An Overview on The Enzyme Production by Fungi and Its Analytical Methods

Abstract

Enzymes, such as proteases, cellulases, and lipases, play major role in various industries and have wide applications. But certain out-dated production techniques are being considered inappropriate due to its high energy demand, environmental pollution, and cost. Fungi are one of the best sources of large group of enzymes which are being used as alternative to toxic chemical catalysts. They are characterized by their high tolerance to temperature, pH, solvents, and substrates. Fungal enzymes are produced in moulds, which are made of hyphae and mycelium cells. The cost-effectiveness and lack of toxicity makes fungal enzyme production attractive as a substitute for traditional chemical and mechanical processes. A review paper on fungal enzymes can contribute to the scientific literature by providing a comprehensive analysis of existing techniques, discussing advancements, offering comparative insights, and exploring applications and implications. The current study focuses on various qualitative and quantitative analytical techniques being used to characterize the enzymes produced specifically by fungi.

Keywords

Introduction

Enzymes are proteins that catalyse different types of biochemical reactions. They are useful in many industrial applications as they reduce energy demand, environmental pollution and costs. Among the most common are proteases, cellulases and lipases. Fungi are a source of these enzymes, which are also used as alternative to toxic chemical catalysts. They are characterized by their high tolerance to temperature, pH and solvents as well as their specificity to substrates (El-Gendi et al., 2021). Fungi are multicellular organisms that live in a wide range of habitats, including the soil and on dead plant matter. They are decomposers and play a crucial role in the cycling of carbon, nitrogen and other elements. A small number of fungal species attack crops causing mildews, rusts and other diseases that can cause substantial monetary losses for farmers. Fungal enzymes are produced in special structures called moulds. A mould is made of fine threads, which are called hyphae. These threads grow in long branches which intertwine to form a network of cells which is called a mycelium. The mycelium can grow into the air and produce spores, which are like seeds and allow the fungus to reproduce. Spores are spread by wind, rain or insects and when they land in new habitats they start to grow and produce more hyphae (El-Gendi et al., 2021). A growing interest in enzyme production from fungi has emerged due to their ability to withstand harsh conditions and produce large amounts of enzymes. In addition, the cost-effectiveness of fungal enzymes and their lack of toxicity make them attractive as a substitute to traditional chemical and mechanical processes. This has resulted in the development of many rational approaches to enhance fungal enzyme activity, such as site-specific mutagenesis and molecular evolution (Maté et al.,2011). A review paper on the qualitative and quantitative analysis of fungal enzymes can contribute to the scientific literature by providing a comprehensive analysis of existing techniques, discussing emerging advancements, offering comparative insights, and exploring the applications and implications of these analyses. It can serve as a valuable resource for researchers, students, and professionals interested in studying fungal enzymes and their diverse roles.

Different classes of enzymes from fungi

Fungi are the dominant source of industrial enzymes, primarily due to their capacity for secreting large numbers of enzymes. They are used for production of technical, food and feed processing enzymes (amylase, glucosidase, protease, chitinase), biocatalysts for chemical synthesis of organic acids and cholesterol-lowering drugs, and for lignocellulose degradation to produce renewable fuels (Naeem et al.,2022). Amylases break down starches and other complex carbohydrates, while proteases break down proteins into their constituent amino acids. Cellulases are enzymes that break down cellulose, while ligninases break down lignin (El-Gendi et al., 2021). In addition to these, fungi produce many other enzymes such as ribonucleases, collagenolytic enzymes, and lipases that have direct antimicrobial activity against different microorganisms such as bacteria, viruses, and fungi (El-Gendi et al., 2021; Wanderley et al., 2017). Enzymes produced by fungi are often classified according to their substrate specificity. This is done based on the presence of carbohydrate-binding modules (CBMs) in the enzyme protein which interact with the glycosidic bond to catalyse degradation or binding. The CBMs may be located at the N- or C-terminus of the enzyme, or in rare cases both. CBM locations and the length of the linker region spanning the catalytic domain can have a significant impact on the efficacy of the enzyme under industrial conditions (Sidar et al.,2020). The process of discovering fungal enzymes for industrial use is often based on experimental laboratory evaluation of multienzyme culture broths that are mixed to contain various concentrations of wild-type fungi secreting enzymes over a range of relevant pH and temperature profiles. The identification of the enzyme of interest is based on the ability to separate the desired protein from the complex mixture under conditions that prevent its degradation by other, unwanted enzymes or by proteases (Lene, 2017). The development of enzyme production strains to deliver desired enzymes is time consuming and expensive. This is especially true when the aim is to develop new blends of enzymes for a given type of biomass conversion. This challenge is expected to increase as the world moves toward a circular bioeconomy where local agro-industrial side streams are upgraded in nearby biorefineries rather than transported long distances for conversion to biofuel (Lene, 2017).

