.2) and was eluted (flow rate 1 mL/min) (Yu and Xu 2018). Following individual purification step, protein estimation of samples was done using 0.3 mg/mL bovine serum albumin as standard with working concentrations ranging from 30 to 150 µg/mL (Lowry et al. 1951).

Assay for pectinase activity

The pectinase activity was assessed using the modified protocol of Miller (1959). The enzyme (500 μL) was mixed with pectin solution (1% w/v, 500 μL prepared in a 0.2 M phosphate buffer, pH 7.2) and considered as ‘test’. Synchronously, suitable ‘buffer blank’ (devoid of the enzyme and substrate) and ‘enzyme blank’ (devoid of the enzyme) were prepared. Both the ‘test’ and ‘blank tubes’ were incubated (37 °C, 30 min). Following incubation, dinitrosalicylic acid reagent (3 mL) was added to all the tubes, incubated in boiling water bath for 15 min and cooled with subsequent addition of potassium sodium tartrate solution (1 mL, 40% w/v). Galacturonic acid (concentration 10 to 100 µg/mL) was used to prepare the standard curve. The absorbance was recorded at 540 nm using a UV‐1800 spectrophotometer (Shimadzu, Japan). One unit of pectinase activity was defined as the amount of enzyme required to release 1.0 μmol galacturonic acid per minute under standard assay conditions and expressed as units per millilitre (U/mL) (El Enshasy et al. 2018).

Molecular mass assay

Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS‐PAGE) was set using a stacking gel (5% w/v) and a separating gel (15% w/v). The vertical electrophoretic unit (Bio-Rad Mini-Protean Tetra Cell) was run at 100 V for 2 h using Tris–glycine buffer (pH 8.3). After overnight staining in Coomassie brilliant blue solution, the gel was destained and the enzyme’s apparent molecular mass determined by referring to that of the protein markers (M_r range, 14.3–158 kDa, New England BioLabs, UK) (Carrasco et al. 2019).

Pectin-zymography was performed in conjunction with SDS-PAGE to test the pectinolytic activity of the partially purified pectinase. The protocol of Ghazala et al. (2015) was followed to perform the zymography. After incubation, the gel was stained with Ruthenium red (0.03% w/v) and the bands of pectinase activity appeared as clear area against a red gel background.

Characterization of pectinase

Effect of pH was studied (at 50 °C) using sodium citrate buffer (pH 4.0, 5.0, 6.0), sodium phosphate buffer (pH 7.0 and 8.0) and Tris–HCl buffer (pH 9.0) (Ahmed and Sohail 2019). Effect of temperature on pectinase activity was determined (at pH 6.0) by incubating the enzyme at varying temperatures (30–100 °C, with a 10 °C increment) (Zehra et al. 2020). Effect of solvents was assessed by pre-incubating the enzyme in toluene, acetone, diethyl ether, methanol, or ethanol (30% v/v, 500 µL) (Magro et al. 2018). Effect of different salts and organic compounds (10 mM, 500 µL): barium chloride, calcium chloride, cobalt chloride, cupric chloride, magnesium chloride, manganese chloride, mercuric chloride, zinc chloride, dimethyl sulfoxide/DMSO, ethylenediaminetetraacetic acid/EDTA or β-mercaptoethanol/BME) was evaluated (Okonji et al. 2019). The assessment of residual activity at the selected pH, temperature and in the presence of solvent involved treatment of the partially purified pectinase for various time durations (0, 15, 30, 45, 60, 90, 120, 150, or 180 min). The maximum reaction rate (V_max) and Michaelis constant (K_m) were recorded from the nonlinear regression plot by varying the pectin concentrations (250–5000 μg/mL). GraphPad Prism software (version 9.2.0) was used for the analysis (Oumer and Abate 2017).

Extraction of anthocyanins

The Bangalore blue grapes (Vitis labrusca) were procured from an outlet of the Horticultural Producers' Cooperative Marketing and Processing Society, Bangalore, India. The healthy berries were surface sterilized using potassium permanganate solution, washed several times in sterile distilled water, following which the grape skin was peeled and air dried.

