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Abstract
The hydrolysis properties of lipase in castor was evaluated using two different substrate forms (tripalmitic glycerides and trioleic glycerides) to catalyze the reaction under different operational conditions. RcLipase was obtained from castor seeds and results show that RcLipase is a conservative serine lipase with a conserved catalytic center (SDH) and a conserved pentapeptide (GXSXG). This enzyme exhibited the greatest activity and tolerance to chloroform and toluene when it was expressed in Pichia pastoris GS115 at 40 ℃ and pH 8.0. Zn and Cu ions exerted obvious inhibitory effects on the enzyme, and displayed good hydrolytic activity for long-chain natural and synthetic lipids. HPLC analysis showed that this enzyme has 1,3 regioselectivity when glycerol tripalmitate and oleic acid are used as substrates. The fatty acid composition in the reaction product was 21.3% oleic acid and 79.1% sn-2 palmitic acid.
Highlights
Abbreviations Symbol Abbreviation meaning RcLipase Sn1,3-specific lipase from Castor bean SDH Conserved catalytic center ‘Serine-Aspartic acid-Histidine GXSXG Conserved pentapeptide ‘glycine-amino acid residues–Serine–amino acid residues–glycine’ GX Conserved pentapeptide ‘glycine–amino acid residues’ GGX Conserved pentapeptide ‘glycine–glycine–amino acid residues’ high performance liquid chromatography (HPLC) High performance liquid chromatography ORF Open reading frame PNPP P-nitrophenyl palmitate SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis CAPSO Cyclohexylamino-1-propanesulphonic acid pNPC P-nitrophenyl caprylate pNPM P-nitrophenyl myristate pNPL P-nitrophenyl laurate ES Enzyme and substrate 1,2-DAG 1,2-Diacylglycerol 1,3-DAG 1,3-Diacylglycerol YNB Yeast nitrogen base LB Lysogeny broth BMGY Buffered glycerol-complex medium BMMY Buffered methanol-complex medium BSM Buffered methanol-complex medium PTM1 Microelement
The catalytic triad of the lipase catalytic center is very conservative, consisting of nucleophilic serine (Ser), aspartic acid (Asp) or glutamic acid (Glu), and histidine (His), and Ser is usually located after the fifth beta fold and before the alpha helix, while Asp or Glu is normally located after the seventh beta fold, with His after the eighth beta fold[6]
The first residue of the oxygen anion cavity is located at the fifth beta fold near the catalytic active center of the Ser residue at the conservative nucleophilic elbow, and is usually located at the X2 position of G-X1-SX2-G, while the second residue of the oxygen anion is usually located at the N end of the lipase on the loop ring between the third beta-chain and the alpha-a-helix[7]
Summary
Abbreviations Symbol Abbreviation meaning RcLipase Sn1,3-specific lipase from Castor bean SDH Conserved catalytic center ‘Serine-Aspartic acid-Histidine GXSXG Conserved pentapeptide ‘glycine-amino acid residues–Serine–amino acid residues–glycine’ GX Conserved pentapeptide ‘glycine–amino acid residues’ GGX Conserved pentapeptide ‘glycine–glycine–amino acid residues’ HPLC High performance liquid chromatography ORF Open reading frame PNPP P-nitrophenyl palmitate SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis CAPSO Cyclohexylamino-1-propanesulphonic acid pNPC P-nitrophenyl caprylate pNPM P-nitrophenyl myristate pNPL P-nitrophenyl laurate ES Enzyme and substrate 1,2-DAG 1,2-Diacylglycerol 1,3-DAG 1,3-Diacylglycerol YNB Yeast nitrogen base LB Lysogeny broth BMGY Buffered glycerol-complex medium BMMY Buffered methanol-complex medium BSM Buffered methanol-complex medium PTM1 Microelement. The first residue of the oxygen anion cavity is located at the fifth beta fold near the catalytic active center of the Ser residue at the conservative nucleophilic elbow, and is usually located at the X2 position of G-X1-SX2-G, while the second residue of the oxygen anion is usually located at the N end of the lipase on the loop ring between the third beta-chain and the alpha-a-helix[7]. Oxygen anion voids are usually divided into GX, GGX, and γ types, among the GX type, X is usually Ser or threonine (Thr) In most cases, it contains a third amino acid, aspartic acid (Asp) or asparagine, which assist in stabilizing the oxygen anion pore through hydrogen bonding. The distribution of fatty acids in formula milk powder is quite different from that in breast m ilk[17]. Breast milk fat substitutes and formula milk powder, which are close to breast milk fat, have become a research hotspot in recent years[19]
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