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A novel galactolipase from a green microalga Chlorella kessleri: purification, characterization, molecular cloning, and heterologous expression

Hashiro, Shuhei, Fujiuchi, Koyu, Sugimori, Daisuke, Yasueda, Hisashi
Applied microbiology and biotechnology 2018 v.102 no.4 pp. 1711-1723
gas chromatography, substrate specificity, polyacrylamide gel electrophoresis, Aspergillus japonicus, acylglycerols, Chlamydomonas reinhardtii, heterologous gene expression, molecular weight, introns, histidine, Escherichia coli, phospholipids, Parachlorella kessleri, aspartic acid, complementary DNA, carboxylic ester hydrolases, molecular cloning, galactolipids, exons, food processing, pH, microalgae, chloroplasts
We have identified an enzyme, galactolipase (ckGL), which hydrolyzes the acyl ester bond of galactolipids such as digalactosyldiacylglycerol (DGDG), in the microalga Chlorella kessleri. Following purification of the enzyme to electrophoretic homogeneity from cell-free extract, the maximum activity toward DGDG was observed at pH 6.5 and 37 °C. ckGL was Ca²⁺-dependent enzyme and displayed an apparent molecular mass of approx. 53 kDa on SDS-PAGE. The substrate specificity was in the order: DGDG (100%) > monogalactosyldiacylglycerol ≈ phosphatidylglycerol (~ 40%) > sulfoquinovosyldiacylglycerol (~ 20%); the enzyme exhibited almost no activity toward glycerides and other phospholipids. Gas chromatography analysis demonstrated that ckGL preferably hydrolyzed the sn-1 acyl ester bond in the substrates. The genomic DNA sequence (5.6 kb) containing the ckGL gene (designated glp1) was determined and the cDNA was cloned. glp1 was composed of 10 introns and 11 exons, and the 1608-bp full-length cDNA encoded a mature ckGL containing 475 amino acids (aa), with a presequence (60 aa) containing a potential chloroplast transit peptide. Recombinant functional ckGL was produced in Escherichia coli. Although the deduced aa sequence of ckGL contained the typical GXSXG motif of serine hydrolases together with conserved histidine and aspartate residues which would form part of the catalytic triad of α/β-hydrolases, ckGL showed no significant overall similarity with known lipases including GLs from Chlamydomonas reinhardtii and Aspergillus japonicus, indicating that ckGL is a novel GL. ckGL, with high specificity for DGDG, could be applicable to food processing as an enzyme capable of improving material textures.