新型GH45家族纤维素酶的智能挖掘及其在里氏木霉中的高效表达

doi:10.19586/j.2095-2341.2024.0189

生物技术进展 ›› 2025, Vol. 15 ›› Issue (2): 287-295.DOI: 10.19586/j.2095-2341.2024.0189

• 研究论文 • 上一篇

新型GH45家族纤维素酶的智能挖掘及其在里氏木霉中的高效表达

王瑾¹(), 白静², 赵晶², 田健², 张伟³, 王苑², 杨浩萌², 石美杉², 张贝贝¹, 徐欣欣³(), 黄火清²()

^1.青岛农业大学动物科技学院，山东青岛 266109
^2.中国农业科学院北京畜牧兽医研究所，北京 100193
^3.中国农业科学院生物技术研究所，北京 100081

收稿日期:2024-11-27 接受日期:2025-01-21 出版日期:2025-03-25 发布日期:2025-04-29
通讯作者: 徐欣欣,黄火清
作者简介:王瑾 E-mail: wjinwj@163.com
基金资助:
国家重点研发计划项目(2022YFA0912300);中国农业科学院基本科研业务费专项(Y2024QC15);国家肉鸡产业技术项目体系(CARS-41-G19)

Intelligent Mining of Novel GH45 Cellulases and its Efficient Expression in Trichoderma reesei

Jin WANG¹(), Jing BAI², Jing ZHAO², Jian TIAN², Wei ZHANG³, Yuan WANG², Haomeng YANG², Meishan SHI², Beibei ZHANG¹, Xinxin XU³(), Huoqing HUANG²()

^1.College of Animal Science and Technology，Qingdao Agricultural University，Shandong Qingdao 266109，China
^2.Beijing Institute of Animal Husbandry and Veterinary Medicine，Chinese Academy of Agricultural Sciences，Beijing 100193，China
^3.Biotechnology Research Institute，Chinese Academy of Agricultural Sciences，Beijing 100081，China

Received:2024-11-27 Accepted:2025-01-21 Online:2025-03-25 Published:2025-04-29
Contact: Xinxin XU,Huoqing HUANG

摘要/Abstract

摘要：

利用生物信息学方法和蛋白性质预测模型挖掘新型GH45家族纤维素酶，实现其在里氏木霉中的高效表达，以期为畜牧饲料加工提供新的酶源。通过Preoptem蛋白模型预测工具结合公共数据库Uniparc，筛选得到Thielaviopsis punctulate来源的GH45家族纤维素酶TpCel45A。将其编码基因进行合成后构建表达载体，通过原生质体转化里氏木霉菌株，表达纯化获得重组TpCel45A蛋白并进行酶学性质测定。结果显示，重组TpCel45A的最适温度为55 ℃，最适pH为5.5，并展现出良好的热稳定性，80 ℃处理15 min后仍保持84%以上的酶活。重组TpCel45A具有宽泛的pH稳定性，在pH 4.0~9.0范围内处理1 h后剩余酶活力达52%以上。动力学参数测定显示，TpCel45A的 K_m为8.04 mg·mL^-1, V_max为19.04 mg·mL^-1·min^-1。k_cat为377.1 s^-1，催化效率k_cat/K_m值为46.9。TpCel45A的挖掘、鉴定及异源表达为纤维素酶的工业应用提供了新的酶源。此外，TpCel45A优异的热稳定性和pH稳定性使其在高温制粒工艺和不同pH条件下具有良好的应用潜力，尤其在饲料加工和生物质转化利用等领域具有重要的应用价值。

关键词: 纤维素酶, GH45, 里氏木霉, 高效表达

Abstract:

Utilizing bioinformatics methods and protein property prediction models to mine novel GH45 family cellulases， and achieving their efficient expression in Trichoderma reesei， in order to provide a new enzyme source for animal feed processing. By using the Preoptem protein model prediction tool combined with the public database Uniparc， the GH45 family cellulase TpCel45A from Thielaviopsispunctulate was screened. We synthesized the encoded gene and construct an expression vector， and transformed it into a strain of Trichoderma reesei using protoplasts. The recombinant TpCel45A protein was obtained through expression and purification， and its enzymatic properties were determined. The research showed that the optimal temperature and pH for recombinant TpCel45A were 55 ℃ and 5.5， respectively， and exhibited good thermal stability. After 15 minutes of treatment at 80 ℃， the enzyme activity remained over 84%. In addition， TpCel45A exhibits good stability within the pH range of 4.0~9.0， with residual enzyme activity exceeding 52%. The determination of kinetic parameters showed that the K_m of the cellulase was 8.04 mg·mL^-1， V_maxis 19.04 mg·mL^-1·min^-1， the k_cat value is 377.1 s^-1， and the catalytic efficiency k_cat/K_m value is 46.9. The excavation， identification， and heterologous expression of TpCel45A provide a new enzyme source for the industrial application of cellulase. In addition， the excellent thermal stability and pH stability of TpCel45A make it have the potential for application in high-temperature granulation and different pH environments， and have important application value in fields such as feed proessing and biomass conversion utilization.