The portion of the air-dried grape skin and the solvents used was in ratio 1:10 w/v. The experimental conditions maintained were as follows; Control (physiological saline + air-dried grape skin sample); set up 1 (acetone + air-dried grape skin sample); set up 2 (partially purified pectinase + air-dried grape skin sample). The grape skins were exposed to the experimental conditions (30 min). Following exposure, the action of the solvent and the partially purified pectinase on the grape skin cells were observed (40 × magnification of a light microscope) (Shanmugavel et al. 2018).

Following 30 min of grape skin exposure, the quantification of the liberated anthocyanin involved passage of aliquots (5 mL) from ‘control’, ‘set up 1’ and ‘set up 2’ tubes through cellulose nitrate membrane (0.22 µm, 142 mm, HiMedia, Mumbai, India) and analysis of anthocyanins at 540 nm using a UV‐1800 spectrophotometer (Shimadzu, Japan) (Sommer and Cohen 2018). Anthocyanin standard solution (1000 μg/mL) was prepared with perchloric acid solution (5% v/v) and the working concentrations ranging from 100–500 μg/mL were used for the preparation of standard curves (Şakar et al. 2008).

Statistical analysis

Experiments were performed in triplicate (n = 3) and data graphically presented (mean ± standard deviation of the mean (S.D.)). The data analysis was done using Microsoft Excel 2010. Statistical analysis was carried out using R software (version 4.0.2). The parameters evaluated as part of enzyme characterization were analysed using one-way analysis of variance (ANOVA). Post hoc analysis, using pairwise t test with Bonferroni p value adjustment, was performed to determine the significant variable within a particular parameter. P-values < 0.05 were considered ‘statistically significant’.

Results and discussion

Purification profile of pectinase

Purification of A. terreus FP6 pectinase was effective with (NH₄)₂SO₄ precipitation (70% saturation) wherein the specific activity increased from 21.58 to 50.30 U/mg. Following ion-exchange chromatography and gel-filtration chromatography, the pectinase’s specific activity was recorded as 305.43 and 450.00 U/mg, respectively. Ion-exchange and gel-filtration chromatography resulted in 14.15 and 20.85-fold increase in specific activity with 17.79 and 11.23% protein recovery, respectively (Table 1).

Table 1.

Step-wise purification summary of pectinase from A. terreus FP6

Steps	Activity (U/mL)	Protein content (mg/mL)	Specific activity (U/mg)	Recovery (%)	Purification fold
Crude enzyme	120.18	5.57	21.58	100.00	1.00
Precipitation	95.06	1.89	50.30	79.10	2.33
Dialysis	73.59	0.51	144.29	61.23	6.69
Ion-exchange chromatography	21.38	0.07	305.43	17.79	14.15
Gel-filtration chromatography	13.50	0.03	450.00	11.23	20.85

The current result is supported by prior literature where A. fumigatus MTCC 2584 pectinase underwent purification using acetone precipitation and gel-permeation chromatography to result in an 18.43-fold purification and 2.98% yield of pectinase with 38.9 IU/mg specific activity (Anand et al. 2016). Another study documented that the crude pectinase of A. fumigatus SKF-2 was purified using ammonium sulphate precipitation and dialysis to facilitate 3.6-fold enzyme purification with 3125.54 U/mg specific activity and 83.81% final yield (Mondal et al. 2020).

Molecular mass assay

As compared to the protein marker run in the SDS-PAGE, the apparent molecular mass of A. terreus FP6 pectinase was approximately 47 kDa. A single band of the purified pectinase indicated that the pectinase was a monomer and was purified to homogeneity. This result was in concurrence with the result obtained in the zymography (Fig. 1).

Fig. 1.

SDS-PAGE and zymography of purified pectinase from A. terreus FP6. Lane M, Protein molecular mass markers (M_r range, 14.3–158 kDa); L1, purified enzyme; L2, Zymography gel showing a band of pectinolysis

According to the observations of Fratebianchi et al. (2017), the molecular mass of the purified Aspergillus sojae pectinase was 47 kDa. Similar to the present observation, A. fumigatus MTCC 2584 pectinase treated by acetone precipitation and gel-permeation chromatography demonstrated an approximate molecular mass of 43 kDa when subjected to SDS-PAGE (Anand et al. 2016).

