**7.2 Metals and chemicals**

The metal ion and chemical reagents had been proved to be one of the critical factors which affected the enzyme activity of xylanases. The effect of metal ions and chemical reagents on the xylanase activities has been determined on various metal ions (Na<sup>+</sup> , K+ , Ca2+, Li+ , Co2+, Cr3+, Ni2+, Cu2+, Mg2+, Fe3+, Zn2+, Pb2+, and Ag+ ) and chemical reagents (SDS, β-mercaptoethanol, ethanol, Triton X-100, and EDTA) at the standard condition. Ca2+, Pb2+, K+ , Mn2+, EDTA, β-ME, Cu2+ and Triton X-100 were reported to enhance enzyme activity by 6.4–29.9% [81], while Fe3+, Cd2+, Hg2+, and Ba2+ completely suppressed the xylanase activity. Besides this, the enzyme had certain ability to resist the Fe2+, Mg2+, Ag2+, SDS, ethanol and SDS. Xylanase activity was not inhibited by chelating reagents such as EDTA and EGTA. Moreover, it is predicted that Ca2+ and Mg2+ ions enhance the enzyme activity by stabilizing the enzyme–substrate complex. In contrast, EDTA is a chelating agent and it removes ions from the enzymes, thus inhibits the enzyme activity [7]. More detailed studies are needed in order to understand the mechanistic effect of metal ions on enzyme activity. Similarly, a xylanase activity isolated from *Planococcus* sp. *SL4* was enhanced by Ca2+ and β-mercaptoethanol. K<sup>+</sup> , Cr3+, Li+ , and Na<sup>+</sup> showed little or no effect on xylanase activity. Ag<sup>+</sup> , Cu2+, Hg2+, Pb2+, Zn2+, Fe3+, and Cr3+ resulted in an almost complete loss of activity [28].

### **7.3 Temperature and pH**

As thermophilic enzymes are preferred over the mesophilic enzymes complements because of high temperatures, which had a great influence on many factors such as decreases contamination risk and viscosity of substrate [82]. In a study carried out with the effect of xylanase activity from *A. niger* DFR-5 on different temperature (between 20 and 60°C). The xylanase activity was increased with increase in temperature with maximum activity of at 40°C. On further increase in


hostility and lethality to most of living organisms due to extreme hot environments is obvious but such factors are counter-productive in the presence of specialized microorganisms. These microorganisms from harsh environments can tolerate high or low temperatures, extremes pH and high concentrations of salts. Based on the unique stability of their enzymes at high temperature, extremes of pH they are expected to be a powerful tool in industrial biotransformation processes that run at harsh conditions. Enzymes derived from extremophiles have great importance to the local enzymes because they can perform industrial processes even under harsh conditions, under which conventional proteins are completely denatured. The extremophile research, the high demands of the biotech industries for tailor-made novel biocatalysts, and the rapid development of new techniques such as genomics, proteomics, metabolomics, directed evolution and gene shuffling will stimulate the

development of new industrial processes on the basis of biocatalysts from

to be an effective in saccharification process.

helpful in the yield production of poultry products.

**305**

Enzymatic hydrolysis of xylan has become attractive due to its biotechnological applications in the food, animal feed, waste treatment, ethanol production, textile, and pulp and paper industries [11]. Xylanases gained its importance for the saccharification process in degradation of lignocellulosic biomass, because xylanases assist the hydrolysis of xylan and ultimately leads to the hydrolysis of hemicellulose and cellulose, to obtain industrially important products [99]. In addition, biofuel production from lignocellulosic biomass has great concern in industrial sectors worldwide. However, biofuel production is still a bottleneck, because the initial conversion of biomass into sugars requires multiple enzymes (including xylanases) with high activity and excellent enzyme properties. Therefore, recent development in biotechnological applications xylanases toward the biomass conversion is on way [100]. Moreover, highly stable enzymes, active under high temperature and wide range of pH, displayed several advantages over the enzymes from mesophilic or neutrophilic origin. For example, highly active xylanases under extreme conditions reduce the contamination risk due to fast reaction rate. In second generation ethanol industries, mild pretreatment of biomass requires to reduce the total costs and sustain the high contents of hemicellulose. However, high dosage of other enzymes is still needed for the complete conversion of hemicellulose. Therefore, the biotechnological application of xylanases especially thermophilic xylanases and its interaction with other enzymes such as cellulases, xylosidases, arabinofuranosidase seems