Key words: cellulase, GH45, Trichoderma reesei, efficient expression

中图分类号:

Q939.9

王瑾, 白静, 赵晶, 田健, 张伟, 王苑, 杨浩萌, 石美杉, 张贝贝, 徐欣欣, 黄火清. 新型GH45家族纤维素酶的智能挖掘及其在里氏木霉中的高效表达[J]. 生物技术进展, 2025, 15(2): 287-295.

Jin WANG, Jing BAI, Jing ZHAO, Jian TIAN, Wei ZHANG, Yuan WANG, Haomeng YANG, Meishan SHI, Beibei ZHANG, Xinxin XU, Huoqing HUANG. Intelligent Mining of Novel GH45 Cellulases and its Efficient Expression in Trichoderma reesei[J]. Current Biotechnology, 2025, 15(2): 287-295.

图/表 7

表1 已发表的GH45家族纤维素酶

Table 1 Published GH45 family cellulases

名称	来源物种	酶学特性	参考文献
STCE1	大孢圆孢菌（Staphylotrichum coccosporum）	最适pH 6.0；最适温度60 ℃	[15]
HrGH45	红鲍螺（Haliotis rufescens）	最适pH 7.0；最适温度45 ℃	[10]
TaCel45	沙质梭孢壳菌（Thielavia arenaria XZ7）	最适温度60 ℃；在90 ℃和100 ℃下处理1 h后，还剩余70%酶活	[11]
FpiGH45	红缘层孔菌（Fomitopsis pinicola）	最适pH 4.0，在pH 3.5~5.5内保持75%活性	[16]
MeCel45A	紫壳菜蛤（Mytilus edulis）	在4 ℃时可保持70%活性	[17]
rNMgh45	粗糙脉孢霉（Neurospora crassa）	最适pH 4.5，最适温度60~70 ℃，在pH 4.0~8.0可维持60%以上酶活，在40~80 ℃可维持60%以上酶活	[18]
TtLPMO9H	喜热梭孢壳（Thermothelomyces thermophilus）	最适pH 9.0~10.0；最适温度85 ℃	[19]
Cel45	嗜热古菌（Thermofilum adornatum）	最适pH 5.5~6.0；最适温度80 ℃	[20]
rEndo2	核盘菌（Sclerotinia sclerotiorum）	最适温度60 ℃，在70 ℃和80 ℃维持10 min后剩余50%活性	[21]
CTendo45	嗜热主壳霉（Chaetomium thermophilum）	70 ℃、pH 4.0条件下活性最高，在70 ℃和80 ℃分别可保留78.9%和65.6%活性	[22]
FpCel45	瘤盖拟层孔菌（Fomitopsis palustris）	80 ℃处理1 h后仍保留91.4%以上活性	[23]

表2 引物名称及序列信息

Table 2 Primers used in this study

名称	序列（5' $→$ 3'）
TpCel45A-F	5-′GCGTCCGGAAGCGGCGTCAC-3′
TpCel45A-R	5-′GTGCGTCAGGCTTTCGCCACGAGCCTA⁃TGAAGCTGTAGGCTGGCACTG-3′
TcbhI-F	5-′GCTCCGTGGCGAAAGCCTG-3′
TrEGIISP-R	5-′GATGCGCAGTCCGCGGTTGAC-3′

表2 引物名称及序列信息

Table 2 Primers used in this study

名称	序列（5' $→$ 3'）
TpCel45A-F	5-′GCGTCCGGAAGCGGCGTCAC-3′
TpCel45A-R	5-′GTGCGTCAGGCTTTCGCCACGAGCCTA⁃TGAAGCTGTAGGCTGGCACTG-3′
TcbhI-F	5-′GCTCCGTGGCGAAAGCCTG-3′
TrEGIISP-R	5-′GATGCGCAGTCCGCGGTTGAC-3′

图1 里氏木霉重组菌株SUS7/TpCel45A的PCR验证注：M—DNA maker；泳道1~22：SUS7—TpCel45A 转化子菌落PCR验证条带；泳道23：阳性质粒对照；泳道24：阴性对照。

Fig. 1 PCR validation of recombinant strain SUS7/TpCel45A of Trichoderma reesei

图2 SDS-PAGE检测纯化和浓缩后的重组TpCel45A注：泳道1—蛋白质分子量marker；泳道2—原始菌株SUS7发酵上清；泳道3—TpCel45A粗酶液；泳道4—纯化后的TpCel45A蛋白。

Fig. 2 SDS-PAGE detection of purified and concentrated recombinant TpCel45A

图3 重组TpCel45A的最适温度和热稳定性A：不同温度下的酶活力；B：60~80 ℃处理后酶活力的变化；C：重组TpCel45A的三维结构图；黄色部分呈现出半胱氨酸的位置

Fig. 3 The optimal temperature and thermal stability of recombinant TpCel45A

图4 重组TpCel45A的最适pH和pH稳定性A：不同pH条件下的酶活力；B：重组TpCel45A的pH 稳定性

Fig. 4 The optimal pH and pH stability of recombinant TpCel45A

图5 重组TpCel45A的动力学参数

Fig. 5 Dynamics parameters of recombinant TpCel45A

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新型GH45家族纤维素酶的智能挖掘及其在里氏木霉中的高效表达

Intelligent Mining of Novel GH45 Cellulases and its Efficient Expression in Trichoderma reesei

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