Enzymatic preparation of diacylglycerols: lipase screening, immobilization, characterization and glycerolysis performance.

Publisher: John Wiley & Sons Country of Publication: England NLM ID: 0376334 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1097-0010 (Electronic) Linking ISSN: 00225142 NLM ISO Abbreviation: J Sci Food Agric Subsets: MEDLINE

Publication: <2005-> : Chichester, West Sussex : John Wiley & Sons
Original Publication: London, Society of Chemical Industry.

Enzymes, Immobilized*/chemistry ; Enzymes, Immobilized*/metabolism ; Lipase*/chemistry ; Lipase*/metabolism ; Diglycerides*/chemistry ; Diglycerides*/metabolism ; Fungal Proteins*/chemistry ; Fungal Proteins*/metabolism ; Enzyme Stability* ; Biocatalysis*; Glycerol/chemistry ; Glycerol/metabolism ; Molecular Docking Simulation ; Eurotiales/enzymology ; Kinetics

Backgrounds: Glycerolysis, with its advantages of readily available raw materials, simple operation, and mild reaction conditions, is a primary method for producing diacylglycerol (DAG). However, enzymatic glycerolysis faces challenges such as high enzyme costs, low reuse efficiency, and poor stability. The study aims to develop a cost-effective immobilized enzyme by covalently binding lipase to pre-activated carriers through the selection of suitable lipases, carriers, and activating agents. The optimization is intended to improve the glycerolysis reaction for efficient DAG production.
Results: Lipase CN-TL (from Thermomyces lanuginosus) was selected through glycerolysis reaction and molecular docking to catalyze the glycerolysis reaction. Optimizing the immobilization method by covalently binding CN-TL to poly(ethylene glycol) diglycidyl ether (PEGDGE)-preactivated resin LX-201A resulted in the preparation of the immobilized enzyme TL-PEGDGE-LX. The immobilized enzyme retained over 90% of its initial activity after five consecutive reactions, demonstrating excellent reusability. The DAG content in the product remained at 84.8% of its initial level, further highlighting the enzyme's potential for reusability and its promising applications in the food and oil industries.
Conclusions: The immobilized lipase TL-PEGDGE-LX, created by covalently immobilizing lipase CN-TL on PEGDGE-preactivated carriers, demonstrated broad applicability and excellent reusability. This approach offers an economical and convenient immobilization strategy for the enzymatic glycerolysis production of DAG. © 2024 Society of Chemical Industry.
(© 2024 Society of Chemical Industry.)