Enhanced enzyme production

Several techniques are recently being developed to enhance the Cellulase production process. Among many matrices used, microbial source is the most cost effective way of making cellulase. Studies have reported many fungal strains with high cellulose production efficiency. Xie et al. (2023) isolated a novel strain of Penicillium oxalicum from fermentation system and optimized the conditions suitable for cellulase production. In another study (Christopher et al., 2023) Penicillium janthinellum was reported to hyper-produce cellulase and the extracellular proteins were studied to understand the increased carbohydrate metabolism. Current researchers are adapting advanced molecular techniques to enhance the production of cellulase. Pant et al. (2022) employed CRISPR/CAS9 technology to advance cellulase engineering in Trichoderma reesei. Li et al. (2022) reported that supplementation of the medium with Strontium ions helped in upregulating the cellulase genes via the calcium signalling transduction pathway, thereby increasing the cellulase production in Trichoderma reesei. The concept of agricultural waste utilization was adopted by Vázquez-Montoya et al. (2020) in cellulase production. They isolated Penicillium funiculosum, Fusarium verticillioides and Cladosporium cladosporioides from the biomass of Moringa oleifera. By using Moringa straw as carbon source in the medium, they increased the cellulase productivity. Interestingly, Vieto et al. (2022) were able to isolate four fungi from the historic oil painting called La Danza kept in the National Theatre of Costa Rica. All isolates, Myxospora, aff. Penicillium, Ustilago and Pestalotiopsis were found to produce cellulase. This in turn confirms their role in biodeterioration of the painting. Microbial xylanases have been reported to be isolated majorly from fungi than any other microorganisms. Around 110 fungal isolates were obtained from saw dust, decaying wood and soil (Ja’afaru, 2013). 17 fungal isolates were found to produce xylanase enzyme. The protein content was studied and the isolates Aspergillus ustus, Trichoderma sp., Trichoderma sp., Aspergillus ustus and Trichoderma viride were found to show highest activity. Dhaver et al. (2022) employed Plackett–Burman Design and Box Behnken Design strategies to produce xylanase from newly isolated strain of Trichoderma harzianum. They reported 4.16 fold increase in the xylanase production. Penicillium oxalicum is known to produce many industrially important enzymes including xylanase. Zhao et al. (2023) studied the role of cxrD gene in regulation of biosynthesis of xylanase by Penicillium oxalicum.  CXRD was found to regulate the expression of major cellulase and xylanase genes and conidiation-regulatory gene brlA under SSF. In another report (Lian et al.,2022) the transcription factor responsible for positive regulation of xylanase and cellulase enzymes in Ganoderma lucidum was characterized. GlSwi6 was found to promote the enzyme production by regulating the Ca2+ signaling. Ellatif et al. (2022) isolated endophytic fungus Trichoderma harzianum from sugar beetroots and optimized the submerged fermentation process and screened the xylanase gene. Apart from this they also evaluated the antifungal efficacy of xylanase, which was found to be significant against pathogenic fungi. Box-Behnken design was used to enhance xylanase and cellulase enzyme production by newly isolated Aspergillus fumigatus strain grown on Stipa tenacissima (alfa grass) biomass (Gares et al.,2023). They reported the potential of alfa as a raw material to produce enzymes without any pretreatment.