The use of xylanases, as accessory enzymes, has shown to improve the hydrolysis of xylan and cellulose, and it has contributed to the reduction of enzyme dosage, but it seems to be a substrate dependent reaction [101]. Recently, less severe pretreatments of lignocellulosic biomass are gaining popularity, thus, hemicellulase characterization and studies regarding synergism of cellulases and xylanases, can contribute to reducing pretreatment severities and enhancing glucose and xylose release [102]. Xylanases are very important in bioconversion of xylan into value added products, such as xylitol. Xylitol is used in soft drinks, candies, ice cream, chewing gum and various pharmaceutical products. Xylitol have key role in sweeten food products and used as a natural sweetener in toothpaste. Xylanases have great importance in the bio-bleaching of wood pulp and in the bioprocessing of textiles. Xylanases are very useful in manufacturing of animal feed. Xylanases reduce the viscosity and enhance the absorption rate by degrading the starch polysaccharides in rich fiber and barley based feeds. Pre-treatment of agricultural silage and grain feed employed with xylanases increases the nutritional value and improves the feed digestion in ruminants. Similarly, feed supplementation combined with xylanases for broiler diets, not only increases growth performance like weight gain but also

extremophiles.

*Xylanase and Its Industrial Applications DOI: http://dx.doi.org/10.5772/intechopen.92156*

**Table 3.**

*List of xylanase producing microbial species/strains with demonstrated pH and temperature.*

temperature, the enzyme activity declined gradually and at 60°C, enzyme exhibited 35.5% of the maximum activity [83]. Similarly, the temperature effect of purified xylanase (xynZF-2) from *A. niger*, was evaluated [84]. The optimum temperature recombinant enzyme was 40°C and the enzyme activity was observed relatively stable on the temperature below 40°C, however enzyme activity decreased rapidly with rise of temperature (above than 40°C). The activity became completely lost on incubation at 50°C for 15 min [84].

Evaluation of the pH stability is a vital part of any enzyme characterization before it can be exploited commercially. Similarly effect of pH had great influence on the xylanase activity. The effect of pH on extracellular xylanase from *A. niger* DFR-5 was evaluated [83]. That suggested the formation of an improper ionic form of the xylanase and/or substrate between pH 4.0 and 5.0 and between pH 5.0 and 6.5. Because the activity of pre-incubated xylanase at pH >6.5 or pH <4.0, was full not recovered at pH 5.0 again. The decline activity at pH above than 6.5 and pH below than 4.0 resulted from irreversible enzyme inactivation [83]. Many researchers have reported the pH stability of xylanase but without interpretation. Xylanase isolated from *Planococcus* sp. SL4 was highly active and stable over the neutral and alkaline pH range from 6 to 11, with maximum activity at pH 7 and more than 60% activity at pH 11 [28]. Xylanase purified from *Arthrobacter* sp. MTCC 5214 was found stable in a narrow pH range of 7.0–8.0 [85]. List of xylanase producing microbial species/strains with demonstrated pH and temperature are given in **Table 3**.

#### **8. Application of xylanases**

Because of their biotechnological characteristics, xylanases are most often produced from microorganisms for commercial applications. The environmental

#### *Xylanase and Its Industrial Applications DOI: http://dx.doi.org/10.5772/intechopen.92156*

hostility and lethality to most of living organisms due to extreme hot environments is obvious but such factors are counter-productive in the presence of specialized microorganisms. These microorganisms from harsh environments can tolerate high or low temperatures, extremes pH and high concentrations of salts. Based on the unique stability of their enzymes at high temperature, extremes of pH they are expected to be a powerful tool in industrial biotransformation processes that run at harsh conditions. Enzymes derived from extremophiles have great importance to the local enzymes because they can perform industrial processes even under harsh conditions, under which conventional proteins are completely denatured. The extremophile research, the high demands of the biotech industries for tailor-made novel biocatalysts, and the rapid development of new techniques such as genomics, proteomics, metabolomics, directed evolution and gene shuffling will stimulate the development of new industrial processes on the basis of biocatalysts from extremophiles.

Enzymatic hydrolysis of xylan has become attractive due to its biotechnological applications in the food, animal feed, waste treatment, ethanol production, textile, and pulp and paper industries [11]. Xylanases gained its importance for the saccharification process in degradation of lignocellulosic biomass, because xylanases assist the hydrolysis of xylan and ultimately leads to the hydrolysis of hemicellulose and cellulose, to obtain industrially important products [99]. In addition, biofuel production from lignocellulosic biomass has great concern in industrial sectors worldwide. However, biofuel production is still a bottleneck, because the initial conversion of biomass into sugars requires multiple enzymes (including xylanases) with high activity and excellent enzyme properties. Therefore, recent development in biotechnological applications xylanases toward the biomass conversion is on way [100]. Moreover, highly stable enzymes, active under high temperature and wide range of pH, displayed several advantages over the enzymes from mesophilic or neutrophilic origin. For example, highly active xylanases under extreme conditions reduce the contamination risk due to fast reaction rate. In second generation ethanol industries, mild pretreatment of biomass requires to reduce the total costs and sustain the high contents of hemicellulose. However, high dosage of other enzymes is still needed for the complete conversion of hemicellulose. Therefore, the biotechnological application of xylanases especially thermophilic xylanases and its interaction with other enzymes such as cellulases, xylosidases, arabinofuranosidase seems to be an effective in saccharification process.

