**2.2 Cloning and expression of chitinase A (Pb-ChiA) gene from** *Paenibacillus* **sp.**

**75**

enzyme solution.

was examined at 10–80°C.

(−20°C) to stop the reaction.

*Paenibacillus* **sp.**

paper [13].

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

that of chitinase A from *Bacillus circulanse* WL-12 [AAA81528.1]).

expression plasmid coded mature Pb-ChiA was named pCold-Pb-ChiA.

pCold-Pb-ChiA was transformed into *Escherichia coli* DH5α, and the transformant was cultured in an LB medium (500 mL) containing ampicillin at 37°C to an OD600 value of 0.4 and established by induction with IPTG (0.1 mM) at 15°C overnight. The cultured *E. coli* cells were collected by centrifugation (9000 × *g*, 20 min, 4°C) and were resuspended in 20 mM Tris–HCl buffer (pH 7.5), sonicated, and centrifuged (9000 × *g*, 20 min, 4°C) again. The supernatant was put onto a HisTrap FF column (GE Healthcare UK Ltd., Little Chalfont, Buckinghamshire, England) equilibrated with 20 mM Tris–HCl buffer (pH 7.5) containing 20 mM imidazol and 300 mM NaCl. The enzyme was eluted with a gradient of 20–300 mM imidazol in the buffer. Active fractions were collected and utilized as the purified

The enzyme activity was determined using soluble chitin (40% deacetylated chitin) as a substrate under various conditions of pH and temperature. The buffer systems were as follows: pH 2.0–4.0 0.2 M glycine-HCl, pH 4.0–6.0 0.2 M acetate buffer, pH 6.0–8.0 0.2 M KH2PO4-K2HPO4, pH 8.0–9.0 0.2 M Tris–HCl, and pH 9.0–11.0 0.2 M glycine-NaOH. The effect of temperature on the enzyme activity

To recognize splitting patterns from hydrolysis products of the purified recombinant enzyme, 5.0 mM *N*-acetylglucosamine oligosaccharide substrates (GlcNAc)2–6 were dissolved in 50 mM sodium acetate buffer (pH 6.0), and aliquots of the enzyme solution were added to 400 μL of each substrate solution. Enzyme reactions were carried out at 37°C for several times, and parts of the reaction mixture were then picked up and mixed with the same volume of chilled acetonitrile

**2.3 Cloning and expression of β-GlcNAcase (Pb-β-GlcNAcase) gene from** 

Analysis of hydrolysis product using HPLC carried out according to a previously

First, forward (5′-GAGGAGGAGGTTCACGGGAGGAGGAGGTTCACGG-3′) and reverse (5′-GCGAGCTGTTGGAGAAGTACTCGTA-3′) primers for PCR were respectively synthesized on the 5′-upstream and 3′-downstream regions of Pb-β-GlcNAcase. Second, to insert the nucleotide sequence of Pb-LPMO in the pCold I expression vector (Takara Bio), forward (5′-GAGCTCGGTACCCTCATGAAGCTTTTTTTT; the *Nde*I

the nucleotide sequences of those genes from *P. barengoltzii* G22 (EOS56884.1) as templates. (The sequence of chitinase gene from *P. barengoltzii* G22 was similar to

First, forward (5′-ATGCATTCGAAGAGAACCCATAGCTTCAC-3′) and reverse (5′-TTATAGCGCCTGAAATAATGCGGGCAC-3′) primers for PCR were synthesized on the regions corresponding to amino acid residues 1–10 and 689–696 of Pb-ChiA, respectively. Second, to insert the nucleotide sequence corresponding to the mature Pb-ChiA in the pColdI expression vector (Takara Bio, Kyoto, Japan), forward (5′-TATACATATGCAACCGAAAGCCGCTGAGGC; the *Nde*I site is underlined) and reverse (5′-TATATCTAGATTATAGCGCCTGAAATAATGCGGG-3′; the *Xba*I site is underlined) primers for PCR were synthesized on regions corresponding to amino acid residues 33–39 and 690–696 of Pb-ChiA, respectively. PCR was carried out in a reaction mixture (20 μL) containing the *Paenibacillus* sp. A-471 genome DNA, 0.5 μM of each primer, and 10 μL of Takara PrimeSTAR Max premix (Takara Bio), according to the protocol of Takara Bio. A DNA fragment of 2.0 kb got by PCR was cloned into the *Nde*I and *Xba*I sites of the pCold I expression vector. The nucleotides of the amplified fragment were made more firm by sequencing after ligation. This

