*3.2.2. Bioconversion of lignocellulose in biofuels*

**3.2. Properties and applications**

ential glycosylation, proteolysis or both [23].

and purifying fibers from other carbohydrates [33].

duces the usage of chlorine as the bleaching agent [33].

*3.2.1. The paper and pulp industries*

state of the remaining lignin [33].

The heterogeneity and complexity of xylan have resulted in a diverse range of xylanases, which differ in their physicochemical properties, structure, mode of action and substrate specificities [10]. As the xylosidic linkages in lignocellulose are neither equivalent nor equally accessible, the production of an enzymatic system with specialized functions is a strategy to achieve superior xylan hydrolysis [28]. Together with the heterogeneous na‐ ture of xylan, the multiplicity of xylanases in microorganisms may be caused by a redun‐ dancy in gene expression. Generally, a single xylanase gene encodes multiple xylanases, and xylanase multiplicity may arise from posttranslational modifications, such as differ‐

258 Sustainable Degradation of Lignocellulosic Biomass - Techniques, Applications and Commercialization

The potential applications of xylanases also include the bioconversion of lignocellulosic material and agro-wastes into fermentative products, the clarification of juices, the improve‐ ment of the consistency of beer and the digestibility of animal feedstocks [28]. One of the most important biotechnological applications of xylanase is its use in pulp bleaching [46]. Xylanases may also be applicable to the production of rayon, cellophane and several chemicals such as cellulose esters (acetates, nitrates, propionates and butyrates) and cellulose ethers (carboxy‐ methyl cellulose and methyl and ethyl cellulose), which are all produced by dissolving pulp

During the past several years, the use of enzymes in paper and pulp bleaching has caught the attention of researchers and industries all over the world. Xylanase enzymes have proven to be a cost-effective means for mills to take advantage of a variety of bleaching benefits [47]. Xylanases and other side-cleaving enzymes have been used in pulp bleaching primarily to reduce lignin and increase the brightness of the pulp [20,46]. The importance of xylanase in the pulp and paper industries is related to the hydrolysis of xylan, which facilitates the release of lignin from paper pulp and, consequently, re‐

Bleaching is the process of lignin removal from chemical pulps to produce bright or completely white finished pulp [1]. Thus, the bleaching of pulp using enzymes or ligninolytic microor‐ ganisms is called biobleaching [48]. This process is necessary due to the presence of residual lignin and its derivatives in the pulping process, which causes the resultant pulp to gain a characteristic brown color. The intensity of this pulp color is related to the amount and chemical

The bleaching of pulp involves the destruction, alteration or solubilization of the lignin, colored organic matter and other undesirable residues on the fibers [33]. Bleaching of kraft pulp usually requires large amounts of chlorine-based chemicals and sodium hydrosulfite, which cause several effluent-based problems in the pulp and paper industries. The use of these chemicals generates chlorinated organic substances, some of which are toxic, mutagenic, persistent, and highly resistant to biodegradation, in addition to causing numerous harmful disturbances in biological systems and forming one of the major sources of environmental pollution [1,33,49]. Currently, second-generation biofuels are the primary products of the bioconversion of lignocellulosic materials. According to Taherzadech and Karimi [54], ethanol is the most important renewable fuel in terms of volume and market value, and following the fossil fuel crisis, it has been identified as an alternative fuel [48]. Despite the primarily first-generation production of ethanol, from sugar and starch, the second-generation production of ethanol has only begun to be tested in pilot plants [55]. And, unlike first-generation biofuels, secondgeneration biofuels do not compete with food production and can provide environmental, economic, and strategic benefits for the production of fuels [56].

Xylanase, together with other hydrolytic enzymes, can be used for the generation of biological fuels, such as ethanol, from lignocellulosic biomass [1,57]. However, enzymatic hydrolysis is still a major cost factor in the conversion of lignocellulosic raw materials to ethanol [56]. In bioethanol fuel production, the first step is the delignification of lignocellulose, to liberate cellulose and hemicellulose from their complex with lignin. The second step is a depolymeri‐ zation of the carbohydrate polymers to produce free sugars, followed by the fermentation of mixed pentose and hexose sugars to produce ethanol [1,58]. Simultaneous saccharification and fermentation is an alternative process, in which both hydrolytic enzymes and fermentative microorganism are present in the reaction [48,59].

