Presentation Information
[P02-210]Regioselectivity Screening of Fungal Lipases for Structured Lipid Production
○John Koh1, Vani Oktavia Sugiharto1, Elvis Teng Chua1, Carol Han1, Yan Ping Lim1, Lay Hiang Ling1, Bo Xue1, Wen Shan Yew1 (1. National University of Singapore (Singapore))
Keywords:
lipase regioslectivity,yeast surface display,whole-cell biocatalyst,structured lipids,acidolysis
Lipases (triacylglycerol hydrolase, EC 3.1.1.3) are a family of enzymes capable of catalysing a diverse range of reactions at lipid–water interfaces, including hydrolysis, esterification, transesterification, acidolysis, alcoholysis, and aminolysis. Catalytic properties such as broad substrate promiscuity and high regioselectivity towards the glycerol backbone of triacylglycerols (TAGs) have rendered them among the most industrially significant enzymes, finding widespread application in food processing, oleochemistry, pharmaceutical synthesis, flavour and fragrance production, and biodiesel manufacturing.
Lipase regioselectivity — the ability to distinguish between the sn-1,3 and sn-2 ester positions of TAGs — is a critical determinant in the enzymatic production of structured lipids (SLs) with tailored nutritional and functional properties. Candida (Pseudozyma) antarctica lipase A (CalA) is one of the few well-characterised lipases demonstrating pronounced sn-2 regioselectivity, making it highly attractive biocatalysts for lipid modification. However, the discovery and functional screening of novel CalA-like lipases remains laborious, largely due to the challenges associated with recombinant protein expression, purification, and immobilisation. Here, we present a yeast surface display system for the rapid functional screening of CalA-like lipases as whole-cell biocatalysts by acidolysis.
A library of CalA-like orthologues was identified through sequence similarity network (SSN) analysis, and were expressed on the surface of Komagataella phaffii (previously known as Pichia pastoris) via fusion to the Flo9 anchor protein. The whole-cell biocatalysts were directly used — without purification or immobilisation — in acidolysis, in which a free fatty acid exchanges an acyl group with an existing ester bond in a triacylglycerol.
This work holds significant promise for the accelerated discovery of biocatalysts for the synthesis of nutritionally valuable SLs, including human milk fat substitutes and medium-chain fatty acid-enriched lipids.
Lipase regioselectivity — the ability to distinguish between the sn-1,3 and sn-2 ester positions of TAGs — is a critical determinant in the enzymatic production of structured lipids (SLs) with tailored nutritional and functional properties. Candida (Pseudozyma) antarctica lipase A (CalA) is one of the few well-characterised lipases demonstrating pronounced sn-2 regioselectivity, making it highly attractive biocatalysts for lipid modification. However, the discovery and functional screening of novel CalA-like lipases remains laborious, largely due to the challenges associated with recombinant protein expression, purification, and immobilisation. Here, we present a yeast surface display system for the rapid functional screening of CalA-like lipases as whole-cell biocatalysts by acidolysis.
A library of CalA-like orthologues was identified through sequence similarity network (SSN) analysis, and were expressed on the surface of Komagataella phaffii (previously known as Pichia pastoris) via fusion to the Flo9 anchor protein. The whole-cell biocatalysts were directly used — without purification or immobilisation — in acidolysis, in which a free fatty acid exchanges an acyl group with an existing ester bond in a triacylglycerol.
This work holds significant promise for the accelerated discovery of biocatalysts for the synthesis of nutritionally valuable SLs, including human milk fat substitutes and medium-chain fatty acid-enriched lipids.
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