嗜热毁丝霉果胶酯酶MtCE12-1的克隆表达及其酶学性质和应用研究

doi:10.13560/j.cnki.biotech.bull.1985.2024-0336

生物技术通报 ›› 2024, Vol. 40 ›› Issue (9): 291-300.doi: 10.13560/j.cnki.biotech.bull.1985.2024-0336

嗜热毁丝霉果胶酯酶MtCE12-1的克隆表达及其酶学性质和应用研究

张曼玉¹^,²(), 董嘉诚¹^,², 苟福凡¹^,², 弓朝晖¹^,², 刘倩², 孙文良², 孔臻³, 郝捷⁴, 王敏¹, 田朝光²()

1.天津科技大学生物工程学院工业发酵微生物教育部重点实验室，天津 300457
2.中国科学院天津工业生物技术研究所低碳合成工程生物学重点实验室，天津 300308
3.中国烟草总公司郑州烟草研究院烟草行业烟草工艺重点实验室，郑州 450001
4.内蒙古昆明卷烟有限责任公司，呼和浩特 010020

收稿日期:2024-04-09 出版日期:2024-09-26 发布日期:2024-10-12
通讯作者: 田朝光，男，博士，研究员，研究方向：真菌合成生物学；E-mail: tian_cg@tib.cas.cn
作者简介:张曼玉，女，研究方向：真菌合成生物学；E-mail: zhangmy@tib.cas.cn
基金资助:
国家重点研发计划(2023YFC3402300);中国烟草总公司重点研发项目(110202202004);烟草工艺重点实验室项目(202022AWCX02)

Cloning, Expression, Characterization and Application of the Pectin Esterase MtCE12-1 from Myceliophthora thermophila

ZHANG Man-yu¹^,²(), DONG Jia-cheng¹^,², GOU Fu-fan¹^,², GONG Chao-hui¹^,², LIU Qian², SUN Wen-liang², KONG zhen³, HAO Jie⁴, WANG Min¹, TIAN Chao-guang²()

1. College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457
2. Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308
3. Key Laboratory of Tobacco Processing, Zhengzhou Tobacco Research Institute of CNTC, Zhengzhou 450001
4. Inner Mongolia Kunming Cigarette Co., Ltd., Hohhot 010020

Received:2024-04-09 Published:2024-09-26 Online:2024-10-12

摘要/Abstract

摘要：

【目的】挖潜新型果胶酯酶的酶资源，在嗜热毁丝霉（Myceliophthora thermophila）中高水平表达烟草生物质诱导显著上调的同源果胶酯酶，对其进行酶学性质研究，并探究该果胶酯酶在协同降解烟草生物质过程中的作用。【方法】利用RT-qPCR的方法分析嗜热毁丝霉在烟草生物质中果胶酯酶MtCE12-1的表达水平，通过2A肽介导的表达筛选系统，在嗜热毁丝霉菌株ATCC 42462中高表达果胶酯酶基因Mtce12-1，对高活性的阳性转化子进行产酶培养和蛋白纯化，表征了果胶酯酶MtCE12-1的酶学性质；以两种烟草生物质烟草压棒和烟草梗丝为底物，通过检测纤维素的剩余含量，分析了与纤维素酶的协同作用效果。【结果】与葡萄糖培养条件相比较，烟草生物质条件下果胶酯酶基因Mtce12-1的转录水平显著上调109-110倍，SDS-PAGE电泳分析、拷贝数和Western检测显示，重组蛋白MtCE12-1成功进行了表达和分泌，表达水平达到464.08 U/mL。该酶在75℃、pH 8.0时表现出最佳酶活力，在50-85℃和pH 7.0-9.0的范围内表现出较好的酶活力，具有良好的热稳定性。将100-300 µg的MtCE12-1添加至降解体系后，烟草生物质中纤维素降解效率分别提高了18.5%-30.7%和14.6%-30.5%。【结论】用2A肽介导的表达系统在嗜热毁丝霉中可以高效表达和纯化制备目标蛋白，碱性果胶酯酶MtCE12-1不仅具有良好的温度稳定性，在烟草生物质降解过程中也具有突出的作用效果，为烟草工业生产应用提供了潜在的优质酶资源。

