Assessment of glycanolytic enzymes producing potentialities of three strains of Penicillium sp

doi:10.21203/rs.3.rs-7383841/v1

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Assessment of glycanolytic enzymes producing potentialities of three strains of Penicillium sp

https://doi.org/10.21203/rs.3.rs-7383841/v1

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Microfungi are part of the microbiome that play a significant role in the decomposition of natural substances by producing various enzymes that have industrial relevance. The primary objective of the study is to determine the glycanolytic enzyme-producing potentialities of three microfungi strains, viz.PDF4, XDF1(i), and XDF7(iii), and to optimize the enzyme production and activity at different pH and temperatures. The strains were characterized by molecular methods. The enzyme production efficacy was tested both qualitatively and quantitatively using glycan molecules, such as carboxymethylcellulose (CMC), birchwood xylan, and pectin. The enzyme activities were evaluated under varying pH and temperature conditions to determine the optimal parameters for maximal enzyme activity. Additionally, the ability of the strains to degrade lignocellulosic substrates, such as sugarcane bagasse and musambi peel, and to synthesize those enzymes was also investigated. The studies revealed that among the strains belonging to Penicillium citrinum strain PDF4 (ITS: OR555780; NL: OR555752), P. citrinum strain XDF1(i) (ITS: OR555782; NL: OR555750), and P. oxalicum strain XDF7 (iii) (ITS: OR555781; NL: OR555751), strain XDF7(iii) was the highest pectinase producer (0.303547 µM/mL/min at pH 3.0), followed by PDF4 with 0.245313 µM/mL/min, and XDF1(i) with 0.205393 µM/mL/min, at pH 4.0 in YP-pectin medium. While XDF7(iii) showed the highest xylanase activity, reaching 0.768501µM/mL/min at pH 5.0, followed by 0.563401 µM/mL/min at pH 6.0. XDF1(i) exhibited the second-highest xylanase activity (0.553409µM/mL/min) at pH 5.0. In contrast, PDF4 recorded 0.343314 µM/mL/min xylanase activity at pH 5.0 in YP-xylan substrates, respectively, while strain XDF7(iii) demonstrated poor production of CMCase (0.01302 µM/mL/min) at pH 3.0, followed by XDF1(i) (0.0121µM/mL/min at pH 3.0) and PDF4 (0.00696 µM/mL/min) at pH 4.0 in YP-CMC medium at 7 days of fermentation at 37°C. The studies further revealed that the strain XDF7(iii) effectively utilized both musambi peel and sugarcane bagasse, and recorded the highest xylanase activity (1.0912764 µM/mL/min) on the 3rd day of incubation on lemon (Musambi) peel and the second-highest activity (0.9921957µM/mL/min) on the 7th day on sugarcane bagasse. While PDF4 produced the highest xylanase activity (0.7687782 µM/mL/min) on the 7th day, XDF1(i) exhibited only moderate xylanase activity on the 3rd day (0.5166818µM/mL/min). In contrast, CMCase activity remained minimal throughout the incubation period with Musambi peel. Thus, the study concludes that these new environmental strains of Penicillium sp. can produce a high amount of industrial enzymes, such as xylanase and pectinase, under standard fermentation conditions. This suggests that they could be utilized as low-cost enzymes from lignocellulose biomasses and also for lignocellulose biomass bioconversion to develop numerous value-added products.

Glycanolytic enzymes

Penicillium citrinum

P. oxalicum

Natural substrates

Fungi secrete several hydrolytic enzymes that degrade natural polymers, such as cellulose, pectin, xylan, and starch. The enzyme cellulases break down the cellulose polymer into β-1,4-glucose residues, the xylanases degrade the linear β-1,4-xylan polymer into xylose, and the pectinase enzyme breaks down the pectins into β-1,4-polygalacturonic acid (Halder and Purakait 2020). These three polymers are the structural components of plant cells and are abundant natural resources that can be used for many value-added products. However, due to their β-linkage, many microbial species are unable to break down this compound. Therefore, searching for microbial species that can degrade these polymers could contribute to the transformation of plant biomass (Beg et al., 2001; Kashyap et al., 2001; de Castro et al., 2010). Among the value-added products, monosaccharides, oligosaccharides, and enzymes have numerous applications in various industries, including the food and pharmaceutical industries, biofuel industries, cosmetic industries, and brewing and distilling industries, among others (FitzPatrick et al., 2010; Chaturvedi and Verma, 2013; Baruah et al., 2018; De Bhowmick et al., 2018; Arthrong et al., 2020; Ashokkumar et al., 2022). It has been observed that applying plant cell wall-degrading enzymes (CWDEs) as catalysts could reduce the cost of hydrolysis, chemical waste generation, and carbon footprint (Velvizhi et al., 2022; Zhao et al., 2023). Therefore, finding new microbial species with glycanolytic properties could significantly impact these concerns. Conventionally, microfungi have superior applications compared to other microbial species due to their higher efficiency, ease of handling, and lower maintenance costs (Mandal et al., 2021).

