Biochemical purification and characterization of a truncated acidic, thermostable chitinase from marine fungus for N-acetylglucosamine production

Bin He; Liyan Yang; Dengfeng Yang; Minguo Jiang; Chengjin Ling; Hailan Chen; Feng Ji; Lixia Pan

doi:10.3389/fbioe.2022.1013313

Biochemical purification and characterization of a truncated acidic, thermostable chitinase from marine fungus for N-acetylglucosamine production

Front Bioeng Biotechnol. 2022 Oct 4:10:1013313. doi: 10.3389/fbioe.2022.1013313. eCollection 2022.

Authors

Bin He^{1

2}, Liyan Yang², Dengfeng Yang², Minguo Jiang³, Chengjin Ling⁴, Hailan Chen¹, Feng Ji⁵, Lixia Pan²

Affiliations

¹ School of Animal Science and Technology, Guangxi University, Nanning, Guangxi, China.
² State Key Laboratory of Non-Food Biomass and Enzyme Technology, Guangxi Key Laboratory of Marine Natural Products and Combinatorial Biosynthesis Chemistry, Guangxi Academy of Sciences, Nanning, Guangxi, China.
³ Guangxi Key Laboratory for Polysaccharide Materials and Modifications, School of Marine Sciences and Biotechnology, Guangxi University for Nationalities, Nanning, China.
⁴ Nanning Dabeinong Feed Technology Co., Ltd., Nanning, Guangxi, China.
⁵ Guangxi Huaren Medical Technolgoy Group, Nanning, Guangxi, China.

Abstract

N-acetylglucosamine (GlcNAc) is widely used in nutritional supplement and is generally produced from chitin using chitinases. While most GlcNAc is produced from colloidal chitin, it is essential that chitinases be acidic enzymes. Herein, we characterized an acidic, highly salinity tolerance and thermostable chitinase AfChiJ, identified from the marine fungus Aspergillus fumigatus df673. Using AlphaFold2 structural prediction, a truncated Δ30AfChiJ was heterologously expressed in E. coli and successfully purified. It was also found that it is active in colloidal chitin, with an optimal temperature of 45°C, an optimal pH of 4.0, and an optimal salt concentration of 3% NaCl. Below 45°C, it was sound over a wide pH range of 2.0-6.0 and maintained high activity (≥97.96%) in 1-7% NaCl. A notable increase in chitinase activity was observed of Δ30AfChiJ by the addition of Mg²⁺, Ba²⁺, urea, and chloroform. AfChiJ first decomposed colloidal chitin to generate mainly N-acetyl chitobioase, which was successively converted to its monomer GlcNAc. This indicated that AfChiJ is a bifunctional enzyme, composed of chitobiosidase and β-N-acetylglucosaminidase. Our result suggested that AfChiJ likely has the potential to convert chitin-containing biomass into high-value added GlcNAc.

Keywords: AlphaFold2; Aspergillus fumigatus; N-acetylglucosamine; N-terminal truncation; acidic chitinase; chitin; β-N-acetylglucosaminidase.