Section 2 Biotechnology

**73**

**Chapter 4**

**Abstract**

Effect of LPMO on the Hydrolysis

of Crystalline Chitin by Chitinase

A and β-*N*-Acetylglucosaminidase

We performed cloning and expression of chitinase A (Pb-ChiA), β-GlcNAcase (Pb-GlcNAcase), and lytic polysaccharide monooxygenase (Pb-LPMO) genes from *Paenibacillus* sp. The analysis of the hydrolysis products indicated Pb-ChiA to be an exo-type chitinase with 10-fold activity toward β-chitin as compared with α-chitin. The sequence of Pb-GlcNAcase was found to be similar to that of β-*N*acetylhexosaminidase from *P. barengoltzii* (99%, WP\_016313754.1). Pb-LPMO was expressed in the *Brevibacillus* expression system. Pb-ChiA was found to have affinity toward crystalline chitin higher than that of Pb-LPMO. Pb-LPMO boosted the activity of Pb-ChiA toward crystalline α-chitin but not toward crystalline β-chitin. When Pb-LPMO (3 μM) was added to the reaction mixture during the hydrolysis of crystalline α-chitin by Pb-ChiA, hydrolysis products at two-fold concentration were obtained. However, the hydrolysis products decreased upon addition of more than

from *Paenibacillus* sp.

*and Shunsuke Sakurai*

3 μM Pb-LPMO to the reaction mixture.

**1. Introduction**

β-D-*N*-acetylhexosaminidase, *Paenibacillus* sp.

*Mitsuhiro Ueda, Kei Nakadoi, Kana Tsukamoto* 

**Keywords:** chitin, chitinase, lytic polysaccharide monooxygenase,

Chitin, which can be easily isolated from crab and shrimp shell waste, is the second most abundant biopolymer on earth. At least 10 gigatons (1 × 1013 kg) of chitin are synthesized and degraded each year in the biosphere. *N*-Acetylchitooligosaccharides (GlcNAc) and chitooligosaccharides (GlcN), which are hydrolyzed products of chitin and chitosan, have various biological roles and many possible future applications [1–3]. GlcNAc and glucosamine are ordinarily utilized for symptom relief in osteoarthritis patients [4]. Hyaluronic acid as heterogeneous polysaccharides contained GlcNAc, and addition of GlcNAc to cultured keratinocytes facilitates the production of hyaluronic acid in a dose-dependent manner [5]. Biotechnological tools for generating enzymes are required for environmentally sustainable production of chitin oligosaccharide, GlcNAc, or both. Multiple chitinases have been characterized and are categorized into four enzyme families, namely,
