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. 2023 Dec 26;14(1):22. doi: 10.1007/s13205-023-03871-x

Agricultural wastes: a new promising source for phenylalanine ammonia-lyase as anticancer agent

Abdulaziz Albogami 1, Deyala M Naguib 2,3,✉
PMCID: PMC10751285  PMID: 38156037

Abstract

The present study aims to investigate the physicochemical characteristics of phenylalanine ammonia-lyase (PAL) extracted from agricultural waste and its potential use as an anticancer agent in comparison to microbial PAL. We extracted and partially purified PAL from agricultural waste sources. We assessed the temperature and pH range of PAL and determined enzyme kinetics parameters including Michaelis constants (Km), maximum velocity (Vmax), and specificity constant values (Vmax/Km). Additionally, we examined the effects of different storage temperatures on PAL activity. In our analysis, we compared the efficacy of agricultural waste-derived PAL with PAL from Rhodotorula glutinis. The results demonstrated that PAL extracted from agricultural waste exhibited significantly higher specific activity (Vmax/Km) compared to its microbial counterpart. The agricultural waste-derived PAL displayed a stronger affinity for phenylalanine, as indicated by a lower Km value than the microbial PAL did. Furthermore, PAL from agricultural waste maintained activity across a broader temperature and pH range (15–75 °C, pH 5–11), in contrast to microbial PAL (20–60 °C, pH 5.5–10). Importantly, the PAL derived from agricultural waste exhibited superior stability, retaining over 90% of its activity after 6 months of storage at room temperature (25 °C), whereas microbial PAL lost more than 70% of its activity under similar storage conditions. In anticancer experiments against various cancer cell lines, agricultural waste-derived PAL demonstrated greater anticancer activity compared to microbial PAL. These findings suggest that PAL sourced from agricultural waste has the potential to be a safe and effective natural anticancer agent.

Keywords: Michaelis constants (Km), Maximum velocity (Vmax), Optimum temperature, Optimum pH, Specificity constant, Rhodotorula glutinis PAL

Introduction

In biological systems, enzymes are the central components of metabolic reactions; they effectively and selectively catalyze various metabolic processes. As such, enzymes wield a significant influence on human health. Recently, there has been growing interest in the use of enzymes within the pharmaceutical industry (Tandon et al. 2021).

Cancer stands out as one of the most perilous and pressing health challenges, demanding the exploration of new, efficacious treatments. Chemotherapy and radiation therapy continue to be the primary modes of cancer treatment, but they carry severe side effects on healthy tissue and often fall short of eliminating cancerous cells. Conversely, targeted cancer treatment has emerged as a promising and well-developed approach with fewer systemic interferences (Weng et al. 2022). Enzymatic therapy for cancer represents one of the most promising techniques within the realm of targeted cancer treatment (Baig et al. 2019). Notably, there has been a resurgence of interest in cancer metabolic treatments, particularly those involving enzyme-mediated amino acid deprivations. The U.S. Food and Drug Administration has approved the use of L-asparaginase in the treatment of acute lymphoblastic leukemia. Additionally, other amino acid-degrading enzymes are effective for cancer therapy, including glutaminase, methionase, lysine oxidase, and phenylalanine ammonia-lyase (Wang et al. 2021).

Phenylalanine ammonia-lyase (PAL) is an enzyme responsible for catalyzing the removal of ammonia from L-phenylalanine, resulting in the production of trans-cinnamic acid. Koukol and Conn originally discovered PAL in Hordeum vulgare in 1961. This enzyme has a well-known role in plant defense mechanisms against various stress conditions and is essential for the biosynthesis of lignin, flavonoids, coumarins, and other secondary metabolites (Barros and Dixon 2020). PAL has found extensive applications in both industry and agriculture and its biological uses have recently garnered significant attention (Kawatra et al. 2020; Besada et al. 2022).