Fungi like Aspergillus, Chaetomium, Clonostachys, Penicillium, and Trichoderma are promising genera that produce CWDEs. A few decades ago, the hyper-cellulolytic filamentous fungus Trichoderma sp., such as T. reesei RUT-C30, was predominantly used in biomass conversion and lignocellulolytic enzyme production (Sreeja-Raju et al., 2020). However, very few species of Penicillium or Penicillium-like fungi have been explored in this direction. Recently, there has been ample evidence that the microfungi Penicillium sp. have greater efficacy than the Trichoderma sp. (Sreeja-Raju et al., 2020; Korotkova et al., 2023). Recently, Sreeja-Raju et al. (2020)demonstrated that P. janthinellum NCIM1366 exhibited enhanced biomass hydrolysis and a higher number of Carbohydrate Active enZymes (CAZymes) with increased induction levels compared toT. reesei RUT-C30. Taylor et al. (2018) compared the cellobiohydrolases (CBHs) from T. reesei (TrCel7A) and P. funiculosum (PfCel7A) and found that PfCel7A exhibits 60% greater performance on biomass than TrCel7A. Industries like Danisco US Inc. in the USA produce food-grade cellulase enzymes from a non-genetically modified P. funiculosum strain, Lzc35(Anonymous 2021). Due to the industrial relevance of these microfungi, whole-genome analysis of certain strains of Penicillium, such as P. oxalicum(Pham et al., 2023), P. ochrochloron RLS11 (genome size 38.06 Mbp with 12,015 protein-coding genes)(Morgan et al., 2022)d parvum (25.8 Mb containing a total of 376 CAZymes (Long et al., 2023) was performed. The whole-genome analysis reveals that Penicillium spp. possess a repertoire of PCWDE-coding genes, including cellulases, numerous hemicellulases, glycosidases, cellobiohydrolases (CBHs), endoglucanases (EGs), and β-glucosidases (BGLs) (Morgan et al., 2022; Long et al., 2023).In comparison, Hu et al. (2016) sequenced the draft genome of Talaromyces verruculosus (P. verruculosum) strain TS63-9 and found that the genome (37.35 Mbp) contains 11,447 predicted protein-coding genes, among which 532 are annotated as CAZymes and 225 are PCWDE. A secretome analysis in P. janthinellum and P. echinulatum, Penicillium sp. Dal 5 found that they contained more CAZymes for biomass hydrolysis (Rai et al., 2016; Schneider et al., 2016; Christopher et al., 2022). Therefore, it is assumed that the species Penicillium, Sarcopodium, and Talaromyces possess the genetic potential for the hydrolysis of lignocellulosic biomass.

Lignocellulose biomass is a cost-effective and renewable energy source within circular economy management(FitzPatrick et al., 2010; Velvizhi et al., 2022). The cost of producing second-generation bioethanol from lignocellulosic biomass is attributed to the enzymes employed in biomass hydrolysis(FitzPatrick et al., 2010; Baruah et al., 2018; De Bhowmick et al., 2018; Ashokkumar et al., 2022; Velvizhi et al., 2022). Therefore, an efficient mixture of polysaccharide-degrading enzymes is needed to convert plant biomass into fermentable sugars for industrial applications. Moreover, selecting new strains of glycanolytic enzymes producing Penicillium sp. and related species, along with their targeted genetic and proteomic analysis, may contribute significantly to the lignocellulose bioconversion industry.

Therefore, searching for potential microfungi that produce suitable glycanolytic enzymes could boost the industries. In this direction, the main objectives of the study were to characterize three microfungal strains [PDF4, XDF1(i), and XDF 7(iii)] for their glycanolytic properties, i.e., cellulolytic, pectinolytic, and xylanolytic activities in different culture conditions, and assess their potentiality in producing such enzymes in various culture conditions.

2.1. Fungal strains and culture conditions

The microfungi strains, viz. Penicillium citrinum strain PDF4 (ITS: OR555780; NL: OR555752), P. citrinum strain XDF1(i) (ITS: OR555782; NL: OR555750), and P. oxalicum strain XDF7 (iii) (ITS: OR555781; NL: OR555751), isolated from the spoiled foods and plant debris, were cultured in selective carbohydrate-enriched media like GYP-Cellulose, GYP-Pectins and GYP-Xylan containing Glucose/Dextrose monohydrate (0.1 g/100 ml), Yeast extract (0.01 g/100 ml), Peptone (0.05 g/100 ml), Manganese sulfate monohydrate (0.05 g/100 ml), Potassium dihydrogen phosphate (0.01 g/100 ml), Sodium nitrate (0.3 g/100 ml), Potassium chloride (0.5 g/100 ml), Calcium chloride (0.01 g/100 ml) and Iron sulfate (0.01 g/100 ml) supplemented with suitable glycan substrate (0.1%, w/v)at 28°C, subcultured at every 7 days. All the strains were maintained in their respective culture plates and stored at 4°C for further study (Mandal et al., 2021).

2.2. The glycanolytic potency of the strains

2.2.1. Enzyme assay: Qualitative test

The production of extracellular glycanolytic enzymes by these strains under submerged cultivation was investigated using a YP medium supplemented with the respective glycan polymer substrates. The optimization steps include studies of substrate concentration, the incubation temperature, and the initial pH of the culture media. The fungal strains were grown separately on YP-cellulose (CMC)/pectin/xylan agar plates. After three to four days of growth, one cup (7 mm) from each culture plate was cut using a sterile cork borer and inoculated into culture tubes, each containing 10 mL of YP-cellulose/pectin/xylan broth with 0.1% (w/v) of the respective substrate. The tubes were incubated at 30°C for 10 days. On the 3rd, 7^th, and 10th days of incubation, the cell-free culture aliquots were collected by centrifuging at 7,683 × g for 10 min at 4°C and used as the enzyme source for detecting glycanolytic enzymes produced by the strains.

