Identification of a putative triacylglycerol lipase from papaya latex by functional proteomics

Latex from Caricaceae has been known since 1925 to contain strong lipase
activity. However, attempts to purify and identify the enzyme were not successful,
mainly because of the lack of solubility of the enzyme. Here, we
describe the characterization of lipase activity of the latex of Vasconcellea
heilbornii and the identification of a putative homologous lipase from
Carica papaya. Triacylglycerol lipase activity was enriched 74-fold
from crude latex of Vasconcellea heilbornii to a specific activity (SA) of 57
lmolÆmin)1Æmg)1 on long-chain triacylglycerol (olive oil). The extract was
also active on trioctanoin (SA = 655 lmolÆmin)1Æmg)1), tributyrin (SA =
1107 lmolÆmin)1Æmg)1) and phosphatidylcholine (SA = 923 lmolÆmin)1Æmg)1).
The optimum pH ranged from 8.0 to 9.0. The protein content of the insoluble
fraction of latex was analyzed by electrophoresis followed by mass
spectrometry, and 28 different proteins were identified. The protein fraction
was incubated with the lipase inhibitor [14C]tetrahydrolipstatin, and a
45 kDa protein radiolabeled by the inhibitor was identified as being a putative
lipase. A C. papaya cDNA encoding a 55 kDa protein was further
cloned, and its deduced sequence had 83.7% similarity with peptides from
the 45 kDa protein, with a coverage of 25.6%. The protein encoded by this
cDNA had 35% sequence identity and 51% similarity to castor bean acid
lipase, suggesting that it is the lipase responsible for the important lipolytic
activities detected in papaya latex.