Abstract
BackgroundLiquid 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.ResultsIn 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.ConclusionsThese 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.
Highlights
Liquid lipases are widely used to convert oil into biodiesel
Semi‐rational design of N‐glycosylation sites in Rhizomucor miehei lipase with propeptide (ProRML) In a previous study, we confirmed that ProRML was N-glycosylated and expressed in P. pastoris and that N-glycan plays a key role in lipase activity and stability
To study the effect of N-glycosylation on ProRML, five N-glycosylation sites were introduced in the α-helix by amino acid substitution: N93 (T93N), N115 (D115N), N183 (L183N), N218 (G218N), and N260 (V260N)
Summary
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. Biodiesel (fatty acid methyl ester, FAME) is an ideal substitute for fossil fuels as a novel renewable energy source, which comprises low-sulphur, low-carbon, and high-hexadecane contents [1]. The activity, thermostability, and methanol tolerance of lipase are the main reasons that hinder its effective application in the biodiesel field, resulting in prolonged reaction times and increased production costs [5]. Improving the specific tolerance and catalytic efficiency of the enzyme is key to further expanding the application of lipase. A semi-rational design approach based on the protein structure is an effective way to improve the properties of enzymes that are based on the modification of several key residues and domains in terms of the structure–function relationship of the enzyme [6,7,8]
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