Microbial Xylanolytic Enzymes

By Pratima Bajpai

Microbial Xylanolytic Enzymes describes the enzyme structure and its interaction with plant cell walls, the properties and production of different enzymes and their applications, and the knowledge gathered on the hydrolysis mechanism of hemicellulose. The knowledge gathered about the hydrolysis mechanism of the hemicelluloses, especially xylans, has greatly promoted the rapid application of these enzymes in new areas. In recent years, there has been a spurt of interest in xylan degrading enzymes due to their applications in several industrial processes, including paper and pulp industries, food and feed industries, biofuel industry, textile industry, chemical and pharmaceutical industry, brewing industry, and more.

Xylan is the principal type of hemicellulose. An enzymatic complex is responsible for the hydrolysis of xylan, but the main enzymes involved are enzymes produced by fungi, bacteria, yeast, algae, protozoans, and more.

• Gives up-to-date authoritative information and cites pertinent research on the synergistic action of xylanolytic enzymes
• Includes studies on xylanase regulation and synergistic action between multiple forms of xylanase
• Covers, in great depth, all aspects of Xylanolytic enzymes
• Includes detailed descriptions on Xylanolytic enzymes as a supplement in animal feed, for the manufacture of bread, food and drinks, textile industry, pulp and paper industry, biofuel industry and production of pharmaceuticals and important chemicals and waste management, etc.
• Challenges future trends in the commercial production and application of xylanases

• Language: English
• Publisher: Academic Press
• Release date: May 29, 2022
• ISBN: 9780323996372

Pratima Bajpai

Dr. Pratima Bajpai is currently working as a Consultant in the field of Paper and Pulp. She has over 36 years of experience in research at the National Sugar Institute, University of Saskatchewan, the Universitiy of Western Ontario, in Canada, in addition to the Thapar Research and Industrial Development Centre, in India. She also worked as a visiting professor at the University of Waterloo, Canada and as a visiting researcher at Kyushu University, Fukuoka, Japan. She has been named among the World’s Top 2% Scientists by Stanford University in the list published in October 2022. This is the third consecutive year that she has made it into the prestigious list. Dr. Bajpai’s main areas of expertise are industrial biotechnology, pulp and paper, and environmental biotechnology. She has contributed immensely to the field of industrial biotechnology and is a recognized expert in the field. Dr. Bajpai has written several advanced level technical books on environmental and biotechnological aspects of pulp and paper which have been published by leading publishers in the USA and Europe. She has also contributed chapters to a number of books and encyclopedia, obtained 11 patents, written several technical reports, and has implemented several processes in Indian Paper mills. Dr. Bajpai is an active member of the American Society of Microbiologists and is a reviewer of many international research journals.

Book preview

Chapter 1: General background on microbial xylanolytic enzymes

Abstract

Xylanases are hydrolytic enzymes which cleave the β-1, 4 backbone of the complex plant cell wall polysaccharide xylan. Xylan is found in large quantities in hardwoods from angiosperms and softwoods from gymnosperms as well as in annual plants. It is typically located in the secondary cell wall of plants, and is also found in the primary cell wall, in particular in monocots. It is the next most abundant renewable polysaccharide after cellulose. Xylanases and associated debranching enzymes produced by a variety of microorganisms including bacteria, actinomycetes, yeast, and fungi bring hydrolysis of hemicelluloses. There has been much industrial interest in xylanases, as a supplement in animal feed, for the manufacture of bread, food and drinks, textiles, bleaching of cellulose pulp, and xylitol production. Use of xylanases could greatly improve the overall economics of processing lignocellulosic materials for the generation of liquid fuels and chemicals.