The assay of the PCWDE of the strains was performed using an agar plate containing 50 µM citrate buffer (pH 5.6–5.8) and 0.1% carboxymethyl cellulose (CMC, pH 5.6–5.8). After 16 hrs of incubation at 28°C, the plates were flooded with a 0.8% Congo red solution (w/v) and destained after 15 min with a 1(M) sodium chloride solution. Clearance zones around the hole appeared and confirmed the presence of cellulase enzyme (CMCase) in the enzyme source, and the diameters were measured. Similarly, the xylanolytic activity and pectinolytic activity were detected with 1% iodine and potassium iodide solution (iodine: potassium iodide, 1:2, w/w) in the agar plate containing 50 µM citrate buffer (pH 5.6 to 5.8) and 0.1% xylan (w/v) and 0.1% pectin (w/v), respectively. The clearance zone appeared around the margin of the holes, indicating the hydrolysis zones. All experiments were repeated three times, and the average values were recorded for analysis.

2.2.2. Enzyme assay: Quantitative test

The enzyme source was taken from the culture aliquot, as stated above. The quantitative glycanolytic activities of the strains on the respective glycan substrates were estimated using the DNS (3,5-dinitrosalicylic acid) method (Wood et al., 2012) and compared to standards, such as glucose, xylose, for CMCase, and pectinase and xylanase, respectively.

Assay of cellulase activities: 200 µL substrate containing 0.1% CMC (w/v) dissolved in 50 µM citrate buffer (pH 5.6–5.8) was used as the substrate for the experiment. 100 µL crude enzyme source from different substrates was added to the reaction mixture and incubated in a water bath at 37°C for 60 min. After that, 300 µL of DNS (3,5-dinitrosalicylic acid) reagent was added to the mixture to stop the reaction. The treated samples were kept in a boiling water bath for 5 minutes. 100 µL 40% Rochelle salt solution was added to the tubes at a lukewarm state. The samples were cooled, and the absorbance was read using a microplate reader (iMark microplate reader, BioRad, USA) at 490 nm. A mixture of 200 µL of CMC solution and 100 µL of YP-CMC broth (without fungal inoculation) was treated similarly as a control. The amount of reducing sugar released was measured from the standard glucose curve (100 µg/ml).

Assay of pectinase activities: 200 µL substrate containing 0.1% pectin (w/v) dissolved in 50 µM citrate buffer (pH 5.6–5.8) was used as the substrate for the experiment, and 100 µL crude enzyme was added to it. This mixture was incubated in a water bath at 37°C for 60 min, and 300 µL of DNS was added to the solution to stop the reaction. After the aforementioned steps, the optical density was measured using a microplate reader (iMark microplate reader, BioRad, USA) at 490 nm. A mixture of 200 µL of pectin solution and 100 µL of YP-Pectin broth (without fungal inoculation) served as the control. The amount of reducing sugar released was measured from the standard curve of glucose (100 µg/ml).

Assay of xylanase activities: 200 µL substrate containing 0.1% birchwood xylan (w/v) dissolved in 50 µM citrate buffer (pH 5.6–5.8) was used as the substrate for the experiment. 100 µL of crude enzyme was added to the substrate and incubated in a water bath at 37°C for 60 min. Then, 300 µL of DNS was added to the solution to stop the reaction. After the steps mentioned above, the optical density was measured using a microplate reader(iMark microplate reader, BioRad, USA) at 490 nm. A mixture of 200 µL of birchwood xylan solution and 100 µL of YP-Xylan broth (without fungal inoculation) served as the control. The amount of reducing sugar released was measured from the standard curve of xylose (100 µg/ml).

2.5. Optimization of pH and temperature for enzyme activity:

2.5.1. Preparation of crude enzyme extract:

Fungal strains were cultured on Yeast Peptone (YP) Agar plates (pH 5.6) at 28°C for 3–4 days. For enzyme production, YP broth media supplemented with selective carbohydrates—carboxymethyl cellulose (CMC), birchwood xylan, and pectin—were prepared and adjusted to a pH of 5.6 ± 0.1. These media were designated as YP-CMC, YP-Xylan, and YP-Pectin, respectively. Following initial growth on YP agar plates, 7.0 mm agar plugs were excised from the plate cultures and inoculated into the YP-CMC, YP-Xylan, and YP-Pectin broths (1 cup/10 mL). The broth cultures were incubated at 28°C for 7 days under static conditions. After the incubation period, soup from the broth cultures was collected and centrifuged to obtain a cell-free crude enzyme extract. The enzyme extracts were stored at 4°C for further analysis.

