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In the present study, the immobilized SMG1-F278N lipase was employed for the esterification of 1,3-propylene glycol with oleic acid. This was the first report of using mono- and diacylglycerol lipase for the production of propylene glycol monoesters (PGME). It was found that immobilized SMG1-F278N preferred 1,3-propylene glycol than 1,2-propylene glycol. Molecular docking of 1,2-propylene glycol and 1,3-propylene glycol into SMG1-F278N suggested that the 1,3-propylene glycol preferentially binds to the active pocket of SMG1-F278N as compared to 1,2-propylene glycol. The maximum 1,3-propylene glycol monooleate content of 70.67% was obtained under the reaction conditions of 1,3-propylene glycol/oleic acid ratio of 5:1 (mol/mol), enzyme loading of 7.5% (w/w, with respect to total substrates), and water addition of 7% (w/w, with respect to total substrates) at 30°C. The present work offers insights into the selectivity of immobilized SMG1-F278N towards 1,2-propylene glycol and 1,3-propylene glycol, and suggests the extended applications of immobilized SMG1-F278N for industrial purpose. Practical applications: To our knowledge, the production of PGME using mono- and diacylglycerol lipases was reported for the first time. This study could contribute to develop potential applications of immobilized SMG1-F278N in industries. 1,2-Propylene glycol/1,3-propylene glycol docked into the catalytic pocket of SMG1-F278N, esterification of 1,2-propylene glycol/1,3-propylene glycol with oleic acid catalyzed by immobilized SMG1-278N and optimization of production of 1,3-propylene glycol monooleate.

T1 lipase is a potential biocatalyst for industrial application since it is highly thermostable and displays optimal activity at 60–75°C. Structural analysis of T1 lipase shows that its lid region undergoes a spatial displacement along with a distinct secondary structure reorganization upon activation. To study structure/function of this atypical lid, we performed site-directed mutagenesis on the hydrophobic residues in the lid region. These residues were mutated to hydrophilic ones and biochemical properties of mutants were investigated. Results showed that F181 might be an important residue for enzyme-substrate binding. Mutants A186S and A190S had 35–50% increase in catalytic efficiencies compared to wild-type T1, without compromising their functions at high temperatures. In general, mutagenesis did not cause large changes to chain-length preference in T1 lipase. Mutants A186S and V187N were inactive towards long-chain pNP esters (p-Nitrophenyl stearate) and V187N showed lower activity towards long-chain triacylglycerols than wt T1, which makes it a potential catalyst in dairy industry. Thermostability of mutants were affected at different extent due to the influence of hydrophobic contact between the lid and the protein core. These findings not only shed light on the lipase structure/function relationship but also lay the framework for further engineering to gain more potent, stable, and selective lipases. Practical applications: A thermostable T1 lipase owns an atypical lid and hydrophobic residues in the lid influence its catalytic properties. Here, we screened mutants A186S and A190S with 35–50% increase in catalytic efficiency, which have industrial application in the modification of fats and oils. Moreover, mutant V187N showed lower activity towards long-chain TAGs than wt T1. This mutant can be a potential biocatalyst in dairy industry which promote generation of short-chain fatty acids from milk fats to increase flavors in dairy products. The lid region of thermostable T1 lipase undergoes a spatial displacement along with a distinct secondary structure reorganization upon activation (PDB ID: 2DSN, 2W22 [16,17]). Mutagenesis on hydrophobic residues in the lid affect catalytic properties of T1 lipase.

Enzymatic activity is important characteristic of enzymes for industrial application, which can be improved by protein engineering. PLA1 is a biocatalyst applied in phospholipid modification for its phospholipase activity and had obtained board attention for its application in oil degumming. Bioinformatics analysis suggested that three charged residues, R81, R84, and E87 located in the lid of the protein, may affect the conformational change of lid thus influence the activity of the enzyme. In the current study, mutagenesis of these residues was conducted with protein engineering. Five mutants such as R81A, R84A, R84M, R84K, and E87Q with higher phospholipase activity were screened out. Biochemical properties analysis showed that all of them had identical optimal pH value with wild-type, while the optimal temperature was decreased to be 50°C and the kcat/KM was improved. Degumming soybean oil, three of five mutants, R81A, R84M, and E87Q, decreased phosphorous content lower than 8.3 mg/kg within 3 h, which was highly improved compared with wild-type. R84M decrease phosphorus content less than 5 mg/kg within 5 h. These findings not only permit optimization of enzyme performance in degumming but also shed light on the application of bioinformatics techniques and protein engineering techniques on industry application. Practical applications: The results support that design proteins according to bioinformatics and protein engineering is desirable and the phospholipase with higher activity is more suitable for oil degumming. The phospholipase activity of PLA1 can be improved by protein engineering based on bioinformatics analysis. With its properties of hydrolyzing sn-1 position ester bond of phospholipids, nonhydratable phospholipids were converted into their hydratable forms, PLA1 can be applied in oil degumming. PLA1 with higher phospholipase activity is more likely to suitable for oil degumming since it could decrease the phosphorous content of oil lower than 10 mg/kg within shorter reaction, which showed great potencial for degumming application.