Functions and Roles of Fungal Enzymes in Various Biological Processes

Fungal enzymes play a key role in many industrial processes and allow for more efficient conversion of raw materials to environmentally friendly main products with a higher overall yield. The use of enzymatic processes reduces the need for fossil fuels and other non-renewable resources, while also lowering energy costs and carbon footprints. Furthermore, the use of enzymes leads to more well-made products that are less toxic and fewer hazardous wastes (Dhevagi et al., 2021).

Fungi are constantly digesting their environment, secreting degradative enzymes and absorbing the building block nutrients that are released. For centuries, humans have endeavoured to harness this innate ability of fungi by recombinantly expressing their enzymes (Dhevagi et al., 2021). The resulting enzymatic cocktails, called biocatalysts, are now used in the food, detergent, paper and other industries. Fungal enzymes include amylases, proteases, lipases, gluco-oxidases, pectinases and tannases. The majority of these are commercialized enzymes (with a signal peptide for secretion) that are produced in large scale fermentation broth without the need to open up the cell that produces them (Dhevagi et al., 2021). One such fungus is Aspergillus oryzae, the most popularly used starch saccharification enzyme for producing starch-based products like soy sauce and sake (Daba et al.,2021). The CAZy (Carbohydrate-Active Enzymes) database shows that members of the Ascomycota, Basidiomycota and Zygomycota divisions have a rich portfolio of lignocellulose-degrading enzymes. However, only a limited number of those enzymes are efficiently converted to commercially viable products in industrial process. This is because of differences in the enzymatic activity of the underlying genes. The genes encoding those enzymes are generally organized into families. Each family is defined by its catalytic function, which is specified using a commission number that specifies the groups of enzymes (oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases) in which it belongs (Lene, 2017).

Qualitative analysis of enzymes from fungi

Qualitative analysis of enzymes from fungi involves the investigation of enzyme activities exhibited by fungal organisms. Fungi are known to produce a wide range of enzymes with diverse functions. Here are some approaches commonly used for qualitative analysis of enzymes from fungi:

Screening on selective media: Fungi can be grown on selective media that contain specific substrates, such as agar plates supplemented with starch, cellulose, chitin, or other complex polymers. After incubation, the plates can be stained or treated with specific reagents to visualize the presence of enzymes that degrade these substrates. Clear zones around the fungal colonies indicate enzymatic activity (Colonia et al., 2014).

Enzyme activity assays: These assays are designed to measure the activity of an enzyme. They often involve monitoring the formation or consumption of a substrate or the release of a product. The presence or absence of enzyme activity can provide qualitative information about the presence of specific enzymes (Bisswanger et al.,2014).

Gel electrophoresis: Gel electrophoresis is a widely used technique to separate and visualize enzymes based on their size and charge. It can provide information about the presence, abundance, and purity of enzymes in a sample (Mishra et al.,2017).

Immunological methods: Immunological techniques, such as enzyme-linked immunosorbent assay (ELISA) or Western blotting, can be used to detect and quantify specific enzymes. These methods rely on the specific binding of antibodies to target enzymes, allowing their qualitative identification and sometimes semi-quantitative analysis (Webster et al.,2018).

Substrate-specific assays: Fungal enzyme activities can be assessed using substrate-specific assays. These assays investigate the substrate specificity of enzymes by exposing them to different substrates and observing the formation of products. For example, cellulase activity can be detected by incorporating carboxymethylcellulose into an Czapek medium and observing for zones of hydrolysis around fungal colonies (Romina et al.,2017).

