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

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.

SUBMITTER: He B

PROVIDER: S-EPMC9578694 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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

He Bin B Yang Liyan L Yang Dengfeng D Jiang Minguo M Ling Chengjin C Chen Hailan H Ji Feng F Pan Lixia L

Frontiers in bioengineering and biotechnology 20221004

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 expresse ...[more]

PMID: 36267443

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Project description:Chitin is a biopolymer of N-acetyl-d-glucosamine with β-1,4-bond and is the main component of arthropod exoskeletons and the cell walls of many fungi. Chitinase (EC 3.2.1.14) is an enzyme that hydrolyzes the β-1,4-bond in chitin and degrades chitin into oligomers. It has been found in a wide range of organisms. Chitinase from Gazyumaru (Ficus microcarpa) latex exhibits antifungal activity by degrading chitin in the cell wall of fungi and is expected to be used in medical and agricultural fields. However, the enzyme's thermostability is an important factor; chitinase is not thermostable enough to maintain its activity under the actual application conditions. In addition to the fact that thermostable chitinases exhibiting antifungal activity can be used under various conditions, they have some advantages for the production process and long-term preservation, which are highly demanded in industrial use. We solved the crystal structure of chitinase to explore the target sites to improve its thermostability. We rationally introduced proline residues, a disulfide bond, and salt bridges in the chitinase using protein-engineering methods based on the crystal structure and sequence alignment among other chitinases. As a result, we successfully constructed the thermostable mutant chitinases rationally with high antifungal and specific activities. The results provide a useful strategy to enhance the thermostability of this enzyme family. IMPORTANCE We solved the crystal structure of the chitinase from Gazyumaru (Ficus microcarpa) latex exhibiting antifungal activity. Furthermore, we demonstrated that the thermostable mutant enzyme with a melting temperature (Tm) 6.9°C higher than wild type (WT) and a half-life at 60°C that is 15 times longer than WT was constructed through 10 amino acid substitutions, including 5 proline residues substitutions, making disulfide bonding, and building a salt bridge network in the enzyme. These mutations do not affect its high antifungal activity and chitinase activity, and the principle for the construction of the thermostable chitinase was well explained by its crystal structure. Our results provide a useful strategy to enhance the thermostability of this enzyme family and to use the thermostable mutant as a seed for antifungal agents for practical use.

Dataset Information

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

Publications

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

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