**3.1 Cloning and sequencing of Pb-ChiA**

The length of the Pb-Chi gene was determined to be 2091 bp. It encodes a protein of 697 amino acids. The mRNA sequence was deposited in the GenBank database (LC573547). The amino acid sequence of Pb-Chi was similar to that of the chitinases from *P. barengoltzii* (99%, AIT70967), *Paenibacillus* sp. FPU-7 (93%, BAM67142.1), and *Brevibacillus* sp. AG162 (71%, WP\_142063498.1). These chitinases belong to the GH family 18, and Pb-Chi may belong to this enzyme family. In addition, all catalytically important residues of chitinases from the GH family 18 [13] were conserved in Pb-Chi (Asp199, Asp201, and Glu203), as shown in *P. barengoltzii, Paenibacillus* sp. FPU-7, and *Brevibacillus* sp. AG162. Moreover, Pb-Chi contains a signal peptide, a catalytic domain, fibronectin type III domains, and a chitin-binding domain, as reported for chitinase A from *Bacillus circulans* WL-12 and the chitinase from *Paenibacillus* sp. FPU-7 [14, 15].

The amino acid sequence of the chitin-binding domain from Pb-Chi was similar to that of the carbohydrate-binding module (CBM) family 12. Chitinase II from *Aeromonas* sp. no. 10S-24, GH family 23 chitinase from *Ralstonia* sp. A-471, and chitinase A from *Bacillus circulans* WL-12 also belong to CBM 12 [16–18]. The aromatic amino acids (tryptophan, phenylalanine, and tyrosine) are present in the binding site of a number of sugar-binding proteins. The interaction of the saccharide with the aromatic residue is determined by their relative position and orientation, which have been found to vary in different sugar-binding proteins [19]. It is reported that CBM family 12 containing GH family 23 chitinase from *Ralstonia* sp. A-471 interacts with the insoluble chitin, but not with soluble chitin [17]. This binding domain can improve the hydrolysis ability against insoluble chitin. Watanabe et al. reported that W687 and Q679 are important amino acid residues during the interaction of saccharides with CBD from *B. circulans* WL-12 [20].

#### **3.2 Expression of Pb-ChiA gene and characterization of recombinant protein**

The molecular mass of the protein rPb-ChiA 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-ChiA at various temperatures and pH values were determined in enzyme assays using soluble chitin as the substrate. The optimum pH and temperature of rPb-Chi were 6.0 (**Figure 1A**) and 50°C (**Figure 1B**), respectively.

To recognized the action modes of purified rPb-Chi, enzyme assays were carried with chitin oligosaccharides of several lengths, such as *N*-acetylchitobiose (GlcNAc)2, *N*-acetylchitotriose (GlcNAc)3, *N*-acetylchitotetraose (GlcNAc)4, *N*-acetylchitopentaose (GlcNAc)5, and *N*-acetylchitohexaose (GlcNAc)6; buffer

#### **Figure 1.**

*Functional properties of purified Pb-ChiA. All reactions were conducted with purified enzyme and soluble chitin as the substrate. (A) Effect of pH on enzyme activity at 37°C in 0.2 M of the following buffers: Glycine-HCl (pH 2.0–4.0), sodium acetate (pH 4.0–6.0), KH2PO4-K2HPO4 (pH 6.0–8.0), Tris-HCl (pH 8.0–9.0), and glycine-HCl (pH 9.0–11.0). (B) Effect of temperature on enzyme activity measured at 20–80°C. The average values of triplicate measurements were used as each activity values.*

**79**

**Figure 2.**

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

aliquots were picked up over time and analyzed by HPLC (**Figure 2**). The main hydrolysis products from (GlcNAc)6 were (GlcNAc)2 (**Figure 2A**), and those from (GlcNAc)5 were (GlcNAc)3 and (GlcNAc)2 (**Figure 2B**). It was suggested that (GlcNAc)3 is further degraded to (GlcNAc)2 and GlcNAc. The major hydrolysis products from (GlcNAc)4 was (GlcNAc)2 (**Figure 2C**) and those from (GlcNAc)3 were (GlcNAc)2 and GlcNAc (**Figure 2D**). In these experiments, GlcNAc2 was not degraded, and the major hydrolysis product of colloidal chitin was GlcNAc2 (data not shown). Mainly, rPb-Chi hydrolyzed the second glycosidic bond from the nonreducing end and/or reducing end, and hydrolysis patterns and products made by rPB-Chi were resemble to those described for chitinase A from *Ralstonias* sp. A-471, chitinase A from *Serratia marcescens*, and chitinase A1 from *B. circulans* WL-12 [14, 21]. The present data suggest that this enzyme is a processive exo-type chitinase.

