**4.1 Testing enzyme activity in vitro**

Lipid metabolism is composed of more than 120 enzymatic reactions. Validation of gene function related to lipid metabolism could be done by testing enzyme activity in vitro. Davies et al. have isolated CnLPAAT protein from immature coconut seeds and tested the LPAAT activity by adding Acyl-CoA and LPA as substrates [10].

Laurate is found enriched in sn-2, which indicates that a laurate-CoApreferring LPAAT is active during endosperm maturation. Davies et al. were able to detect such an enzyme from this tissue, which allowed Knutzon et al. [11] to perform protein purification and cloning of a cDNA encoding the 299-amino acid CLP protein from coconut. When expressed in *E. coli*, and using 12:0-LPA as an acceptor, this enzyme preferred medium-chain CoAs over 18:1-CoA as acyl donors. This is a direct evidence that in coconut endosperm, not only had the common fatty acid biosynthesis pathway been modified to produce almost entirely saturated medium chains but at least one enzyme of lipid biosynthesis (LPAAT) had been modified.
