**3.8 Optimum ratio of Pb-LPMO, Pb-ChiA, and Pb-GlcNAcase during the hydrolysis of crystalline chitin**

To understand the effect of Pb-GlcNAcase on the hydrolysis of crystalline chitin, we added 0.1–10 mM Pb-LPMO to the reaction solution containing

**Figure 5.**

*Optimum ratio of Pb-LPMO and Pb-ChiA during the hydrolysis of crystalline chitin. Blue bar: GlcNAc content; reddish brown bar: (GlcNAc)2 content; gray bar: Total GlcNAc content. Total GlcNAc content: (GlcNAc)2 (reddish brown bar) content was converted to twice the GlcNAc content. Total GlcNAc content = GlcNAc (blue bar) content + converted GlcNAc content.*

**83**

biomass.

crystalline chitin hydrolysis.

**4. Conclusion**

**Figure 6.**

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

1 mM Pb-ChiA and 3 mM Pb-LPMO (**Figure 6**). With increasing concentration of Pb-GlcNAcase, the concentration of released GlcNAc increased, but the concentration of released (GlcNAc)2 decreased. It is possible that (GlcNAc)2 was hydrolyzed by Pb-GlcNAcase. The concentration (approximately 0.8 mM) of released products (**Figure 6**, gray bar) when Pb-GlcNAcase was added was almost the same as that obtained from the reaction of only Pb-ChiA and Pb-LPMO (**Figure 5**). Therefore, Pb-GlcNAcase has no effect on the hydrolysis of crystal chitin but only contributes to the degradation of chitin oligosaccharides. Wang et al. reported that only β-*N*-GlcNAcase plays a role in the hydrolysis of chitinoligosaccharides and transglycosylation [24]. Hemsworth et al. reported that it will become increasingly important to understand how cellulase and LPMO enzymes synergize with one another in biomass breakdown [10]. It found that synergization of chitinase and LPMO enzyme is also very important in the

*content = GlcNAc (blue bar) content + converted GlcNAc content.*

*Optimum ratio of Pb-LPMO, Pb-ChiA, and Pb-GlcNAcase during the hydrolysis of crystalline chitin. Blue bar: GlcNAc content; reddish brown bar: (GlcNAc)2 content; gray bar: Total GlcNAc content. Total GlcNAc content: (GlcNAc)2 (reddish brown bar) content was converted to twice the GlcNAc content. Total GlcNAc* 

Pb-ChiA had a higher affinity toward crystalline chitin than Pb-LPMO. Pb-LPMO boosts the activity of Pb-ChiA toward crystalline α-chitin but not toward crystalline β-chitin. For the efficient hydrolysis of crystalline chitin, addition of 3 μM Pb-LPMO to the reaction mixture containing 1 μM Pb-ChiA was optimal. When Pb-LPMO (3 μM) was added to the reaction mixture during the hydrolysis of crystalline α-chitin by Pb-ChiA, two-fold higher concentration hydrolysis products were obtained. It was found that Pb-GlcNAcase is involved only in the degradation of chitin oligosaccharides. Synergization of Pb-ChiA and Pb-LPMO was shown during crystalline chitin decomposition. LPMO contributes to the efficiency of chitinase used in the conversion of chitin

*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 6.**

*Molecular Biotechnology*

**crystalline chitin**

**hydrolysis of crystalline chitin**

**3.6 Expression of Pb-LPMO gene and characterization of recombinant protein**

The molecular mass of the recombinant protein rPb-LPMO was 45 kDa, as determined by SDS-PAGE (data not shown), in good agreement with that predicted from the amino acid sequence. The activity of rPb-LPMO was determined in

enzyme assays using α-chitin or β-chitin as the substrate. After incubation, oxidized