#### *3.2.3. The pharmaceutical, food and feed industries*

Xylanase, together with pectinase, carboxymethylcellulase and amylase, can be used for the clarification of juices because the turbidity observed is due to both pectic materials and other materials suspended in a stable colloidal system [60]. Xylanase may also improve the extraction of coffee, plant oils, and starch [25]. The xylose resulting from xylan depolymerization may also be converted to xylitol, a valuable sweetener that has applications in both the pharma‐ ceutical and food industries [61-63].

and method, the interlaboratory standard variation of the results decreased from 108% to 17% from the mean. Others researchers use the 4-o-methylglucuronoxylan covalently dyed with Remazol Brilliant Blue (RBB xylan) as substrate, and the xylanase is assayed based on the release of the dyed fragments [77]. There are also available some commer‐ cial methods for xylanase assays, as the fluorescence-based method EnzChek® Ultra Xy‐ lanase Assay Kit (Invitrogen, Carlsbad, CA) or the Xylazyme tablet (Megazyme, Bray, Ireland), which employs azurine-crosslinked arabinoxylan (AZCLArabinoxylan) as sub‐

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Microorganisms, in particular, have been regarded as a good source of useful enzymes because they multiply at extremely high rates and synthesize biologically active products that can be controlled by humans. In recent years, there has been a phenomenal increase in the use of enzymes as industrial catalysts. These enzymes offer advantages over the use of conventional chemical catalysts for numerous reasons: they exhibit high catalytic activity and a high degree of substrate specificity, they can be produced in large amounts, they are highly biodegradable, they pose no threat to the environment and

In this context, microbial xylanases are the preferred catalysts for xylan hydrolysis, due to their high specificity, mild reaction conditions, negligible substrate loss and side prod‐ uct generation. Xylanases derived from microorganisms have many potential applications in the food, feed, and paper pulp industries [10,12,78]. Complete xylanolytic enzyme sys‐ tems, which including all of these activities, have been found to be widespread among fungi [20,24], actinomycetes [79] and bacteria [12], and some of the most important xyla‐ nolytic enzyme producers include *Aspergillus, Trichoderma, Streptomyces, Phanerochaetes, Chytridiomycetes, Ruminococcus, Fibrobacteres, Clostridia* and *Bacillus* [12,78,80,81]. The eco‐ logical niches of these microorganisms are diverse and widespread and typically include environments where plant materials accumulate and deteriorate, as well as in the rumen

Although there have been many reports on microbial xylanases since the 1960s, the prime focus has been on plant pathology related studies [84]. Only during the 1980's did the use of xylanases for biobleaching begin to be tested [85]. Since 1982, several microorganisms, including fungi and bacteria, have been reported to readily hydrolyze xylans by synthesizing 1,4-β-D endoxylanases (E.C. 3.2.18) and β-xylosidases (EC.3.2.1.37) [86]. Table 1 presents a list

The production of xylanases must be improved by finding more potent fungal or bacterial strains or by inducing mutant strains to excrete greater amounts of the enzymes. Moreover, the level of microbial enzyme production is influenced by a variety of nutritional and phys‐ iological factors, such as the supply of carbon and nitrogen, physical circumstances and

of some of the xylanase-producing microorganisms and their activities.

strate and its hydrolysis by xylanase produces water soluble dyed fragments.

**3.4. Producing microorganisms**

they are economically viable [4].

of ruminants [78,82,83].

chemical conditions [98].

In the bakery industry, xylanase may improve the quality of bread, by increasing the bread's specific volume. In combination with amylases, this characteristic was enhanced, as observed upon the introduction of *Aspergillus niger* var. *awamori* [64]. According to Collins *et al*. [65], psychrophilic enzymes may be suitable for use in the baking industry as they are generally optimally active at the temperatures most frequently used for dough preparation (at or below 35 °C). These enzymes could also be used as more effi‐ cient baking additives than the currently used commercial mesophilic enzymes, which are optimally active at higher temperatures.