Effect of pH on pectinase activity and stability

The susceptibility of respective enzyme towards the changing pH establishes the connection between cellular activities and pH. Enzymes are naturally functional within a certain pH limit and the total cellular enzyme activity is thus an intricate operation of the environmental pH. Besides determining the pH optima of an enzymatic reaction, the enzyme stability at a particular pH for significant time duration is also crucial for its industrial applications (Sharma and Singh 2016).

The A. terreus FP6 pectinase activity was low at extremes of pH. Enzyme activity improved as the pH increased from 4 to 6 and reached a maximum (15.8 U/mL) at pH 6 (p < 0.001). The enzyme functioning gradually decreased beyond pH 6, with the lowest value (4.3 U/mL) being recorded at pH 9 (Fig. 2a). The enzyme stability results showed, A. terreus FP6 pectinase retained its activity (80.3%) at pH 6 till 90 min, beyond which the residual activity steadily decreased. The enzyme underwent denaturation after 180 min, with 38.4% residual activity (Fig. 2b). Highly significant reduction in pectinase activity was noted between 90 and 120 min (p < 0.0001) possibly due to the irreversible change the amino acids underwent at the active site.

Fig. 2.

a Effect of pH on the A. terreus FP6 pectinase activity, b residual pectinase activity at pH 6.0, when incubated for different time durations. Data represent mean ± S.D. (n = 3); p < 0.05

A change in the reaction mixture’s pH influences the enzyme activity both reversibly (due to protonation of amino acids of an enzyme) and irreversibly (due to alteration in the structurally important groups of amino acids), where the irreversible changes are lethal (Ahmed and Sohail 2019). The reduction in A. terreus FP6 pectinase activity at extremes of pH (pH 8.0 and 9.0) might have resulted from the disintegration of ionic bonds preserving the enzyme tertiary structure. Correspondingly, the enzyme possibly lost its configuration (conformation of active site), affecting its interaction with the substrate (D'Souza et al. 2020).

Additionally, the variations in the A. terreus FP6 pectinase activity in the presence of different buffers indicate a change in the enzyme secondary structure. The obtained results highlight that at extremes of pH there was an increase in random coil that decreased the alpha helix and beta sheets thus possibly explaining the reduction in enzyme activity due to the distortion of the enzyme secondary structure. At pH 6, the A. terreus FP6 pectinase experienced the highest secondary structure stability (Poondla et al. 2017).

The active site of pectinase contains His₂₂₃ and Asp₂₀₁ residues where His₂₂₃ acts as proton donor, while Asp₂₀₁ acts as a nucleophilic site. Pectinase action on its substrate is initiated by the formation of multiple hydrogen bonds between the substrate (on the side of susceptible glycosidic bond) and the enzyme. This result in distortion and strain in the enzyme active site to locate the substrates. Catalytic site of His₂₂₃ transfers a proton to the susceptible glycosidic bond, leading to the breakdown of the glycosidic bond, releasing the first product and also the formation of new covalent bond between substrate and active site from nucleophile Asp₂₀₁. The other Asp₂₀₂ residue releases the second product and restores the enzyme active site by locating a water molecule for the nucleophilic attack. Therefore, pH condition affects in ionization of His and Asp (Palanivelu 2006).

In the present study, the increase in pectinase activity between pH 4.0 to 6.0 denotes a gradual increase in the ionization of the active site of the enzyme leading to a higher number of ionized carboxyl groups. The generation of high number of ionized carboxyl groups favour easier enzyme–substrate binding. Since for the A. terreus FP6 pectinase the highest activity was recorded at pH 6.0, it can be predicted that at this particular pH the number of H⁺ derived from the ionization of carboxylic group was maximum to form enzyme-substrates complex. On the other hand, beyond pH 6 the pectinase activity decreased due to the inability of many carboxylic groups to protonize the substrate (Roosdiana et al. 2013).

Previous research reported that optimum pH for pectinase activity of A. fumigatus was at pH 4.0 and 5.0, and residual activity values remained above 50% at these pH (de Medeiros Bezerra Jácome et al. 2020). The enzyme activity being low under alkaline conditions confirms the acidic nature of the A. terreus FP6 pectinase and indicates its wide application in beverage and food industries.