Induction assays: Fungi can be cultured in liquid media with specific inducers that trigger the production of certain enzymes. For instance, cellulase production in fungi can be induced by growing them in media containing cellulose or its derivatives. After incubation, the presence of cellulase can be detected using substrate-specific assays or other techniques (Ellilä et al.,2017).

Inhibition studies: Enzyme inhibitors can be used to determine the presence of specific enzymes. Inhibition studies involve exposing the enzyme to a known inhibitor and observing changes in enzyme activity. The presence of inhibition can indicate the presence of the enzyme targeted by the inhibitor (Ramsay et al.,2017).

pH and temperature profiling: Enzymes often exhibit specific pH and temperature optima for activity. By subjecting the enzyme to a range of pH or temperature conditions, researchers can determine the optimal conditions for enzyme activity, which can provide qualitative insights into the identity of the enzyme (Bisswanger et al.,2014).

Mass spectrometry: Mass spectrometry can be employed for the identification and characterization of enzymes by analysing their molecular weight and fragmentation patterns. This technique can provide qualitative information about the presence and identity of enzymes in a sample (Dueñas et al.,2023).

Zymography: Zymography is a technique that allows the visualization of enzymatic activities on gel electrophoresis. Fungal enzymes can be separated on polyacrylamide gels containing substrates specific to their activities, such as casein for proteases or cellulose for cellulases. After electrophoresis, the gel is incubated under appropriate conditions to allow enzyme activity, and the resulting bands or zones of activity can be visualized (Dojnov et al.,2017).

Molecular techniques: Molecular approaches, such as polymerase chain reaction (PCR) or gene expression analysis, can provide qualitative information about the presence or expression of specific genes encoding fungal enzymes. PCR can be used to amplify and detect specific gene sequences associated with known enzymes, while gene expression analysis can assess the transcription levels of enzyme-encoding genes under different conditions (Deepak et al.,2007).

Metabolomic profiling: Metabolomics, which involves the comprehensive analysis of small molecules in a biological sample, can provide qualitative information about the presence or absence of specific enzymatic activities. Metabolomic techniques, such as ultra-high pressure liquid chromatography coupled to high resolution mass spectrometry (UPLC-HRMS), can be used to identify and quantify metabolites that are indicative of certain enzyme activities for screening large fungal populations (Witte et al.,2022). It is important to note that qualitative analysis alone may not provide detailed quantitative information about enzyme concentrations. For quantitative analysis, additional techniques, such as enzyme kinetics or spectrophotometry, are typically employed. These techniques, along with others specific to the targeted enzyme of interest, can be employed to qualitatively analyse enzymes produced by fungi and provide insights into their functional capabilities.

Quantitative analysis of enzymes from fungi

Quantitative analysis of enzymes from fungi involves the measurement and quantification of enzyme activities, concentrations, and other quantitative parameters related to fungal enzyme function. Here are some commonly used methods for quantitative analysis of enzymes from fungi:

Enzyme activity assays: Enzyme activity assays provide a quantitative measure of enzyme activity in fungal extracts. These assays involve monitoring the rate of substrate conversion or product formation over a specific time period. The results are typically expressed in units such as international units (IU) or moles per minute (Bisswanger et al.,2014).

Protein concentration determination: Quantifying the protein concentration in fungal extracts is important for normalizing enzyme activity data. Methods such as the Bradford assay, bicinchoninic acid (BCA) assay, or enzyme-linked immunosorbent assay (ELISA) can be used to quantitatively measure protein levels in fungal samples. These assays rely on the specific binding of dyes, metals, or antibodies to proteins, which can be measured spectrophotometrically or fluorometrically (Mishra et al.,2017).

Spectrophotometry: Spectrophotometric assays are based on the measurement of absorbance or changes in absorbance of a substrate or product at specific wavelengths. Enzyme-catalysed reactions can be monitored using spectrophotometers, and the rate of reaction can be determined by measuring the change in absorbance over time. The results can be used to calculate enzyme activity or kinetic parameters (Roskoski et al.,2007).