Xylanase may also improve the nutritional properties of agricultural silage and grain feed. The use of this enzyme in poultry diets showed that the decrease in weight gain and feed conversion efficiency in rye-fed broiler chicks has been associated with intestinal viscosity [66]. The incorporation of xylanase from *Trichoderma longibrachiatum* into the rye-based diet of broiler chickens reduced intestinal viscosity, thus, improving both the weight gain of the chicks and their feed conversion efficiency [67].

Xylanases can also be used in cereals as a pretreatment for arabinoxylan-containing substrates, as arabinoxylans are partly water soluble and result in a highly viscous aqueous solution. This high viscosity of cereal grain water extract may lead to brewing problems, by decreasing the rate of filtration or haze formation in beer. Additionally, it is unfavorable in the cereal grains used in animal feeding [68,69].

The enzymatic hydrolysis of xylan may also result in oligomers known as xylooligosac‐ charides (XOs), which may be used in pharmaceutical, agriculture and feed products. XOs have prebiotic effects, as they are neither hydrolyzed nor absorbed in the upper gastrointestinal tract, and they affect the host by selectively stimulating the growth or ac‐ tivity of one or a number of bacteria in the colon, thus improving health [70-72]. Among their key physiological advantages are the reduction of cholesterol, maintenance of gas‐ trointestinal health, and improvement of the biological availability of calcium. They also inhibit starch retrogradation, improving the nutritional and sensory properties of food [73]. For the production of XOs, the enzyme complex must have low exoxylanase or βxylosidase activity, to prevent the production of high amounts of xylose, which has in‐ hibitory effects on XO production [74,75].

#### **3.3. Xylanase assays**

The xylanase activity is often assayed based on measurement of reducing sugar released during the course of hydrolysis of xylan, by DNS or Nelson-Somogyi methods. Due to absent of standardization, Bailey *et al.* [76] compared the measurement of xylanase activi‐ ty by twenty different laboratories. According to the author, the major source of varia‐ tion between apparent xylanase activities was probably the substrate chosen, although small differences in protocols were also significant. After standardization of substrate and method, the interlaboratory standard variation of the results decreased from 108% to 17% from the mean. Others researchers use the 4-o-methylglucuronoxylan covalently dyed with Remazol Brilliant Blue (RBB xylan) as substrate, and the xylanase is assayed based on the release of the dyed fragments [77]. There are also available some commer‐ cial methods for xylanase assays, as the fluorescence-based method EnzChek® Ultra Xy‐ lanase Assay Kit (Invitrogen, Carlsbad, CA) or the Xylazyme tablet (Megazyme, Bray, Ireland), which employs azurine-crosslinked arabinoxylan (AZCLArabinoxylan) as sub‐ strate and its hydrolysis by xylanase produces water soluble dyed fragments.

#### **3.4. Producing microorganisms**

also be converted to xylitol, a valuable sweetener that has applications in both the pharma‐

260 Sustainable Degradation of Lignocellulosic Biomass - Techniques, Applications and Commercialization

In the bakery industry, xylanase may improve the quality of bread, by increasing the bread's specific volume. In combination with amylases, this characteristic was enhanced, as observed upon the introduction of *Aspergillus niger* var. *awamori* [64]. According to Collins *et al*. [65], psychrophilic enzymes may be suitable for use in the baking industry as they are generally optimally active at the temperatures most frequently used for dough preparation (at or below 35 °C). These enzymes could also be used as more effi‐ cient baking additives than the currently used commercial mesophilic enzymes, which

Xylanase may also improve the nutritional properties of agricultural silage and grain feed. The use of this enzyme in poultry diets showed that the decrease in weight gain and feed conversion efficiency in rye-fed broiler chicks has been associated with intestinal viscosity [66]. The incorporation of xylanase from *Trichoderma longibrachiatum* into the rye-based diet of broiler chickens reduced intestinal viscosity, thus, improving both the weight gain of the chicks and

