**3.3 Cloning and sequencing of Pb-GlcNAcase**

The length of the Pb-GlcNAcase gene was determined to be 1926 bp. It encodes a protein of 642 amino acids. The mRNA sequence was deposited in the GenBank database (LC573548). The amino acid sequence of Pb-GlcNAcase was similar to that of the β-*N*-acetylhexosaminidase from *P. barengoltzii* (99%, WP\_016313754.1), β-*N*-GlcNAcase from *Fontibacillus panacisegetis* (63%, WP\_091231148.1), and β-*N*acetylhexosaminidase from *Gorillbacterium massiliense* (49%, WP\_040950886.1). These β-*N*-GlcNAcase and β-*N*-acetylhexosaminidases belong to the GH family 20, and so Pb-GlcNAcase may belong to this enzyme family. In addition, all catalytically important residues of GlcNAcase from the GH family 20 [22] were conserved in Pb-GlcNAcase (Asp228 and Glu229).

#### **Figure 2.**

*HPLC analysis of products of hydrolysis of chitin oligosaccharides (GlcNAc)3 to (GlcNAc)6 by recombinant Pb-ChiA. The hydrolysis products of (A) (GlcNAc)6, (B) (GlcNAc)5, (C) (GlcNAc)4, and (D) (GlcNAc)3 by the enzyme were detected by HPLC, as described in the materials and methods. Lines: GlcNAc (dark-blue line), (GlcNAc)2 (red line), (GlcNAC)3 (yellowish olive-green line), (GlcNAc)4 (purple line), (GlcNAc)5 (light blue line), and (GlcNAc)6 (mustard-yellow line).*

## **3.4 Expression of Pb-GlcNAcase gene and characterization of recombinant protein**

The molecular mass of the recombinant protein rPb-GlcNAcase was 70 kDa, as determined by SDS-PAGE (data not shown), in good agreement with that predicted from the amino acid sequence. The activity of rPb-GlcNAcase at various temperatures and pH values were determined in enzyme assays using *p*NP-GlcNAc as the substrate. The optimum pH of rPb-GlcNAcase was 6.0 (**Figure 3A**), while the optimal temperature for rPb-Chi activity was 50°C (**Figure 3B**); these values were similar to those of rPb-ChiA.

To recognized the action modes of purified rPb-GlcNAcase, enzyme assays were carried with chitin oligosaccharides of several lengths, such as (GlcNAc)2, (GlcNAc)3, (GlcNAc)4, (GlcNAc)5, and (GlcNAc)6; buffer aliquots were picked up over time and analyzed by HPLC (**Figure 4**). The main hydrolysis products of (GlcNAc)6 were GlcNAc and (GlcNAc)5 (**Figure 4A**), those of (GlcNAc)5 were GlcNAc and (GlcNAc)4 (**Figure 4B**), and those of (GlcNAc)4 and (GlcNAc)3 were GlcNAc (**Figure 4C, D**). In these experiments, (GlcNAc)2 was degraded to GlcNAc (**Figure 4E**). In addition, the specific activities of (GlcNAc)2 and (GlcNAc)3 were higher than those of (GlcNAc)4–6 (**Table 1**). The present data indicate that this enzyme is an exo-type carbohydrate hydrolase.

#### **3.5 Cloning and sequencing of Pb-LPMO**

The length of the Pb-LPMO gene was determined to be 1350 bp. It encodes a protein of 449 amino acids. The amino acid sequence of Pb-LPMO was the same or very similar to that of the LPMO from *Paenibacillus* sp. (100%, WP\_028538775) and *P. barengoltzii* (99%, WP\_016312786) and the chitin-binding domain from *Paenibacillus* sp. oral taxon 786 (96%, WP\_009222736). As these LPMOs belong to the AA family 10, Pb-LPMO may also belong to this enzyme family. In addition, catalytically important residues of LPMO from the AA family 10 [9] were conserved in Pb-LPMO (His38, His123) as well as the residues of LPMO from *P. barengoltzii* (WP\_016312786), *Paenibacillus* sp. (WP\_028538775), and *S. marcescens* BJL200 (AAU882020.1). Moreover, Pb-LPMO contains a signal peptide, an LPMO domain, two fibronectin type III domains, and a chitin-binding domain, as reported for the LPMOs of *P. barengoltzii* (WP\_016312786) and *Paenibacillus* sp. (WP\_028538775).

#### **Figure 3.**

*Functional properties of purified Pb-GlcNAc. All reactions were conducted with purified enzyme and pNp-GlcNAc as the substrate. (A) Effect of pH on enzyme activity at 37°C in various buffers. The buffer systems comprised x mL of 0.2 M boric acid and 0.05 mM citrate acid, and (200–x) mL of 0.1 M Na3PO4*∙*12H2O (pH 3.0–9.0). (B) Effect of temperature on enzyme activity measured at 20–70°C. The average values of triplicate measurements were used as activity values.*

