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

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

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 solution.

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 10–80°C.

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 described above.

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 of each substrate solution.