Xylanases can also be used in cereals as a pretreatment for arabinoxylan-containing substrates, as arabinoxylans are partly water soluble and result in a highly viscous aqueous solution. This high viscosity of cereal grain water extract may lead to brewing problems, by decreasing the rate of filtration or haze formation in beer. Additionally, it is unfavorable in the cereal grains

The enzymatic hydrolysis of xylan may also result in oligomers known as xylooligosac‐ charides (XOs), which may be used in pharmaceutical, agriculture and feed products. XOs have prebiotic effects, as they are neither hydrolyzed nor absorbed in the upper gastrointestinal tract, and they affect the host by selectively stimulating the growth or ac‐ tivity of one or a number of bacteria in the colon, thus improving health [70-72]. Among their key physiological advantages are the reduction of cholesterol, maintenance of gas‐ trointestinal health, and improvement of the biological availability of calcium. They also inhibit starch retrogradation, improving the nutritional and sensory properties of food [73]. For the production of XOs, the enzyme complex must have low exoxylanase or βxylosidase activity, to prevent the production of high amounts of xylose, which has in‐

The xylanase activity is often assayed based on measurement of reducing sugar released during the course of hydrolysis of xylan, by DNS or Nelson-Somogyi methods. Due to absent of standardization, Bailey *et al.* [76] compared the measurement of xylanase activi‐ ty by twenty different laboratories. According to the author, the major source of varia‐ tion between apparent xylanase activities was probably the substrate chosen, although small differences in protocols were also significant. After standardization of substrate

ceutical and food industries [61-63].

are optimally active at higher temperatures.

their feed conversion efficiency [67].

used in animal feeding [68,69].

hibitory effects on XO production [74,75].

**3.3. Xylanase assays**

Microorganisms, in particular, have been regarded as a good source of useful enzymes because they multiply at extremely high rates and synthesize biologically active products that can be controlled by humans. In recent years, there has been a phenomenal increase in the use of enzymes as industrial catalysts. These enzymes offer advantages over the use of conventional chemical catalysts for numerous reasons: they exhibit high catalytic activity and a high degree of substrate specificity, they can be produced in large amounts, they are highly biodegradable, they pose no threat to the environment and they are economically viable [4].

In this context, microbial xylanases are the preferred catalysts for xylan hydrolysis, due to their high specificity, mild reaction conditions, negligible substrate loss and side prod‐ uct generation. Xylanases derived from microorganisms have many potential applications in the food, feed, and paper pulp industries [10,12,78]. Complete xylanolytic enzyme sys‐ tems, which including all of these activities, have been found to be widespread among fungi [20,24], actinomycetes [79] and bacteria [12], and some of the most important xyla‐ nolytic enzyme producers include *Aspergillus, Trichoderma, Streptomyces, Phanerochaetes, Chytridiomycetes, Ruminococcus, Fibrobacteres, Clostridia* and *Bacillus* [12,78,80,81]. The eco‐ logical niches of these microorganisms are diverse and widespread and typically include environments where plant materials accumulate and deteriorate, as well as in the rumen of ruminants [78,82,83].

Although there have been many reports on microbial xylanases since the 1960s, the prime focus has been on plant pathology related studies [84]. Only during the 1980's did the use of xylanases for biobleaching begin to be tested [85]. Since 1982, several microorganisms, including fungi and bacteria, have been reported to readily hydrolyze xylans by synthesizing 1,4-β-D endoxylanases (E.C. 3.2.18) and β-xylosidases (EC.3.2.1.37) [86]. Table 1 presents a list of some of the xylanase-producing microorganisms and their activities.

The production of xylanases must be improved by finding more potent fungal or bacterial strains or by inducing mutant strains to excrete greater amounts of the enzymes. Moreover, the level of microbial enzyme production is influenced by a variety of nutritional and phys‐ iological factors, such as the supply of carbon and nitrogen, physical circumstances and chemical conditions [98].


enzymes with regards to the carbon sources present in the medium are influenced by the availability of precursors for protein synthesis. Therefore, in some fungi, growing the cells on xylan uncontaminated by cellulose under a lower nitrogen/carbon ratio may be a possible strategy for producing xylanolytic systems free of cellulases [106]. Another major problem associated with fungi is the reduced xylanase yield in fermenter studies. Agitation is normally used to maintain the medium homogeneity, but the shearing forces in the fermenter can disrupt the fragile fungal biomass, leading to the reported low productivity. Higher rates of agitation