2.5..2. Optimization of pH for enzyme activity:

To determine the optimal pH for enzyme activity, a range of 100 mM buffer solutions was prepared, covering pH values from 3 to 11. Carbohydrate substrates—carboxymethyl cellulose (CMC), birchwood xylan, and pectin were dissolved in different buffer systems according to the pH range: citrate buffer was used for pH 3, 4, and 5; phosphate buffer for pH 6, 7, and 8; and carbonate-bicarbonate buffer for pH 9, 10, and 11. For the enzyme assay, 280 µL of the substrate-buffer solution was dispensed into microcentrifuge tubes. Subsequently, 20 µL of the crude enzyme extract was added, mixed thoroughly, and incubated under the following conditions: For xylanase and pectinase activity, incubation was performed in a water bath at 37°C for 1 hr. For CMCase (cellulase) activity: incubation in a BOD incubator at 37°C for 16–20 hrs due to the recalcitrant nature of CMC. After incubation, enzyme activity was assessed using the DNSA (3,5-dinitrosalicylic acid) method for estimating reducing sugars. DNSA reagent was added to each reaction mixture according to the standard protocol, and the absorbance was measured at 490 nm using a microplate reader(iMark microplate reader, BioRad, USA). The subsequent enzyme activities (µM/mL/min) were calculated from the resulting data.

2.5.3. Optimization of temperature for enzyme activity:

To determine the optimum temperature for enzyme activity, substrate-buffer systems were prepared specific to each enzyme assay. For the xylanase assay, birchwood xylan was dissolved in 100 mM citrate buffer (pH 5.0); for the pectinase assay, pectin was dissolved in 100 mM citrate buffer(pH 5.6); and for the CMCase assay, carboxymethyl cellulose(CMC) was dissolved in 100 mM citrate buffer (pH 3.0 and pH 4.0).

For the assay, 280 µL of the respective substrate-buffer solution was taken into sterile microcentrifuge tubes, followed by the addition of 20 µL of crude enzyme extract. The mixtures were incubated at 27°C, 37°C, and 47°C for 60 minutes in a water bath for the xylanase and pectinase assays. In contrast, for CMCase assays, samples were incubated under identical temperature conditions in a BOD incubator for 16–20 hrs due to the complex and recalcitrant nature of the CMC substrate.

Post-incubation, enzymatic activity was quantified using the 3,5-dinitrosalicylic acid (DNSA) method to estimate the concentration of reducing sugars. The absorbance was recorded at 490 nm using a microplate reader(iMark microplate reader, BioRad, USA). CMCase, xylanase, and pectinase activities (µM/mL/min) were calculated from the obtained data.

2.3. Enzymatic activity of the strains on natural substrates:

The estimation of CMCase, Xylanase, and Pectinase activities utilizing natural carbohydrate sources as substrates was determined by the following methods. Fungal strains were initially cultivated on Yeast Peptone (YP) agar plates, pH 5.6, and incubated at 28°C for 3–4 days to promote active mycelial development. For enzyme production, YP broth was supplemented with natural lignocellulosic substrates, including musambi peel (MP) and sugarcane bagasse (SB). Following sufficient mycelial growth, 7.0 mm agar cups were excised from 3–4 day-old plate cultures and transferred (1 cup/10 mL broth) into 100 mL Erlenmeyer flasks containing YP broth enriched with 5% natural substrates (pH 5.6). The flasks were incubated at 28°C under shaking conditions (100 RPM) to facilitate substrate degradation and enhance enzyme secretion. Culture aliquots were harvested on the 3rd, 7^th, and 10th days of incubation. The harvested broths were centrifuged to obtain cell-free crude enzyme extracts, which were stored at 4°C for subsequent enzymatic assays. For the determination of enzyme activity, specific substrates were prepared using carboxymethyl cellulose (CMC), pectin, and birchwood xylan in their respective buffers, and assayed as described above. The corresponding enzyme activities (µM/mL/min) were subsequently calculated from the resulting data.

2.4. Statistical analysis:

All the experiments were executed in triplicate. The box plot, PCA, and One-way ANOVA were used to assess significant differences using past software (https://past.en.lo4d.com/windows).

3.1. Microfungal strains and their phylogenetic positions:

The fungal strains and their systematic position are shown in Fig. 1. It has been noted that the strain PDF4 (ITS: OR555780; NL: OR555752), and strain XDF1(i) (ITS: OR555782; NL: OR555750) belong to the genus Penicillium citrinum, though they are distantly placed in the phylogenetic tree, and the strain XDF7 (iii) (ITS: OR555781; NL: OR555751) belongs to P. oxalicum.

3.2. Qualitative and quantitative assays for glycanolytic enzymes: cellulase, pectinase, and xylanase.

During fermentation, the strains produced different glycanolytic enzymes, i.e., cellulase (CMCase), pectinase, and xylanase (Fig. S1a). The qualitative enzymatic assays show that the diameter of substrate hydrolysis increased with increasing incubation time. The strains PDF4 and XDF7(iii) produced a high amount of CMCase, while the lowest was for pectinase and xylanase (Fig. S1b). Meanwhile, the XDF1(i) strain showed moderate enzymatic activity.

The quantitative assays for glycanolytic enzymes show a significant difference in their enzyme activities compared to the qualitative tests.

