Draft:Xylanase

Xylanase
Xylanase is an enzyme that catalyses the hydrolysis of Xylan, a major component of hemicellulose found in plant cell walls. Xylan is a complex polysaccharide consisting of a backbone of xylose residues linked by β-1,4-glycosidic bonds, often with various substituents. Xylanases break down xylan into simpler sugars like xylose and xylooligosaccharides.. .These enzymes are produced by a wide range of microorganisms, including bacteria, fungi, and some yeast species. Xylanases have significant applications in various industries, such as pulp and paper, food, animal feed, and biofuels

History
1970s-1980s: Early research on xylanases began in the mid-1970s at the Department of Chemical Enzymology of Moscow State University. . Studies focused on understanding the mechanism, kinetics, and synergistic interactions of xylanases and other carbohydrases in the enzymatic conversion of cellulose and plant biomass.

By the 1990s, environmental concerns, especially those related to the pulp and paper industry, spurred the search for chlorine-free bleaching methods. Xylanases were identified as potential agents for pretreating kraft pulps to facilitate bleaching without the use of harmful chlorine. This period also saw the cloning and sequencing of the xylanase gene from Bacillus subtilis, which enabled the recombinant production of the enzyme, a significant advancement for industrial applications.

As the 21st century progressed, the applications of xylanase continued to diversify. The enzyme was explored for its potential in producing xylooligosaccharides from lignocellulosic biomass, which have value-added properties such as antioxidant effects .Additionally, xylanases were considered as non-antibiotic growth promoters in animal feed, reflecting a growing interest in sustainable and health-conscious agriculture.

Microorganisms, including fungi like Trichoderma sp. and Aspergillus sp., yeast such as Cryptococcus sp., and bacteria like Streptomyces sp., have been recognized for their ability to produce microbial xylanase. The production of microbial enzymes like xylanase not only meets industrial demands but also supports economic growth by creating employment opportunities and saving foreign exchange reserves that would otherwise be spent on importing industrial enzymes.

Today, xylanase continues to be a focus of scientific research, with studies delving into the structural and functional properties of the enzyme to optimize its use in various biotechnological processes,. Its applications span the beverage and bakery industries, paper production, and even poultry feed additives, showcasing the enzyme's broad impact on both industry and the economy.

Classification
Xylanases have been classified in at least three ways: based on the molecular weight and isoelectric point, the crystal structure and kinetic properties, or the substrate specificity and product profile. The favorable system for the classification of xylanases is based on the primary structure and comparison of the catalytic domains .According to the CAZy database (http://www.cazy.org), xylanases (EC3.2.1.8) are related to glycoside hydrolase (GH) families 5, 7, 8, 9, 10, 11, 12, 16, 26, 30, 43, 44, 51, and 62. Among those, xylanases in GH 10 and 11 are the two families that have been thoroughly studied.

GH family 10 is composed of endo-1,4-β-xylanases and endo-1,3-β-xylanases (EC 3.2.1.32). Members of this family are also capable of hydrolyzing the aryl β-glycosides of xylobiose and xylotriose at the aglyconic bond. Furthermore, these enzymes are highly active on short xylooligosaccharides, thereby indicating small substrate-binding sites. Family 11 is composed only of xylanases (EC 3.2.1.8), leading to their consideration as “true xylanases,” as they are exclusively active on d-xylose-containing substrates. Among all xylanases, endoxylanases are the most important due to their direct involvement in cleaving the glycosidic bonds and in liberating short xylooligosaccharides. Although several Bacillus species secrete high levels of extracellular xylanase, filamentous fungi secret high amounts of extracellular xylanase, which are often accompanying cellulolytic enzymes—for example as in species of Trichoderma, Penicillium, and Aspergillus.

Fungal xylanases are produced by a range of fungi, including species of Aspergillus, Trichoderma, and Penicillium. These fungi are known for their ability to secrete large amounts of xylanase, which can be used to treat kraft pulp, improving its brightness and delignification,. Aspergillus nige r, for example, is a prolific producer of xylanase and has been used in various studies to optimize the degradation of biomass waste. Trichoderma species are also notable for their xylanase production, often used in combination with other fungal species to achieve efficient biomass breakdown.

Bacterial xylanases are produced by both mesophilic and thermophilic bacteria, including genera such as Bacillus, Streptomyces, and Clostridium. These bacteria can produce xylanases that function at a wide range of temperatures, which is beneficial for various industrial processes, ,. For instance, Bacillus species have been used in animal feed to reduce digesta viscosity and improve nutrient absorption. Streptomyces, an actinomycete, is another significant producer of xylanases, with species like Streptomyces olivaceoviridis E-86 being studied for their ability to degrade xylan substrates efficiently.

Actinomycete xylanases, particularly from Streptomyces and Thermomonospora species, are known for their robustness and stability, which make them suitable for industrial applications where harsh conditions are prevalent. The actinomycete Thermomonospora fusca, for example, produces thermostable xylanases that have been studied for their potential in continuous culture systems. Streptomyces species not only produce xylanases but also exhibit a broad spectrum of antifungal activities, which can be beneficial in agricultural settings.

