Ruiguo Cui,Xinyi Che,Lilang Li,Dongxiao Sun-Waterhouse,Jia Wang,Yonghua Wang
Lipase MAJ1 from Janibacter sp. is an sn-1,3 specific lipase for preparing long-medium-long (LML) type structured triacylglycerols (TAGs), but low optimum reaction temperature limited its application. Based on the structure built with Alphafold2 and MD simulation, we performed mutagenesis on the lid domain. The mutants L171A, A182P and P192C-T100C increased the optimum temperatures by 5 °C, 5 °C and 10 °C, respectively. The combination of three mutations (MAJ1 M4) increased the optimum temperature by 10 °C and a 5.5-fold increase in activity on tricaprylin. The residual activity of MAJ1 M4 was 70% after 60 min incubation at 45 °C, compared to no residual activity determined for MAJ1 wild-type in the same condition. The production of LML with the immobilized lipase MAJ1 M4-catalyzed interesterification was 57.1 mol% in 24 h compared with 44.3 mol% produced by MAJ1 wild-type. Moreover, the immobilized MAJ1 M4 retained 93% productivity after 13 cycles of synthesis. This mutagenesis design would pave the way for the application of MAJ1 in the food industry.
Ruiguo Cui,Long Xu,Dongming Lan,Bo Yang,Yonghua Wang
Our study offers a novel sn -1,3 specific lipase MAJ1 from marine member Janibacter sp. strain HTCC2649 for preparing long-medium-long (LML) type structured triacylglycerols (TAGs). Firstly, the resin ECR1030 was selected as a suitable support for the immobilization of lipase MAJ1. An efficient synthesis of LML-type structured TAGs by the immobilized lipase MAJ1-catalyzed interesterification of methyl palmitate and tricaprylin was studied in a solvent-free system. The reaction conditions, including substrate molar ratio, temperature and enzyme loading, were optimized. Under the optimum conditions (immobilized lipase MAJ1 of 45 U/g, substrate molar ratio of 4:1, temperature of 35 °C, reaction time of 24 h), the structured TAGs with double long chains (DLCST) were obtained in a yield of 44.3 mol%. Secondly, multi-dimensional mass spectrometry-based shotgun lipidomics (MDMS-SL) was employed to quantify each TAG positional isomer in DLCST. The content of 1,3-dipalmitoyl-2-capryloyl- sn -glycerol in DLCST was 97.6% determined by the MDMS-SL technology.
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