Heterologous Expression and Characterization of a Novel Chitinase Derived from Talaromyces flavus

doi:10.19586/j.2095-2341.2025.0100

Current Biotechnology ›› 2026, Vol. 16 ›› Issue (2): 380-387.DOI: 10.19586/j.2095-2341.2025.0100

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Heterologous Expression and Characterization of a Novel Chitinase Derived from Talaromyces flavus

Yang WANG(), Songni YU(), Yahua LI, Hongquan XIAN()

College of Life Sciences，Qingdao Agricultural University，Shandong Qingdao 266109，China

Received:2025-08-06 Accepted:2025-10-22 Online:2026-03-25 Published:2026-04-27
Contact: Hongquan XIAN

Abstract

Abstract:

Talaromyces flavus is a mycoparasitic fungus， and the chitinases it producs play an important role in the mycoparasitic process， showing high application value in biological control of plant diseases and chitin degradation. In this study， intronless chitinase genes were cloned from T. flavus using PCR technology， expressed in the Pichia pastoris system， and the characteristics of chitinases were analyzed. The results showed that the ORF sizes of chitinase genes 04446 and 06441 were 1 032 and 1 029 bp， respectively. The P. pastoris expression vectors pPIC9K/04446 and pPIC9K/06441 were successfully constructed， and highly expressed yeast engineering strains GS115/04446-45 and GS115/06441-57 were obtained； the purified protein Tfch04446 expressed by 04446 had a molecular weight of 36.8 kD， and the protein Tfch06441 expressed by 06441 had a molecular weight of 37.2 kD. The optimal reaction temperature of chitinase Tfch04446 was 45 ℃， while that of chitinase Tfch06441 was 50 ℃. The optimal reaction pH was 6 for both Tfch04446 and Tfch06441. For Tfch04446， the K_m value was 2.27 mg·mL^-1 and the maximum reaction rate （V_max） was 0.085 mg·mL^-1·min^-1； for Tfch06441， the K_m value was 1.52 mg·mL^-1 and the V_max was 0.067 mg·mL^-1·min^-1. Regarding metal ions， Ca²⁺ and Ba²⁺ exerted a promoting effect on the activity of chitinase Tfch04446， while NH₄⁺ showed a certain degree of inhibitory effect. For chitinase Tfch06441， Ba²⁺ had a promoting effect on its activity， whereas Na⁺， K⁺， Ca²⁺ and NH₄⁺ all exhibited inhibitory effects on it. The research results laid a foundation for the development and utilization of T. flavus and its chitinases.

Key words: Talaromyces flavus, chitinase, intronless gene, gene expression, Pichia pastoris, enzymatic property

CLC Number:

Q939.9

Yang WANG, Songni YU, Yahua LI, Hongquan XIAN. Heterologous Expression and Characterization of a Novel Chitinase Derived from Talaromyces flavus[J]. Current Biotechnology, 2026, 16(2): 380-387.

Figures/Tables 12

Table 1 Primers for chitinase gene amplification

引物名称	序列（5'→3'）	酶切位点类别
04446s	5'-CGCCCTAGGATGCGTTTCTCAACTCTCG-3'	AvrⅡ
04446A	5'-ATTTGCGGCCGCTTAGTGGTGGTGGTGGTGGTGGCAGGCGCTGCC-3'	NotⅠ
06441S	5'-GGAATTCATGCATTTCTCCACCGTTAT-3'	EcoRⅠ
06441A	5'-ATTTGCGGCCGCTTAGTGGTGGTGGTGGTGGTGACAAACTTTACCCTTCGTC-3'	NotⅠ

Fig. 1 Verification of PCR amplification products of chitinase genes 04446 and 06441

Fig. 2 Detection of double digestion products of recombinant plasmids pMD18-T/04446 and pMD18-T/06441

Fig. 3 G418 plate screening of multicopy transformants

Fig. 4 PCR identification of yeast inverters

Fig. 5 SDS-PAGE analysis of chitinases with Tfch04446 and Tfch06441

Fig. 6 The optimal reaction temperature of Tfch04446 and Tfch06441

Fig. 7 The optimal reaction pH of Tfch04446 and Tfch06441

Table 2 Effect of different ions on Tfch04446 activity

离子	相对活性/%	离子	相对活性/%
H₂O	100.00	Zn²⁺	0.00
K⁺	95.00	Ag⁺	102.50
Ca²⁺	144.50	Mg²⁺	102.20
NH₄⁺	51.90	Ba²⁺	375.00
Mn²⁺	127.00	Cu²⁺	112.50
Fe²⁺	0.00	Na⁺	115.00

Table 3 Effect of different ions on Tfch06441 activity

离子	相对活性/%	离子	相对活性/%
H₂O	100.00	Zn²⁺	0.00
K⁺	53.00	Ag⁺	123.00
Ca²⁺	53.20	Mg²⁺	75.00
NH₄⁺	59.60	Ba²⁺	125.00
Mn²⁺	80.90	Cu²⁺	0.00
Fe²⁺	0.00	Na⁺	48.00

Fig. 8 The process curve of chitinase Tfch04446 and Tfch06441 enzymatic hydrolysis reaction

Fig. 9 Lineweaver-Burk curve of chitinase Tfch04446 and Tfch06441

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Heterologous Expression and Characterization of a Novel Chitinase Derived from Talaromyces flavus

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