Research Article

Purification and Characterization of α-Amylase Produced from Penicillium citrinum

Mehak Iftikhar1, Memuna G. Shahid1, Zohaib Anjum1, Awais Anjum1, Ikram-ul-Haq2, Nazish Mazhar Ali3 and Reham Shakeel1

1Department of Botany, Government College University, Lahore, Pakistan; 2Department of Biotechnology, Government College University, Lahore, Pakistan; 3Department of Zoology, Government College University, Lahore, Pakistan.

Received | September 25, 2025; Accepted | May 18, 2026; Published | March 28, 2026

*Correspondence | Mehak Iftikhar, Department of Botany, Government College University, Lahore, Pakistan; Email: [email protected]

Citation | Iftikhar, M., M.G. Shahid, Z. Anjum, A. Anjum, I. Haq, N.M. Ali and R. Shakeel. 2026. Purification and characterization of α-amylase produced from Penicillium citrinum. Pakistan Journal of Weed Science Research, 32(1): 62-68.

DOI | https://dx.doi.org/10.17582/journal.pjwsr/2026/32.1.62.68

Keywords | α-Amylase, Potato peel, OFAT, Purification, Characterization

Copyright: 2026 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Enzymes are biological catalysts that initiate chemical processes within living organisms. They are typically created by living things found in the different cells in extremely tiny concentrations (approximately 0.1%) (Djekrif-Dakhmouche et al., 2006). A hydrolytic enzyme called α-amylase breaks down α-1, 4-glycosidic bonds in starch and similar polysaccharides to produce glucose, maltotriose, and maltose (Farooq et al., 2019). Due to its wide range of uses in food, textiles, paper, detergents, fermentation, and medicines, it makes for around 25% of the worldwide industrial enzyme market (Kubilay et al., 2010). Because of their high production efficiency, stability, and ease of scaling, microbial α-amylases especially those derived from bacteria and fungi are preferred in industry (Reddy et al., 2003).

Filamentous fungi, such as A. niger and A. oryzae, are key producers of extracellular enzymes, including α-Amylase, under SSF (solid-state fermentation) is due to their capacity to colonize and penetrate solid substrates (Jiby et al., 2016). Penicillium citrinum is particularly appealing because to its fast growth, Generally Recognized as Safe (GRAS) status, and ability to release hydrolytic enzymes in acidic environments. The literature on optimal enzyme synthesis, purification, and characterization specifically from P. citrinum is still scarce, despite the fact that numerous researches have assessed α-amylase production from fungal species. Their hydrolytic efficiency and tolerance to acidic pH make them ideal for enzyme production (Ertan et al., 2006).

The expanding industrial need for thermostable and pH-tolerant enzymes emphasizes the need to investigate new microbial sources and optimize culture conditions to increase enzyme production and performance. P. citrinum, an underutilized but promising species, has the potential to produce high-activity α-amylase for industrial starch breakdown and bioprocessing (Jujjavarapu and Dhagat, 2019; Silaban et al.,2020). Optimization of fermentation parameters such as temperature, pH, incubation time, and nutrient composition significantly enhances enzyme yield. Submerged and surface fermentation techniques are widely used, with surface culture involving microbial growth on liquid surfaces followed by enzyme extraction (Jensen et al., 2002). Advanced optimization has improved the production of high-purity α-amylases for industrial and pharmaceutical applications (Elyasi et al., 2020).

In this study, Penicillium citrinum was employed for α-amylase production under optimized fermentation conditions. Enzyme characterization included analyses of temperature and incubation effects, and enzyme activity was determined using spectrophotometry. Salt precipitation can help to partially purify the enzyme. Determine the enzyme’s activity under a variety of physicochemical conditions. The project aims to provide baseline data to improve fungal α-amylase production and develop cost-effective strategies for industrial enzyme applications (Silaban et al., 2020; Ullah et al., 2025).

Materials and Methods

Sample collection

In this investigation, α-amylase was produced using potato peels as a substrate. They were collected from Lays, Pepsi-Cola International (Pvt) Ltd, located in Lahore, Pakistan.

Obtainment of fungal organism

The fungus species, Penicillium citrinum, was obtained from Cell Culture Insight Laboratory, Department of Botany GC University Lahore, Pakistan.

Maintenance of fungal culture

PDA and MEA slants were used to maintain the fungal culture of Penicillium citrinum. The media was prepared, sanitized, and placed into aseptic test tubes for solidification.

Inoculation of fungal strains

The Penicillium citrinum strain was inoculated on PDA slants, incubated at 25℃ for 7 days, and the best-growing slants were kept as parent cultures. PDA slants with excellent growth were chosen for further subculture and experimentation.

Fermentation technique

α-amylase was produced from Penicillium citrinum by surface culture fermentation using a modified medium that contained potato peel, sucrose, yeast extract, and different salts, with a pH of 5.5. A spore suspension of Penicillium citrinum was aseptically introduced into the medium after it had been autoclaved. For the best enzyme synthesis, the fermentation was incubated for five to seven days at 25°C.

