Cloning, expression, purification, and mutant construction of the chitinase PbCHI2 gene from Photobacterium sp.

Authors

Zhe Wang, Ziqi Zhao, Lin Pan, Yawen Zhang, Zhuo Guo, Weiye Yang, Liangshan Feng, Xiaohui Wang

Published in

Archives of microbiology. Volume 207. Issue 11. Pages 272. Sep 20, 2025. Epub Sep 20, 2025.

Abstract

Chitin, the second most abundant biopolymer on Earth, exhibits broad application potential in medicine, cosmetics, and agriculture through its degradation products (chito-oligosaccharides). Here, we characterized a cold-adapted chitinase (PbCHI2) secreted by Photobacterium sp. LG-29 isolated from deep-sea sediments. The PbCHI2 gene was cloned into an expression vector and heterologously expressed in Escherichia coli BL21(DE3), followed by protein structure prediction and molecular docking to identify key residues for mutagenesis. The enzyme showed optimal activity at 35 °C while retaining functionality at 4 °C, confirming its cold-adaptation. Notably, it maintained > 60% activity at 60 °C (substrate: 4-MU-GlcNAc3) and > 70% residual activity after 3 h at 45 °C, demonstrating remarkable thermostability. PbCHI2 exhibited pH-dependent activity, with peak performance at pH 8.0 and > 70% activity at pH 11. Metal ion effects revealed Mn²⁺, Ca²⁺, and Mg²⁺ as activators, whereas Fe^2+/3+, Zn²⁺, Cu²⁺, and Cd²⁺ acted as inhibitors. Surprisingly, urea, Sodium Dodecyl Sulfate (SDS), and Dithiothreitol (DTT) enhanced activity at specific concentrations. Site-directed mutagenesis within a 5Å radius of the active site yielded mutants with improved catalytic efficiency, validated by HPLC analysis of degradation products (primarily GlcNAc2 and GlcNAc), suggesting exochitinase and β-N-acetylglucosaminidase activities. Structural analysis classified PbCHI2 as a GH18 family chitinase, featuring a ChtBD3 domain and PKD domain(s). To our knowledge, this is the first comprehensive study on a chitinase from Photobacterium sp., providing insights into its cold-adaptation mechanism and laying a foundation for engineering industrially relevant chitinases.

PMID:
40974411
Bibliographic data and abstract were imported from PubMed on 20 Sep 2025.

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