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Xylanases have been reported in *Bacillus*, *Streptomyces* and other bacterial genera that do not have any role related to plant pathogenicity [86]. The extreme thermophile *Rhodothermus marinus* has been reported to produce α-L-arabinofuranosidase [107], and two different polypeptides with α-arabinofuranosidase activity from *Bacillus polymyxa* were characterized

Bacteria, just like many other industrial enzymes, have fascinated researchers due to their alkaline-thermostable xylanase-producing trait [33]. The optimum pH of bacterial xylanases are, in general, slightly higher than the optimal pH of fungal xylanases [109], which is a suitable characteristic in most industrial applications, especially the paper and pulp industries. Noteworthy producers of high levels of xylanase activity at an alkaline pH and high temper‐ ature are *Bacillus* spp. [33]. When considering only temperature, a handful of xylanases that show optimum activity at higher temperatures have been reported from various microorgan‐ isms. These include *Geobacillus thermoleovorans*, *Streptomyces sp.* S27, *Bacillus firmus*, *Actinoma‐ dura sp.* strain Cpt20 and *Saccharopolyspora pathunthaniensis* S582, all of which produce xylanases that show activity between 65 and 90 °C [8]. One xylanase, reported from *Thermo‐ toga sp.* [110], has been shown to exhibit a temperature optima between 100 and 105 °C.

Xylanases are produced by either solid-state or submerged fermentation [5]. Although most xylanase manufacturers produce these enzymes using submerged fermentation (SmF) techniques (nearly for 90% of the total xylanase sales worldwide) [2], the enzyme productivity via solid-state fermentation (SSF) is normally much higher than that of submerged fermenta‐ tion [5]. The growing interest in using solid-state fermentation (SSF) techniques to produce a wide variety of enzymes, including xylanases from fungal origins, is primarily due to the

The advantages of SSF processes over SmF include a low cultivation cost for the fermentation, lower risk of contamination [1], improved enzyme stability, mimicking the natural habit of the fungus, production of enzymes with higher specific activities, generation of a protein-enriched byproduct, and easier downstream processing of the enzymes produced [112]. SSF conditions are especially suitable for the growth of fungi, as these organisms are able to grow at relatively

may also lead to hyphal disruption, further decreasing the xylanase activity [50].

at the gene level for the production of α-arabinofuranosidases [108].

**3.5. Production of xylanases under SSF and SmF**

economic and engineering advantages of this process [111].

*3.4.2. Bacteria*

**Table 1.** Review of xylanases-producing microorganisms.

#### *3.4.1. Fungi*

Filamentous fungi are particularly interesting producers of xylanases and other xylandegrading enzymes because they excrete the enzymes into the medium and their enzyme levels are much higher than those of yeast and bacteria. In addition to xylanases, fungi produce several auxiliary enzymes required for the degradation of substituted xylan [2].

The fungal genera *Trichoderma*, *Aspergillus*, *Fusarium*, and *Pichia* are considered great producers of xylanases [99]. White-rot fungi have also been shown to produce extracellular xylanases that act on a wide range of hemicellulosic materials, are useful as food sources [100] and produce metabolites of interest to the pharmaceutical, cosmetic, and food industries [78]. White-rot basidiomycetes normally secrete large amounts of these enzymes to degrade lignocellulosic materials. For example, *Phanerochaete chrysosporium* produces high levels of αglucuronidase [101], and *Coriolus versicolor* produces a complex xylanolytic combination of enzymes [102]. Xylanase is also produced by *Cuninghamella subvermispora* when growing on plant cell-wall polysaccharides or on wood chips [103].