Production of α-amylase enzyme from Penicillium citrinum

Mycelial growth was weighed and collected after five to seven days of incubation. It was then dried and crushed in acetone. The mycelium was centrifuged to extract the intracellular α-amylase, and the leftover media was centrifuged to acquire the extracellular α-amylase.

Determination of α-amylase

Amylase activity was determined using the method described by Kubilay et al. (2010). To determine α-amylase activity, a glucose standard curve was created by serially diluting glucose and measuring its optical density at 540 nm. The enzyme activity was measured by incubating the supernatant with starch and DNS solution, then measuring optical density at 540nm (Hiteshi and Gupta, 2014). In fermentation conditions, one unit of -Amylase is produced as one mol of α-amylase (1U/min), which is the unit of enzyme activity U/ml.

Partial purification of α-amylase enzyme

The enzyme was refined using Solid-State Fermentation with sodium sulfate and centrifugation at 10,000 rpm. To assess pure α-amylase absorbance, Sodium citrate buffer (pH 5.5) was used to dissolve the precipitate and Optical density was measured at 540 nm (Prakash et al., 2010).

Characterization of α-amylase enzyme

The effect of temperature, pH, and substrate concentration on α-amylase activity was investigated by incubating the enzyme with varying conditions (temperatures: 25–60°C, pH: 3.5–7, substrate concentrations: 1–50 mM) and measuring activity using common procedures. Each experiment was repeated three times for accuracy.

Statistical analysis

Standard error (SE) was determined in enzyme assays to compare variability among replicates. Mean activity values were plotted against treatment conditions to create graphical representations. Error bars show ±SD. Steel and Torrie’s (1996) techniques were used to conduct all statistical analyses.

Results

Production of α-amylase with Penicillium citrinum: Table 1 displays the production of α-amylase in a fermentation medium using Penicillium citrinum. With extracellular (0.978 U/ml) and intracellular (1.230 U/ml) activity, sample A had the highest α-amylase production, whereas sample B had the lowest with extracellular (0.273 U/ml) and intracellular (0.240 U/ml) activity.

 

Table 1: Production of α-amylase with Penicillium citrinum.

S. No.	Samples	Extracellular α-amylase activity (mg/ml)	Intracellular α-amylase activity (mg/ml)
1	Sample A	0.978 ± 0.004	1.230±0.1
2	Sample B	0.273±0.004	0.240±0.04
3	Sample C	0.281±0.003	0.250±0.01

 

Effect of pH and temperature on α-amylase production

The maximum activity at various pH levels was in 10 mM sodium acetate buffer at pH 5.5 (0.327 ± 0.005 U/mL), followed by pH 6 (0.271 ± 0.011 U/mL). The optimal amylase activity was found at 37 ºC (0.398 ± 0.019 U/mL) and 40 ºC (0.382 ± 0.016 U/mL). The standard deviation is indicated by the symbol ±, and each result is the mean of three replicates (Figure 1).

 

 

Effect of carbon, nitrogen sources and inorganic salts on α-amylase enzyme production

Maximum production of α-amylase was observed with sucrose (1.518±0.1 U/ml extracellular) and fructose (2.547±0.1 U/ml intracellular) as carbon sources, peptone (0.420±0.004 U/ml extracellular, 0.301±0.001 U/ml intracellular) as a nitrogen source, and 2g KH₂PO₄ (0.372±0.002 U/ml extracellular) and 3g KH₂PO₄ (0.372±0.002 U/ml extracellular) and 1.190±0.004 U/ml intracellular) as phosphate sources, and 1.5g NaCl (0.340±0.003 U/ml extracellular) and 0.75g NaCl (0.585±0.004 U/ml intracellular) as sodium sources, the highest α-amylase production was observed Figure 2.

Effect of metal ions on α-amylase production

Metal ion concentrations (MgSO₄, MnSO₄, ZnSO₄, and FeSO₄) had varying effects on α-Amylase production. The highest extracellular and intracellular α-Amylase activities were observed at 1g of MgSO₄, 1g of MnSO₄, 0.5g of ZnSO₄, and 0.25g of FeSO₄, while the lowest α-Amylase activities were observed at 0.25g of MgSO₄, 1.5g of MnSO₄, 1.5g of ZnSO₄ Figure 3.

Partial purification of α-amylase

The partial purification of α-Amylase is displayed in Table 2, where sample D yielded the highest yield (1.270±0.002 U/ml) and sample C yielded the lowest yield (0.138±0.002U/ml). The mean±standard deviation of three replicates is represented by each value.

 

Table 2: Partial purification of α-amylase.