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

*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*

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

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

in Pb-GlcNAcase (Asp228 and Glu229).

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

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

*Molecular Biotechnology*

database (LC573547). The amino acid sequence of Pb-Chi was similar to that of the chitinases from *P. barengoltzii* (99%, AIT70967), *Paenibacillus* sp. FPU-7 (93%, BAM67142.1), and *Brevibacillus* sp. AG162 (71%, WP\_142063498.1). These chitinases belong to the GH family 18, and Pb-Chi may belong to this enzyme family. In addition, all catalytically important residues of chitinases from the GH family 18 [13] were conserved in Pb-Chi (Asp199, Asp201, and Glu203), as shown in *P. barengoltzii, Paenibacillus* sp. FPU-7, and *Brevibacillus* sp. AG162. Moreover, Pb-Chi contains a signal peptide, a catalytic domain, fibronectin type III domains, and a chitin-binding domain, as reported for chitinase A from *Bacillus circulans* WL-12

The amino acid sequence of the chitin-binding domain from Pb-Chi was similar to that of the carbohydrate-binding module (CBM) family 12. Chitinase II from *Aeromonas* sp. no. 10S-24, GH family 23 chitinase from *Ralstonia* sp. A-471, and chitinase A from *Bacillus circulans* WL-12 also belong to CBM 12 [16–18]. The aromatic amino acids (tryptophan, phenylalanine, and tyrosine) are present in the binding site of a number of sugar-binding proteins. The interaction of the saccharide with the aromatic residue is determined by their relative position and orientation, which have been found to vary in different sugar-binding proteins [19]. It is reported that CBM family 12 containing GH family 23 chitinase from *Ralstonia* sp. A-471 interacts with the insoluble chitin, but not with soluble chitin [17]. This binding domain can improve the hydrolysis ability against insoluble chitin. Watanabe et al. reported that W687 and Q679 are important amino acid residues during the interaction of

**3.2 Expression of Pb-ChiA gene and characterization of recombinant protein**

To recognized the action modes of purified rPb-Chi, enzyme assays were carried with chitin oligosaccharides of several lengths, such as *N*-acetylchitobiose (GlcNAc)2, *N*-acetylchitotriose (GlcNAc)3, *N*-acetylchitotetraose (GlcNAc)4, *N*-acetylchitopentaose (GlcNAc)5, and *N*-acetylchitohexaose (GlcNAc)6; buffer

*Functional properties of purified Pb-ChiA. All reactions were conducted with purified enzyme and soluble chitin as the substrate. (A) Effect of pH on enzyme activity at 37°C in 0.2 M of the following buffers: Glycine-HCl (pH 2.0–4.0), sodium acetate (pH 4.0–6.0), KH2PO4-K2HPO4 (pH 6.0–8.0), Tris-HCl (pH 8.0–9.0), and glycine-HCl (pH 9.0–11.0). (B) Effect of temperature on enzyme activity measured at 20–80°C. The average* 

*values of triplicate measurements were used as each activity values.*

The molecular mass of the protein rPb-ChiA 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-ChiA at various temperatures and pH values were determined in enzyme assays using soluble chitin as the substrate. The optimum pH and temperature of rPb-Chi were 6.0 (**Figure 1A**) and 50°C (**Figure 1B**),

and the chitinase from *Paenibacillus* sp. FPU-7 [14, 15].

saccharides with CBD from *B. circulans* WL-12 [20].

**78**

**Figure 1.**

respectively.

aliquots were picked up over time and analyzed by HPLC (**Figure 2**). The main hydrolysis products from (GlcNAc)6 were (GlcNAc)2 (**Figure 2A**), and those from (GlcNAc)5 were (GlcNAc)3 and (GlcNAc)2 (**Figure 2B**). It was suggested that (GlcNAc)3 is further degraded to (GlcNAc)2 and GlcNAc. The major hydrolysis products from (GlcNAc)4 was (GlcNAc)2 (**Figure 2C**) and those from (GlcNAc)3 were (GlcNAc)2 and GlcNAc (**Figure 2D**). In these experiments, GlcNAc2 was not degraded, and the major hydrolysis product of colloidal chitin was GlcNAc2 (data not shown). Mainly, rPb-Chi hydrolyzed the second glycosidic bond from the nonreducing end and/or reducing end, and hydrolysis patterns and products made by rPB-Chi were resemble to those described for chitinase A from *Ralstonias* sp. A-471, chitinase A from *Serratia marcescens*, and chitinase A1 from *B. circulans* WL-12 [14, 21]. The present data suggest that this enzyme is a processive exo-type chitinase.
