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. 2023 May 29;21(6):332.
doi: 10.3390/md21060332.

Biochemical Properties of a Cold-Active Chitinase from Marine Trichoderma gamsii R1 and Its Application to Preparation of Chitin Oligosaccharides

Jianrong Wang  1   2 , Mujin Zhu  1 , Ping Wang  1 , Wei Chen  1
Affiliations

Biochemical Properties of a Cold-Active Chitinase from Marine Trichoderma gamsii R1 and Its Application to Preparation of Chitin Oligosaccharides

Jianrong Wang et al. Mar Drugs. .

Abstract

The enzymatic degradation of different chitin polymers into chitin oligosaccharides (COSs) is of great significance given their better solubility and various biological applications. Chitinase plays a pivotal role in the enzymatic preparation of COSs. Herein, a cold-adapted and efficient chitinase (ChiTg) from the marine Trichoderma gamsii R1 was purified and characterized. The optimal temperature of ChiTg was 40 °C, and the relative activity at 5 °C was above 40.1%. Meanwhile, ChiTg was active and stable from pH 4.0 to 7.0. As an endo-type chitinase, ChiTg exhibited the highest activity with colloidal chitin, then with ball-milled and powdery chitin. In addition, ChiTg showed high efficiency when hydrolyzing colloidal chitin at different temperatures, and the end products were mainly composed of COSs with one to three degrees of polymerization. Furthermore, the results of bioinformatics analysis revealed that ChiTg belongs to the GH18 family, and its acidic surface and the flexible structure of its catalytic site may contribute to its high activity in cold conditions. The results of this study provide a cold-active and efficient chitinase and ideas for its application regarding the preparation of COSs from colloidal chitin.

Keywords: Trichoderma gamsii; chitin oligosaccharides; chitinase; cold-adapted.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
SDS-PAGE analysis of purified ChiTg. M: protein marker, 1: crude culture, 2: sample from ultrafiltration, and 3: purified ChiTg.
Figure 2
Figure 2
The characterization of purified ChiTg. Optimum temperature (A), thermostability (B), stability of ChiTg at 40 and 45 °C (C), optimum pH (D), and pH stability (E).
Figure 3
Figure 3
TLC analysis of the hydrolytic process of ChiTg toward (GlcNAc)2 (A), (GlcNAc)3 (B), (GlcNAc)4 (C), and (GlcNAc)5 (D).
Figure 4
Figure 4
Preparation of COSs from colloidal chitin. Optimization of different ratios of enzyme to substrate (A), concentration of substrate (B), reaction pH (C), reaction time (D), and reaction temperature (E). HPLC analysis of hydrolysates from large-scale reaction (F).
Figure 5
Figure 5
Sequence alignment of ChiTg with already crystallized chitinase. The listed sequences include Chit42 from Trichoderma harzianum (S78423.1), ChiCr from Clonostachys rosea (ABV57861.1), and ChiAf from Aspergillus fumigatus Af293 (XP_747065.1).
Figure 6
Figure 6
The overall structure of ChiTg.
Figure 7
Figure 7
Homology modeling structure of ChiTg. Interactions between ChiTg and (GlcNAc)2 (A), (GlcNAc)3 (B), (GlcNAc)4 (C), and (GlcNAc)5 (D). The amino acids residues colored in yellow play an important role in catalytic hydrolysis of substrates.
Figure 8
Figure 8
The potential factors related to the cold adaptation of ChiTg. Surface electrostatic potential of ChiTg (A). The surface coloring is based on the electrostatic potential, with a gradient from red (electronegative) to blue (electropositive). B-factor changes over the backbone of ChiTg (B). Tertiary structure alignment between ChiTg and thermostable chitinase Chit1 from Thermomyces lanuginosu s(C). The amino acid residues on the surface colored in red and yellow are from Chit1 and ChiTg, respectively.
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Supplementary concepts