Evaluation of lignocellulatic activity of enzymes from microwave-irradiated Pleurotus sajor-caju cultivated with wheat straw

Abstract

The production of lignocellulytic enzymes by microwave-radiated Pleurotus sajor-caju was assayed. Wheat straw was employed as substrate to P. sajor-caju for production of laccase, manganese peroxidase (MnPase), filter-paperase (FPase), carboxmethyl cellulase (CMCase), and cellulase (as evaluated using microcrystalline cellulose). P. sajor-caju exposed to 10 s of microwave radiation (MR) showed maximum growth with colony radius of 7.17 ± 0.45 cm, while with increasing the exposure time up to 50 s the growth decreased up to 2.67 ± 0.22 cm. Moreover, it failed to grow at 80 s of exposure time. Cellulase, MnPase, FPase, CMCase, and laccase activities were induced to 37 ± .0.54, 49 ± 2.36, 189 ± 2.12, 0.37 ± 0.06, and 1.58 ± 0.03 U/mL compared to that at control 31 ± 0.25, 46 ± 1.25, 177 ± 1.65, 0.28 ± 0.03, and 1.37 ± 0.12 U/mL, respectively as a result of P. sajor-caju exposure to 10 s of MR. As the exposure time increased, these enzymes activity decreased. Different levels of moisture with surfactant (polysorbate 80) were applied to optimize the enzymes activities at 10 s of exposure time. The optimum activities 3.15 ± 0.23, 0.62 ± 0.06, 269 ± 5.36, 65 ± 1.63, and 48 ± 0.98 U/mL were recorded for cellulase, MnPase, FPase, CMCase, and laccase, respectively at 70% of moisture and 0.15 mL/L of polysorbate 80.

Full Article

Evaluation of Lignocellulatic Activity of Enzymes from Microwave-irradiated Pleurotus sajor-caju Cultivated with Wheat Straw

Aisha M. H. Al-Rajhi,^a,* Abdulrahman S. Bazaid,^b,c Ahmed M. Abdulfattah,^d T. M. Abdelghany,^e Abdel-Rahman M. Shater,^f and Samy Selim ^g,*

The production of lignocellulytic enzymes by microwave-radiated Pleurotus sajor-caju was assayed. Wheat straw was employed as substrate to P. sajor-caju for production of laccase, manganese peroxidase (MnPase), filter-paperase (FPase), carboxmethyl cellulase (CMCase), and cellulase (as evaluated using microcrystalline cellulose). P. sajor-caju exposed to 10 s of microwave radiation (MR) showed maximum growth with colony radius of 7.17 ± 0.45 cm, while with increasing the exposure time up to 50 s the growth decreased up to 2.67 ± 0.22 cm. Moreover, it failed to grow at 80 s of exposure time. Cellulase, MnPase, FPase, CMCase, and laccase activities were induced to 37 ± .0.54, 49 ± 2.36, 189 ± 2.12, 0.37 ± 0.06, and 1.58 ± 0.03 U/mL compared to that at control 31 ± 0.25, 46 ± 1.25, 177 ± 1.65, 0.28 ± 0.03, and 1.37 ± 0.12 U/mL, respectively as a result of P. sajor-caju exposure to 10 s of MR. As the exposure time increased, these enzymes activity decreased. Different levels of moisture with surfactant (polysorbate 80) were applied to optimize the enzymes activities at 10 s of exposure time. The optimum activities 3.15 ± 0.23, 0.62 ± 0.06, 269 ± 5.36, 65 ± 1.63, and 48 ± 0.98 U/mL were recorded for cellulase, MnPase, FPase, CMCase, and laccase, respectively at 70% of moisture and 0.15 mL/L of polysorbate 80.

DOI: 10.15376/biores.19.4.8882-8893

Keywords: Lignocellulatic Enzymes; Pleurotus sajor-caju; Wheat Straw; Surfactant

Contact information: a: Department of Biology, College of Science, Princess Nourah bint Abdulrahman University, P.O. Box 84428, Riyadh 11671, Saudi Arabia; b: Department of Medical Laboratory Science, College of Applied Medical Sciences, University of Ha’il, Hail 55476, Saudi Arabia; c: Medical and Diagnostic Research Center, University of Ha’il, Hail 55473, Saudi Arabia; d: Department of Medical Laboratory Sciences, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah 21589, Saudi Arabia; e: Botany and Microbiology Department, Faculty of Science, Al-Azhar University, Cairo 11725, Egypt; f: Biology Department, College of Science, Jazan University, Jazan 82817, Saudi Arabia; g: Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Jouf University, Sakaka, Saudi Arabia;

* Corresponding authors: amoalrajhi@pnu.edu.sa (A.M.H.A.), sabdulsalam@ju.edu.sa (S.S.)