Optimal Temperature
Xylanases can be categorized based on their optimal temperature for activity. Mesophilic xylanases operate efficiently at moderate temperatures, typically around 40-50°C, making them suitable for processes that occur at or near room temperature,. On the other hand, thermophilic xylanases exhibit optimal activity at higher temperatures, above 60°C, which is advantageous for industrial processes that require high temperatures, as these enzymes are more stable and can maintain activity under these conditions. For instance, a study demonstrated that a C-terminal proline-rich sequence in GH10 ruminal xylanases broadens the optimal temperature range, enhancing their utility in processes requiring higher operational temperatures.

Optimal pH
The activity of xylanases is also highly dependent on the pH of the environment. Most xylanases have an optimal pH in the acidic to neutral range (pH 4-7). However, alkaline xylanases, which have an optimal pH above 8, have been identified and are particularly useful in industries that operate under alkaline conditions. Engineering efforts have been made to shift the optimal pH of GH11 xylanases towards more alkaline values, expanding their applicability in various industrial settings.

Substrate Specificity
Xylanases vary in their specificity towards different xylan substrates and substituents. This specificity is crucial for the efficient breakdown of xylan into its constituent sugars. For example, a study on a class of long-horned-beetle-derived xylanases revealed unique substrate specificities, highlighting the diversity among xylanases in their approach to xylan hydrolysis. Understanding and manipulating this specificity can lead to more efficient degradation of specific biomass types.

Catalytic Mechanism
The catalytic mechanism of xylanases involves two conserved glutamate residues in the active site that facilitate either a retaining or an inverting mechanism for cleaving the β-1,4-xylosidic linkages in xylan. In the retaining mechanism, one glutamate acts as a nucleophile, forming a covalent glycosyl-enzyme intermediate, while the other acts as an acid/base catalyst. In the inverting mechanism, the two glutamates act as general acid and base, facilitating a direct nucleophilic attack by a water molecule. Structural studies and site-directed mutagenesis have provided insights into the roles of active site residues in substrate recognition, binding, and catalysis.

Production of Xylanase
The production of xylanase involves several steps, from microbial fermentation using various substrates to the enzyme's extraction, purification, and formulation for specific applications. This process can be broadly categorized into four main stages: solid-state fermentation (SSF), submerged fermentation (SmF), extraction and purification, and formulation for specific applications.

Solid-State Fermentation for Xylanase Production
Figure 4Schematic diagram of Solid-State Fermentation for Xylanase Production

Solid-state fermentation (SSF) is a process where microorganisms grow on solid materials in the absence or near-absence of free water. This method is particularly suitable for fungi, such as Penicillium and Aspergillus species, which are prolific producers of xylanase. SSF mimics the natural habitat of these fungi, allowing them to produce high enzyme yields. Agricultural residues like wheat bran, rice straw, and corn cobs are commonly used as substrates due to their high cellulose and hemicellulose content, which serve as carbon sources for microbial growth. For instance, Penicillium crustosum has shown remarkable xylanase production using pretreated water hyacinth biomass in SSF, highlighting the potential of utilizing agricultural waste for enzyme production.

Submerged Fermentation of Xylanase Production
Submerged fermentation (SmF) involves growing microorganisms in a liquid nutrient medium under controlled conditions. This method is widely used for bacterial xylanase production, including species of Bacillus and Streptomyces, due to the ease of controlling fermentation parameters such as pH, temperature, and aeration,. SmF is characterized by faster microbial growth rates and shorter fermentation times compared to SSF. A study on Trichoderma harzianum demonstrated efficient xylanase production under SmF by optimizing culture conditions, showcasing the method's effectiveness for producing thermostable xylanases suitable for industrial applications.

Xylanase Extraction and Purification
After fermentation, xylanase needs to be extracted and purified from the culture medium. The extraction process often involves the use of aqueous two-phase systems (ATPS) or membrane filtration techniques to separate the enzyme from other cellular components. Purification steps may include ultrafiltration, chromatography, or precipitation methods to achieve the desired purity level. For example, an ATPS based on polyethylene glycol and sodium citrate has been used to extract xylanase with high efficiency and purification factor.

Xylanase Formulation for Specific Application
The final step in the production process is formulating the purified xylanase for specific industrial applications. This involves stabilizing the enzyme in a suitable form, such as liquid, powder, or granules, and may include the addition of stabilizers or other additives to enhance shelf life and activity under application conditions. The formulation depends on the intended use of the xylanase, whether for the pulp and paper industry, biofuel production, food and feed applications, or textile processing

Industrial Application
The versatility of xylanases has led to their extensive use in the pulp and paper, food, animal feed, biofuel, and textile industries, among others.