Nicholson RA and Marangoni AG, Enzymatic glycerolysis converts vegetable oils into structural fats with the potential to replace palm oil in food products. Nature Food 1:684–692 (2020).
Zhong N, Chen W, Liu L and Chen H, Immobilization of Rhizomucor miehei lipase onto the organic functionalized SBA‐15: their enzymatic properties and glycerolysis efficiencies for diacylglycerols production. Food Chem 271:739–746 (2019).
Gibon V, Enzymatic interesterification of oils. Lipid technology 23:274–277 (2011).
Kadhum AAH and Shamma MN, Edible lipids modification processes: a review. Crit Rev Food Sci Nutr 57:48–58 (2017).
Sun S and Li K, Biodiesel production from phoenix tree seed oil catalyzed by liquid lipozyme TL100L. Renew Energy 151:152–160 (2020).
Elgharbawy AS, Sadik WA, Sadek OM and Kasaby MA, Glycerolysis treatment to enhance biodiesel production from low‐quality feedstocks. Fuel 284:118970 (2021).
Mamtani K, Shahbaz K and Farid MM, Glycerolysis of free fatty acids: a review. Renew Sustain Energy Rev 137:110501 (2021).
Lee Y‐Y, Tang T‐K, Phuah E‐T, Tan C‐P, Wang Y, Li Y et al., Production, safety, health effects and applications of diacylglycerol functional oil in food systems: a review. Crit Rev Food Sci Nutr 60:2509–2525 (2020).
Tang KHD, Lock SSM, Yap P‐S, Cheah KW, Chan YH, Yiin CL et al., Immobilized enzyme/microorganism complexes for degradation of microplastics: a review of recent advances, feasibility and future prospects. Sci Total Environ 832:154868 (2022).
Garmroodi M, Mohammadi M, Ramazani A, Ashjari M, Mohammadi J, Sabour B et al., Covalent binding of hyper‐activated Rhizomucor miehei lipase (RML) on hetero‐functionalized siliceous supports. Int J Biol Macromol 86:208–215 (2016).
Abaházi E, Lestál D, Boros Z and Poppe L, Tailoring the spacer arm for covalent immobilization of Candida Antarctica lipase b—thermal stabilization by bisepoxide‐activated aminoalkyl resins in continuous‐flow reactors. Molecules 21:767 (2016).
Palomo JM, Muñoz G, Fernández‐Lorente G, Mateo C, Fernández‐Lafuente R and Guisán JM, Interfacial adsorption of lipases on very hydrophobic support (octadecyl–Sepabeads): immobilization, hyperactivation and stabilization of the open form of lipases. J Mol Catal B: Enzym 19:279–286 (2002).
Pota G, Bifulco A, Parida D, Zhao S, Rentsch D, Amendola E et al., Tailoring the hydrophobicity of wrinkled silica nanoparticles and of the adsorption medium as a strategy for immobilizing lipase: an efficient catalyst for biofuel production. Microp Mesop Mat 328:111504 (2021).
Chen W, He L, Song W, Huang J and Zhong N, Encapsulation of lipases by nucleotide/metal ion coordination polymers: enzymatic properties and their applications in glycerolysis and esterification studies. J Sci Food Agric 102:4012–4024 (2022).
Dos Santos JC, Rueda N, Torres R, Barbosa O, Goncalves LR and Fernandez‐Lafuente R, Evaluation of divinylsulfone activated agarose to immobilize lipases and to tune their catalytic properties. Process Biochem 50:918–927 (2015).
Rodrigues RC, Virgen‐Ortíz JJ, Dos Santos JC, Berenguer‐Murcia Á, Alcantara AR, Barbosa O et al., Immobilization of lipases on hydrophobic supports: immobilization mechanism, advantages, problems, and solutions. Biotechnol Adv 37:746–770 (2019).
Feng K, Huang Z, Peng B, Dai W, Li Y, Zhu X et al., Immobilization of aspergillus Niger lipase onto a novel macroporous acrylic resin: stable and recyclable biocatalysis for deacidification of high‐acid soy sauce residue oil. Bioresour Technol 298:122553 (2020).
Mateo C, Palomo JM, Fernandez‐Lorente G, Guisan JM and Fernandez‐Lafuente R, Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb Technol 40:1451–1463 (2017).
Kim S, Thiessen PA, Bolton EE, Chen J, Fu G, Gindulyte A et al., PubChem substance and compound databases. Nucleic Acids Res 44:D1202–D1213 (2016).
Ravindranath PA, Forli S, Goodsell DS, Olson AJ and Sanner MF, AutoDockFR: advances in protein‐ligand docking with explicitly specified binding site flexibility. PLoS Comput Biol 11:e1004586 (2015).
Zhang H, Secundo F, Sun J and Mao X, Advances in enzyme biocatalysis for the preparation of functional lipids. Biotech Adv 61:108036 (2022).
Sun X, Zou S, Xie X, Wang Y and Zhang Z, Immobilization of Lecitase® Ultra for production of flaxseed oil‐based diacylglycerols by glycerolysis and its operational stability. LWT 183:114964 (2023).
