关键词: 宏基因组, 果胶酸裂解酶, 毕赤酵母, 苎麻, 脱胶

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