Fungal xylanases are generally associated with celluloses [104]. On cellulose these strains produce both cellulase and xylanase, which may be due to traces of hemicellulose present in the cellulosic substrates [105]; however, selective production of xylanase may be possible using only xylan as the carbon source. The mechanisms that govern the formation of extracellular enzymes with regards to the carbon sources present in the medium are influenced by the availability of precursors for protein synthesis. Therefore, in some fungi, growing the cells on xylan uncontaminated by cellulose under a lower nitrogen/carbon ratio may be a possible strategy for producing xylanolytic systems free of cellulases [106]. Another major problem associated with fungi is the reduced xylanase yield in fermenter studies. Agitation is normally used to maintain the medium homogeneity, but the shearing forces in the fermenter can disrupt the fragile fungal biomass, leading to the reported low productivity. Higher rates of agitation may also lead to hyphal disruption, further decreasing the xylanase activity [50].

## *3.4.2. Bacteria*

**Microorganisms Xylanases Cultivation**

*Thermomyces lanuginosus SD-21*

*Neocallimastix sp.* strain

*Streptomycessp.* strain Ib

*Paecilomyces themophila*

**Table 1.** Review of xylanases-producing microorganisms.

plant cell-wall polysaccharides or on wood chips [103].

L2

24D

J18

*3.4.1. Fungi*

**conditions**

8,237 IU/g pH 6.0; 40 °C Corn cob and wheat bran

1.13 IU/mL 50 °C Avicel (PH 105) from Serva

18,580.0 IU/g pH 6.9; 50 °C Wheat straw and yeast

Filamentous fungi are particularly interesting producers of xylanases and other xylandegrading enzymes because they excrete the enzymes into the medium and their enzyme levels are much higher than those of yeast and bacteria. In addition to xylanases, fungi produce

The fungal genera *Trichoderma*, *Aspergillus*, *Fusarium*, and *Pichia* are considered great producers of xylanases [99]. White-rot fungi have also been shown to produce extracellular xylanases that act on a wide range of hemicellulosic materials, are useful as food sources [100] and produce metabolites of interest to the pharmaceutical, cosmetic, and food industries [78]. White-rot basidiomycetes normally secrete large amounts of these enzymes to degrade lignocellulosic materials. For example, *Phanerochaete chrysosporium* produces high levels of αglucuronidase [101], and *Coriolus versicolor* produces a complex xylanolytic combination of enzymes [102]. Xylanase is also produced by *Cuninghamella subvermispora* when growing on

Fungal xylanases are generally associated with celluloses [104]. On cellulose these strains produce both cellulase and xylanase, which may be due to traces of hemicellulose present in the cellulosic substrates [105]; however, selective production of xylanase may be possible using only xylan as the carbon source. The mechanisms that govern the formation of extracellular

several auxiliary enzymes required for the degradation of substituted xylan [2].

1,447.0 IU/mL pH 7.5; 28 °C Tomato pomace [96]

extract

*Streptomyces sp.* P12–137 27.8 IU/mL pH 7.2; 28 °C Wheat bran and KNO3 [88]

*Penicillium clerotiorum* 7.5 IU/mL pH 6.5; 30 °C Wheat bran [91]

*Aspergillus niger* PPI 16.0 IU/mL pH 5.0; 28 °C Oat and urea [92]

*Cochliobolus sativus Cs6* 1,469.4 IU/g pH 4.5; 30 °C Wheat straw and NaNO3 [94] *Bacillus circulans* D1 8.4 IU/mL pH 9.0; 45 °C Bagasse hydrolysates [95]

*Penicillium canescens* 18,895 IU/g pH 7.0; 30 °C Soya oil cake and casein

262 Sustainable Degradation of Lignocellulosic Biomass - Techniques, Applications and Commercialization

*Penicillium fellutanum* 39.7 IU/mL 30 °C Oat spelt xylan, urea,

*Acremonium furcatum* 33.1 IU/mL 30 °C Oat spelt xylan, urea,

**Media Reference**

[87]

[89]

[90]

[90]

[93]

[97]

peptone

and (NH4)2SO4

peptone and yeast extract

peptone and yeast extract

(Heidelberg, Germany)

Xylanases have been reported in *Bacillus*, *Streptomyces* and other bacterial genera that do not have any role related to plant pathogenicity [86]. The extreme thermophile *Rhodothermus marinus* has been reported to produce α-L-arabinofuranosidase [107], and two different polypeptides with α-arabinofuranosidase activity from *Bacillus polymyxa* were characterized at the gene level for the production of α-arabinofuranosidases [108].