Targeted cancer therapies depend on metabolic differences that distinguish normal cells from cancerous ones. Cancer cells require a substantial metabolic supply of amino acids to support their rapid multiplication, and one of the amino acids they cannot produce themselves is L-phenylalanine. Since cancer cells cannot synthesize phenylalanine and cannot utilize phenylpyruvate as an alternative for growth, PAL plays a crucial role in inhibiting tumor growth by reducing the amount of external phenylalanine in these cells (Kawatra et al. 2020). Abell and his colleagues were pioneers in reporting the effectiveness of PAL both in vitro and in vivo as an anticancer agent against murine tumors in 1973. Subsequently, Babich et al. (2013) identified PAL as a promising candidate for breast cancer and prostate cancer therapy. More recently, Yang et al. (2019) demonstrated the effectiveness of PAL in the treatment of colorectal cancer.

The primary source of therapeutic enzyme production is microbial fermentation. However, recent clinical use of microbial therapeutic enzymes has revealed several disadvantages, including hypersensitivity, cytotoxicity, and a short circulating half-life. Moreover, the production of anti-enzyme antibodies stands out as a particularly serious drawback, often leading to treatment discontinuation (Fonseca et al. 2021). Plant-derived enzymes may hold the key to overcoming these limitations (Mohammadi et al. 2020).

Agriculture and industry generate vast amounts of waste annually, posing significant environmental challenges unless we find practical ways to address them (Adejumo and Adebiyi 2020). Numerous biotechnological endeavors have explored the valorization of agricultural waste due to its rich content of valuable active chemical components (Naguib and Tantawy 2019; Puglia et al. 2021; Al-Shaaibi et al. 2022; Capanoglu et al. 2022; Ditsawanon et al. 2022; Singh et al. 2022). One such emerging initiative involves the recovery of proteins bound within agricultural waste, highly valued by the food, chemical, and pharmaceutical sectors. The recovery and subsequent modification of these proteins have garnered considerable research attention (Yusree et al. 2021).

In light of these considerations, this study aims to investigate the physicochemical characteristics of PAL extracted from agricultural waste and explore its potential as an anticancer agent against various cancer cell lines in comparison to microbial PAL.

Materials and methods

Phenylalanine ammonia-lyase extraction and assay

Phenylalanine ammonia-lyase extraction from agricultural wastes

Agricultural wastes, including wheat straw, rice husk, soybean bagasse, corn bagasse, and peanut husk, were sourced for this study. Wheat straw, rice husk, soybean bagasse, and corn bagasse were acquired from a farm under the Agricultural Administration in Awlad Sakr, Sharqia Governorate, Egypt. Peanut husks were collected by purchasing peanuts from the local market in Egypt. The waste materials underwent thorough washing with sterilized tap water as an initial step, followed by drying to inhibit microbial activity. Subsequently, the cleaned and dried agricultural wastes were processed into a fine powder. A known quantity of the agricultural waste powder was then incubated in an extraction buffer at a ratio of 1:100 (w:v). The extraction buffer consisted of 50 mM Tris–HCl, 10 mM 2-mercaptoethanol, 1 mM EDTA, and 2.5% polyvinylpyrrolidone-40, with a pH of 8.8. After centrifuging the mixture for 20 min, the clear supernatant was partially desalted in aliquots using an Amicon Ultra-15 Centrifugal Filter Unit with a membrane nominal mass limit of 50 kDa. These aliquots served as crude enzyme sources. The obtained enzyme extract was further precipitated with 60% ammonium sulfate at 4 °C for 20 min on a stirrer. The mixture was then centrifuged at 10,000 g at 4 °C for 30 min to isolate the precipitated protein. The resulting pellet was resuspended in 100 mM ice-cold Tris–HCl (pH 8.8). The enzyme extract was subsequently dialyzed against 100 mM Tris–HCl (pH 8.8) at 4 °C for 24 h, with changing the buffer every 2 h during the first 6 h of the incubation period. The dialysate solution was used as a partially purified PAL (Phenylalanine Ammonia-Lyase). Enzyme activity and protein content were determined in both the crude enzyme and the purified enzyme extract (Şirin et al. 2016).

Enzyme assay

The assay reaction mixture contained 100 mM Tris–HCl, 40 mM l-phenylalanine, and an aliquot of the enzyme (0.5 mL) in a total volume of 1 mL at pH 8.8. The reaction mixture was incubated at 37 °C for 30 min and stopped with the addition of 50 μL of 4 M HCl. Trans-cinnamate production was then evaluated by the change in absorbance at 290 nm to determine PAL activity. The PAL-specific activity was expressed in U mg-1 protein. One unit is defined as the enzyme that catalyzes the deamination of 1µM of L-phenylalanine to trans-cinnamate and ammonia per minute (Goldson et al. 2008).