Effect of temperature on pectinase activity and stability

The molecular kinetic energy of an enzyme‐mediated reaction increases with any elevation of the reaction temperature because of the involvement of random collision of substrate molecules and enzyme. However, with rising temperature, the vibrational energy of enzymes increases, putting pressure on the hydrogen and ionic bonds (weaker forces of interaction), thereby changing the configuration of active site (Zhou and Pang 2018).

The A. terreus FP6 pectinase activity was assessed across a temperature range and was steady between 40 °C and 60 °C. An improvement in enzyme performance was observed beyond 40 °C, reaching a maximum of 19.7 U/mL at 50 °C (p < 0.001). At temperatures lesser than the optimum, the enzyme activity was less (with minimum product generation). Beyond 60 °C, a gradual decrease in pectinase activity was recorded (Fig. 3a). At higher temperatures (70–100 °C), sufficient thermal energy was generated to disrupt the intramolecular interactions (dipole–dipole attractions, hydrogen bonding, hydrophobic forces, and ionic interactions) between the polar groups and non-polar species in the enzyme interior. The disruption of these intramolecular forces resulted in the conversion of enzyme structure from their native forms (secondary and tertiary) to randomly coiled entities. This, in turn, possibly altered the active site's conformation to an extent that the specific substrate binding could no longer be facilitated (Robinson 2015).

Fig. 3.

a Effect of temperature on the A. terreus FP6 pectinase activity, b residual pectinase activity at 50 °C, when incubated for different time durations. Data represent mean ± S.D. (n = 3); p < 0.05

An enzyme’s ability to resist thermal unfolding when its substrate is absent is a reflection of its thermal stability. The optimal working ranges of the A. terreus FP6 pectinase were understood based on the evaluation of its thermal stability. Results illustrated that after 90 min, the pectinase possessed 79.1% of its activity. Only 38.8% of enzyme activity remained after 180 min (Fig. 3b). Highly significant reduction in pectinase activity was noted between 90 and 120 min (p < 0.0001). The significant decrease in the residual activity between 90- and 120-min exposure at 50 °C hints at the conversion of native form of the enzyme to the randomly coiled form.

In accordance with the present result, an earlier finding reported that A. sojae exopolygalacturonase displayed activity over a temperature range of 25–75 °C with 55 °C as temperature optima. The increase in temperature and time facilitated in overall improvement in the stability until 55 °C and 20 min after which a decline was observed (Dogan and Tari 2008). Kaur et al. (2021) showed that A. niger pectinase demonstrated an optimum activity at 50 ºC and was able to retain its thermostability till 98 h. Enzymes with thermostability and tolerance are desirable in varied industrial systems and thus the thermotolerant nature of A. terreus FP6 pectinase favours its commercial applications.

Effect of solvents on pectinase activity and stability

Enzyme catalysis in the presence of non-polar solvents has the advantages of better recovery and reusability of enzymes (even without immobilization) and is being increasingly used for numerous enzyme-mediated applications. With the usage of substrates having greater solubility in organic solvents, higher enzyme activity, enhanced thermostability, energy-efficient and minimal risk of microbial contamination is recorded (Kumar et al. 2016).

While analyzing the effect of solvent on the function of partially purified pectinase from A. terreus FP6, it was observed that highly non-polar solvents like toluene and diethyl ether (with less dielectric constants, i.e., ε values of 2.4, and 4.3, respectively) enhanced the pectinase activity. Maximum pectinase activity (26.4 U/mL) was observed with toluene (p < 0.001). On the contrary, the less non-polar solvents, i.e., acetone, ethanol, and methanol (with ε values 21, 25, and 33, respectively) decreased A. terreus FP6 pectinase activity. This decrease was more significant (10.1 U/mL) with methanol (p < 0.0001; Fig. 4a). Study of the enzyme stability against toluene revealed that A. terreus FP6 pectinase retained its activity (87.6%) till 30 min, beyond which the residual activity gradually decreased (Fig. 4b). The reduction of activity was highly significant between 30 and 45 min (p < 0.0001) probably due to the highest deactivation that the enzyme suffered in the presence of toluene.

