黄蓝状菌来源的新型几丁质酶的异源表达及鉴定

doi:10.19586/j.2095-2341.2025.0100

生物技术进展 ›› 2026, Vol. 16 ›› Issue (2): 380-387.DOI: 10.19586/j.2095-2341.2025.0100

• 研究论文 • 上一篇

黄蓝状菌来源的新型几丁质酶的异源表达及鉴定

王洋(), 于松尼(), 李雅华, 咸洪泉()

青岛农业大学生命科学学院，山东青岛 266109

收稿日期:2025-08-06 接受日期:2025-10-22 出版日期:2026-03-25 发布日期:2026-04-27
通讯作者: 咸洪泉
作者简介:王洋 E-mail : wy767466064@163.com
于松尼 E-mail : ysndyx000@163.com第一联系人：并列第一作者
基金资助:
中央引导地方科技发展资金(YDJX2025106);山东省重点研发计划项目(2022CXGC020710)

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

摘要/Abstract

摘要：

黄蓝状菌为菌寄生真菌，其产生的几丁质酶不仅在菌寄生过程中发挥着重要作用，也在植物病害生防和几丁质降解方面具有很高的应用价值。利用PCR技术从黄蓝状菌中克隆出无内含子的几丁质酶基因，采用毕赤酵母表达系统表达几丁质酶，并分析了几丁质酶的特性。结果表明,几丁质酶基因04446、06441开放阅读框大小分别为1 032和1 029 bp，成功构建了毕赤酵母表达载体pPIC9K/04446和pPIC9K/06441，获得高效表达的酵母工程菌株GS115/04446-45和GS115/06441-57；纯化的04446基因表达蛋白Tfch04446为36.8 kD、06441基因表达蛋白Tfch06441为37.2^{kD。几丁质酶Tfch04446最适反应温度为45 ℃，Tfch06441最适反应温度为50 ℃，且两者最适宜反应pH均为6。Tfch04446的K_m为2.27 mg·mL^-1，最大反应速度V_max为0.085 mg·mL^-1?min^-1，Tfch06441的K_m为1.52 mg·mL^-1，最大反应速度V_max为0.067 mg·mL^-1?min^-1。此外，金属离子Ca²⁺、Ba²⁺对几丁质酶Tfch04446活性有促进作用，NH₄⁺有一定的抑制作用；Ba²⁺对几丁质酶Tfch06441活性有促进作用，Na⁺、K⁺、Ca²⁺、NH₄⁺对其有抑制效果。研究结果可为黄蓝状菌及其几丁质酶的开发及利用奠定基础。}

关键词: 黄蓝状菌, 几丁质酶, 无内含子基因, 基因表达, 毕赤酵母, 酶学性质

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

中图分类号:

Q939.9

王洋, 于松尼, 李雅华, 咸洪泉. 黄蓝状菌来源的新型几丁质酶的异源表达及鉴定[J]. 生物技术进展, 2026, 16(2): 380-387.

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.

图/表 12

表1 几丁质酶基因扩增引物

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

图1 几丁质酶基因04446、06441 PCR扩增产物验证注：M—DL2000 maker；1—几丁质酶基因04446；2—几丁质酶基因06441。

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

图2 重组质粒pMD18-T/04446、pMD18-T/06441双酶切产物检测注：M—DL2000 maker；1—AvrⅡ、NotⅠ双酶切pMD18-T/0446；2—EcoRⅠ、NotⅠ双酶切pMD18-T/06441。

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

图3 多拷贝转化子的G418平板筛选A：04446酵母转化子G418平板筛选；B：06441酵母转化子G418平板筛选

Fig. 3 G418 plate screening of multicopy transformants

图4 酵母转化子的PCR鉴定A：04446酵母转化子；M—DL5000 marker；1—04446s、04446A为引物的PCR扩增产物；2—5'AOX1、3'AOX1为引物的PCR扩增产物；B：06441酵母转化子；3—06441s、06441A为引物的PCR扩增产物；4—以5'AOX1、3'AOX1为引物的PCR扩增产物

Fig. 4 PCR identification of yeast inverters

图5 几丁质酶Tfch04446、Tfch06441的SDS-PAGE分析注：M—蛋白分子量标准；1—Tfch04446纯化后的蛋白；2—Tfch06441纯化后的蛋白。

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

图6 Tfch04446、Tfch06441最适反应温度

Fig. 6 The optimal reaction temperature of Tfch04446 and Tfch06441

图7 Tfch04446、Tfch06441最适反应pH

Fig. 7 The optimal reaction pH of Tfch04446 and Tfch06441

表2 不同离子对Tfch04446活性的影响

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

表3 不同离子对Tfch06441活性的影响

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

图8 几丁质酶Tfch04446、Tfch06441酶解反应进程曲线

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

图9 几丁质酶Tfch04446、Tfch06441的Lineweaver-Burk曲线

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

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黄蓝状菌来源的新型几丁质酶的异源表达及鉴定

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

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