Improved methanol tolerance of Rhizomucor miehei lipase based on N‑glycosylation within the α-helix region and its application in biodiesel production

Abstract

Background Liquid lipases are widely used to convert oil into biodiesel. Methanol-resistant lipases with high catalytic activity are the first choice for practical production. Rhizomucor miehei lipase (RML) is a single-chain α/β-type protein that is widely used in biodiesel preparation. Improving the catalytic activity and methanol tolerance of RML is necessary to realise the industrial production of biodiesel. Results In this study, a semi-rational design method was used to optimise the catalytic activity and methanol tolerance of ProRML. After N-glycosylation modification of the α-helix of the mature peptide in ProRML, the resulting mutants N218, N93, N115, N260, and N183 increased enzyme activity by 66.81, 13.54, 10.33, 3.69, and 2.39 times than that of WT, respectively. The residual activities of N218 and N260 were 88.78% and 86.08% after incubation in 50% methanol for 2.5 h, respectively. In addition, the biodiesel yield of all mutants was improved when methanol was added once and reacted for 24 h with colza oil as the raw material. N260 and N218 increased the biodiesel yield from 9.49% to 88.75% and 90.46%, respectively. Conclusions These results indicate that optimising N-glycosylation modification in the α-helix structure is an effective strategy for improving the performance of ProRML. This study provides an effective approach to improve the design of the enzyme and the properties of lipase mutants, thereby rendering them suitable for industrial biomass conversion.

Rational Design of Lipase ROL to Increase Its Thermostability for Production of Structured Tags

“…Although the methanol tolerance of both ROL mutants is lower than that of Eversa (80% v / v ), we see a strong correlation between the thermostability and methanol tolerance of the ROL mutants. Such correlation has been observed in many other enzymes including lipases [ 23 , 24 , 25 , 26 , 27 ]. This finding suggests that further improvement to the thermostability of ROL might increase their tolerance towards organic solvent, which have the potential to be used for biodiesel production.…”

Fungal lipases as biocatalysts: A promising platform in several industrial biotechnology applications

“…Using the commercial lipase Novozyme ® 435, edible waste oil was used as raw material to produce biodiesel via enzymecatalyzed regeneration (Sorte et al, 2020). Moreover, M. Tian et al (2021) proved that Rhizomucor miehei lipase, which is a single-chain α/β-type protein, was found to be widely used in biodiesel production due to its catalytic activity and methanol tolerance. One year later, the same authors have used a semirational directed evolution method combined with N-glycosylation to enhance catalytic activity and methanol tolerance (M. Tian et al, 2022).…”

“…In such cases, recombinant forms are favored to conquer the demand of purification protocols (Lotti & Alberghina, 2007). For example, some new results have shown that N-glycosylation modification in the α-helix structure of Rhizomucor miehei lipase proved to be an effective strategy for improving the performance of this lipolytic enzyme in biodiesel applications (M. Tian et al, 2021).…”

Biomanufacturing Biotinylated Magnetic Nanomaterial via Construction and Fermentation of Genetically Engineered Magnetotactic Bacteria

“…The heightened awareness of actively mitigating climate change, reducing pollution, and improving food security has driven the need for biomanufacturing. Biosynthesis provides a key platform for bioproduction [ 1 ], including green synthesis of biodiesel [ 2 , 3 , 4 ], ethanol [ 5 , 6 ], organic acids [ 7 , 8 ], and biopolymers [ 9 ]. Compared with those, fossil-based to bio-based material production represents a more significant challenge in biosynthesis technology and high-volume output [ 1 ].…”