PAL from Rhodotorula glutinis (microbial PAL) was purchased from Sigma–Aldrich to be used in the comparison of PAL obtained from agricultural wastes.

Physiochemical characterization of PAL

Optimum pH and temperature

The optimum pH for the PAL activity from the agricultural wastes and the microbial PAL was determined by assaying the activity at different pH values at 30 °C.

The optimum temperature for PAL activity was determined by measuring the activity of the enzyme at different temperatures at pH 8.5.

Storage stability

The PAL obtained from the agricultural wastes and the microbial PAL were stored for 6 months at different temperatures (room temperature of 25 °C, cooling at 10 °C, and freezing at − 18 °C). PAL activity was determined after the storage period. The activity of the fresh enzyme was taken as 100%.

Kinetic parameters

The values of Michaelis constants (Km) and maximum velocity (Vmax) were determined using phenylalanine as substrate in the range of 0.1–30 mM. Kinetic parameters were determined from the Lineweaver–Burk plot.

Anticancer activity of PAL

The anticancer activity of PAL from different agricultural wastes and the microbial PAL was tested using the MTT test, as described previously by Mosmann (1983). Breast cancer cell line (MCF-7), colon cancer cell line (HCT116), cervical cancer cell line (HELLA), gastric cancer cell line (CLS-145), esophageal cancer cell line (KYSE-410), pancreatic cancer cell line (AsPC-1), and liver cancer cell line (HepG2) were obtained from Cell Line Service (Germany). The anticancer activity was determined in terms of the IC50 of the enzymes.

Statistical analysis

The statistical analysis was performed by SPSS software (version 14). Data were expressed as mean ± standard error. A comparison of the mean between each phenylalanine ammonia-lyase (PAL) from agricultural wastes and the PAL from Rhodotorula glutinis was done using a paired t test, P < 0.01 (Levesque 2007).

Results and discussion

Extraction and assay of PAL

The protein content in the purified enzyme extract from agricultural waste varied among different types. Among the agricultural wastes, peanut husks exhibited the highest protein content in the purified enzyme extract, whereas rice and wheat straw demonstrated the lowest protein content (see Table 1). It is worth noting that agricultural wastes are known for their richness in polyphenols and lignin, with PAL (Phenylalanine Ammonia-Lyase) being a crucial enzyme in the biosynthesis pathway of lignin and other polyphenols in plants (Barros and Dixon 2020). Consequently, the extracted protein from agricultural wastes contains enzymes associated with the biosynthesis of these compounds (Cheng et al. 2020). The variations in lignin content are because of the differences in PAL content and its activity (Barros and Dixon 2020). For instance, peanut husks have been reported to contain approximately 36.1% lignin (Pączkowski et al. 2021), whereas rice and wheat straws have a lignin content of around 15% (Guangjun et al. 2018). Moreover, Adhikari et al. (2019) have previously highlighted the potential of peanut shells as a valuable source of pharmaceutical compounds.

Table 1.

Protein content (mg/mL), phenylalanine ammonia-lyase (PA) activity (U/mL), and PAL-specific activity (U/mg protein) in the crude and purified enzyme extract from different agricultural wastes