Bacteria, just like many other industrial enzymes, have fascinated researchers due to their alkaline-thermostable xylanase-producing trait [33]. The optimum pH of bacterial xylanases are, in general, slightly higher than the optimal pH of fungal xylanases [109], which is a suitable characteristic in most industrial applications, especially the paper and pulp industries. Noteworthy producers of high levels of xylanase activity at an alkaline pH and high temper‐ ature are *Bacillus* spp. [33]. When considering only temperature, a handful of xylanases that show optimum activity at higher temperatures have been reported from various microorgan‐ isms. These include *Geobacillus thermoleovorans*, *Streptomyces sp.* S27, *Bacillus firmus*, *Actinoma‐ dura sp.* strain Cpt20 and *Saccharopolyspora pathunthaniensis* S582, all of which produce xylanases that show activity between 65 and 90 °C [8]. One xylanase, reported from *Thermo‐ toga sp.* [110], has been shown to exhibit a temperature optima between 100 and 105 °C.

#### **3.5. Production of xylanases under SSF and SmF**

Xylanases are produced by either solid-state or submerged fermentation [5]. Although most xylanase manufacturers produce these enzymes using submerged fermentation (SmF) techniques (nearly for 90% of the total xylanase sales worldwide) [2], the enzyme productivity via solid-state fermentation (SSF) is normally much higher than that of submerged fermenta‐ tion [5]. The growing interest in using solid-state fermentation (SSF) techniques to produce a wide variety of enzymes, including xylanases from fungal origins, is primarily due to the economic and engineering advantages of this process [111].

The advantages of SSF processes over SmF include a low cultivation cost for the fermentation, lower risk of contamination [1], improved enzyme stability, mimicking the natural habit of the fungus, production of enzymes with higher specific activities, generation of a protein-enriched byproduct, and easier downstream processing of the enzymes produced [112]. SSF conditions are especially suitable for the growth of fungi, as these organisms are able to grow at relatively low water activities, contrary to most bacteria and yeast, which will not proliferate under these culture conditions [113].

An increasing number of publications have described numerous xylanases from several sources and the cloning, sequencing, mutagenesis and crystallographic analysis of these enzymes [12]. The available amino acid sequence data, X-ray crystallographic data, molecular dynamics and computational design of xylanases provide information that authenticates the relationship between the structure and function of xylanases. All of these methods aid in the design of xylanases that are required in industrial processes, such as improvement of the

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To attempt these processes for commercial purposes, genes encoding several xylanases have been cloned in homologous and heterologous hosts [12,48]. Recombinant xylanases have shown equivalent or better properties than the native enzymes, and the xylanase genes from anaerobic microorganisms have also been expressed successfully in hosts that can be employed

*Escherichia coli* is known for its ease of manipulation, inexpensive growth conditions, sim‐ ple techniques required for transformation and accumulation of high levels of product in the cell cytoplasm; therefore, this organism has become the most widely used expression host [117]. Despite *Escherichia coli's* use as a good cloning host for recombinant proteins, it does not provide efficient and functional expression of many xylanases [23,24], and not all genes are easily expressed in *E. coli* [117]. This problem may be due to the repetitive appearance of rare codons and the requirement for specific translational modifications, such as disulfide-bond formation and glycosylation [23]. Therefore, this microorganism is useful for the detailed study of xylanase gene structure and for the improvement of the

*Lactobacillus* species and *Bacillus subitilis* have been attractive hosts for the production of heterologous proteins, obtaining higher expression levels than *E. coli* [23,118]. *B. subtilis* and *Lactobacillus* are gram-positive and perform N-glycosilation [119]. Their primary in‐ terest in industry and research, is due to the fact that are non-toxic and are generally recognized as safe (GRAS) [23,118]. Members of the genus *Bacillus*, unlike *E. coli*, do not contain endotoxins (lipopolysaccharides), which are difficult to remove from many pro‐ teins during the purification process. The secretory production could also be advanta‐