关键词: 嗜热毁丝霉, 果胶酯酶, 基因表达, 酶学性质, 烟草生物质

Abstract:

【Objective】 To explore a novel pectin esterase enzyme, a homologous pectin esterase significantly up-regulated in tobacco biomass induction was highly expressed in Myceliophthora thermophila. The enzymatic properties of this pectin esterase were investigated, along with its role in assisting the degradation of tobacco biomass. 【Method】 RT-qPCR was used to analyze the expression of the pectin esterase gene Mtce12-1 in M. thermophila under the condition of tobacco biomass. Using a viral 2A peptide-mediated expression screening system, the pectin esterase gene Mtce12-1 was highly expressed in the M. thermophila wild-type strain ATCC 42462. The positive transformants overexpressing MtCE12-1-His-2A-GFP were subjected to recombinant enzyme production and protein purification, and the enzymatic properties of the pectin esterase MtCE12-1 were characterized. 【Result】 The transcription level of the pectin esterase gene Mtce12-1 was significantly up-regulated by about 109-110 folds under tobacco biomass conditions as compared to glucose condition. The sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, copy number determination and Western blotting indicated that the recombinant proteins were successfully expressed and secreted in M. thermophila, with expressions reaching as the 464.08 U/mL. The MtCE12-1 presented the highest activity at 75℃ and pH 8.0. It had good enzymatic activity in the range of 50-80℃ and pH 7.0-9.0 and demonstrated excellent thermal stability. The addition of 100-300 µg of MtCE12-1 to the degradation system resulted in an increase in cellulose degradation efficiency in tobacco bar and tobacco stem by 18.5%-30.7% and 14.6%-30.5%, respectively. 【Conclusion】 The use of the 2A peptide-mediated expression system in M. thermophila facilitates efficient target protein expression and purification. The alkaline pectin esterase MtCE12-1 not only shows good temperature stability but also demonstrates exceptional effectiveness in tobacco biomass degradation, providing potential high-quality enzyme resources for tobacco industry applications.

Key words: Myceliophthora thermophila, pectin esterase, gene expression, enzymatic properties, tobacco biomass

张曼玉, 董嘉诚, 苟福凡, 弓朝晖, 刘倩, 孙文良, 孔臻, 郝捷, 王敏, 田朝光. 嗜热毁丝霉果胶酯酶MtCE12-1的克隆表达及其酶学性质和应用研究[J]. 生物技术通报, 2024, 40(9): 291-300.

ZHANG Man-yu, DONG Jia-cheng, GOU Fu-fan, GONG Chao-hui, LIU Qian, SUN Wen-liang, KONG zhen, HAO Jie, WANG Min, TIAN Chao-guang. Cloning, Expression, Characterization and Application of the Pectin Esterase MtCE12-1 from Myceliophthora thermophila[J]. Biotechnology Bulletin, 2024, 40(9): 291-300.

图/表 6

表1 扩增靶序列引物

Table 1 Primers for amplifying the target sequence

名称 Primer	序列 DNA sequence（5'-3'）	用途 Application
Mtce12-1-RT-F	GACGACATTGTAGTGATC	基因表达分析
Mtce12-1-RT-R	TAGTGGTTGAAGGTGTAG	基因表达分析
actin-RT-F	AACGCTCCTGCCTTCTAC	基因表达分析
actin-RT-R	GTAACACCATCACCAGAGTC	基因表达分析
Mtce12-1-F	GCCAGTTTCGTTCTTCAGAACTAGTATGCGACCGTGGTCGACTCT	基因克隆
Mtce12-1-R	GTGGTGGTGGTGGTGGTGGATATCGAACACCTGAGGCACCGGGG	基因克隆
Ptef1-F	CTTCGACCCCTCCTCAAATCTTCTT	基因鉴定
TtrpC-R	GAGCTATTAAATCACTAGAAGGCAC	基因鉴定
Mtalp1-ko-F	TTCTGGCCTGCCCTTTTCTTTCAAC	基因鉴定
Mtalp1-ko-R	GCCCCTTCTTCCGAAAGGGGAGGTA	基因鉴定

图1 在烟草生物质培养条件下果胶酯酶MtCE12-1基因表达情况

Fig. 1 Expression of the Mtce12-1 gene for pectin esterase under conditions of the glucose and tobacco biomass