Fig. 4.

a Effect of solvents on the A. terreus FP6 pectinase activity, b residual pectinase activity in presence of toluene, when incubated for different time durations. Data represent mean ± S.D. (n = 3); p < 0.05

Literatures suggest that for many enzymes, the rates of reaction are greater in non-polar solvents than in polar solvents. In the present study, A. terreus FP6pectinase was reported to demonstrate higher activities in highly non-polar solvents possibly because such solvents affected the thermodynamic activity coefficient (ground-state free energy) of the substrate. Even if the physical nature of these solvents (density and viscosity) did not affect the enzyme–substrate interaction, the activation energy level (different for various solvents) was probably low due to the differences in the ground-state free energy, which ultimately favoured the pectinase activity in highly non-polar solvents (Kumar et al. 2016). The disulfide bond and amino acid residue(s) located on the enzyme surface is closely related to the secondary structure and in the present study played an important role in conferring stability towards non-polar solvents. Therefore, the conformational mobility of the enzyme at such low water content was restricted indicating that the enzyme was more rigid under non-polar solvent system (Karimi et al. 2016).

In corroboration with the present result, a previous literature documented that for A. fumigatus, the rise in pectinase activity was more significant (approximately 35%) with n-hexane than with other non-polar solvents. Methanol and ethanol at 20% solvent concentration significantly decreased the enzyme activity (Okonji et al. 2019). Different manufacturing processes involving enzyme usage are performed in the presence of various solvents where the enzyme stability is essential for functionality. The stability of A. terreus FP6 pectinase towards diverse solvents advocates its possible industrial utilization.

Effect of activators and inhibitors on pectinase activity

A good number of enzymes require specific metal ions during their catalysis for the correct orientation of their substrates and electron exchange in redox reactions (to stabilize negative charges). Moreover, in such reactions metal ions for their ability to be present in higher concentrations at neutral pH, behave similar to proton (as Lewis’s acids) and in some instances are more effective than the proton. As an adaptation to awkward environmental conditions and remain stable, enzymes often take benefit of many metal ions available (Prejanò et al. 2020).

In this research, among the metal ions, Cu²⁺ and Co²⁺ had a positive influence on A. terreus FP6 pectinase activity. Though Ba²⁺, Mn²⁺, Mg²⁺, Hg²⁺, and Zn²⁺ moderately decreased the pectinase activity, the same was strongly inhibited by Ca²⁺. A decrease (13.4, 12.3, and 10.1 U/mL, respectively) in pectinase activity by BME, DMSO, and EDTA was observed (Fig. 5).

Fig. 5.

Effect of activators and inhibitors on the A. terreus FP6 pectinase activity. Data represent mean ± S.D. (n = 3); p < 0.05

In the presence of Cu²⁺ and Co²⁺, the elevation in A. terreus FP6 pectinase activity may be a result of the charge neutralization on pectin molecule by these ions, thereby reducing the repulsion between pectin and the overall negative charge of the enzyme. Inhibition by the salt of divalent cations like Ca²⁺ and Mg²⁺ may result in the cross-linking of homogalacturonan chain leading to lesser substrate availability to the enzyme. Additionally, this may affect the cation binding to the side chain of amino acid (involved in the binding or catalysis of the substrate). Inhibition of A. terreus FP6 pectinase activity by Ca²⁺ also suggests that the pectinase is not a polymethylgalacturonate lyase as Ca²⁺ is essential for polymethylgalacturonate lyase activity. Mercuric ions decreased the enzyme catalysis by binding to sulfhydryl groups in amino acids thereby damaging the overall enzyme structure (Ajsuvakova et al. 2020). The A. terreus FP6 pectinase displaying its highest activity in the presence of Cu²⁺, moderately decreased activity in the presence of Mn²⁺and Zn²⁺, and inhibition of activity in the presence of Ca²⁺ and Hg²⁺ indicate the enzyme to be a polygalacturonase. Similar results pertaining to the characterization of polygalacturonase from A. fumigatus MTCC 2584 was reported earlier (Anand et al. 2016). Additionally, the A. terreus FP6 pectinase being a polygalacturonase can be hypothesized from the fact that the molecular mass of the enzyme is 47 kDa while that from A. fumigatus MTCC 2584 was 43 kDa.