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

*Paenibacillus* sp. A-471 was used as a DNA donor. *Paenibacillus* sp. A-471 is closely related to *Paenibacillus barengoltzii* G22 strain. The genome sequence of *P. barengoltzii* G22 strain has been identified [13]. To clone the chitinase, β-*N*acetylglucosaminidase, and LPMO genes from *Paenibacillus* sp. A-471, we used

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

*Molecular Biotechnology*

deconstruction [10].

**2. Materials and methods**

**2.1 Enzyme and protein measurements**

families 18, 19, 23, and 48 [6, 7]. The glycoside hydrolase (GH) families 18 and 19 chitinases have almost no amino acid sequence similarities and have entirely different three-dimensional structures. Ra-ChiC from *Ralstonia* sp. A-471 was the first chitinase of the GH family 23 [7] and has been categorized to goose-type lysozyme. However, although Ra-ChiC shows chitinase activity, no activity toward peptidoglycan has been described [7]. β-GlcNAcase (exo-type enzyme) belongs to GH family 20. Chitin oligosaccharides such as *N*-acetylchitobiose and *N*-acetylchitotriose are good substrates for β-GlcNAcase [8]. Lytic polysaccharide monooxygenases (LPMOs, auxiliary activity [AA] family 10) are copper-dependent and cleave polysaccharide chains embedded in the crystalline regions of the substrate that are generally inaccessible to glycoside hydrolases [9]. Hemsworth et al. reported that fungi in particular makes considerable use of LPMOs in biomass degradation, producing more LPMO enzymes than cellulases [10]. Enzyme cocktails contained cellulases and LPMO enzymes can be utilized in the bio-refinery for lignocellulose

To understand the synergic activity of LPMO during the hydrolysis of crystalline chitin by chitinase A (GH family 18) and β-GlcNAcase from *Paenibacillus* sp., cloning and expression of chitinase A (Pb-ChiA), β-GlcNAcase (Pb-GlcNAcase), and LPMO (Pb-LPMO) genes from *Paenibacillus* sp. were carried out in this study.

The chitinase activity was assayed by determining the reducing sugars released from soluble chitin. The activity was assayed according to a previously paper [11]. The β-GlcNAcase activity was measured by determining the release of *p*-nitrophenol from *p*NP-GlcNAc. The enzyme activity required to form 1 μmol *p*-nitrophenol was regarded as one unit. The enzyme activity was measured according to a standard assay method. The reaction mixtures comprised 40 μL of 1 mM *p*NP-GlcNAc solution in 0.1 M acetate buffer (pH 6.0) and 20 μL of enzyme solution. After the reaction mixtures were incubated for 15 min at 37°C, the enzyme

The LPMO activity was measured by detecting the oxidized products released from chitin. Solutions of α or β-chitin (5 mg/mL), Pb-LPMO (1.0 mM), ascorbic acid (1.0 mM), and 20 mM acetate buffer (pH 5.0) were mixed to 1.0 mL volume. The reaction mixture was incubated at 37°C for 24 h. The oxidized products were then applied onto a TSK-gel Amide-80 column (4.6 × 250 mm, Tosoh Co., Tokyo, Japan) and eluted with 70% acetonitrile at a flow rate of 0.7 mL/min; the products

**2.2 Cloning and expression of chitinase A (Pb-ChiA) gene from** *Paenibacillus* **sp.**

*Paenibacillus* sp. A-471 was used as a DNA donor. *Paenibacillus* sp. A-471 is closely related to *Paenibacillus barengoltzii* G22 strain. The genome sequence of *P. barengoltzii* G22 strain has been identified [13]. To clone the chitinase, β-*N*acetylglucosaminidase, and LPMO genes from *Paenibacillus* sp. A-471, we used

reaction was stopped by adding 200 μL of 0.5 M Na2CO3 solution.

