**2.4 Cloning and expression of lytic polysaccharide monooxygenase (Pb-LPMO) gene from** *Paenibacillus* **sp.**

pBIC3 forward (5′-AGTTCCGCATTCGCTCACGGCTGGGTGGACGG-3′) and pBIC reverse (5′-CATCCTGTTAAGCTTTTATTTCAGCAGCCACAA-3′) primers for PCR were synthesized on the regions corresponding to amino acid residues 38–43 and 445–449 of Pb-LPMO, respectively. PCR was carried out in a reaction mixture (20 μL) containing the *Paenibacillus* genome DNA, 0.5 μM of each primer, and 10 μL of Takara PrimeSTAR Max premix (Takara Bio), using the same thermocycling conditions as the Section 2.2. A DNA fragment of approximately 1.4 kb obtained by PCR was cloned into the pBIC3 expression vector. The nucleotides of the amplified fragment were confirmed by sequencing after ligation. This expression-plasmid-coded Pb-LPMO was named pBIC-Pb-LPMO.

pBIC-Pb-LPMO was transformed to *Brevibacillus choshinensis* according to the protocol of the *Brevibacillus* expression system (Takara Bio). The transformant was cultured using TMNm medium (10 g/L glucose, 10 g/L Bacto Soytone, 5 g/L tuna extract, 2.0 g/L Bacto Yeast Extract, 10 mg/L FeSO4∙7H2O, 10 mg/L MnSO4∙4H2O, and 1.0 mg/L ZeSO4∙7H2O) with neomycin (final concentration of 50 μg/mL) at 30°C for 2 days. After cultivation, the culture medium was centrifuged at 20,000 × *g* for 15 min at 4°C. The supernatant was added to 80% saturated (NH4)2SO4. A pellet was recovered by centrifugation at 20,000 × *g* for 15 min at 4°C.

**77**

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

The pellet was then redissolved in 20 mM Tris-HCl buffer (pH 7.5) and dialyzed against 20 mM Tris-HCl buffer (pH 7.5) for 1 day. The supernatant was applied to a chitin resin column (1 × 4 cm, Biorad Laboratory, America) equilibrated with 20 mM Tris-HCl buffer (pH 7.5). The enzyme was eluted with 20 mM acetic acid. Active fractions were dialyzed with 20 mM acetate buffer (pH 5.0) and used as the

**2.5 Optimum ratio of Pb-LPMO and Pb-ChiA during the hydrolysis of** 

Pb-LPMO (0.01, 0.1, 0.5, 1.0, 3.0, 5.0, 10, and 30 μM), 1 μM Pb-ChiA, and 1.0 mM ascorbic acid in 20 mM acetate buffer (pH 5.0) were incubated with α-chitin at 37°C for 24 h. After the reaction, the reaction mixture was boiled for 10 min to terminate the enzyme reaction. The samples were mixed with the same volume of acetonitrile. The resultant solution was applied onto a column of TSK-GEL Amide 80 (4.6 × 250 mm, Tosoh Co., Tokyo, Japan) and eluted with 70% acetonitrile at a flow rate of 0.7 mL/min, and the products were monitored by absorbance measurement at 210 nm. In this experiment, we estimated the concentration of GlcNAc, (GlcNAc)2, and total sugars (GlcNAc). The concentration of (GlcNAc)2 in total sugars was calculated as twice the concentration of GlcNAc.

**2.6 Optimum ratio of Pb-LPMO, Pb-ChiA, and Pb-GlcNAcase during the** 

total sugars was calculated as the concentration of two GlcNAc.

We incubated 3.0 μM Pb-LPMO, 1 μM Pb-ChiA, Pb-GlcNAcase (0, 0.1, 1.0, 3.0, 5.0, and 10 μM), and 1.0 mM ascorbic acid in 20 mM acetate buffer (pH 5.0) with α-chitin at 37°C for 24 h. The reaction mixture was then boiled for 10 min to terminate the enzyme reaction. The samples were mixed with the same volume of acetonitrile. The resultant solution was applied onto the same column and conditions as mentioned in Section 2.4. In this experiment, we estimated the concentration of GlcNAc, (GlcNAc)2, and total sugars (GlcNAc). The concentration of (GlcNAc)2 in

**2.7 Additional effect of Pb-LPMO during the hydrolysis of crystalline chitin by** 

We incubated 1 μM Pb-ChiA and 1.0 mM ascorbic acid with or without 3 μM Pb-LPMO in 20 mM acetate buffer (pH 5.0) with α-chitin at 37°C for 24 h. The reaction mixture was then boiled for 10 min to terminate the enzyme reaction. The samples were mixed with the same volume of acetonitrile. The resultant solution was applied onto the same column and conditions mentioned in Section 2.4. In this experiment, we investigated the total concentration of GlcNAc, (GlcNAc)2, and total sugars (GlcNAc). The concentration of (GlcNAc)2 in total sugars was calcu-

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

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

**hydrolysis of crystalline chitin**

lated as twice the concentration of GlcNAc.

