Enhanced activity and stability of Rhizomucor miehei lipase by mutating N-linked glycosylation site and its application in biodiesel production. (15th November 2021)

• Record Type: Journal Article
• Authors: Tian, Miao; Fu, Junying; Wang, Zhiyuan; Miao, Changlin; Lv, Pengmei; He, Dong; Li, Zhibing; Liu, Tao; Li, Ming; Luo, Wen
• Is Part Of: Fuel. Volume 304(2021)
• Publication Date: 2021-11-15
• Subjects: Rhizomucor miehei lipase -- N-linked glycosylation site -- Thermostability -- Methanol tolerance -- Biodiesel; Fuel -- Periodicals; Coal -- Periodicals; Coal; Fuel; Periodicals; 662.6
• DOI: 10.1016/j.fuel.2021.121514
• Languages: English
• ISSNs: 0016-2361

Abstract

Highlights: The deglycosylation of RML enhanced activity and stability for biodiesel production. The enzyme activity of N9P was 22 times higher than that of WT. The mutant N59H retained 69.66 % activity after incubation in 60% methanol for 1 h. N59H achieved 99.84 % FAME yield in one-pot production of biodiesel. Mutations to basic amino acids were conducive to improving the properties of RML. Abstract: Engineering a lipase with high activity, thermostability, and methanol resistance is of great significance for biodiesel production. To this end, a semi-rational evolutionary method involving site-directed saturation mutagenesis at the N-linked glycosylation site of Rhizomucor miehei lipase has been reported. The enzyme activity of all mutants was improved, particularly N9P, the hydrolytic activity of which was 22 times that of the wild-type Rhizomucor miehei lipase with propeptide. Thermostability and tolerance of all the mutants in 30% methanol were improved, except for N9P. Upon mutation, when the asparagine (N) residue at position 59 was substituted with the charged basic amino acids histidine and lysine, the mutants N59H and N59K were obtained, respectively, both of which showed better performance than the other mutants. During biotransformation of colza oil to biodiesel with one-shot addition of methanol, N59H yielded 87.83% of fatty acid methyl esters in 24 h, a yield significantly higher than obtained using non-glycosylated N9A/N59A (9.49%). Owing to its excellent methanol tolerance and productivity, the genetically engineered mutant N59H has excellent potential for commercial one-pot biodiesel production.