CMCase:

The time course optimisation study revealed that XDF7(iii) reached the peak CMCase activity (0.032286833 µM/mL/min) on the 7th day of incubation in YP-CMC medium. PDF4 attained maximal CMCae activity (0.034298978 µM/mL/min) on the 7th day of incubation in medium YP pectin medium, with comparable activity levels (0.028771361 µM/mL/min in YP-CMC on the 7th day of incubation. XDF1(i) recorded its maximum CMCase activity (0.031454221 µM/mL/min) on day 7 in medium YP pectin (Fig. 2a).

Pectinase:

XDF7(iii) demonstrated the maximal pectinase activity (0.049642011 µM/mL/min on the 7th day of their growth in YP-pectin medium. Similarly, PDF4 achieved the highest levels of pectinase activity (0.033781464 µM/mL/min) on 7th day of incubation in YP-pectin medium. XDF1(i) attained peak pectinase activity (0.03680763 µM/mL/min) on the 7th day of its growth in YP-CMC medium, with comparable pectinase activity levels in YP-xylan (0.02921002 µM/mL/min) and YP-pectin (0.027664744 µM/mL/min) media on 7th day of incubation(Fig. 2b).

Xylanase:

The time course activity of xylanase revealed that almost all strains attained peak xylanase activity when in YP-xylan medium (Fig. 2c). PDF4 exhibited peak xylanase activity (0.016180421 µM/mL/min on the 3rd day of incubation. XDF7(iii) showed peak activity of 0.018234197 µM/mL/min on 10th day of growth in YP-xylan medium with comparable levels of activity(0.015902884 µM/mL/min) on 7th day of growth in the same medium. Similarly, XDF1(i) achieved maximal xylanase activity (0.015486578 µM/mL/min) on the 10th day of incubation in YP-xylan medium. One-way ANOVA test for the data sets is the sum of squares between groups:0.000152044, with df 2.0, within groups: 0.00759106, with df 78, Mean square between groups 7.60219E-05, and within the group with 9.73213E-05, F 0.7811. p = 0.4614. Welch F test in the case of unequal variances: F = 0.6892, df = 51.46, p = 0.5066

3.3. pH optimum for the enzymatic activity of the strains

Effect of pH on CMCase activity in different fungal strains:

The pH optimization experiment revealed that the tested strains exhibited minimal CMCase activity under highly acidic conditions, specifically at pH 3.0 to 4.0. Enzyme activity progressively decreased with increasing pH (Fig. 3a). At higher pH levels (6–10), CMCase activity was either absent or undetectable, suggesting that the enzyme is susceptible to alkaline conditions. Among the strains cultured in YP-CMC broth, strain XDF7(iii) demonstrated the highest CMCase activity (0.01302 µM/mL/min) at pH 3.0, followed by XDF1(i) (0.0121µM/mL/min at pH 3.0) and PDF4 (0.00696 µM/mL/min) at pH 4.0. Furthermore, when the strains were grown in YP-xylan and YP-pectin media, a marginal reduction in CMCase activity was observed, indicating substrate-specific regulation of enzyme expression and activity under varying environmental conditions.

Effect of pH on Pectinase activity in different fungal strains:

The pH range experiment revealed that YP-pectin is the most effective medium for inducing pectinase activity in the tested fungal strains. The results indicate that pectinase activity is optimal at acidic pH levels, particularly between pH 3.0 and 4.0, with a marked decline in enzyme activity observed as the pH increases (Fig. 3b). Among the tested strains, strain XDF7(iii) was the highest pectinase producer, showing an activity of 0.303547 µM/mL/min at pH 3.0. followed by PDF4 with 0.245313 µM/mL/min, and XDF1(i) with 0.205393 µM/mL/min, at pH 4.0.

Effect of pH on Xylanase activity in different fungal strains:

The pH range experiment demonstrated that the fungal strains exhibit maximum xylanase activity predominantly at acidic pH levels, particularly between 5.0 and 6.0. Among the tested media, YP-xylan was identified as the most suitable substrate for xylanase production, whereas YP-CMC and YP-pectin induced only negligible xylanase activity. Among the fungal strains, XDF7(iii) showed the highest xylanase activity, reaching 0.768501µM/mL/min at pH 5.0, followed by 0.563401 µM/mL/min at pH 6.0. XDF1(i) exhibited the second-highest xylanase activity (0.553409µM/mL/min) at pH 5.0. While PDF4 recorded 0.343314 µM/mL/min xylanase activity at pH 5.0 (Fig. 3c), it was also observed that the xylanase activity of the strains declined with increasing pH.

Temperature optimum for the enzymatic activity of the strains:

CMCase:

Among the temperatures tested, 37°C was found to be the optimal temperature for CMCase activity (Fig. 4a). Additionally, YP-CMC medium was determined to be the most favourable medium for supporting this CMCase activity. At pH 3.0, XDF7(iii) demonstrated the highest CMCase (0.013017µM/mL/min) followed by XDF1(i) (0.012 µM/mL/min and PDF4(0.0048 µM/mL/min). At 27°C and 47°C, CMCase activity of the fungal strains was either below detectable limits or completely absent.