Keywords

Animal feed; Bacteria; Chemicals; Food industry; Fungi; Industrial application; Industry; Liquid fuel; Pulp and paper industry; Textile; Xylan; Xylanase; Yeast

1.1. Introduction on enzymes

Enzymes are the catalytic keystone of metabolism. So, they are the focus of extensive research worldwide in the biological community, as well as with chemical engineers, process designers, process engineers, and researchers working in other scientific fields. According to MarketsandMarkets, the industrial enzymes market is estimated to be valued at USD 5.9 billion in 2020 and is projected to reach USD 8.7 billion by 2026, recording a CAGR of 6.5%, in terms of value. The rising environmental concerns and increase in demand for bioethanol and advancements in R&D activities led to the growth of the industrial enzymes market (https://www.marketsandmarkets.com/PressReleases/industrial-enzymes.asp). Increasing product demand from the end-use industries, for instance, biofuel, home cleaning, food, animal feed, and beverage, is projected to fuel the industry growth in the near future. The market is expected to be driven by increasing demand for carbohydrases and proteases in the food and beverage applications, particularly in the developing countries of Asia Pacific, such as India, China, and Japan. Furthermore, growth in the developed countries can be attributed to increasing industrial development, along with the developments in the nutraceutical sector. Such factors have boosted the product demand on a large scale. The cost of production and the enzyme yield are considered the main problems in commercial use. The advancements in enzyme engineering and green chemistry and the introduction of genetically engineered enzymes have increased the industrial usage. Factors such as the multifunctional benefits of industrial enzymes across various applications and the technological innovations to reduce the consumption of chemicals contribute to the growth of the industrial enzymes market (Bajpai, 2018c).

Microbial enzymes show remarkable potential for various applications. Over the years because of their incredible features, enzymes are occupying the central stage of all the biochemical and industrial processes (Bajpai, 2018c). Enzymes are environment-friendly and are considered the best option to polluting chemical technologies as these can perform innumerable biochemical reactions under ambient conditions. At present, the enzymes are replacing chemical additives in several food applications. Enzymes are considered as safe natural additives. The usage of enzymes is becoming a need of the hour, as they promote effects similar to those of chemical additives. Enzymatic treatment provides the same level of output as it is obtained through traditional methods which use harsh chemicals. So, use of enzymes at various industrial levels is gaining momentum.

1.2. Xylan

Organic wastes from renewable forest and agricultural residues contain cellulose, hemicelluloses, and lignin (Fig. 1.1). The exact percentage of these components is found to vary from source to source (Gray et al., 2006; Singla et al., 2012). Hemicelluloses are embedded in the cell walls of plants with pectin. Hemicelluloses contain xylan, which is a heteropolysaccharide substituted with monosaccharides such as D-galactose, D-mannoses, L-arabinose, and organic acids such as acetic acid, glucuronic acid, ferulic acid, interwoven together with help of glycosidic and ester bonds (Ota et al., 2013; Collins et al., 2005; Ahmed et al., 2007; Motta et al., 2013; Sharma, 2017; Walia et al., 2017).

In plants, xylans or the hemicelluloses are situated between the lignin and the collection of cellulose fibers underneath. Consistent with their structural chemistry and side-group substitutions, the xylans seem to be interspersed, interwined, and covalently linked at various points with the overlying ‘sheath’ of lignin, while producing a coat around underlying strands of cellulose via hydrogen bonding. The xylan layer with its covalent linkage to lignin and its non-covalent interaction with cellulose may be important in maintaining the integrity of the cellulose in situ and in helping protect the fibers against degradation to cellulases (Uffen, 1997; Biely, 1985, 1997; Joseleau et al., 1992).

Figure 1.1  Structure of lignocellulosic biomass. Reproduced from Wang et al. (2019). https://doi.org/10.1186/s42825-019-0003-y. This Figure is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/).

Xylan, one of the hemicelluloses, is a polymer of xylose and represents more than 20%–40% of plant biomass, making it second most plentiful polysaccharide in world. Xylans are mostly found in secondary walls of the plant cell wall. It consists of a linear polymer of β-(1,4)-linked xylose residues substituted with acetyl, glucuronic acid (GlcA), 4-O-methylglucuronic acid (Me-GlcA), and arabinose residues (Emilie et al., 2014). This structure can be degraded by xylanolytic enzymes. 1,4-endoxylanases play a most important role in degradation of xylan backbone by catalyzing the random hydrolysis of xylosidic linkages. On the other hand, β-xylosidases release xylosyl residues by endwise attack on xylooligosaccharides in xylan (Sharma and Kumar, 2013).