INTRODUCTION

Lignocellulose is a major constituent of woody and decayed plant materials. Various extracellular enzymes generated from white rot fungi (WRF), such cellobiose dehydrogenase (CDHase), lignin peroxidase (LiPase), laccase, and manganese peroxidase (MnPase), are associated with the breakdown of lignin and its derivatives (Reyes et al. 2021; Al-Rajhi et al. 2022a). Each year, a large amount of straw is generated from wheat cultivation. While some is utilized as feed for animals or as a substrate to cultivate edible mushrooms, the remainder can be regarded as a huge underutilized resource of energy (Devi et al. 2024). Globally, a significant amount of lignocellulosic remains from agriculture (such as wheat straw) and wood (such as wood chips), and different wastes of industry have accumulated as a result of the growing agro-industrial activities. The ecosystem is contaminated by these substances (Elgueta and Diez 2010; Sánchez and Montoya 2020). It is crucial to use lignocellulose residues to increase WRF’s ability to produce extracellular phenoloxidase enzymes (Omoni et al. 2022; Abiola-Olagunju et al. 2024). Maintaining sustainable social development may depend on making full use of lignocellulosic resources. Enzymatic hydrolysis of these wastes to produce valuable compounds and solvents is the most promising method (Shankar et al. 2024). The past 20 years have seen a great deal of study in this field. Two sub-processes make up the conversion: first, lignocellulosic materials are hydrolysed to produce fermentable sugars, and then those sugars are fermented to produce the desired products (Singh et al. 2024).

Lignocellulolytic enzymes serve numerous roles in biotechnological processes in the chemical, fuel, brewery, food, wine, textile, and pulp, dyes degradation and paper industries (Xu et al. 2023; Pham et al. 2024). In fungi, extracellular enzymes are constitutively produced in different amounts, and they are affected by many typical fermentation factors such as medium constituents, carbon/nitrogen ratio, temperature, pH, and aeration level (Abdel Ghany et al. 2018; Al Abboud et al. 2022; Bakri et al. 2022; An et al. 2023; Aza and Camarero 2023), besides metal ions, particularly Cu ²⁺ (Akpinar and Ozturk 2017; Abdel Ghany et al. 2020; Al-Rajhi et al. 2022b). The occurrence of Mn²⁺ is known to induce the production of MnPase in many WRF, but Mn²⁺ lowers LiPase titers (Li et al. 2022). The content of moisture is a critical agent on enzymes production (Sosa-Martínez et al. 2023). A reduction in enzyme yield can be caused by higher moisture levels due to steric hindrance of the producer strain. The bioavailability of less soluble substrates for the fungi can be increased by surfactants, especially polysorbate-80 (Sun et al. 2018). Many investigators have mentioned that some natural inductive compounds such as phenols and flavonoids are present in lignocellulosic wastes including rice bran, oak sawdust, wheat straw, and wheat bran; these substrates can be used to induce the fungal lignocellulatic enzymes secretion (Wang et al. 2015; Wang et al. 2019; Hermosilla et al. 2020). Also, olive leaves contain hydroxytyrosol and oleuropein which act as inducers for laccase production by white rot fungi (Yuan et al. 2015).

Electromagnetic radiation includes several forms, including microwave radiation (MR). During MR, the target samples are heated by the thermal energy generated from electromagnetic energy, and subsequently interacts with the ingredients of the target samples (de la Hoz et al. 2005). Fungal growth is regulated and inhibited via numerous chemical, physical, and biological methods. However, MR is one of the greatest physical techniques to manage of fungal pathogens and decaying wood fungi (Ahmed and Amein 2023). According to Poonia et al. (2021) the viability of treated white and brown rot fungi namely Trametes versicolor and Rhodonia placenta by microwave radiation decreased the dependency on exposure time with complete inhibition at 180 s. In another study, Mahdi et al. (2021) mentioned that the exposed bacteria and fungi to radiation of microwave for 45 s were completely killed. Poonia et al. (2021) utilized MR to repress the growth of Serpula lacrymans (brown rot fungus), which is important in European indoor rot inside thick wooden beams (Poonia et al. 2021). Several properties such as rapid reaction time and low reactivity with applied biomass were associated with MR. The authors are proposing the hypothesis in this work that MR at low contact time may enhance the activity of used fungus to degrade wheat straw with lignocellulotic enzymes production. Therefore, the main purposes of this investigation were to evaluate the influence of microwave radiation on the white rot fungus Pleurotus sajor-caju, in addition to polysorbate 80 to enable their efficient hydrolysis of lignocellulosics biomass from wheat straw to produce lignocellulotic enzymes.

