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

作者：Hashiro Shuhei; Fujiuchi Koyu; Sugimori Daisuke^*; Yasueda Hisashi^*

来源：Applied Microbiology and Biotechnology, 2018, 102(4): 1711-1723.

DOI：10.1007/s00253-017-8713-7

• Chlorella kessleri
• Galactolipase
• Characterization
• Genomic structure
• cDNA cloning
• Heterologous expression

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 degrees 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 approximate to phosphatidylglycerol (similar to 40%) > sulfoquinovosyldiacylglycerol (similar to 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 alpha/beta-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.

• 出版日期2018-2