Microplate spectrophotometric method for regioselective lipase screening using structured triglycerides with punicic...

Area of Science:

• Biocatalysis and Enzyme Technology
• Lipase Engineering and Application

Background:

• Lipase regioselectivity is critical for producing structured lipids and other industrial applications.
• Existing methods for assessing lipase regioselectivity are often inefficient or lack high-throughput capabilities.

Purpose of the Study:

• To develop a continuous, high-throughput assay for screening lipase regioselectivity at sn-1,3 and sn-2 positions on triacylglycerols (TAGs).
• To characterize the regioselectivity of lipases, including novel findings on specific enzymes.

Main Methods:

• A continuous assay measuring specific hydrolysis rates at primary (sn-1,3) and secondary (sn-2) positions of TAG derivatives.
• The assay utilizes the absorbance ratio of released punicic acid from specific TAG substrates (sn-POP and sn-OPO).
• Validation with known lipases and in silico analysis (molecular docking) to understand structural determinants of regioselectivity.

Main Results:

• The assay successfully differentiates between sn-1,3 and sn-2 regiospecific lipases.
• Recombinant Lipase 4 from Candida rugosa (rCRLip4) unexpectedly demonstrated significant sn-2 regioselectivity, surpassing rCALA.
• Structural analysis provided insights into the regioselectivity differences between CRLip4 and CRLip1.

Conclusions:

• The developed continuous assay is a versatile tool for screening various lipase regioselectivities (sn-1,3, sn-2, or non-regioselective).
• This assay has significant potential for optimizing biocatalytic processes, particularly in the production of structured lipids.
• The discovery of rCRLip4's sn-2 regioselectivity opens new avenues for enzyme engineering and industrial applications.