EXPERIMENTAL

Fungus and Wheat Straw Used

Pleurotus sajor-caju (obtained from Professor Abdelghany TM, Al Azhar University, Egypt) was cultivated for 15 days at 28 °C on the surface of Malt agar in petri dishes. Mycelium Agar plugs containing active mycelia (6 mm in diameter) were cut and used for fungus propagation. Dried wheat straw (WS) was collected from agriculture areas in Saudi Arabia and then cut into 2 to 3 mm lengths.

Fungal Growth and Production of Lignocellulytic Enzymes at Different Times of Microwave Radiation

The cultivated P. sajor-caju on malt agar plates at 8 days was exposed to a household microwave oven (Type Zanussi, 230 V, 50 Hz, 2450 MHz, 1100 W, China) for 10, 30, 50, or 80 sec. From each plate, a disc (6 mm) of exposed fungus to microwave ray was used to inoculate a uniform malt agar media and incubated at 28 °C. The developed colony of the fungus was measured after 8 days. The well growth mycelium colonies on malt agar plates were plugged (9 mm in diameter) from the growing colonies and applied to inoculate on wheat straw as a substrate. The flasks containing the fungus inoculum were incubated for 14 days at 28 °C, and then laccase, MnPase, CMCase, FPase, cellulase (evaluated based on microcrysalline cellulose degradation), and extracellular protein were determined.

Enzymes Preparation

In a 500 mL conical flask, 10 grams of the wheat straw waste were added. After adding different levels (50 to 80 %) of moistening agent besides different concentration (0.05 to 0.2 mL/L) of polysorbate 80 (Tween 80) to the substrate, it was autoclaved for 30 min at 121 °C. Two 6 mm fungus discs were utilized to inoculate every flask. For 15 days, they were incubated at 28 °C. Following the incubation period, each flask received 100 mL of distilled water, and the flasks were shaken for 60 min at 200 rpm. Using muslin cloth on a glass funnel, the entire contents of the flask were filtered into a dry, clean flask. The filtrates underwent a 10-min cooling centrifugation at 80,000 rpm. Crude enzymes were made from the supernatant solutions. The crude enzymes were directly applied in some experiments, while other quantity of enzymes was kept for short times in a refrigerator at 4 °C.

Tests for Cellulase Activity

Before describing various tests for enzyme activity, it is important to clarify that three of them, namely the “CMCase”, “FPase”, and “MCCase” tests, are all different means of determining the activity of one class of enzymes, the cellulase class. The CMCase test employs a soluble derivative of cellulose, whereas the “MCCase” test employs crystalline cellulose. The “FBase” can be regarded as intermediate in character, since the cellulose fibers within the filter paper will contain both crystalline and non-crystalline cellulose domains.

Carboxymethyl cellulase (CMCase) test and protein quantity detection

As described in Wang et al. (2008), 1 mL of pH 5.0 sodium acetate buffer was mixed with 1 mg of carboxymethyl cellulose. An aliquot of 1 mL of the supernatant (enzyme) was placed in a clean, dry tube along with 1 mL of 1 percent CMCase in acetate buffer. For thirty min, the tube was kept at 62 °C, after which the released reducing sugar was recorded using the Miller (1959) DNS method, with the absorbance being measured at 540 nm. The blank was one mL of distilled water rather than one mL of supernatant (enzyme). Using the glucose standard curve, the concentration of the resulting reducing sugar was ascertained. The micromole of glucose released/mL of culture filtrate/min is one unit of CMCase. The detected quantity of soluble proteins in the supernatant containing the fungus filtrate of medium growth was performed according to protocol of Lowry (1951).

Filter-paperase (FPase) test

An aliquot of 1 mL of the enzyme-containing supernatant (pH 4.8) and 2 mL of 0.1M citrate buffer pH 4.8, containing 0.05 g of filter paper (Whatman No. 1) (Gadgil et al. 1995). For 60 min, the tube was incubated at 50 °C. The micromole of glucose released/mL of filtrate per/min is equivalent to one unit of FPase.

Microcrystalline cellulose (MCC)-ase test

Avicell MCC (2.0 g) was mixed with 100 mL of sodium phosphate buffer (SPB) with pH 6.6 according to method of Li and Gao (1997). An aliquot of one mL of the enzyme-containing supernatant was combined with 1 mL of 2% MCC in SPB in a clean, dry tube. For two hours, the reaction mixture was incubated at 40 °C. One micromole of glucose liberated/mL of filtrate/min was defined as one unit of MCCase.