Sample Protein content (mg/mL) PAL activity (U/mL) PAL-specific activity (U/mg protein)
Sorghum bagasse Crude enzyme extract 34.283 ± 2.102* 70.283 ± 1.932* 2.050 ± 0.129
Purified enzyme extract 3.291 ± 0.534* 73.435 ± 2.034* 22.314 ± 1.044*
Sugar cane bagasse Crude enzyme extract 40.230 ± 1.243* 74.019 ± 2.125* 1.840 ± 0.0913
Purified enzyme extract 3.302 ± 0.685* 76.583 ± 2.009* 23.193 ± 1.583*
Peanut husk Crude enzyme extract 51.174 ± 1.543* 172.340 ± 3.102* 3.368 ± 0.329*
Purified enzyme extract 6.124 ± 0.863* 173.554 ± 3.029* 28.340 ± 0.957*
Rice straw Crude enzyme extract 9.892 ± 0.143* 3.102 ± 0.647* 0.314 ± 0.001*
Purified enzyme extract 0.872 ± 0.053* 3.921 ± 0.938* 4.496 ± 0.142*
Wheat straw Crude enzyme extract 9.524 ± 0.520* 3.092 ± 1.019* 0.325 ± 0.035*
Purified enzyme extract 0.805 ± 0.025* 3.703 ± 0.892* 4.600 ± 0.673*
R. glutinis 19.587 ± 0.501 47.009 ± 1.203 2.400 ± 0.472
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Values are given as means of three replicates ± standard error. Values followed by asterisk are significantly different from R. glutinis PAL according to paired t test at P value 0.01

Table 1 illustrates that the extraction process of PAL from agricultural wastes is notably more efficient compared to PAL extracted from R. glutinis, a microbial source. Specifically, the specific activity of PAL extracted from agricultural wastes significantly surpasses that of R. glutinis, indicating that a given quantity of the extracted protein contains more PAL units than the microbial counterpart. This finding aligns with previous research, as Mohammadi et al. (2020) reported that therapeutic enzymes derived from plant sources tend to outperform their microbial counterparts. Similarly, Al-Hazmi and Naguib (2022) found that asparaginase from plant sources exhibits greater activity compared to its microbial counterpart. It is important to note that the efficacy of therapeutic enzymes depends on their kinetics and biochemical characteristics (Mohutsky and Hall 2021).

Physiochemical characterization of PAL

The physiochemical characteristics of an enzyme undoubtedly play a significant role in its activity and efficiency. In the present study, we investigated the impact of temperature and pH on PAL activity. As depicted in Fig. 1, PAL extracted from agricultural waste exhibited a broader temperature tolerance compared to its microbial counterpart. The temperature range for PAL from agricultural waste spanned from 15°C to 70°C, with an optimal temperature around 40°C. In contrast, microbial PAL displayed a narrower temperature range, ranging from 20°C to 60°C, with an optimal temperature of approximately 30°C. These findings are consistent with previous research suggesting that phenylalanine ammonia-lyase in plants can remain active at temperatures as high as 80°C (Eissa and Ibrahim 2018). Figure 2 presents the results regarding the pH sensitivity of PAL. PAL extracted from agricultural waste demonstrated a broader pH tolerance, ranging from pH 5 to 11, with an optimal pH level of around 8.5. On the other hand, microbial PAL had a narrower pH range, spanning from pH 5.5 to 10, with an optimal pH of around 8. These results are in line with the research conducted by Hyun et al. (2011), who reported an optimal pH range for PAL activity between 8 and 9.

Fig. 1.

Fig. 1

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Effect of different temperature degrees on phenylalanine ammonia-lyase (PAL) activity from different agricultural wastes (wheat straw, rice husk, soybean bagasse, corn bagasse, and peanut husk) in comparison with PAL from Rhodotorula glutinis. Points represent the mean of three replicates. The error bars represent the standard deviation

Fig. 2.

Fig. 2

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Effect of different pH degrees on phenylalanine ammonia-lyase (PAL) activity from different agricultural wastes (wheat straw, rice husk, soybean bagasse, corn bagasse, and peanut husk) in comparison with PAL from Rhodotorula glutinis. Points represent the mean of three replicates. The error bars represent the standard deviation

Besides temperature and pH, other crucial factors influence enzyme efficiency, with the Michaelis constant (Km) being one of the most significant determinants. Km serves as a valuable indicator of the affinity between an enzyme and its substrate, with an increase in Km associated with reduced susceptibility to inhibition by competitive inhibitors and enhanced affinity between the enzyme and substrate (Seibert and Tracy 2021). In Table 2 and Fig. 3, it is evident that all PAL enzymes extracted from agricultural waste exhibit lower Michaelis–Menten constants (Km) compared to their microbial counterparts. Additionally, the maximum velocity (V max) of PAL from agricultural waste surpasses that of microbial PAL. The Km values for agricultural waste PAL range from 0.191 to 0.313 mM, while microbial PAL has a Km of 1.476 mM. These findings corroborate with research by Hyun et al. (2011), who reported Km values for PAL between 0.011 mM and 1.7 mM phenylalanine. It is important to note that lower Km values in therapeutic enzymes correspond to higher efficiency (Mohutsky and Hall 2021). The low Km values of therapeutic amino acid-degrading enzymes contribute to the reduction of endogenous amino acid levels to safe levels (Beckett and Gervais 2019).