S. No.	Samples	Purified α-amylase enzyme
1	Sample A	0.553±0.002
2	Sample B	0.355± 0.001
3	Sample C	0.138±0.002
4	Sample D	1.270±0.002

 

Discussion

Amylases are enzymes that hydrolyze starch molecules to produce glucose-based polymers. They are widely employed in sectors such as medicines, food processing, and fermentation. Penicillium citrinum produces α-amylase efficiently during surface culture fermentation, supporting previous reports that filamentous fungi are reliable sources of hydrolytic enzymes due to their ability to penetrate solid substrates and secrete high levels of extracellular enzymes. Kathiresan and Manivannan (2006) published similar findings, demonstrating significant amylolytic activity in Penicillium species isolated from varied biological settings. Our findings further show that P. citrinum can be successfully grown on agro-industrial waste materials like potato peels, making the procedure both cost-effective and environmentally friendly.

Penicillium citrinum was used to purify and characterize α-amylase using surface culture fermentation. Table 1 shows the maximum levels of α-amylase production and mycelial development. This study’s ideal pH for enzyme activity was 5.5, which is consistent with research by Saranraj and Stella (2013) and Gupta et al. (2003), which found that fungal α-amylases usually show their highest catalytic efficiency in acidic to slightly acidic conditions. The enzyme retained half of its activity at 60°C after 15 minutes, was most active at 40°C, and continued to be active between 40 °C and 70°C. These results align with those of Ogbonna et al. (2018) (Figure 1).

Initially, different carbon sources (sucrose, glucose, fructose, maltose, and galactose) were investigated in the fermentation medium to determine how they affected α-amylase production and mycelial growth. Sucrose (2.5g) was discovered to be the best carbon source for maximizing enzyme yield (Figure 2). Faiq et al. (2025) found that sucrose concentration in fermentation media plays a crucial role in producing α-amylase. Similarly, fructose increased intracellular enzyme synthesis, implying that intracellular and extracellular amylases follow different regulatory processes. The minimal reaction to organic nitrogen sources is consistent with the findings of Sun et al. (2010), who discovered that nitrogen supplies have a stronger influence on fungal biomass than enzyme induction. This could explain why peptone stimulated moderate enzyme production but did not significantly increase synthesis.

Furthermore, the study investigated the impact of organic nitrogen sources on fungal growth and enzyme synthesis. The study found that organic nitrogen did not significantly increase α-amylase output (Figure 2). Sun et al. (2010) found that nitrogen supplies can boost fungal growth but may not directly contribute to α-amylase synthesis.

 

Inorganic salts (KH₂PO₄) and cofactors (MgSO₄, MnSO₄, ZnSO₄, NaCl, and FeSO₄) were investigated further. The enzyme production was maximum with 3g KH₂PO₄ (Figure 2), 1.25g ZnSO₄, and 0.25g MgSO₄ (Figure 3). Inorganic salts were also important for enzyme control. In our work, KH₂PO₄, a key buffering and metabolic salt, dramatically boosted α-amylase production. This is similar with previous findings by Kammoun et al. (2008), who showed that phosphate enhances fungal metabolism and enzyme biosynthesis. The highest α-amylase yield was found at 1g MnSO₄ and 1g FeSO₄ (Figure 3). MnSO₄ had the least effect in increasing enzyme synthesis compared to other salts. Sindhu (2005) found that using an adjusted medium with MgSO₄ and FeSO₄, which function as cofactors during fungal fermentation, increased α-amylase production considerably.

Penicillium citrinum α-amylase was purified in a single step using ammonium sulfate precipitation (Table 2). Similar purification patterns have been reported for Aspergillus and Penicillium enzymes (Sazzad and Sabita, 2008), demonstrating the efficacy of this technique for downstream processing. Although single-step precipitation produced partially purified enzyme, the activity levels imply that additional purification stages, such as ion-exchange chromatography, could improve enzyme purity for industrial uses.

Conclusions

Penicillium citrinum has the ability to manufacture more α-amylase by surface culture fermentation, whereas fungi and bacteria also produce the enzyme. Optimizing fungal strains and fermentation conditions can increase yield, with temperature and incubation time having a substantial impact on enzyme activity and stability. The study recommends the OFAT method as a realistic way to increase α-Amylase production. Purification was restricted to ammonium sulfate; enzyme kinetics and industrial-scale parameters were not investigated. Future studies should include chromatographic purification, kinetic profiling, and bioreactor optimization.

Novelty Statement

The present work describes the first detailed purification and biochemical characterization of alpha-amylase from Penicillium citrinum showing its unique kinetic and physicochemical properties with promising industrial applicability

Author’s Contribution

Mehak Iftikhar: Orignal idea, experimental work, Analysis.

Memuna G. Shahid: Research Supervisor, editor, analysis, experimentataion.

Zohaib Anjum: Analysis, experimental work.

Awais Anjum and Reham Shakeel: Analysis.

Ikram-ul-Haq: Editor, statistical analysis.

Nazish Mazhar Ali: Editor.

Generative AI and AI assisted technology statement

The authors declare that no generative AI and AI assisted technology was used in the creation of this manuscript.

Conflict of interest

The authors have declared no conflict of interest.

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