HPLC (High-Performance Liquid Chromatography): HPLC can be utilized for the quantitative analysis of enzymes and their substrates or products. Enzyme reactions can be monitored by separating and quantifying the components using a suitable chromatographic column and detector. This method allows for precise quantification and identification of enzymatic species (Gaurav et al.,2017).

Radioisotope assays: Radioisotope-labelled substrates can be employed to quantitatively measure enzyme activity. The incorporation or release of a radioactive isotope can be detected and quantified using techniques like liquid scintillation counting or autoradiography. Radioactive assays offer high sensitivity but require proper handling of radioactive materials (Zeng et al., 2015).

Fluorescence-based assays: Fluorescence techniques can provide sensitive and quantitative measurements of enzyme activity. Fluorogenic substrates or probes can be used to monitor enzymatic reactions, and the resulting fluorescence intensity can be measured using fluorometers. Fluorescence resonance energy transfer (FRET) assays can also be employed to study enzyme-substrate interactions (Ishikawa-Ankerhold et al., 2012).

Mass spectrometry: Mass spectrometry can be used for quantitative analysis of enzymes and their products. By measuring the mass-to-charge ratio of ions, mass spectrometry enables the identification and quantification of enzyme-catalysed reactions. Isotope dilution mass spectrometry (IDMS) is a precise method that utilizes isotopically labelled internal standards for accurate quantification (Villanueva et al., 2014).

Enzyme kinetics: Enzyme kinetic studies can provide quantitative information about the rate of enzyme-catalysed reactions and their kinetic parameters. Techniques such as Michaelis-Menten kinetics, Lineweaver-Burk plots, or steady-state kinetics can be used to determine parameters such as maximum velocity (Vmax), Michaelis constant (Km), and catalytic efficiency (kcat/Km) (Bisswanger et al., 2014).

Molecular methods: Molecular techniques such as quantitative PCR (qPCR) or gene expression analysis can provide quantitative data on the expression levels of genes encoding fungal enzymes. These methods involve the amplification and quantification of specific gene transcripts or their corresponding cDNA. The results can be used to infer enzyme expression levels and activity (Deepak et al.,2007).

Metabolomic profiling: Metabolomics can offer quantitative insights into the activities of fungal enzymes by measuring the levels of metabolites involved in enzyme-catalysed reactions. Techniques such as mass spectrometry or NMR spectroscopy can be employed to identify and quantify metabolites, providing quantitative data on the overall metabolic activity and enzyme function in fungi (Wishart et al., 2019).

Stable isotope labelling: Stable isotope labelling techniques can be utilized to quantitatively assess enzyme activities in fungi. By incorporating isotopically labelled substrates or precursors, the metabolic fate and conversion of these compounds can be quantified using mass spectrometry or other analytical techniques (Beckeret al., 2008).

These above-mentioned methods, along with appropriate controls and calibration standards, enable the quantitative analysis of enzymes from fungi, facilitating a deeper understanding of their activities, regulation, and roles in fungal metabolism and physiology.

Future Directions in Fungal Enzyme Biotechnology

The field of fungal enzyme biotechnology is rapidly expanding, with many researchers exploring the vast potential of fungal enzymes in various applications. The discovery of novel enzymes with unique properties and the engineering of enzymes with new functions and catalytic activities will be central to future developments in this field. With advances in high-throughput sequencing technologies, we can expect to see a continued increase in our understanding of the diversity and capabilities of fungal enzymes. As fungal enzyme biotechnology continues to expand, it has the potential to contribute significantly to various industries and address some of the world's most pressing environmental challenges.

CONCLUSION

Fungal enzymes are a fascinating group of biocatalysts that have numerous applications in various industries. Quantitative and qualitative analysis techniques allow researchers to explore the properties of fungal enzymes and design optimal production methods. While challenges in production exist, there are many solutions available to overcome them. As research progresses, we will likely see more discoveries and developments utilizing enzymes from fungi in biotechnology and medicine.

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