Table 2.

Kinetic parameters of agricultural wastes or microbial phenylalanine ammonia-lyase

Parameter
Enzyme		 Km Vmax Vmax/Km
Sorghum bagasse 0.191 ± 0.001 27.322 ± 0.562 143.047*
Sugar cane bagasse 0.313 ± 0.010 28.490 ± 0.950 91.022*
Peanut husk 0.297 ± 0.005 33.003 ± 1.029 111.121*
Rice straw 0.244 ± 0.009 5.559 ± 0.213 22.783*
Wheat straw 0.286 ± 0.004 6.353 ± 0.120 22.213*
R. glutinis 1.476 ± 0.009 1.701 ± 0.300 1.152
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Values are given as means of three replicates ± standard error. Values followed by asterisk are significantly different from R. glutinis PAL according to paired t test at P value 0.01

Fig. 3.

Fig. 3

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Lineweaver–Burk plot curve relating the velocity phenylalanine ammonia-lyase (PAL) from different agricultural wastes (wheat straw, rice husk, soybean bagasse, corn bagasse, and peanut husk) and PAL from Rhodotorula glutinis to phenylalanine concentrations

Beyond differences in kinetic parameters (Km and Vmax), another pivotal factor in determining enzyme efficiency is the specificity constant, defined as the ratio Vmax/Km. The specificity constant offers a measure of an enzyme's efficiency in converting substrate into products, representing the duration of the transition state. As the specificity constant increases, the transition state duration decreases, indicating improved efficiency in converting substrate into products (Park 2022). Notably, PAL extracted from agricultural waste exhibits a significantly higher specificity constant compared to microbial PAL (see Table 2). This higher specificity constant underscores the efficiency of plant therapeutic enzymes and provides a potential solution to address the limitations associated with therapeutic enzymes of microbial origin, as previously suggested by Mohammadi et al. (2020).

Another critical factor influencing the utility of enzymes in therapy is the impact of storage conditions on enzyme activity, as highlighted by Biswas et al. (2021). In our study, we investigated the effect of storing PAL from various sources over 6 months at different temperatures. As depicted in Fig. 4, PAL extracted from agricultural waste exhibits remarkable stability, retaining over 95 percent of its activity when stored at room temperature. However, it does experience some activity loss when stored at colder temperatures, such as 10°C or freezing conditions (− 18°C). In contrast, microbial PAL demonstrates greater resilience at freezing temperatures, maintaining its activity levels. This finding aligns with earlier research indicating that plant-derived PAL can endure temperature increases but is less tolerant of temperature decreases, while microbial PAL exhibits better tolerance to cooling temperatures (Hyun et al. 2011).

Fig. 4.

Fig. 4

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Effect of different storage temperatures on phenylalanine ammonia-lyase (PAL) activity from different agricultural wastes (wheat straw, rice husk, soybean bagasse, corn bagasse, and peanut husk) in comparison with PAL from Rhodotorula glutinis. Columns represent the mean of three replicates. The error bars represent the standard deviation

