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

作　　者：张曼玉 董嘉诚 苟福凡 弓朝晖 刘倩[2] 孙文良[2] 孔臻[3] 郝捷 王敏 田朝光[2] 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(College of Biotechnology,Tianjin University of Science and Technology,Tianjin 300457;Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308;Key Laboratory of Tobacco Processing,Zhengzhou Tobacco Research Institute of CNTC,Zhengzhou 450001;Inner Mongolia Kunming Cigarette Co.,Ltd.,Hohhot 010020)

机构地区：[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

出　　处：《生物技术通报》2024年第9期291-300,共10页Biotechnology Bulletin

基　　金：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)。

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RTqPCR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100-300μg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。【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 100-300μg 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 resourc

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 

分 类 号：Q78[生物学—分子生物学] TS414[农业科学—烟草工业]