Enhanced delignification of lignocellulosic substrates by Pichia GS115 expressed recombinant laccase

The Journal of General and Applied Microbiology

Atul P. Kolte¹  Vidya Pradeep Kumar²  Arindam Dhali³ 

[1] Animal Nutrition Division, National Institute of Animal Nutrition and Physiology;Bioenergetics and Environmental Sciences Division, National Institute of Animal Nutrition and Physiology;Sir M. Visvesvarya Institute of Technology

关键词: Laccase gene;    lignocellulose;    Pichia pastoris;    recombinant;    synthetic codon;   

DOI  :  10.2323/jgam.2017.11.006

学科分类：微生物学和免疫学

来源: Applied Microbiology, Molecular and Cellulrar Biosciences Research Foundation

【 摘 要 】

Utilization of energy-rich crop residues by ruminants is restricted by the presence of lignin, which is recalcitrant to digestion. Application of lignin degrading enzymes on the lignocellulosic biomass exposes the cellulose for easy digestion by ruminants. Laccases have been found to be considerably effective in improving the digestibility by way of delignification. However, laccase yields from natural hosts are not sufficient for industrial scale applications, which restricts their use. A viable option would be to express the laccase gene in compatible hosts to achieve higher production yields. A codon-optimized synthetic variant of Schizophyllum commune laccase gene was cloned into a pPIC9K vector and expressed in P. pastoris GS115 (his4) under the control of an alcohol oxidase promoter. Colonies were screened for G418 resistance and the methanol utilization phenotype was established. The transformant yielded a laccase activity of 344 U·mL–1 after 5 days of growth at 30°C (0.019 g·mL–1 wet cell weight). The laccase protein produced by the recombinant Pichia clone was detected as two bands with apparent molecular weights of 55 kDa and 70 kDa on SDS-PAGE. Activity staining on native PAGE confirmed the presence of bioactive laccase. Treatment of five common crop residues with recombinant laccase recorded a lignin loss ranging between 1.64% in sorghum stover, to 4.83% in finger millet, with an enhancement in digestibility ranging between 8.71% in maize straw to 24.61% in finger millet straw. Treatment with recombinant laccase was effective in enhancing the digestibility of lignocellulosic biomass for ruminant feeding through delignification. To date, a number of hosts have been adventured to produce laccase in large quantities, but, to our knowledge, there are no reports of the expression of laccase protein from Schizophyllum commune in Pichia pastoris, and also on the treatment of crop residues using recombinant laccase for ruminant feeding.

【 授权许可】

Unknown