Protein contents were assayed using Micro BCA protein assay kits (ThermoFisher Scientific) with bovine serum albumin as the standard. Protein concentrations of Pb-LPMO, Pb-ChiA, and Pb-GlcNAcase were calculated from absorbance measurement at 280 nm and the protein extinction coefficient accord-

were monitored by absorbance measurement at 210 nm.

ing to the method of Gill and von Hippel [12].

**74**

the nucleotide sequences of those genes from *P. barengoltzii* G22 (EOS56884.1) as templates. (The sequence of chitinase gene from *P. barengoltzii* G22 was similar to that of chitinase A from *Bacillus circulanse* WL-12 [AAA81528.1]).

First, forward (5′-ATGCATTCGAAGAGAACCCATAGCTTCAC-3′) and reverse (5′-TTATAGCGCCTGAAATAATGCGGGCAC-3′) primers for PCR were synthesized on the regions corresponding to amino acid residues 1–10 and 689–696 of Pb-ChiA, respectively. Second, to insert the nucleotide sequence corresponding to the mature Pb-ChiA in the pColdI expression vector (Takara Bio, Kyoto, Japan), forward (5′-TATACATATGCAACCGAAAGCCGCTGAGGC; the *Nde*I site is underlined) and reverse (5′-TATATCTAGATTATAGCGCCTGAAATAATGCGGG-3′; the *Xba*I site is underlined) primers for PCR were synthesized on regions corresponding to amino acid residues 33–39 and 690–696 of Pb-ChiA, respectively. PCR was carried out in a reaction mixture (20 μL) containing the *Paenibacillus* sp. A-471 genome DNA, 0.5 μM of each primer, and 10 μL of Takara PrimeSTAR Max premix (Takara Bio), according to the protocol of Takara Bio. A DNA fragment of 2.0 kb got by PCR was cloned into the *Nde*I and *Xba*I sites of the pCold I expression vector. The nucleotides of the amplified fragment were made more firm by sequencing after ligation. This expression plasmid coded mature Pb-ChiA was named pCold-Pb-ChiA.

pCold-Pb-ChiA was transformed into *Escherichia coli* DH5α, and the transformant was cultured in an LB medium (500 mL) containing ampicillin at 37°C to an OD600 value of 0.4 and established by induction with IPTG (0.1 mM) at 15°C overnight. The cultured *E. coli* cells were collected by centrifugation (9000 × *g*, 20 min, 4°C) and were resuspended in 20 mM Tris–HCl buffer (pH 7.5), sonicated, and centrifuged (9000 × *g*, 20 min, 4°C) again. The supernatant was put onto a HisTrap FF column (GE Healthcare UK Ltd., Little Chalfont, Buckinghamshire, England) equilibrated with 20 mM Tris–HCl buffer (pH 7.5) containing 20 mM imidazol and 300 mM NaCl. The enzyme was eluted with a gradient of 20–300 mM imidazol in the buffer. Active fractions were collected and utilized as the purified enzyme solution.

The enzyme activity was determined using soluble chitin (40% deacetylated chitin) as a substrate under various conditions of pH and temperature. The buffer systems were as follows: pH 2.0–4.0 0.2 M glycine-HCl, pH 4.0–6.0 0.2 M acetate buffer, pH 6.0–8.0 0.2 M KH2PO4-K2HPO4, pH 8.0–9.0 0.2 M Tris–HCl, and pH 9.0–11.0 0.2 M glycine-NaOH. The effect of temperature on the enzyme activity was examined at 10–80°C.

To recognize splitting patterns from hydrolysis products of the purified recombinant enzyme, 5.0 mM *N*-acetylglucosamine oligosaccharide substrates (GlcNAc)2–6 were dissolved in 50 mM sodium acetate buffer (pH 6.0), and aliquots of the enzyme solution were added to 400 μL of each substrate solution. Enzyme reactions were carried out at 37°C for several times, and parts of the reaction mixture were then picked up and mixed with the same volume of chilled acetonitrile (−20°C) to stop the reaction.

Analysis of hydrolysis product using HPLC carried out according to a previously paper [13].
