Abstract: 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 pPIC9 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 pH 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 Km and Vmax were determined to be 0.26 mg/mL and 488.40 μmol/min·mg, respectively. EDTA and metal ions Cd2+, Zn2+, Mn2+, Cu2+ and Fe3+ showed strongly inhibitory effects on PELA activity, while the enzyme activity was enhanced by 1 mmol/L Ca2+ and 2 mmol/L K+. The recombinant PELA was applied to treat the ramie fiber. The percentage of weight loss was reached 9.2% after 4 h treatment. The whiteness, dispersionramie and softness of ramie fiber were enhanced significantly. The PELA from metagenome has a good potential application for ramie degumming.

Key words: metagenome, pectate lyase, Pichia pastoris, ramie, degumming

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pPIC9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/mL。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/mL和488.40 μmol/min·mg。EDTA及金属离子Cd2+、Zn2+、Mn2+、Cu2+、Fe3+能够高度抑制酶的活性，1 mmol/L Ca2+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。

关键词: 宏基因组, 果胶酸裂解酶, 毕赤酵母, 苎麻, 脱胶