To understand the effect of Pb-LPMO on the hydrolysis of crystalline chitin, we added 0.01–30 μM Pb-LPMO to the reaction solution containing 1 μM Pb-ChiA. (**Figure 5**). With increasing concentration of Pb-LPMO, the concentration of the hydrolysis products (GlcNAc and (GlcNAc)2) increased. When 1–10 μM of Pb-LPMO was added to the reaction mixture, products at higher concentration were obtained as compared with other concentrations. However, the addition of more than 10 μM Pb-LPMO decreased the concentration of the hydrolysis products in the reaction mixture. For efficient hydrolysis of crystalline chitin, addition of 3 μM Pb-LPMO to the reaction mixture containing 1 μM Pb-ChiA is optimal. The released products were approximately two-fold higher upon the addition of Pb-LPMO. Vaaje-Kolstad et al. reported that CBP21 (LPMO) strongly promotes hydrolysis of crystalline β-chitin by chitinase A and C from *S. marcescens* [23]. In this study, Pb-LPMO was found to efficiently promote hydrolysis of crystalline chitin.

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

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

To understand the effect of Pb-GlcNAcase on the hydrolysis of crystalline chitin, we added 0.1–10 mM Pb-LPMO to the reaction solution containing

*Optimum ratio of Pb-LPMO and Pb-ChiA during the hydrolysis of crystalline chitin. Blue bar: GlcNAc content; reddish brown bar: (GlcNAc)2 content; gray bar: Total GlcNAc content. Total GlcNAc content: (GlcNAc)2 (reddish brown bar) content was converted to twice the GlcNAc content. Total GlcNAc* 

*content = GlcNAc (blue bar) content + converted GlcNAc content.*

products were detected by HPLC (data not shown).

**82**

**Figure 5.**

*Optimum ratio of Pb-LPMO, Pb-ChiA, and Pb-GlcNAcase during the hydrolysis of crystalline chitin. Blue bar: GlcNAc content; reddish brown bar: (GlcNAc)2 content; gray bar: Total GlcNAc content. Total GlcNAc content: (GlcNAc)2 (reddish brown bar) content was converted to twice the GlcNAc content. Total GlcNAc content = GlcNAc (blue bar) content + converted GlcNAc content.*

1 mM Pb-ChiA and 3 mM Pb-LPMO (**Figure 6**). With increasing concentration of Pb-GlcNAcase, the concentration of released GlcNAc increased, but the concentration of released (GlcNAc)2 decreased. It is possible that (GlcNAc)2 was hydrolyzed by Pb-GlcNAcase. The concentration (approximately 0.8 mM) of released products (**Figure 6**, gray bar) when Pb-GlcNAcase was added was almost the same as that obtained from the reaction of only Pb-ChiA and Pb-LPMO (**Figure 5**). Therefore, Pb-GlcNAcase has no effect on the hydrolysis of crystal chitin but only contributes to the degradation of chitin oligosaccharides. Wang et al. reported that only β-*N*-GlcNAcase plays a role in the hydrolysis of chitinoligosaccharides and transglycosylation [24]. Hemsworth et al. reported that it will become increasingly important to understand how cellulase and LPMO enzymes synergize with one another in biomass breakdown [10]. It found that synergization of chitinase and LPMO enzyme is also very important in the crystalline chitin hydrolysis.

### **4. Conclusion**

Pb-ChiA had a higher affinity toward crystalline chitin than Pb-LPMO. Pb-LPMO boosts the activity of Pb-ChiA toward crystalline α-chitin but not toward crystalline β-chitin. For the efficient hydrolysis of crystalline chitin, addition of 3 μM Pb-LPMO to the reaction mixture containing 1 μM Pb-ChiA was optimal. When Pb-LPMO (3 μM) was added to the reaction mixture during the hydrolysis of crystalline α-chitin by Pb-ChiA, two-fold higher concentration hydrolysis products were obtained. It was found that Pb-GlcNAcase is involved only in the degradation of chitin oligosaccharides. Synergization of Pb-ChiA and Pb-LPMO was shown during crystalline chitin decomposition. LPMO contributes to the efficiency of chitinase used in the conversion of chitin biomass.

*Molecular Biotechnology*