Anticancer effect of PAL

The significance of PAL as a therapeutic enzyme has been steadily growing in recent times. It has potential in the treatment of various conditions such as phenylketonuria, tyrosinemia, and cancer, as well as its applications in antimicrobial production and health supplements (Kawatra et al. 2020). In our current study, we have demonstrated the effectiveness of PAL against cancer cell lines, with all PAL sources exhibiting a low IC50 (less than 0.5 mg/ml). Notably, PAL derived from agricultural waste displayed a significantly lower IC50 compared to microbial sources. It is important to note that the IC50 of PAL varied when tested against different cancer cell lines, as presented in Table 3. These findings align with research conducted by Babich et al. (2013), who reported the efficacy of PAL against breast cancer and prostate cancer cell lines, with notable differences in IC50 values across various cell lines due to their varying sensitivities to phenylalanine deprivation. Similarly, Yang et al. (2019) documented the effectiveness of PAL against colorectal cancer, and Şirin and Aslım (2019) reported the anticancer potential of PAL from plant sources against colorectal cancer cell lines. The mechanism behind PAL’s anticancer activity lies in the fact that phenylalanine is one of the few amino acids essential for auxotrophic cancer cells. PAL catalyzes the degradation of phenylalanine, rapidly reducing the intracellular exogenous phenylalanine levels, which leads to the cessation of cancer cell growth. Malignant cells are unable to utilize phenylpyruvate as a growth alternative, while normal cells can, making PAL a promising tool in cancer regression (Kawatra et al. 2020). Additionally, PAL can inhibit the mitotic activity of different cancer cell lines (Babich et al. 2013).

Table 3.

IC50 (µg) from agricultural wastes or microbial phenylalanine ammonia-lyase against different cancer cell lines

Enzyme source
Cancer cell line		 R. glutinis Sorghum bagasse Sugar cane bagasse Peanut husk Rice straw Wheat straw
Breast cancer (MCF-7) 297.432 ± 2.394 29.392 ± 3.293* 38.203 ± 1.289* 30.108 ± 2.192* 110.235 ± 3.203* 108.480 ± 2.394*
Cervical cancer (HELLA) 376.294 ± 4.304 45.381 ± 2.384* 60.320 ± 3.293* 50.361 ± 3.281* 194.307 ± 4.391* 194.556 ± 4.203*
Colon cancer (HCT116) 235.190 ± 3.293 21.291 ± 4.293* 32.469 ± 4.109* 28.304 ± 1.394* `98.402 ± 2.039* 93.203 ± 3.298*
Esophagus cancer (KYSE-410) 502.346 ± 1.293 48.102 ± 4.002* 71.204 ± 3.102* 59.362 ± 1.304* 227.178 ± 2.119* 223.293 ± 2.394*
Gastric cancer (CLS-145) 466.193 ± 5.405 46.153 ± 5.294* 65.290 ± 5.304* 54.024 ± 5.495* 213.239 ± 4.293* 210.384 ± 1.204*
Liver cancer (HepG2) 431.201 ± 3.102 44.320 ± 1.375* 62.194 ± 5.002* 51.293 ± 3.019* 197.293 ± 5.394* 195.392 ± 5.302*
Pancreatic cancer (AsPC-1) 315.910 ± 2.192 31.293 ± 3.728* 39.019 ± 4.192* 33.390 ± 2.183* 163.295 ± 3.482* 160.782 ± 3.220*
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Values are given as means of three replicates ± standard error. Values followed by asterisk are significantly different from R. glutinis PAL according to paired-t test at P value 0.01

Conclusion and future perspectives

Phenylalanine ammonia-lyase (PAL) is emerging as a promising therapeutic enzyme, originally known for its role in plant defense mechanisms. While microbial sources, particularly fungi, have been used for PAL production, they come with certain disadvantages, including sensitivity to microbial products. This study introduces an effective PAL sourced from inexpensive agricultural waste. The PAL extracted from agricultural waste demonstrates superior activity compared to its microbial counterpart, featuring enhanced kinetic parameters such as Vmax, Km, and the specificity constant. Additionally, it exhibits a wider range of tolerance in terms of temperature and pH compared to microbial PAL. Consequently, PAL from agricultural waste holds potential as a natural and effective anticancer candidate against various forms of cancer. Further research is essential to delve deeper into the characterization of different PALs obtained from various agricultural waste sources. In vivo studies are also imperative to assess the effectiveness of PAL from agricultural waste as an anticancer agent and its impact on normal living cells. These future investigations will contribute to a comprehensive understanding of the potential therapeutic applications of PAL derived from agricultural waste.

Funding

This paper is self-funding and we did not take any funds from any organization or person.

Data availability

Data will be made available on a reasonable request.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This research article does not contain any studies with human participants or animals performed by any of the authors.

Consent for publication

Not applicable.

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