*3.6.1. Expression in bacteria*

low water activities, contrary to most bacteria and yeast, which will not proliferate under these

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

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

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].

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

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

culture conditions [113].

in applications aimed at improving animal feed [113].

**3.6. Cloning and expression of xylanases**

all of the characteristics required by industry [8].

vital for ensuring the viability of the process [23].

*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 enzymes via protein engineering [35].

*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‐ geous in industrial production [33].

#### *3.6.2. Expression in yeast*

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

*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 the industrial production of xylanases at low costs [35]. *Pichia pastoris* has also emerged as an excellent host for the commercial production of xylanases due to very high expression under its own promoters [35]. However, the success of this methylotrophic yeast, similar to *Hansenula polymorpha*, is reached with the promoters of alcohol oxidase, an enzyme involved in the methanol-utilization pathway [23]. Therefore, these promoters have limited use at the large scale due to the health and fire hazards of methanol [35].

**Author details**

Campinas, Brazil

**References**

F. L. Motta1\*, C. C. P. Andrade2

University of Campinas, Campinas, Brazil

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\*Address all correspondence to: flopesmotta@gmail.com

and M. H. A. Santana1

1 Development of Biotechnological Processes Laboratory, School of Chemical Engineering,

A Review of Xylanase Production by the Fermentation of Xylan: Classification, Characterization and Applications

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2 Bioprocess Engineering Laboratory, Food Engineering Department, University of Campinas,

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#### *3.6.3. Expression in filamentous fungi*

Filamentous fungi are capable producers of xylanases, via both heterologous and homologous gene expression, and reach high expression yields with their own promoters [35]. Filamentous fungi have already undergone intricate strain improvement for high-level protein secretion and are feasible when using the native xylanase-expressing machinery for functional expres‐ sion of foreign xylanases from remote sources. The xylanase gene from *P. griseofulvum* has been successfully expressed in *A. oryzae* [120].
