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Xylanases: Sources, Production, and Purification Strategies

Mariana Delgado-Garcia1, Lizeth G. Campos-Muzquiz2, Rocio G. Castillo-Godina2, Sendar D. Nery-Flores2, Lissethe Palomo-Ligas2, Adriana C. Flores-Gallegos2, Beatriz del C. Cutiño-Laguna2, and Raul Rodriguez-Herrera2

1Technological Institute of Superior Studies of Monterrey, Campus Guadalajara, School of Engineering and Sciences, Av. General Ramón Corona 2514, Nuevo México, Zapopan, 45138 Jalisco, México

2Universidad Autónoma de Coahuila, School of Chemistry, Blvd. V. Carranza y Jose Cárdenas s/n, Col. Republica, Saltillo, Coahuila 25280, México

1.1 Introduction

The cell wall of the plant is composed of different lignocellulosic compounds, being the xylan the main compound of hemicellulose. This structure consists of xylose united by ß-1,4-glycosidic bonds and different branches of a-D-glucuronide, arabinose, galactose, acetate, methyl glucuronic acid, and other simple sugars [1, 2]. Xylanase is a group of hydrolytic enzymes involved in the hydrolysis of xylan to convert it into monosaccharides and xylooligosaccharides. The xylanase system is constituted by glycosyl hydrolases (endo-xylanases, exo-xylanases, ß-D-xylosidases, a-glucuronidase, and a-L-arabinofuranosidases) and esterases [3].

The heterogeneous composition of hemicellulose hampers the complete depolymerization by a single enzyme, requiring the action of both glycosyl hydrolases and esterases [4]. Each enzyme of the xylanase group contributes to xylan degradation in a specific way: endo-xylanase randomly cleaves the xylan; exo and endo xylanases acting on the xylan backbone and producing short-chain oligomers; ß-D-xylosidases cleaves xylose monomers, a-L-arabinofuranosidases removes the side groups, a-D-glucuronidases, and acetylxylan esterases remove acetyl and phenolic side branches and act synergistically on the complex polymer [4, 5]. The most common natural sources of xylanases are produced by different biological systems such as bacteria, protozoans, fungi, plants, and mollusks. Actually, it has been reported that xylanases have been identified from lignocellulose-degrading microbiota from cow rumen and, the termite hindgut. There are two strategies applied to date for microbial xylanase production, either using native microorganisms or genetic engineering modified microorganisms [3, 4].

Xylanase production from nonmodified fungi and bacteria must use proper microorganisms, which should produce acceptable yields and should not produce toxins or any other unsought products [6]. Xylanases can be produced by hydrolysis of xylan by the microorganisms that express the enzyme gene. Nonetheless, due to the xylan complexity, the production of this enzyme from different microorganisms on a large-scale is hard because one of the main problems is the presence of other enzymes. This problem is also present during the purification steps, increasing costs. Hence, one alternative is the use of modified strains for large-scale xylanase production [7, 8].

In the case of bacteria, the alkaline-thermostable xylanase-producing trait is useful in most industrial applications since it reduces the steps due to the higher pH level required for the optimal growth and activity of the microorganism [9]. Xylanases require N-glycosylation as one of the most important posttranslational modifications; therefore, not all bacterial expression hosts are suitable, such as Escherichia coli, which lacks the pgl gene, to produce this modification. Because of this, other alternative expression hosts are Bacillus subtilis and Lactobacillus sp. [10, 11].

Filamentous fungi are an important option to produce high amounts of xylanases in comparison to yeast and bacteria [12]. A problem associated with fungal xylanases is cellulase excretion; therefore, an operational process to obtain xylanolytic systems free of cellulases is very important in this case [13]. Another major problem associated with fungi is the reduced xylanase yield in fermenter studies, principally for the agitation that promotes fungal disruption, leading to low productivity [14].