The influence of inhibitors on enzyme activity also provides crucial knowledge of the primary and secondary structures of the protein. The result obtained in this paper suggests that EDTA as a chelating agent bound non-specifically to the A. terreus FP6 pectinase, removed metal ions and decreased pectinase activity (Abdulrachman et al. 2017). DMSO being a dipolar, aprotic solvent (dielectric constant of 46.45) besides causing partial unfolding of the enzyme, disrupted the hydrogen bonds present between the water molecules and enzyme by competing with the protein hydrogen bond acceptors for hydrogen bond donors. Thus, DMSO decreased the enzyme catalysis by affecting the dynamic properties of the enzyme (Nandi et al. 2018). BME reduced the disulfide bonds and acted as a biological antioxidant. The inhibition by BME suggests the lack of vulnerable disulfide bond holding the pectinase structure together, thus highlighting that the A. terreus FP6 pectinase is a monomeric enzyme. The result is in perfect accordance with the observation made during the molecular mass determination of A. terreus FP6 pectinase by SDS-PAGE (Adalberto et al. 2016).

In agreement to the present result, a previous research suggested that metal ions influenced the A. niger MCAS2 pectinase activity, where Cd²⁺, Mg²⁺, Ba²⁺, and Fe³⁺ stimulated the pectinase activity, while Na⁺, Pb²⁺, Zn²⁺, and Ca²⁺ acted as inhibitors. The maximum inhibition was observed by Ca²⁺ followed by Na⁺ (Khatri et al. 2015). In another study, the lethal effect of BME on A. tamarii pectinase was recorded (Munir et al. 2020). Therefore, presence of regulatory molecules plays a vital role in the enzyme adaptability.

Enzyme kinetics

In this research, for the maximum pectinase activity (24.9 U/mL), the optimum pectin concentration was recorded as 3000 µg/mL. The catalytic activity became constant when substrate concentration increased beyond 3000 µg/mL (Fig. 6). The reason for the graph being rectangular hyperbolic is that, beyond a certain concentration any increase in availability of substrate did not affect the reaction rate since the concentration of substrate was no longer acting as a limiting factor. With the constant depletion of substrate, the enzyme catalysis ceases and accelerates with the replenishment of the substrate when more products are formed by the collision of substrate molecules with the enzyme. The enzyme thus became saturated and functioned at its maximum possible rate (Robin et al. 2018; Wang et al. 2020).

Fig. 6.

Michaelis–Menten kinetics of the A. terreus FP6 pectinase. Data represent mean ± S.D. (n = 3); p < 0.05

The best fit value obtained from the GraphPad Prism software (version 9.2.0) was used to evaluate the affinity of the A. terreus FP6 pectinase towards pectin and the K_m and V_max values were 0.002 mM and 27.39 U/mL, respectively. These results are significant as the small K_m value indicates a high enzyme affinity towards the substrate. The relation between reaction velocity and the substrate concentration when analyzed with non-regression analysis revealed that the regression coefficient (R²) was 0.9654. This denotes a positive correlation between the substrate concentrations and enzyme activity readings. Likewise, the K_m and V_max of A. niger AN07 pectinase for polygalacturonic acid as substrate were 2.6 mg/L and 181.8 µmol/mL/min, respectively (Patidar et al. 2017). Therefore, the low K_m value (0.002 mM) of obtained from the non-linear plot indicates the high efficiency of the A. terreus FP6 pectinase.

Extraction of anthocyanins

The constant need for natural pigments from various industries lead to the application of different methods for their extraction like solvent extraction, supercritical CO₂ extraction, etc. As an alternate means of pigment extraction from the highly coloured fruit and or vegetable tissues, cell wall degrading enzymes are increasingly been used. A combination of cellulolytic and pectinolytic enzymes resulting in the pore formation in cell wall of plants and subsequent release of pigments can hence be used (Samanta 2021).