Pectinase:

Among the tested temperatures, viz. at 27°C, 37°C, and 47°C, both 27°C and 37°C supported moderate levels of pectinase activity, with YP-pectin as the most suitable medium at pH 5.6 (Fig. 4b). The strain PDF4 exhibited pectinase activity of 0.14258 µM/mL/min and 0.14222 µM/mL/min at 27°C and 37°C, respectively. XDF7(iii) showed 0.138431 µM/mL/min and 0.179781 µM/mL/min pectinase activity at 27°C and 37°C, respectively. XDF1(i) showed 0.138002 µM/mL/min and 0.145084 µM/mL/min pectinase activity at 27°C and 37°C, respectively. Pectinase activity was markedly reduced at 47°C, suggesting that elevated temperatures adversely affect the enzyme's catalytic efficiency.

Xylanase:

Among the three tested temperatures, viz. 27°C, 37°C, and 47°C, the temperature of 37°C was found to be the most favorable, and YP-xylan was identified as the most suitable medium for xylanase enzyme activity (Fig. 4c and d). At 37°C temperature and pH 5.0, XDF7(iii) exhibited the highest xylanase activity of 0.768501µM/mL/min, followed by XDF1(i) (0.5534093µM/mL/min) and PDF4 (0.343314µM/mL/min). In contrast, at 27°C, xylanase activity was significantly reduced across all strains. The enzyme activity at this temperature ranged approximately from 0.089 to 0.104 µM/mL/min. At 27°C and pH 5.0, PDF4 exhibited the highest enzyme activity (0.1049996µM/mL/min) followed by XDF7(iii) (0.0929735µM/mL/min) and XDF1(i) (0.0895508µM/mL/min). At 47°C, xylanase activity in all tested strains was either below the detectable limit or completely absent, indicating a potential loss of enzyme stability or function at higher temperatures.

Enzymatic activity of the strains on natural substrates:

CMCase:

In the case of CMCase, all strains exhibited very low enzyme activity on Lemon (Musambi) peel, while no detectable activity was observed on Sugarcane bagasse across all incubation periods(Fig. 5a). Among the tested strains, XDF7(iii) emerged as the highest CMCase producer, showing peak activity(0.011714µM/mL/min) on the 3rd day of incubation on Musambi peel. This was followed by PDF4 (0.011388 µM/mL/min) on the 7th day. XDF1(i) showed the highest amount of CMCase activity on the 7th day of incubation, measuring 0.011598 µM/mL/min.

Pectinase:

All fungal strains utilised both musambi peel and sugarcane bagasse for pectinase production, with a general preference for musambi peel. Among the tested strains, the XDF7(iii) ranked the highest in pectinase production, with the highest activity recorded on the 3rd day (0.857629 µM/mL/min) of incubation utilising musambi peel, followed by the 7th (0.8159919µM/mL/min) and 10th day (0.7125442 µM/mL/min) of growth on the same substrate (Fig. 5b). With sugarcane bagasse, moderate levels of pectinase were also produced; however, enzyme activity declined over time, with the highest activity noted on the 3rd day (0.5945022µM/mL/min) of incubation.XDF1(i) demonstrated considerable pectinase activity, achieving peak enzyme levels (0.8106979µM/mL/min) on the 7th day of incubation with musambi peel. When grown on sugarcane bagasse, the highest pectinase activity (0.5000687 µM/mL/min) was observed on the 10th day.PDF4 also proved to be a notable pectinase producer on musambi peel, with peak activity (0.7362957 µM/mL/min) on the 3rd day of incubation. When grown on sugarcane bagasse, pectinase activity peaked on the 10th day (0.5608782 µM/mL/min).

Xylanase:

All five fungal strains demonstrated varying levels of xylanase production when grown on natural lignocellulosic substrates, with differences observed in both substrate preference and incubation time.XDF7(iii) utilized both musambi peel and sugarcane bagasse effectively. It recorded the highest xylanase activity (1.0912764µM/mL/min) among all strains on the 3rd day of incubation on lemon (Musambi) peel and the second-highest activity (0.9921957µM/mL/min) on the 7th day on sugarcane bagasse (Fig. 5c). Additionally, it maintained good enzyme activity on the 3rd (0.9194809 µM/mL/min)and 10th day (0.9405737 µM/mL/min) on sugarcane bagasse, as well as on the 7th day (0.8481538 µM/mL/min) on musambi peel. However, a decline was noted by the 10th day, with a measurement of 0.6394458 µM/mL/min. PDF4 also demonstrated good substrate utilization efficiency, showing a preference for lemon (Musambi) peel. It recorded its highest xylanase activity (0.7687782 µM/mL/min) on the 7th day of growth on the lemon (Musambi) peel substrate followed by 0.7079975 µM/mL/min xylanase activity on 3rd day of growth on sugarcane bagasse, XDF1(i) exhibited only moderate activity on lemon (Musambi) peel, with the 3rd day (0.5166818µM/mL/min) yielding the highest output, followed by the 7th day (0. 5081707µM/mL/min). A sharp decline in xylanase activity (0.2088005 µM/mL/min) was observed on 10th day of growth. Sugarcane bagasse was not a suitable substrate for XDF1(i), as xylanase activity remained consistently low throughout all incubation days. The combined box plot (Fig. 5d), the matrix plot (Fig. 5e), and the PCA (Fig. 5f) analyses of the data sets also project the same observation, focusing on the strains XDF7(iii), which emerged as a strong xylanase producer in the natural substrates musambi peel and sugarcane bagasse. This production level was also higher than that of single-carbohydrate sources, such as pectins and birchwood xylan. The one-way ANOVA test for the data sets yields a sum of squares between groups of 0.084567, with a degree of freedom (df) of 4.0, and a sum of squares within groups of 1.09144, with a df of 70. The mean square between groups is 0.0211417, and the mean square within groups is 0.015592. The F-value is 1.356. p = 0.2581.