Laccase Test

Laccase activity was tested according to Garzillo et al. (2001). Two mM 2,2 azino-bis 3-ethyl benzo-thiazoline-6 sulforicacide (ABTS) was mixed with McIlvaine buffer (pH 5) in a 1 mL reaction mixture. One hundred microliters of centrifuged extracellular supernatants were added to the assay mixture. Through observation of the absorbance at 420 nm at 30 °C, the enzymatic activity was calculated in IU.

Manganase Peroxidase (MnPase) Test

In a one cm quartz cuvette, MnPase activity was measured. In a McIlvaine buffer (pH 5.0), the 1 mL reaction mixture contained one mM Mn²⁺ and two mM ABTS. Initiating the peroxidase activity, 0.4 mM H₂O₂ was added to the assay mixture after 100 µL of centrifuged extra cellular fluids (supernatants) were added (Garzillo et al. 2001). Using a spectrophotometer (JENWAY, Model 6300, EU) at 30 °C, the absorbance change at 420 nm (ABTS), =36 mM^-1cm^-1, was monitored to estimate the enzymatic activity in IU.

Statistical Examination

Standard deviation (± SD) was estimated from the calculation of the mean three replicates. Subsequently, the Tukey-Kramer honestly significant difference (HSD) test was employed. The level of significance was 5%.

RESULTS AND DISCUSSION

The production of lignocellulytic enzymes on WS as a cheap, renewable substrate has become well known. In this work, WS was employed as substrate for production of value-added enzymes using the fungus P. sajor-caju. In a previous study (Biswas et al. 2019) it was stated that WS is a suitable substrate for MnPase and lipase production as well as other enzymes. Table 1 shows the effect of MR on growth of P. sajor-caju. The outcomes of fungus growth reflected that as the fungus was exposed to different times of MR, the enzyme production increased up to 7.17 ± 0.45 mm at 10 s, while after this exposure time the growth decreased to 4.53 ± 0.32 and 2.67 ± 0.22 mm at 30 and 50 s. Furthermore, the fungus failed to grow at exposure time 80 s. All of these results were compared with untreated fungus, where the colony growth was 6.66 ± 0.28 mm with HSD at 5% of 0.816 among all growths at the different exposure times. The obtained findings were in agreement with outcomes of Poonia et al. (2021), where the growth of brown and white rot fungi namely Rhodonia placenta and Trametes versicolor, respectively, decreased based to the applied exposure time of MW. Górny et al. (2007) mentioned that the influence of microwave radiation on fungal and bacterial viability depended on the time of exposure and power density of radiation.

Table 1. Effect of Different Exposure Times of Microwave Radiation on the Growth of P. sajor-caj

± Standard deviation of average 3 repetitions of results

The effect of microwave radiation as a physical effect on development of fungi and their lignocellulatic enzymes activity was selected due to several advantages such as non-contact and volumetric heating, rapid reaction period, and little reactant consumption (Bichot et al. 2022). According to other studies, the generated electromagnetic waves from microwaves penetrate easily and rapidly inside the applicable biomass or substrate (Beneroso et al. 2017; Bundhoo 2018). Enzymes activity of P. sajor-caju was evaluated under different times of exposure to MR (Table 2). Exposure to short time 10 s of microwave irradiation induced all the examined enzymes namely MCCase, MnPase, FPase, CMCase, and laccase, where the activities were 37 ± .0.54, 49 ± 2.36, 189 ± 2.12, 0.37 ± 0.06, and 1.58 ± 0.03 U/mL compared to that at control 31 ± 0.25, 46 ± 1.25, 177 ± 1.65, 0.28 ± 0.03, and 1.37 ± 0.12 U/mL, respectively. MR at 30 and 50 s of MR repress the enzymes production by P. sajor-caju. Since at 50 s, the values of activities were 19 ± 0.65, 27 ± 0.65, 124 ± 3.25, 0.21 ± 0.05, and 0.78 ± 0.09 U/mL for MCCase, MnPase, FPase, CMCase, and laccase, respectively. The effect of MR was studied by Zhu et al. (2006) on the enzymatic hydrolysis of rice straw. Their results showed that the level of initial hydrolysis was greatly speeded up, but the yield then declined slightly. Improving the enzymatic saccharification of grain stillage by Phanerochaete chrysosporium was reported by pretreated via microwave-assisted hydrothermal treatment (Ren et al. 2020).