For the extraction of anthocyanins, black grape skin was considered. Compared to the pomace, grape skin is a rich source of anthocyanins (due to the high concentration of chromatophores). Following the 30 min treatment, the microscopic observation revealed different visible changes. The pectinase treatment on the grape skins showed shrinkage in cell size and complete loss of pigment. This pattern was similar to the result obtained during the acetone treatment. The grape skin treated with physiological saline (control) showed no visible discolouration (Fig. 7).

Fig. 7.

Microscopic view of morphological changes in grape skin. a Physiological saline treated (control), b acetone treated, c pectinase treated. The double headed arrows in a, b and c indicate the cell size. Note that in ‘b’ and ‘c’, there is shrinkage in cell size. The single headed arrows indicate the shrinkage of pigment containing vacuoles (appearing as black dots)

The results from the present study are in agreement with another study, where pectinase produced by A. tamarii MTCC 515 showed the liberation of pigment from the grape skin and its subsequent discolouration following 24 h of enzymatic treatment (Shanmugavel et al. 2018). Among enzyme preparations, namely pectinase (from Aspergillus niger P2611), Viscozyme L (from Aspergillus aculeatus V2010), and cellulase (from Trichoderma reesei ATCC 26,921), Vicozyme L, and pectinase significantly facilitated the higher recovery of carotenoids from red capsicum (Nath et al. 2016).

The spectrophotometric analysis in the present study revealed that recovery of anthocyanin from grape skin after 30-min treatment with acetone and pectinase were 5.8 mg/mL and 5.2 mg/mL, respectively. Siddiq et al. (2018) reported similar results where, 11.49 mg/100 mL of anthocyanin from blueberry (Vaccinium corymbosum L.) juice was extracted using Pectinix (commercial enzyme).

The fact that a short duration (i.e., 30 min) of the enzymatic treatment resulted in substantial loss of pigment and shrinkage of the cells indicated the high efficiency of the pectinase as compared to those cited in the literature. Enzyme-assisted extraction is a greener approach towards pigment extraction from plant tissues. Such a technique ensures faster extraction, highest recovery, reduced or no solvent usage, and lesser energy usage when compared to the existing non-enzymatic methods. Non-solvent-based extraction improves the bioactive compounds yield by the disruption of plant cell walls and thereby releasing the bound pigments, phenolics, and flavonoids that would otherwise be unavailable and or lost in press residues (Calderón-Oliver and Ponce-Alquicira 2021).

Conclusions

The result of the present research elucidated that purification of A. terreus FP6 pectinase facilitated substantial increase in its specific activity. The optimal pectinase action at pH 6.0, 50 °C and stability towards various solvents are noteworthy features to advocate the industrial applications of the enzyme. Besides its low K_m value, the faster extraction of anthocyanins and shrinkage of the cells by the pectinase, as revealed by the in vitro plant pigment extraction assay, support its future use as a promising bioactive molecule for the beverage and food industry. However, further studies should be initiated towards understanding the amino acid composition of the pectinase which may improve its activity and stability.

Significance of the study

First report on purification and characterization of pectinase from Aspergillus terreus.

The purified enzyme is a thermostable and solvent-tolerant acid pectinase.

The low K_m value of the pectinase indicates the high enzyme affinity towards pectin as a substrate.

Application of the purified pectinase for anthocyanin extraction favoured a shorter duration (30 min) of pigment extraction as compared to those cited in the existing literature.

The shrinkage in cell size and complete loss of pigment from the grape cells was similar to the result obtained during the acetone treatment and advocates enzyme-assisted extraction to be a greener approach for pigment extraction from plant tissues.

Author contributions

SB designed the study and interpreted the data. AD contributed to the enzyme characterization studies. RB, DM and NVK performed the experiments. All authors have seen and approved the final manuscript and its contents, and are aware of the responsibilities connected to the authorship.

Funding

Not applicable.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Code availability

GraphPad Prism software (version 9.2.0); R software (version 4.0.2).

Declarations

Conflict of interest

The authors declare that they have no conflict of interest in the publication.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Publication status

The work has not been published elsewhere, either completely, in part, or in another form. The manuscript has not been submitted to another journal and will not be published elsewhere.