Heterologous protein expression in yeast systems is highly attractive because they provide additional benefits over bacterial expression systems. Among these benefits are the ability to perform eukaryotic post-translational modifications, the ability to grow to very high cell densities and the ability to secrete proteins into the fermentation media. Moreover, yeast are

*Saccharomyces cerevisiae* secretes high amounts of xylanases into the culture medium. Because it has already been established as an industrial microorganism, it can be used conveniently for

stability of xylanases at higher temperatures and alkaline pHs [8].

in the fermentation industry [35].

enzymes via protein engineering [35].

geous in industrial production [33].

free of toxins and the majority have GRAS status [23].

*3.6.2. Expression in yeast*

*3.6.1. Expression in bacteria*

On the contrary, submerged fermentation allows better control of the conditions during fermentation [114]. The submerged fermentation of aerobic microorganisms is a well-known and widely used method for the production of cellulase and xylanase [115]. In general, SmF is the preferred method of production when the preparations require more purified enzymes, whereas synergistic effects from a battery of xylan-degrading enzymes can easily be found in preparations obtained by SSF using complex substrates, though the latter is commonly sought in applications aimed at improving animal feed [113].

The choice of the substrate is of great importance for the selection of the fermentation process and the successful production of xylanases. In this context, purified xylans can be excellent substrates because the low molecular weight compounds derived from them are the best xylanase inducers. The use of such substrates has led to increased yields of xylanase production and a selective induction of xylanases, with concomitantly low cellulase activity in a number of microorganisms. However, for large-scale processes other alternatives have to be considered due to the cost of such substrates. Some lignocellulolytic substrates such as barley husk, corn cobs, hay, wheat bran or straw have been compared in relation to pure substrates, and many have performed significantly better than isolated xylans (or celluloses) with respect to the yields of xylanase in large-scale production processes. Solid-state fermentation processes are practical for complex substrates, including agricultural, forestry and food processing residues and wastes, which are used as inducing carbon sources for the production of xylanases [113]. The use of abundantly available and cost-effective agricultural residues, such as wheat bran, corn cobs, rice bran, rice husks, and other similar substrates, to achieve higher xylanase yields via SSF allows the reduction of the overall manufacturing cost of biobleached paper. This has facilitated the use of this environmentally friendly technology in the paper industry [1].

#### **3.6. Cloning and expression of xylanases**

To meet specific industrial needs, an ideal xylanase should have specific properties, such as stability over a wide range of pH values and temperatures, high specific activity, and strong resistance to metal cations and chemicals [116]. Other specifications include cost-effectiveness, eco-friendliness, and ease of use [32]. Therefore, most of the reported xylanases do not possess all of the characteristics required by industry [8].

Native enzymes are not sufficient to meet the demand, due to low yields and incompatibility of the standard industrial fermentation processes [35]. Therefore, molecular approaches must be implemented to design xylanases with the required characteristics [8]. Heterologous expression is the main tool for the production of xylanases at the industrial level [35]. Protein engineering (alteration or modification of existing proteins) by recombinant DNA technology could be useful in improving the specific characteristics of existing xylanases [8]. Genetic engineering and recombinant DNA technology allow the large-scale expression of xylanases in homologous or heterologous protein-expression hosts. As industrial applications require cheaper enzymes, the elevation of expression levels and efficient secretion of xylanases are vital for ensuring the viability of the process [23].

An increasing number of publications have described numerous xylanases from several sources and the cloning, sequencing, mutagenesis and crystallographic analysis of these enzymes [12]. The available amino acid sequence data, X-ray crystallographic data, molecular dynamics and computational design of xylanases provide information that authenticates the relationship between the structure and function of xylanases. All of these methods aid in the design of xylanases that are required in industrial processes, such as improvement of the stability of xylanases at higher temperatures and alkaline pHs [8].

To attempt these processes for commercial purposes, genes encoding several xylanases have been cloned in homologous and heterologous hosts [12,48]. Recombinant xylanases have shown equivalent or better properties than the native enzymes, and the xylanase genes from anaerobic microorganisms have also been expressed successfully in hosts that can be employed in the fermentation industry [35].
