3.3. Identification of isolate
In Fig. 3, a fungus was isolated and identified based on cultural characteristics and sporulation as Aspergillus sp. Based on primary and secondary screening, Aspergillus sp. was identified as a potent strain and was chosen for further investigation. Initially, the colonies are made up of a compact yellow felt. The colonies then turn brown due to the production of conidiophores. The reverse side ranges from cream to yellow. And the hyphae had radiate conidial heads, and no teleomorph stage was observed, but there was heavy sporulation, the hyphae were septate and hyaline, and the conidia were globose to subglobose. Aspergillus sp. strains are promising sources of pectinase enzyme. Several previous studies have shown that Aspergillus sp. is important in the commercial production of polygalacturonase enzyme (Maciel et al., 2013, El Enshasy et al., 2018).
3.4. Production of Polygalacturonase
After solid-state fermentation, the crude enzyme was extracted. In 150 mL of crude extract, protein content and polygalacturonase enzyme activity were determined. The protein content was determined to be 13.74 mg/ml. The activity of polygalacturonase was determined to be 94.6 U/ml. The specific activity was found to be 6.89 U/mg. Mucorgenevensis has a maximum reported polygalacturonase activity of 5 U/mL (Alves et al., 2002), Penicillium viridicatum has an activity of 18 U/mL (Gomes et al., 2009), and Mucorcircinelloides has an activity of 9.15 U/mL (Thakur, 2010).
3.5. Enzyme Purification
PGase of the screened Aspergillus sp was purified from 500 mL of SSF crude extract (Table 3). Purification was carried out in two stages. First, ammonium sulphate precipitation was used to separate proteins with varying solubilities. The polygalacturonase enzyme was initially purified by adding solid ammonium sulphate. Ammonium sulphate precipitation increased the enzyme's specific activity from 6.89 U/mg to 12.42 U/mg. Polygalacturonase can be precipitated with 0–90% ammonium sulphate depending on the source of the enzyme (Mohammed et al., 2010; Shalom et al., 2017). Second, the sample was analyzed on two columns of chromatography, the first of which yielded 2.93 mg/ml of protein produced from 80 mL enzyme extract and purified 3.58 folds by Sephadex G -200, and the second of which yielded specific activity of 24.66 U/mL. The Sephacryl S-100 column aided in final fold purification, with yields of 9.93% and 20.97%, respectively. Previous researchers have found significant variations in yield and purification. Satapathy et al. (2021) found Aspergillus parvisclerotigenus to be 2.10-fold purified with a yield of 2.21%. Penicillium oxalicum was purified 29.9 times and yielded 17.1%, according to Cheng et al. (2016). Penicillium oxalicum was purified 28 times and yielded 57%, according to Almowallad et al. (2022). For Aspergillus fumigatus, Anand et al. (2016) reported 18.43-fold purification and 2.98% yield. These factors are primarily determined by the strain as well as the purification techniques used.
Table 3
Purification of polygalacturonase from Aspergillus sp
Purification steps | Collected Volume (mL) | Total Protein (mg/mL) | Total enzyme Activity (U/mL) | Specific Activity (U/mg) | Purification fold | Yield (%) |
Crude extract | 150 | 13.74 | 94.6 | 6.89 | 1 | 100 |
(NH4)2 SO4 precipitation | 80 | 2.93 | 36.4 | 12.42 | 1.8 | 38.48 |
SephadexG-200 | 12 | 1.16 | 28.6 | 24.66 | 3.58 | 30.23 |
Sephacryl S-100 | 1 | 0.29 | 19.84 | 68.41 | 9.93 | 20.97 |
3.6. Characterization of the enzyme
Effect of Temperature on the activity of polygalacturonase
Temperature is a critical factor in both microbial growth and product formation. The temperature of incubation has a significant impact on microbial growth rate, enzyme secretion, enzyme inhibition, and protein denaturation (Adeyefa and Ebuehi, 2020). The effect of reaction temperature on polygalacturonase activity is depicted in Fig. 4. The pectinase activity was detected at temperatures ranging from 30°C to 60°C, with 55°C being the optimal temperature, followed by 50°C and 45°C. The finding demonstrated that polygalacturonase activity increased with increasing temperature until the optimal temperature was reached. Meanwhile, polygalacturonase activity dropped dramatically above 60°C. The present study results are in line with those revealed by Kaur et al. (2004), who found that exo-polygalacturonase produced from thermophilic mould Sporotrichum thermophile was optimally active at 55°C. The present study's findings are in agreement with those of Kaur et al. (2004), who found that exo-polygalacturonase produced by the thermophilic mould Sporotrichum thermophile was most active at 55°C. Previous research has found that the optimal temperature for PGase activity produced by Aspergillus awamori NRC-F18 and Rhizomucor pusillus was 55oC. The decrease in enzyme activity at high temperatures is attributed to the denaturation of the enzymes (Almowallad et al., 2022).
Effect of Substrate specificity on the activity of polygalacturonase
Purified pectinase's affinity for substrates was determined, as shown in Fig. 5. When polygalacturonic acid was used as a substrate, the PGases specificity was highest. The enzyme has 100%, 51.02%, 16.07%, 8.47%, and 45.67% relative activity against polygalacturonic acid, pectin, xylan, galactose, and cellulose, respectively. Siddiqui et al. (2012) reported a similar observation in which polygalacturonic acid was used as a substrate in the substrate specificity study and 8.34 U/mL of polygalacturonase activity (100% relative activity) was obtained.
Effect of pH on the activity of polygalacturonase
The initial pH of the fermentation medium is critical in determining metabolite synthesis levels. The stability of the microbial metabolite is also affected by the medium's hydrogen ion concentration (Adeyefa and Ebuehi, 2020). pH is important in pectinase production because it promotes and regulates extracellular enzyme synthesis by microorganisms, particularly fungi (Siddiqui et al., 2012). Fungi, particularly Aspergillus species, have been shown to thrive in acidic or slightly alkaline environments (Yusuf, 2019). In Fig. 6, the present study found that pH 5 was the optimum pH for polygalacturonase activity (100% relative activity) produced by Aspergillus sp. The results agreed with those of Aminzadeh et al. (2020), who found that Polygalacturonase from Tetracoccosporium sp. was more active at acidic pH of 5. Polygalacturonase from Aspergillus fumigatus with 5 (Wang et al., 2015) with 5, Penicillium oxalicum CZ1028 with 5 (Cheng et al., 2016), Thermoascus aurantiacus (da Silva Martins et al., 2012) with 5, Penicillium oxalicum AUMC 4153 with 5 ((Almowallad et al., 2022), Aspergillus tubingensis with 5 (Tai et al., 2012).
Effect of divalent cations on enzyme activity
The effects of different metal ions were studied using a concentration of 1 mM of each metal ion in the reaction solution (Fig. 7). Only Ca+ 2 was found to increase PG activity among all metal ions, whereas Zn+ 2 inhibited enzyme activity. Metal ions may typically act as PGase's moral character endorsement to naturally inspire the process (Robinson, 2015). In the energetic location, the bending ability of the enzyme capacity typically has differential elasticity (Mohamad et al., 2015).