Kulkarni NH, Muley AB, Bedade DK and Singhal RS, Cross‐linked enzyme aggregates of arylamidase from Cupriavidus oxalaticus ICTDB921: process optimization, characterization, and application for mitigation of acrylamide in industrial wastewater. Bioprocess Biosyst Eng 43:457–471 (2020).
Li G, Chen J, Ma X, Zhang Z, Liu N and Wang Y, Enzymatic preparation and facile purification of medium‐chain, and medium‐and long‐chain fatty acid diacylglycerols. LWT 92:227–233 (2018).
Wang X, He L, Huang J and Zhong N, Immobilization of lipases onto the halogen & haloalkanes modified SBA‐15: enzymatic activity and glycerolysis performance study. Int J Biol Macromol 169:239–250 (2021).
Silva JMF, Dos Santos KP, Dos Santos ES, Rios NS and Gonçalves LRB, Immobilization of Thermomyces lanuginosus lipase on a new hydrophobic support (streamline phenyl™): strategies to improve stability and reusability. Enzyme Microb Technol 163:110166 (2023).
Noro J, Castro TG, Cavaco‐Paulo A and Silva C, Substrate hydrophobicity and enzyme modifiers play a major role in the activity of lipase from Thermomyces lanuginosus. Cat Sci Technol 10:5913–5924 (2020).
Liu D‐M, Chen J and Shi Y‐P, Advances on methods and easy separated support materials for enzymes immobilization. TrAC Trends Anal Chem 102:332–342 (2018).
Zhao X, Qi F, Yuan C, Du W and Liu D, Lipase‐catalyzed process for biodiesel production: enzyme immobilization, process simulation and optimization. Renew Sustain Energy Rev 44:182–197 (2015).
Zhong N, Li Y, Cai C, Gao Y, Liu N, Liu G et al., Enhancing the catalytic performance of Candida Antarctica lipase B by immobilization onto the ionic liquids modified SBA‐15. Eur J Lipid Sci Technol 120:1700357 (2018).
Zhao X, Zhao F and Zhong N, Production of diacylglycerols through glycerolysis with SBA‐15 supported Thermomyces lanuginosus lipase as catalyst. J Sci Food Agric 100:1426–1435 (2020).
Barbosa O, Torres R, Ortiz C and Fernandez‐Lafuente R, The slow‐down of the CALB immobilization rate permits to control the inter and intra molecular modification produced by glutaraldehyde. Process Biochem 47:766–774 (2012).
Shi XY, Guo ZH and Chen J, Cellulose filter paper immobilized α‐glucosidase and its application to screening inhibitors from traditional Chinese medicine. J Sep Sci 45:2724–2733 (2022).
Naga N, Sato M, Mori K, Nageh H and Nakano T, Synthesis of network polymers by means of addition reactions of multifunctional‐amine and poly (ethylene glycol) diglycidyl ether or diacrylate compounds. Polymers 12:2047 (2020).
Chai H‐Y, Lam S‐M and Sin J‐C, Green synthesis of magnetic Fe‐doped ZnO nanoparticles via Hibiscus rosa‐sinensis leaf extracts for boosted photocatalytic, antibacterial and antifungal activities. Mater Lett 242:103–106 (2019).
Miao Q, Zhang C, Zhou S, Meng L, Huang L, Ni Y et al., Immobilization and characterization of pectinase onto the cationic polystyrene resin. ACS Omega 6:31683–31688 (2021).
Ladole MR, Mevada JS and Pandit AB, Ultrasonic hyperactivation of cellulase immobilized on magnetic nanoparticles. Bioresour Technol 239:117–126 (2017).
Li J, Shi X, Qin X, Liu M, Wang Q and Zhong J, Improved lipase performance by covalent immobilization of Candida Antarctica lipase B on amino acid modified microcrystalline cellulose as green renewable support. Colloids Surf B Biointerfaces 235:113764 (2024).
Guo H, Lei B, Yu J, Chen Y and Qian J, Immobilization of lipase by dialdehyde cellulose crosslinked magnetic nanoparticles. Int J Biol Macromol 185:287–296 (2021).
Feng T, Shi J, Yue K, Xia J, Yan L, Suo H et al., Covalent organic framework immobilized lipase for efficient green synthesis of 1, 3‐dioleoyl‐2‐palmitoylglycerol. Molecular Cata 552:113671 (2024).
Cao Y‐P, Zhi G‐Y, Han L, Chen Q and Zhang D‐H, Biosynthesis of benzyl cinnamate using an efficient immobilized lipase entrapped in nano‐molecular cages. Food Chem 364:130428 (2021).

2024A04J3254 Bureau of Science and Information of Guangzhou; 32272341 National Natural Science Foundation of China; 2022B0202010003 Department of Science and Technology of Guangdong Province

Keywords: catalytic stability; diacylglycerol; glycerolysis; lipase immobilization

0 (Enzymes, Immobilized)
EC 3.1.1.3 (Lipase)
0 (Diglycerides)
0 (Fungal Proteins)
PDC6A3C0OX (Glycerol)

Thermomyces lanuginosus

Date Created: 20240911 Date Completed: 20241211 Latest Revision: 20241211

20241211

10.1002/jsfa.13872

39258418