1.3. Xylanases

Xylanases are hydrolytic enzymes consisting of endo-1,4-β-xylanase (EC 3.2.1.8) and β-xylosidase (EC 3.2.1.37). Xylanases catalyze the hydrolysis of xylan which is a main component in plant cell wall. Endo-1,4-β-xylanase along with assisting hydrolytic enzymes (Table 1.1) such as α-glucuronidase, α-L-arabinofuranosidase, acetylxylan esterase, and phenolic acid (ferulic and p-coumaric acid) esterase hydrolyze xylan (present in hemicelluloses of plants) to monomeric sugars. Of these, endo-1,4-β-xylanase (often referred as xylanase) is mainly important, as it breaks the main backbone of xylan for hydrolysis. This enzyme catalyzes the hydrolysis of β-1,4 linkages in xylan. The role of the assisting enzymes comes into play depending on the nature of the side chain with which the xylan is associated. These side chains actually help to completely breakdown xylan to its monomer, xylose.

Table 1.1

Based on Zabel and Morrell (2020).

"Xylanases were first reported in 1955 and were originally termed as pentosanases.

They were recognized by the International Union of Biochemistry and Molecular Biology in 1961 and were assigned the enzyme code EC 3.2.1.8. They have been referred to by various names. Their commonly used synonymous terms include:

➢ Xylanase

➢ Endoxylanase

➢ Endo-1,4-β-D-xylanase

➢ β-1,4-xylanase

➢ β-xylanase

However, the official name is endo-1,4-β-xylanase.

Based on amino acid sequence similarities and hydrophobic cluster analysis, xylanases fall mainly into two glycosyl hydrolase families, family 10 (>30kDa with low pI values) and family 11 (<30kDa and high pI values), although other families, including families 5, 7, 8, and 43, also contain some xylanolytic enzymes.

Xylanase secretions are often accompanied by cellulolytic enzymes, e.g., as in the species of Trichoderma, Penicillium, and Aspergillus" (Thomas et al., 2017; Collins et al., 2005).

"Xylans are present in the cell wall and in the middle lamella of plant cells. This term covers a range of noncellulose polysaccharides composed, in various proportions, of monosaccharide units such as D-xylose, D-mannose, D-glucose, L-arabinose, D-galactose, D-glucuronic acid, and D-galacturonic acid. Classes of hemicellulose are named according to the main sugar unit. Thus, when a polymer is hydrolyzed and yields xylose, it is a xylan; in the same way, hemicelluloses include mannans, glucans, arabinans, and galactans (Whistler and Richards, 1970; Viikari et al., 1994; Uffen, 1997; Ebringerova, 2005).

In nature, wood hemicelluloses hardly ever consist of just one type of sugar. Usually they are complex structures made of more than one polymer, the most common being glucuronoxylans, arabinoglucuronoxylans, glucomannans, arabinogalactans, and galactoglucomannans (Haltrich et al., 1996; Sunna and Antranikian, 1997; Kulkarni et al., 1999; Subramaniyan and Prema, 2002). The amount of each component varies from species to species and even from tree to tree. Therefore, hemicellulose is not a well-defined chemical compound, but a class of polymer components of plant fibers, with properties peculiar to each one. Hemicelluloses mainly comprise xylans, which are degraded by xylanolytic enzymes. These enzymes are produced mainly by microorganisms—bacteria, fungi, actinomycetes, and yeast (Hrmova et al., 1984; Liu et al., 1998, 1999; Gilbert and Hazlewood, 1993; Sunna and Antranikian, 1997; Ball and McCarthy, 1989; Beg et al., 2000; Bajpai, 1997, 2009; Motta et al., 2013; Ota et al., 2013; Emilie et al., 2014; Gray et al., 2006; Singla et al., 2012; Ahmed et al., 2007; Bhardwaj et al., 2019) and take part in the breakdown of plant cell walls, along with other enzymes that hydrolyze polysaccharides, and also digest xylan during the germination of some seeds, for example, in the malting of barley grain. Xylanases also can be found in marine algae, protozoans, crustaceans, insects, snails, and seeds of land plants (Sunna and Antranikian, 1997). Among microbial sources, filamentous fungi are especially interesting as they secrete these enzymes into the medium and their xylanase levels are very much higher than those found in yeasts and bacteria" (Bajpai, 2014).