Pulp and Paper Industry
Traditionally, the bleaching process in paper production involves the use of harsh chemicals such as chlorine and hydrogen peroxide, which can have detrimental effects on the environment. , . However, the introduction of xylanases as a bio-bleaching agent has revolutionized this process. By selectively degrading the xylan component of the pulp, xylanases facilitate the removal of lignin, thereby reducing the amount of chemicals required for bleaching,. This not only minimizes the environmental impact but also improves the overall quality and brightness of the pulp

Food Industry
In the food industry, xylanases have found numerous applications, particularly in the baking sector. The addition of xylanases to bread dough has been shown to improve its handling properties, texture, and shelf life,. By hydrolyzing the xylan present in the dough, xylanases help in the redistribution of water, leading to enhanced dough stability and reduced stickiness. Moreover, the release of xylooligosaccharides during the baking process can contribute to the formation of a desirable crust color and flavor[. Xylanases are also employed in the processing of fruits and vegetables, where they aid in the clarification of juices and wines by reducing the viscosity and improving the overall quality of the final product

Animal Feed Industry
The presence of non-starch polysaccharides, such as xylan, in animal feed can lead to reduced nutrient digestibility and increased viscosity of the feed, which can negatively impact animal performance. . The incorporation of xylanases into animal feed has been found to alleviate these issues by breaking down the xylan component, thereby improving nutrient absorption and reducing the viscosity of the feed. This, in turn, leads to better feed conversion efficiency and enhanced animal growth

Biofuel Industry
The production of bioethanol and other biofuels from lignocellulosic biomass requires the efficient saccharification of the biomass into fermentable sugars. . Xylanases play a crucial role in this process by hydrolyzing the xylan component of the hemicellulose fraction, thus increasing the accessibility of cellulose for subsequent enzymatic hydrolysis. The synergistic action of xylanases with other enzymes, such as cellulases, has been shown to significantly improve the overall yield of fermentable sugars, making the biofuel production process more economically viable

Textile Industry
Xylanases have found applications in the processing of plant fibers, such as linen and ramie. These fibers often contain a significant amount of hemicellulose, which can interfere with the dyeing and finishing processes. The treatment of these fibers with xylanases helps in the removal of the hemicellulosic component, leading to improved fiber properties, such as increased softness, brightness, and dye uptake. Additionally, the use of xylanases reduces the requirement for harsh chemical treatments, making the textile processing more eco-friendly.

The diverse applications of xylanases in various industries have made them an invaluable tool in biotechnology. From improving the efficiency and sustainability of the pulp and paper industry to enhancing the nutritional value of animal feed and facilitating the production of biofuels, xylanases have demonstrated their immense potential. As the demand for eco-friendly and sustainable processes continues to grow, the use of xylanases is expected to expand further, driving innovation and advancements in various industrial sectors.

Optimum Storage Conditions
Proper storage of xylanase preparations is essential to maintain their activity and stability, as well as to prevent potential hazards associated with enzyme degradation or contamination. The optimum storage conditions for xylanases may vary depending on the specific enzyme formulation and the manufacturer's recommendations.

In general, xylanases should be stored in a cool, dry place, away from direct sunlight and sources of heat,. The recommended storage temperature for most xylanase preparations is between 4°C and 25°C. Some formulations may require refrigeration to maintain their activity and stability over extended periods.

It is also crucial to ensure that xylanase preparations are stored in tightly sealed containers to prevent moisture absorption and contamination. Exposure to moisture can lead to enzyme degradation and loss of activity, as well as the growth of microorganisms that may pose health risks.

The shelf life of xylanase preparations can vary depending on the specific formulation and storage conditions. Manufacturers typically provide information on the expected shelf life of their products, which can range from several months to a few years,. It is important to follow the manufacturer's guidelines and to use the enzyme preparation before its expiration date to ensure optimal performance and safety.

Potential Hazards
One of the primary concerns when working with xylanases is the potential for allergic reactions in sensitive individuals. Like other enzymes and proteins, xylanases may cause allergic reactions upon inhalation or skin contact. Symptoms of an allergic reaction may include respiratory difficulties, such as asthma or rhinitis, as well as skin irritation or dermatitis. In rare cases, severe allergic reactions, such as anaphylaxis, may occur.

To minimize the risk of allergic reactions, proper handling and protective measures should be implemented when working with xylanase preparations. This includes wearing appropriate personal protective equipment (PPE), such as gloves, safety glasses, and respiratory protection, especially when handling powdered or aerosolized forms of the enzyme. Additionally, adequate ventilation and dust control measures should be in place to reduce the risk of inhalation exposure.

While xylanases are generally considered safe and non-toxic, it is essential to be aware of potential hazards associated with their use, particularly the risk of allergic reactions in sensitive individuals. Proper handling and protective measures, including the use of appropriate PPE and adequate ventilation, should be followed to minimize these risks. Additionally, proper storage of xylanase preparations, in a cool, dry place and in tightly sealed containers, is crucial to maintain their activity, stability, and safety. By following these guidelines and the manufacturer's recommendations, the industrial use of xylanases can be carried out safely and effectively.