Microfungi grow on diverse substrates to meet their nutritional requirements. To utilize such diverse substrates, they employ different hydrolytic enzymes and have a wide range of metabolic pathways for substrate utilization. The study shows that the strains PDF4, XDF1(i), and XDF7(iii) could assimilate a wide range of sugars, with slow utilization of lactose, sodium gluconate, and alpha-methyl-d-glucoside. This difference established that the strains had unique sugar utilization pathways similar to those of all other microfungi, such as Aspergillus spp. and Thermomyces spp. (Mandal et al., 2021; Maheshwari et al., 2000). Utilizing a wide range of sugars indicates their ability to hydrolyze polymers, which is essential for survival in natural habitats. Arnthong et al. (2020) showed that among the 297 fungal strains, they identified four fungal strains with potential cellulase-producing capabilities, belonging to P. oxalicum (AG452, AG496, AG498, and AG559). They demonstrated that P. oxalicum AG452 yielded the highest glucose after saccharifying pretreated sugarcane trash, cassava pulp, and coffee silverskin. Chein et al. (2019) isolated 85 strains from peanut kernels and identified them using ITS5/4 and beta-tubulin (Bt2a/2b) primer sets, finding 12 strains of P. citrinum that produced a significant amount of glycanolytic enzymes. The present study shows that the current strains produce more than one type of glycanolytic enzyme in a single substrate, which could be beneficial in hydrolyzing complex biomasses, as observed in the hyper-cellulolytic P. funiculosum, which produces accessory enzymes to facilitate the complete saccharification of sugarcane bagasse (Ogunyewo et al., 2021). Batista et al. (2022) screened the production of hydrolases (amylases, cellulases, and pectinases) from the Amazonian palm (Euterpe precatoria) species' endophytic fungi and Penicillium sp. L3 was the best producer of amylase.

The fermentation time is a crucial factor for enzyme production in a microbial system. The cost-effectiveness relies on it. The time-course optimisation study indicated that among the strains, PDF4 exhibited peak CMCase activity on the 10th day of incubation, while strains XDF1(i), and XDF7(iii) reached their highest activity on the 7th day. All strains consistently showed maximum pectinase activity on the 7th day of incubation. In contrast, xylanase production displayed apparent temporal differences: PDF4 reached peak activity on the 3rd day, and strains XDF1(i) and XDF7(iii) on the 10th day of incubation. Therefore, the time course study showed a fast to moderate fermentation time requirement for all the strains, indicating their suitability for large-scale enzyme production.

The pH optimisation study demonstrated that all three enzymes exhibited varying degrees of activity across the tested pH range. Pectinase and xylanase exhibit optimum activity under acidic conditions, specifically, pectinase activity peaks between pH 3.0 and 4.0, with YP-pectin medium emerging as the most potent medium for pectinase production. Within this range, XDF7(iii) had the most significant enzyme activity. In parallel, strains also had substantial xylanase activity in an acidic environment (pH 5.0–6.0). YP-xylan was identified as the most suitable medium for producing xylanase.XDF7(iii), XDF1(i), and PDF4 were observed to have a notably high xylanase activity at pH 5.0, while XDF7(iii) maintained a considerable amount of xylanase activity at pH 6.0. The strains exhibited negligible CMCase activity across the entire pH range. The present study also revealed that cellulase production is not directly related to the effect of substrate on hydrolytic performance, as compared to P. brasilianum IBT 20888 (Jørgensen and Olsson, 2006). Prasanna et al. (2016) reported the production of cellulolytic enzymes in a Czapek-Dox medium supplemented with 0.5% (w/v) cellulose by Penicillium sp. in submerged shake culture conditions. The acidophilic xylanase production was reported from Penicillium sp. 40 (Kimura et al., 2000; Knob and Carmona,2010) and alkaliphilic xylanase from the alkali-tolerant P. citrinum strain (Dutta et al., 2007). Many reports indicate that high acidic pH (pH 3.0) and high basic pH (pH 12.0) may impact growth rate and enzyme production (Kimura et al., 2000; Dutta et al., 2007). Han et al. (2017)reported improved cellulase productivity of P. oxalicum RE-10 using a repeated fed-batch fermentation strategy, and the maximum filter paper activity (FPA) (12.69 U/ml) was observed during submerged batch fermentation compared to single-batch flask culture (8.61 U/ml). Dos Reis et al. (2013) also reported the use of a repeated fed-batch fermentation strategy to increase cellulase productivity from 105.75 U/L/h in batch fermentation to 158.38 U/L/h.

