融合荧光蛋白的纤维素酶表达与性质分析  

Expression and characterization of cellulases fusing fluorescent protein

作　　者：周姗娜 胡艳梅 乔琦茗 李华南 江正兵 ZHOU Shanna;HU Yanmei;QIAO Qiming;LI Huanan;JIANG Zhengbing(State Key Laboratory of Biocatalysis and Enzyme Engineering,School of Life Sciences,Hubei University,Wuhan 430062,China)

机构地区：[1]湖北大学生命科学学院省部共建生物催化与酶工程国家重点实验室,湖北武汉430062

出　　处：《生物加工过程》2023年第2期184-190,共7页Chinese Journal of Bioprocess Engineering

基　　金：湖北省技术创新专项(2020BBA056、2019ABA117);湖北省中央领导地方科技发展专项(2018ZYYD034);武汉市科技局应用基础研究(20190207010111496);湖北省教育厅科学技术研究计划青年人才项目(201910701301002)。

摘　　要：纤维素酶是能特异性分解纤维素的一系列酶,被广泛应用于食品加工处理、衣物洗涤、农业及造纸等行业。纤维素酶通过不同的水解方式协同作用将纤维素水解为寡糖或可发酵糖。在将纤维素分解成寡糖的过程中,外切葡聚糖酶(CBH)和内切葡聚糖酶(EG)发挥了重要作用。目前关于两种酶协同作用机制和顺序尚未有明确的解释。本研究将嗜热毛壳菌来源的cbh和葡萄穗霉来源的eg分别与绿色荧光蛋白基因gfp和红色荧光蛋白基因mcherry进行融合,并电转入毕赤酵母X33进行异源表达,随后对这两种融合纤维素酶CBH-GFP和EG-MCHERRY进行了性质分析。结果表明:两种融合荧光蛋白的纤维素酶基因在毕赤酵母X33中实现了分泌表达。纯化的CBH-GFP和EG-MCHERRY的蛋白浓度与荧光强度呈正相关关系。对羧甲基纤维素钠(CMC-Na)、磷酸膨胀纤维素(PASC)、滤纸(FP)、微晶纤维素(Avicel)的活性测试结果显示,CBH-GFP对CMC-Na、PASC、Avicel均有催化活性,而EG-MCHERRY对CMC-Na、PASC具有催化活性,CBH-GFP对PASC有最高比酶活(1.1 U/mg);EG-MCHERRY对CMC-Na有最高比酶活(1.9 U/mg),对比原始纤维素酶CBH和EG对不同底物的活性,融合荧光蛋白的纤维素酶能保持85%以上的酶活。CBH-GFP和EG-MCHERRY最适反应温度分别为55和50℃,最适反应pH分别为6和5,与原始纤维素酶的相应性质一致。EG-MCHERRY和CBH-GFP蛋白浓度与其荧光强度呈线性正相关且性质相近,同时在pH为6、温度为50℃的情况下均能保持80%以上的酶活,因此可用于后续的纤维素酶协同反应机制的研究。Cellulases is are series of enzymes that can specifically decompose cellulose,and widely used in various industries such as food processing,laundry,agriculture,papermaking.Cellulases can synergistically hydrolyze cellulose into oligosaccharides or fermentable sugars in different ways.In the process of breaking down cellulose into oligosaccharides,exoglucanase(CBH) and endoglucanase(EG) play an important role.Currently,the mechanisms of synergy of these two enzymes has not been well explained.In this study,the cbh from Chaetomium thermophila and the eg from Stachybotrys chartarum were fused with the green fluorescent protein gene gfp and red fluorescent protein gene mcherry,respectively.The fusion genes were electrotransformed into Pichia pastoris X33 for heterologous expression.Subsequently,the properties of the two fusion cellulase CBH-GFP and EG-MCHERRY was analyzed.Results show that the two fusion genes were expressed in Pichia pastoris X33 by secretion.The concentration of CBH-GFP and EG-MCHERRY were positively correlated with their fluorescence.The activity test on carboxymethyl cellulose sodium(CMC-Na),phosphoric acid swollen cellulose(PASC),filter paper(FP),microcrystalline cellulose(Avicel) showed that CBH-GFP was active on CMC-Na,PASC,Avicel;EG-MCHERRY had catalytic activity on CMC-Na and PASC.CBH-GFP had the highest activity on PASC(1.1 U/mg);EG-MCHERRY had the highest activity on CMC-Na(1.9 U/mg).Comparing the enzymatic activity of the original cellulases CBH and EG on different substrates,CBH-GFP and EG-MCHERRY maintained 85% of the enzymatic activity.The optimal reaction temperatures of CBH-GFP and EG-MCHERRY were 55 and 50 ℃,and the optimal reaction pH was 6 and 5 respectively,which were consistent with the corresponding properties of cellulases without fusing fluorescent protein.The protein concentration of CBH-GFP and EG-MCHERRY was linearly positively correlated with their fluorescence intensity and their properties are similar.They could both maintain more than 80% activity at pH 6 and 50 ℃,

关 键 词：纤维素酶 绿色荧光蛋白-外切葡聚糖酶 红色荧光蛋白-内切葡聚糖酶 荧光蛋白 毕赤酵母 协同催化 

分 类 号：Q556[生物学—生物化学]