Some examples of xylanases-producing fungus used in industry are Penicillium canescens, Streptomyces sp. P12-137, Thermomyces lanuginosus SD-21, Penicillium fellutanum, Penicillium sclerotiorum, Acremonium furcatum, Aspergillus niger PPI, Neocallimastix sp. Strain L2, Cochliobolus sativus Cs6, Bacillus circulans D1, Streptomyces sp. strain Ib 24D, and Paecilomyces themophila J18. The substrate used by these microorganisms for fermentation is derived from cereals as soya, wheat, corn, and oat [15-25]. On the other hand, yeasts are good expression hosts due to their ability to perform eukaryotic posttranslational modifications, high cell density growth, and secretion of proteins into fermentation media [26, 27]. Some yeasts used for xylanase production are Saccharomyces cerevisiae and Pichia pastoris [28, 29]

Plants are also used for xylanases production, using bio-farming. The requirements for this objective are (i) high-level expression, (ii) stability and functionality of enzymes to be expressed, and (iii) easy purification. In planta expression of lignocellulose-digesting enzymes from mesophilic bacteria and fungi can compromise plant biomass production because of autohydrolysis of cell walls and others such as growth, yield, germination, fertility and susceptibility of the host to disease [30]. There are enzymes that can be used during the lignocellulose pretreatment without losing their enzymatic activity for their hypo-thermophilic capacity [31].

Recently, there has been much industrial interest on xylanases, from native microorganisms and recombinant hosts for different applications. For example, in the baking industry, endo-1,4-ß-xylanase from Aspergillus oryzae, B. subtilis, and Trichoderma longibrachiatum is used for bread making, the production of maize starch and alcohol through fermentation. Particularly, in the bread industry, the uses of xylanase are intended for flexibility and stabilization of dough (breaking down polysaccharides) and improve gluten strength. This impacts the sensory perception of bread [32].

In the animal nutrition industry, xylanases from Acidothermus cellulolyticus and Neocallimastix patriciarum are used to reduce feed conversion rate and enhance the digestibility of cereal feeds in poultry and ruminant [33, 34]. Lactobacillus xylanases depolymerize hemicellulose, making silage more stable and digestible by cattle [35]. The most common uses of xylanases have been used in the paper and pulp industry for the benefits of the quality of the products as purity, bright, and more permeability of fiber surface and diffusion during the bleaching processes [36-38]. Due to the current crisis of energy, the utilization of lignocellulosic agents is considered as sustainable biomass to produce nonfossil fuels. These biomasses should be hydrolyzed for bioethanol production from agricultural waste such as corncob, chili residue, rice straw, banana peel, apple pomace, and others [3, 39-42].

Xylanases have an important role in hydrolyzing the xylan and generate value-added products, such as xylitol. Xylitol is a sweetener used in soft drinks, candies, ice cream, chewing gum, and various pharmaceutical products as a natural sweetener in toothpaste [43]. Other uses have been explored, e.g., extracellular xylanase from a culture of Aspergillus carneus M34 and used to treat xylooligosaccharide. Feruloyl xylooligosaccharides showed antioxidative capacity in a cell model of ultraviolet B (UVB)-induced oxidative damage, demonstrating the potential of xylanases use in photo-protectant preparation [44].

1.2 Sources, Production, and Purification Strategies

Xylanases can be obtained in a large number of biologic systems such as fungi, bacteria (Bacillus pumilus, B. subtilis, Bacillus amyloliquefaciens, Bacillus cereus, B. circulans, Bacillus megatorium, Bacillotherus licheniformis, Bacillotherus sp., Streptomyces roseiscleroticus, Streptomyces cuspidosporus, Streptomyces actuosus, Pseudonomas sp., Clostridium absonum, and Thermoactinomyces thalophilus), yeasts, and seaweed [45]. Some other organisms such as mycorrhizae, actinomycetes, protozoa, insects, crustaceans, snails, and some plant seeds during the germination phase have been identified as xylanase sources [46]. Filamentous fungi being the main producers of xylanolytic enzymes, compared to other microorganisms [47]. In this way, xylanases have different applications, according to the source of production and some studies have focused on optimizing enzyme production, mainly from more powerful fungal and bacterial strains or through mutant strains for higher enzyme production [26, 48-50].

Fungal strains are important producers of xylanases due to their high yield and extracellular release of enzymes. They also show greater xylanase activity than yeast and...