Most of the xylanases producing microorganisms are saprotrophs which require these enzymes for plant degradation and liberation of xylose which is a main carbon source for cell metabolism. Others are plant pathogens requiring hemicellulose degradation for plant cell infection. Trichoderma or Aspergillus species are well-studied xylan-degrading organisms. Most of the xylan-degrading enzymes have been identified, characterized, and also expressed in other xylanase-negative organisms, for example Escherichia coli or Saccharomyces cerevisiae. Interest in xylanases from diverse sources has increased significantly in the last 2decades. Numerous patents have been issued for various xylanase sources, uses, or production methods.

According to Wong et al. (1988), xylanases may be classified mainly by three ways (Table 1.2). Xylanases are of interest for several reasons. On the one hand, they are clearly involved in providing sources of carbon and energy for the organisms that produce them. They act in the same fashion for hosts harboring xylanase-producing organisms, and they are involved in the growth, maturation, and ripening of cereals and fruits. Moreover, xylanases appear to be involved in the invasion of plants and fruits by pathogens. On the other hand, xylan-degrading enzyme systems have great potential in several biotechnological applications. Diverse forms of these enzymes exist, displaying varying folds, mechanism of action, substrate specificities, hydrolytic activities and physicochemical characteristics (Bajpai, 1997, 2009). Several reports and articles mention the isolation of newer microbial species for xylanase production. This shows an ever-increasing interest by the scientific community in this field.

Table 1.2

Based on Wong et al. (1988).

Xylanase enzymes have a wide range of industrial applications (Bajpai, 1997, 1999, 2004, 2006, 2009, 2013, 2015, 2018a,b,c; Bajpai and Bajpai, 1997; Bedford and Classen, 1992, 1993; Maat et al., 1992; Wong and Saddler, 1992a,b; Kuhad and Singh, 1993; Biely, 1985, 1991, 1997; Kapoor et al., 2001; Puchart et al., 1999; Sharma, 1987; Zeikus et al., 1991; McCleary, 1986; Woodward, 1984; Campbell et al., 1991; Fengler and Marquardt, 1988; Grahm and Inborr, 1992; Groot Wassink et al., 1989; Linko et al., 1989; Poutanen and Puls, 1988; Pettersson and Aman, 1989; Harris and Ramalingam, 2010; Viikari et al., 1986, 1994; Sprössler, 1997; Keskin et al., 2004). There has been a lot of industrial interest in using xylanases in pulp and paper industry, textile industry, manufacture of food and beverages, animal feed, pharmaceuticals, and production of bioethanol. Because of their biotechnological characteristics, xylanases are most often produced from microorganisms for industrial applications.

Let's briefly take a look at some most promising industrial application of xylanases.

Xylanases are being used in the biobleaching of pulp and in the bioprocessing of textiles. In fact, treatment of cellulosic pulps with xylanases selectively removes residual xylan. Xylanases can degrade the hemicellulosic content present in the pulp without having an effect on cellulose. Treatment with enzyme enhances physical properties of paper which includes viscosity, tensile strength, breaking length, and tear strength. Furthermore, bleaching with xylanases softens the fibers. Due to this, chemical bleaching becomes easier.

Xylanases are also used for converting xylan into high value-added products. As the enzymatic hydrolysis of xylan produces xylose, different fermentations may occur and a range of compounds may result from these reactions. One of the most important such products is xylitol. It is used as a natural sweetener in various pharmaceutical products and in toothpaste. Xylitol is also used to sweeten food products, for instance, candy, chewing gum, soft drinks, and ice