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

**3. Results and discussion**

**Pb-ChiA**

purified enzyme solution.

**crystalline chitin**

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

*Molecular Biotechnology*

solution.

10–80°C.

described above.

of each substrate solution.

**gene from** *Paenibacillus* **sp.**

site is underlined) and reverse (5′-GATTACCTATCTAGATTACATCGACAGCGA-3′; the *Xba*I site is underlined) primers for PCR were synthesized on the region corresponding to amino acid residues 1–5 and 638–641 of Pb-β-GlcNAcase, respectively. PCR was carried out in a reaction mixture (20 μL) containing the *Paenibacillus* genome DNA with first amplified PCR fragment, 0.5 μM of each primer, and 10 μL of Takara PrimeSTAR Max premix (Takara Bio), using the same thermocycling conditions as the Section 2.2. A DNA fragment of approximately 2.0 kb obtained by PCR was cloned into the *Xho*I and *Xba*I sites of the pCold I expression vector according to the protocol of InFusion cloning (Takara Bio). The nucleotides of the amplified fragment were confirmed by sequencing after InFusion cloning. This expression-plasmid-coded Pb-β-GlcNAcase was named pCold-Pb-β-GlcNAcase. Transformation and purification of Pb-β-GlcNAcase used the same method as those of Pb-Chi. Active fractions were dialyzed with 20 mM phosphate buffer (pH 7.0) and used as the purified enzyme

The enzyme activity was determined using *p*NP-GlcNAc as a substrate under various conditions of pH and temperature. 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 2.0–12.0). The effect of temperature on enzyme activity was examined at

The activity of the purified recombinant protein was tested using soluble chitin, *p*NP-GlcNAc, *p*NP-GalNAC, and (GlcNAc)2–6. In the case of *p*NP substrates, the released *p*-nitrophenol was detected by absorbance measurement at 405 nm. The hydrolysis products of (GlcNAC)2–6 were assayed by HPLC and measured as

HPLC analysis of the hydrolysis products of chitin oligosaccharides (GlcNAc)2–6 was performed by the same method as described in Section 2.2. To investigate the cleavage patterns from the hydrolysis products of the purified enzyme (130 units/ mL substrate: pNP-GlcNAc), 5.0 mM (GlcNAc)2–6 was dissolved in 50 mM sodium acetate buffer (pH 6.0), and aliquots of the enzyme solution were added to 400 μL

**2.4 Cloning and expression of lytic polysaccharide monooxygenase (Pb-LPMO)** 

pBIC3 forward (5′-AGTTCCGCATTCGCTCACGGCTGGGTGGACGG-3′) and pBIC reverse (5′-CATCCTGTTAAGCTTTTATTTCAGCAGCCACAA-3′) primers for PCR were synthesized on the regions corresponding to amino acid residues 38–43 and 445–449 of Pb-LPMO, respectively. PCR was carried out in a reaction mixture (20 μL) containing the *Paenibacillus* genome DNA, 0.5 μM of each primer, and 10 μL of Takara PrimeSTAR Max premix (Takara Bio), using the same thermocycling conditions as the Section 2.2. A DNA fragment of approximately 1.4 kb obtained by PCR was cloned into the pBIC3 expression vector. The nucleotides of the amplified fragment were confirmed by sequencing after ligation. This expres-

pBIC-Pb-LPMO was transformed to *Brevibacillus choshinensis* according to the protocol of the *Brevibacillus* expression system (Takara Bio). The transformant was cultured using TMNm medium (10 g/L glucose, 10 g/L Bacto Soytone, 5 g/L tuna extract, 2.0 g/L Bacto Yeast Extract, 10 mg/L FeSO4∙7H2O, 10 mg/L MnSO4∙4H2O, and 1.0 mg/L ZeSO4∙7H2O) with neomycin (final concentration of 50 μg/mL) at 30°C for 2 days. After cultivation, the culture medium was centrifuged at 20,000 × *g* for 15 min at 4°C. The supernatant was added to 80% saturated (NH4)2SO4. A pellet was recovered by centrifugation at 20,000 × *g* for 15 min at 4°C.

sion-plasmid-coded Pb-LPMO was named pBIC-Pb-LPMO.

**76**

The pellet was then redissolved in 20 mM Tris-HCl buffer (pH 7.5) and dialyzed against 20 mM Tris-HCl buffer (pH 7.5) for 1 day. The supernatant was applied to a chitin resin column (1 × 4 cm, Biorad Laboratory, America) equilibrated with 20 mM Tris-HCl buffer (pH 7.5). The enzyme was eluted with 20 mM acetic acid. Active fractions were dialyzed with 20 mM acetate buffer (pH 5.0) and used as the purified enzyme solution.