The use of xylanases, as accessory enzymes, has shown to improve the hydrolysis of xylan and cellulose, and it has contributed to the reduction of enzyme dosage, but it seems to be a substrate dependent reaction [101]. Recently, less severe pretreatments of lignocellulosic biomass are gaining popularity, thus, hemicellulase characterization and studies regarding synergism of cellulases and xylanases, can contribute to reducing pretreatment severities and enhancing glucose and xylose release [102]. Xylanases are very important in bioconversion of xylan into value added products, such as xylitol. Xylitol is used in soft drinks, candies, ice cream, chewing gum and various pharmaceutical products. Xylitol have key role in sweeten food products and used as a natural sweetener in toothpaste. Xylanases have great importance in the bio-bleaching of wood pulp and in the bioprocessing of textiles.

Xylanases are very useful in manufacturing of animal feed. Xylanases reduce the viscosity and enhance the absorption rate by degrading the starch polysaccharides in rich fiber and barley based feeds. Pre-treatment of agricultural silage and grain feed employed with xylanases increases the nutritional value and improves the feed digestion in ruminants. Similarly, feed supplementation combined with xylanases for broiler diets, not only increases growth performance like weight gain but also helpful in the yield production of poultry products.

temperature, the enzyme activity declined gradually and at 60°C, enzyme exhibited 35.5% of the maximum activity [83]. Similarly, the temperature effect of purified xylanase (xynZF-2) from *A. niger*, was evaluated [84]. The optimum temperature recombinant enzyme was 40°C and the enzyme activity was observed relatively stable on the temperature below 40°C, however enzyme activity decreased rapidly with rise of temperature (above than 40°C). The activity became completely lost on

*List of xylanase producing microbial species/strains with demonstrated pH and temperature.*

**Species Strain pH Temperature References** *Caldicoprobacter algeriensis* TH7C1(T) 11.0 70°C [26] *Microcella alkaliphila* JAM-AC0309 8.0 65°C [86] *Planococcus* sp. SL4 7.0 70°C [28] *Geobacillus* sp. WBI 7.0 65°C [87] *Caldicellulosiruptor* sp. F32 6.6 75°C [88] *Actinomadura* sp. Cpt20 10.0 80 °C [89] *Thermofilum pendens* Tpbgl 3.5 95°C [90] *Sulfolobus solfataricus* MT4 7.0 90°C [91] *Thermococcus zilligii* AN1 6.0 75°C [92] *Penicillium occitanis* Pol6 3.0 65°C [93] *Saccharopolyspora pathumthaniensis* S582 6.5 70°C [94] *Paecilomyces thermophila* J18 7.0 75°C [95] *Bispora* sp. MEY-1 4.5 85°C [96] *Malbranchea cinnamomea* S168 6.5 80 °C [97] *Penicillium oxalicum* B3–11(2) 5.0 50 °C [98]

Evaluation of the pH stability is a vital part of any enzyme characterization before it can be exploited commercially. Similarly effect of pH had great influence on the xylanase activity. The effect of pH on extracellular xylanase from *A. niger* DFR-5 was evaluated [83]. That suggested the formation of an improper ionic form of the xylanase and/or substrate between pH 4.0 and 5.0 and between pH 5.0 and 6.5. Because the activity of pre-incubated xylanase at pH >6.5 or pH <4.0, was full not recovered at pH 5.0 again. The decline activity at pH above than 6.5 and pH below than 4.0 resulted from irreversible enzyme inactivation [83]. Many

researchers have reported the pH stability of xylanase but without interpretation. Xylanase isolated from *Planococcus* sp. SL4 was highly active and stable over the neutral and alkaline pH range from 6 to 11, with maximum activity at pH 7 and more than 60% activity at pH 11 [28]. Xylanase purified from *Arthrobacter* sp. MTCC 5214 was found stable in a narrow pH range of 7.0–8.0 [85]. List of xylanase producing microbial species/strains with demonstrated pH and temperature are

Because of their biotechnological characteristics, xylanases are most often pro-

duced from microorganisms for commercial applications. The environmental

incubation at 50°C for 15 min [84].

*Biotechnological Applications of Biomass*

given in **Table 3**.

**304**

**Table 3.**

**8. Application of xylanases**

Conclusively, the xylanases have potential applications in a wide range of industrial processes, covering all sectors of industrial enzymes markets. Arguably, these are attractive for research studies and deeper investigation with aims to explore dividends and utility of these useful products in industrial context in relation to industrial enzymes producing organisms.