图2 嗜热毁丝霉重组表达果胶酯酶MtCE12-1 A：表达载体构建以及重组表达MtCE12-1的示意图；B：荧光显微镜观察重组菌株OE-MtCE12-1-GFP的孢子和菌丝荧光，B中的标尺为 10 µm

Fig. 2 Recombinant expressed pectin esterase MtCE12-1 in M. thermophile A: Schematic illustration of the expressed vector and recombinant expression of MtCE12-1. B: Microscopic fluorescence imaging of conidia and mycelia of the overexpressing strain OE-MtCE12-1-GFP. Scale bar 10 μm

图3 SDS-PAGE电泳、Western-blotting和拷贝数检测重组蛋白MtCE12-1的表达 A：上清液SDS-PAGE电泳分析 M: 蛋白Marker，泳道CK为空载体pAN52-1N转入宿主菌株的阴性对照，其它泳道为重组菌株OE-MtCE12-1-GFP；B：Western-blotting检测分析，泳道CK为阴性对照，其它泳道为重组菌株OE-MtCE12-1-GFP；C：Mtce12-1在重组菌株中的基因拷贝数分析；D：纯化后的蛋白SDS-PAGE电泳分析, M: 蛋白Marker，泳道1为重组菌株OE-MtCE12-1-GFP蛋白上清液，泳道2为MtCE12-1纯化样品

Fig. 3 Detecting the expression of recombinant protein MtCE12-1 by using the SDS-PAGE electrophoresis, Western blotting, and copy number A: SDS-PAGE analysis of secreted proteins from the CK and OE-MtCE12-1-GFP strains after 4-day of growth. M: the protein molecular weight markers. CK is used as the negative control for the transformation of the empty vector pAN52-1N into the host wild-type strain. B: Western blotting of culture supernatants from the strains and probed with anti-His antibody. C: Assay of Mtce12-1 copy number in overexpressing strains by RT-qPCR. D: SDS-PAGE analysis of the purified MtCE12-1. M: the protein molecular weight markers; Lane 1: the culture supernatants of the OE-MtCE12-1-GFP strain T8; Lane 2: the purified enzyme of MtCE12-1

图4 果胶酯酶MtCE12-1的酶学性质 A：pH对MtCE12-1酶活力的影响；B：温度对MtCE12-1酶活力的影响；C：pH对MtCE12-1酶活力稳定性的影响；D：温度对MtCE12-1酶活力稳定性的影响；E：底物特异性

Fig. 4 Enzymatic properties of pectin esterase MtCE12-1 A: Effect of pH on the enzyme activity of MtCE12-1. B: Effect of temperature on the enzyme activity of MtCE12-1. C: Effect of pH on the enzyme activity of MtCE12-1. D: Effect of temperature on the stability of MtCE12-1. E: Substrate specificity of MtCE12-1

图5 果胶酯酶MtCE12-1与纤维素酶协同降解烟草废弃生物质 A：MtCE12-1与纤维素酶协同降解烟草压棒；B：MtCE12-1与纤维素酶协同降解烟草梗丝。其中条件1为不添加酶液的对照组，条件2为只添加纤维素酶，条件3为添加100 μg MtCE12-1和纤维素酶，条件4为添加200 μg MtCE12-1和纤维素酶，条件5为添加300 μg MtCE12-1和纤维素酶。不同字母表示具有统计学意义（Tukey’s HSD, P < 0.05）

Fig. 5 Synergistic degradation of tobacco waste biomass by pectin esterase MtCE12-1 and cellulase A: Synergistic degradation of tobacco bar by MtCE12-1 and cellulase. B: Synergistic degradation of tobacco stem by MtCE12-1 and cellulase. 1: Control（no enzyme）; 2: only cellulase; 3: 100 μg MtCE12-1 and cellulase; 4: 200 μg MtCE12-1 and cellulase; 5: 300 μg MtCE12-1 and cellulase. Different letters are statistically significant（Tukey’s HSD, P < 0.05）

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嗜热毁丝霉果胶酯酶MtCE12-1的克隆表达及其酶学性质和应用研究

Cloning, Expression, Characterization and Application of the Pectin Esterase MtCE12-1 from Myceliophthora thermophila

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