Table 2. Effect of Different Times of MR on Cellulytic Enzymes and Extracellular Protein Produced by P. sajor-caju by Solid State Fermentation of Wheat Straw (± Standard deviation of average 3 repetitions of results)

Table 3. Production of Lignocellulytic Enzymes on Wheat Straw Amended with P. sajor-caju

± Standard deviation of average 3 repetitions of results

The exposed fungus culture to 10 s of MR with cultivated certain optimum condition was studied for the optimization of enzymes production (laccase, MnPase, FPase, MCCase, and extracellular protein). The variables used were volume of moisting agent polysorbate 80 was added as surfactant to help in secretion of enzymes on solid state fermentation. There has been some investigation showing that lignocellulytic enzymes secretions by Pleurotus ostreatus depend on the producer of fungi, substrate composition, and cultivation condition (da Silva et al. 2019; Zhang et al. 2023). The maximum activities of studied enzymes were noted at 0.10 mL/L of polysorbate 80 and moisture 50%, at 0.15 mL/L of polysorbate 80, and moisture 60% and 70%. At 80% moisture, the maximum activities of all studied enzymes were recorded at 0.10 mL/L of polysorbate 80. Generally, 70% of moisture and 0.15 mL/L of polysorbate 80 represented the optimum conditions for enzymes activities (3.15 ± 0.23, 0.62 ± 0.06, 269 ± 5.36, 65 ± 1.63, and 48 ± 0.98 U/mL for MCCase, MnPase, FPase, CMCase, and laccase, respectively). The outcomes of Zhang et al. (2023) designate that it was probable to reach important activities of some enzymes, namely LiP and cellulase utilizing Pleurotus ostreatus with lignocellulosic biomass fermentation under solid state condition by aiding exogenous inducers of surfactants. Generally, according to published reports, the microwave-irradiated biomasses were affected by several associated parameters of microwave irradiation; from these parameters the hydration condition and the polarity of the reaction substrate which subsequently effect the enzymatic productivity (Chen et al. 2020). Moreover based on another study, microwave heating appears to possess an influence on the stereo-selectivity of enzymes (Mazumder et al. 2004). A direct microwave energy absorption by the enzyme polar substrates result in a greater reactivity of the chemical functional groups included in the enzymatic reaction. Other mechanisms associated with change of enzyme activity caused my microwaves were reported (Habinshuti et al. 2020; Deng et al. 2022). For instance, the activity of thermally unstable enzymes was affected via treatment by microwave because of heat denaturation. Treatment by microwave may cause some protein sites to become more susceptible to enzymatic hydrolysis due to molecular rearrangement and unfolding of proteins. In tables (2 and 3), there is a relation between the activity of enzymes and the detection amount of proteins, where the high activity of enzymes at all treatments was accompanied by a high detected amount of protein and vice versa.

In the present paper, polysorbate 80 at a specific concentration increased the enzymes activity. According to Shrestha et al. (2023), surfactant giving an appropriate membrane composition for enzymes to join with the substrate can be expected to increase their activity. Polysorbate 80 modifies the structure of fungal cell membranes to encourage the excretion of ligninolytic enzymes (Rodrigues et al. 2008). However, at high concentrations of polysorbate 80 above 0.15 mL/L, the activity of enzymes was decreased, maybe due to that surfactant at these concentrations affecting the permeability of cell membrane, leading to blockage the enzymes secretion as mentioned previously (Ahlawat et al. 2009). Laccase yield from Pleurotus sajor-caju was increased from 33.5 to 50 times when growth medium amended with 7.5% mL/v of polysorbate 80 compared to its yield in medium without polysorbate 80 (Teodoro et al. 2018). He et al. (2023) documented that the addition of polysorbate 80 promoted the increasing in temperature during the composting process that a companied by accelerate the lignocellulose degradation and decline the phytotoxicity as well as increase the lignocellulytic enzymes. Further investigations are required to determine the effect of suitable conditions such as pH, temperature, fermentation process, and some metals in combination with the effect of MR on the lignocellulatic enzymes activity as well as the application of lignocellulatic enzymes on the industrial scale.

CONCLUSIONS

1. The investigation concluded that low exposure time (10 s) of microwave radiation has better potential of application to induce lignocellulotic enzymes of P. sajor-caju using WS biomass.
2. Moisture level of 70% and 0.15 mL/L of polysorbate 80 considered the optimum conditions for the secretions of lignocellulotic enzymes.

ACKNOWLEDGMENTS

The authors would like to acknowledge Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2024R217), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

FUNDING

This research was funded by Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2024R217), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

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Article submitted: August 1, 2024; Peer review completed: August 31, 2024; Revised version received: September 2, 2024; Accepted: September 8, 2024; Published: October 4, 2024.

DOI: 10.15376/biores.19.4.8882-8893