**81**

**Table 1.**

*Substrate specificity of Pb-GlcNAcase.*

**Figure 4.**

*Effect of LPMO on the Hydrolysis of Crystalline Chitin by Chitinase A…*

*HPLC analysis of products of hydrolysis of chitin oligosaccharides (GlcNAc)3 to (GlcNAc)6) by recombinant Pb-GlcNAcase. The degradation products of (A) (GlcNAc)6, (B) (GlcNAc)5, (C) (GlcNAc)4, (D) (GlcNAc)3, and (E) (GlcNAc)2 by the enzyme were detected by HPLC, as described in the materials and methods. Lines: GlcNAc (dark-blue line), (GlcNAc)2 (red line), (GlcNAC)3 (yellowish olive-green line), (GlcNAc)4 (purple* 

**Substrate Activity (units/ mg protein)**

Soluble chitin ND pNP-GlcNAc 111 × 10−3 pNP-GalNAc 73.7 × 10−3 (GlcNAc)2 10.2 (GlcNAc)3 12.7 (GlcNAc)4 5.8 (GlcNAc)5 3.8 (GlcNAc)6 3.7 *The average values of triplicate measurements were used as each activity value.*

*line), (GlcNAc)5 (light-blue line), (GlcNAc)6 (mustard-yellow line).*

*DOI: http://dx.doi.org/10.5772/intechopen.93761*

*Effect of LPMO on the Hydrolysis of Crystalline Chitin by Chitinase A… DOI: http://dx.doi.org/10.5772/intechopen.93761*

#### **Figure 4.**

*Molecular Biotechnology*

similar to those of rPb-ChiA.

**3.5 Cloning and sequencing of Pb-LPMO**

**protein**

hydrolase.

**3.4 Expression of Pb-GlcNAcase gene and characterization of recombinant** 

The molecular mass of the recombinant protein rPb-GlcNAcase was 70 kDa, as determined by SDS-PAGE (data not shown), in good agreement with that predicted from the amino acid sequence. The activity of rPb-GlcNAcase at various temperatures and pH values were determined in enzyme assays using *p*NP-GlcNAc as the substrate. The optimum pH of rPb-GlcNAcase was 6.0 (**Figure 3A**), while the optimal temperature for rPb-Chi activity was 50°C (**Figure 3B**); these values were

To recognized the action modes of purified rPb-GlcNAcase, enzyme assays were carried with chitin oligosaccharides of several lengths, such as (GlcNAc)2, (GlcNAc)3, (GlcNAc)4, (GlcNAc)5, and (GlcNAc)6; buffer aliquots were picked up over time and analyzed by HPLC (**Figure 4**). The main hydrolysis products of (GlcNAc)6 were GlcNAc and (GlcNAc)5 (**Figure 4A**), those of (GlcNAc)5 were GlcNAc and (GlcNAc)4 (**Figure 4B**), and those of (GlcNAc)4 and (GlcNAc)3 were GlcNAc (**Figure 4C, D**). In these experiments, (GlcNAc)2 was degraded to GlcNAc (**Figure 4E**). In addition, the specific activities of (GlcNAc)2 and (GlcNAc)3 were higher than those of (GlcNAc)4–6 (**Table 1**). The present data indicate that this enzyme is an exo-type carbohydrate

The length of the Pb-LPMO gene was determined to be 1350 bp. It encodes a protein of 449 amino acids. The amino acid sequence of Pb-LPMO was the same or very similar to that of the LPMO from *Paenibacillus* sp. (100%, WP\_028538775) and *P. barengoltzii* (99%, WP\_016312786) and the chitin-binding domain from *Paenibacillus* sp. oral taxon 786 (96%, WP\_009222736). As these LPMOs belong to the AA family 10, Pb-LPMO may also belong to this enzyme family. In addition, catalytically important residues of LPMO from the AA family 10 [9] were conserved in Pb-LPMO (His38, His123) as well as the residues of LPMO from *P. barengoltzii* (WP\_016312786), *Paenibacillus* sp. (WP\_028538775), and *S. marcescens* BJL200 (AAU882020.1). Moreover, Pb-LPMO contains a signal peptide, an LPMO domain, two fibronectin type III domains, and a chitin-binding domain, as reported for the LPMOs of *P. barengoltzii* (WP\_016312786) and *Paenibacillus* sp. (WP\_028538775).

*Functional properties of purified Pb-GlcNAc. All reactions were conducted with purified enzyme and pNp-GlcNAc as the substrate. (A) Effect of pH on enzyme activity at 37°C in various buffers. The buffer systems comprised x mL of 0.2 M boric acid and 0.05 mM citrate acid, and (200–x) mL of 0.1 M Na3PO4*∙*12H2O (pH 3.0–9.0). (B) Effect of temperature on enzyme activity measured at 20–70°C. The average values of* 

**80**

**Figure 3.**

*triplicate measurements were used as activity values.*

*HPLC analysis of products of hydrolysis of chitin oligosaccharides (GlcNAc)3 to (GlcNAc)6) by recombinant Pb-GlcNAcase. The degradation products of (A) (GlcNAc)6, (B) (GlcNAc)5, (C) (GlcNAc)4, (D) (GlcNAc)3, and (E) (GlcNAc)2 by the enzyme were detected by HPLC, as described in the materials and methods. Lines: GlcNAc (dark-blue line), (GlcNAc)2 (red line), (GlcNAC)3 (yellowish olive-green line), (GlcNAc)4 (purple line), (GlcNAc)5 (light-blue line), (GlcNAc)6 (mustard-yellow line).*


#### **Table 1.**

*Substrate specificity of Pb-GlcNAcase.*