The fungal strains exhibited substrate-specific preferences for enzyme production when grown on natural lignocellulosic materials. XDF7(iii), and PDF4 utilised both musambi peel (MP) and sugarcane bagasse(SB) effectively and had significant xylanase activity. In contrast, XDF1(i) displayed considerable Xylanase activity utilizing musambi peel, with minimal xylanase activity observed when grown on sugarcane bagasse. All the fungal strains were capable of producing pectinase, utilising both musambi peel and sugarcane bagasse, with higher pectinase activity observed on musambi peel, indicating it as the more favorable substrate for pectinase production. CMCase activity was consistently low across all strains. Detectable CMCase activity was only observed when the fungal strains were cultivated on musambi peel. No CMCase activity was detected in cultures grown on sugarcane bagasse. While Rai et al. (2016) reported that Penicillium sp. strain Dal 5 under a shake flask on CWR (cellulose, wheat bran and rice straw) medium produced appreciably higher levels of endoglucanase (35.69 U/ml), β-glucosidase (4.20 U/ml), cellobiohydrolase (2.86 U/ml), FPase (1.2 U/ml) and xylanase (115U/ml) compared to other Penicillium strains reported in literature. Dos Santos et al. (2020) reported a strain of P. digitatum RV 06 produced the maximum CMCase (1.6 U/ml) in a stationary liquid culture of 1% lactose, pH of 5.0, at 25°C for 5 days, while Xue et al. [2016] reported the co-production of glucose and xylooligosaccharides (XOS) by P. oxalicum EU2106 in the sugarcane bagasse (SB), and the maximum yields of glucose and XOS were 34.43 ± 0.32g and 5.96 ± 0.09 g per 100 g raw SB. In comparison to the above literature, it can be hypothesized that optimizing culture conditions might enhance enzyme production from the present strains.

From the above observations, it can be concluded that among the new strains of Penicillium sp., viz. P. citrinum strain PDF4, P. citrinum strain XDF1(i), and P. oxalicum strain XDF7(iii); the strain XDF7(iii) showed the highest pectinase activity, followed by PDF4 and XDF1(i). In comparison, XDF7(iii) showed the highest xylanase activity, followed by XDF1 (i) and PDF4. These pectinases and xylanases are acidophilic enzymes. All the strains demonstrated poor production of CMCase activity. Moreover, natural substrate utilization revealed that the strains displayed prominent xylanase and pectinase activities, with minimal CMCase activity. Musambi peel was proven to be the most effective inducer of pectinase, while sugarcane bagasse supported both xylanase and pectinase activities, but failed to induce any CMCase activity. Therefore, exploiting abundant, low-cost agro-residues such as Musambi peel and sugarcane bagasse could be used for large-scale enzyme production from these strains. Therefore, employing these carbohydrate-active enzymes (CAZymes) from the current strains could lead to the generation of value-added products and the production of bioethanol from these low-cost agro-residues. Further studies are underway to determine the optimum enzyme production and cost-effective purification for global applications.

CZA

Czapek dox Agar

CMC

Carboxymethyl cellulose

CTAB

Cetytrimethyle ammonium bromide

CZB

Czapek dox broth

EDTA

Ethylenediamine tetra acetic acid

PCR

Polymerase chain reaction

PDA

Potato Dextrose Agar

PDF

Pectin Degrading Fungus

PVP

Polyvinyl pyrrolidone

SDS

Sodium dodecylesulfate

Yeast Peptone

XDF

Xylan Degrading Fungus

Competing Interests:

The authors declare that they have no conflict of interest.

Consent to Publish:

Not applicable

Consent to participate:

Not applicable.

Funding:

The authors did not receive support from any organization for the submitted work.

Authors' contribution:

All authors contributed to the conception and design of the study. Conceptualization: Vivekananda Mandal; Methodology: Nabanita Kundu, Dilruba Khatun, Ashutosh Kundu; Formal analysis and investigation: Nabanita Kundu, Dilruba Khatun, Ashutosh Kundu; Writing - original draft preparation: Nabanita Kundu, Dilruba Khatun; Writing - review and editing: Vivekananda Mandal; Funding acquisition: Vivekananda Mandal; Resources: Vivekananda Mandal; Supervision: Vivekananda Mandal. All authors read and approved the final manuscript.

Acknowledgments:

We sincerely acknowledge the BOOST (Biotechnology based Opportunities Offered to Science & Technology Departments) equipment grant support (BOOST program 2017–2018 (Ref. No. 1089/BT (Estt)/1P-07/2018; dated: 24.01.2019) to the department by the West Bengal Department of Science & Technology and Biotechnology, Government of West Bengal, for the infrastructure development of the department.

Data Availability Statement:

The data supporting this study's findings are available from the corresponding author upon reasonable request.

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Assessment of glycanolytic enzymes producing potentialities of three strains of Penicillium sp

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1. Fungal strains and culture conditions

2.2. The glycanolytic potency of the strains

2.2.1. Enzyme assay: Qualitative test

2.2.2. Enzyme assay: Quantitative test

2.5. Optimization of pH and temperature for enzyme activity:

2.5.1. Preparation of crude enzyme extract:

2.5..2. Optimization of pH for enzyme activity:

2.5.3. Optimization of temperature for enzyme activity:

2.3. Enzymatic activity of the strains on natural substrates:

2.4. Statistical analysis:

3. Results

3.1. Microfungal strains and their phylogenetic positions:

3.2. Qualitative and quantitative assays for glycanolytic enzymes: cellulase, pectinase, and xylanase.

3.3. pH optimum for the enzymatic activity of the strains

Discussion

Conclusion

Abbreviations

Declarations

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Data Availability Statement:

References

Supplementary Files

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