宏基因组来源果胶酸裂解酶在毕赤酵母中的表达及应用  

Expression and Application of a Pectate Lyase from Metagenome in Pichia pastoris

作　　者：董俊帅 秦星[2] 张志伟[1] 张宇宏[2] 张伟[2] DONG Junshuai;QIN Xing;ZHANG Zhiwei;ZHANG Yuhong;ZHANG Wei(College of Forestry, Shanxi Agricultural University, Shanxi Taigu 030801, China;Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China)

机构地区：[1]山西农业大学林学院,山西太谷030801 [2]中国农业科学院生物技术研究所,北京100081

出　　处：《生物技术进展》2018年第3期237-245,共9页Current Biotechnology

基　　金：国家863计划项目(2012AA022207)资助

摘　　要：果胶酸裂解酶可用于含果胶废水的处理、纸浆漂白以及棉麻纺织品的生物精炼等。基于高通量宏基因组测序技术,从富含果胶土壤宏基因组中挖掘得到一个果胶酸裂解酶基因pela。将pela连接表达载体p PIC9转化毕赤酵母Pichia pastoris GS115。在3 L发酵罐水平,甲醇诱导10 h后,培养基中果胶酸裂解酶活力达到10.8 U/m L。重组PELA的最适温度为45℃,最适pH为9.0,在pH 7.5~11.0具有良好的稳定性。PELA的比活力为244.12 U/mg,以聚半乳糖醛酸为底物时催化反应的Km和Vmax分别为0.26 mg/m L和488.40μmol/min·mg。EDTA及金属离子Cd^(2+)、Zn^(2+)、Mn^(2+)、Cu^(2+)、Fe^(3+)能够高度抑制酶的活性,1 mmol/L Ca^(2+)和2 mmol/L K^+对酶活力有促进作用。将重组PELA作用于苎麻纤维4 h后,纤维质量损失率达到9.2%,苎麻纤维分散度和白度都明显提高。结果表明从宏基因组来源的果胶酸裂解酶PELA在苎麻脱胶中具有良好的应用潜力。Pectate lyase can be used for the treatment of wastewater containing pectin,pulp bleaching,and bio-refining of cotton and linen textiles. In this study,a pectate lyase gene pela was obtained from pectin-rich soil based on high throughput metagenomic sequencing technology. The pela was ligated into p PIC9 vector and transformed into Pichia pastoris GS115. The PELA activity was 10. 8 U/mL after induction with methanol for 10 h in 3 L fermentor. The optimal temperature and p H of recombinant PELA were 45℃ and 9. 0,respectively. PELA has good stability at pH 7. 5 - 11. 0. The specific activity of the recombinant enzyme was determined to be 244.12 U/mg. Use polygalacturonic acid as substrate,the kinetic parameters Kmand Vmaxwere determined to be 0.26 mg/mL and 488.40 μmol/min·mg,respectively. EDTA and metal ions Cd^2＋,Zn^2＋,Mn^2＋,Cu^2＋ and Fe^3＋ showed strongly inhibitory effects on PELA activity,while the enzyme activity was enhanced by 1 mmol/L Ca^2＋and2 mmol/L K^＋. The recombinant PELA was applied to treat the ramie fiber. The percentage of weight loss was reached 9.2% after 4h treatment. The whiteness, dispersionramie and softness of ramie fiber were enhanced significantly. The PELA from metagenome has a good potential application for ramie degumming.

关 键 词：宏基因组 果胶酸裂解酶 毕赤酵母 苎麻 脱胶 

分 类 号：Q55[生物学—生物化学] Q78