Immobilization, Regiospecificity Characterization and Application of Aspergillus oryzae Lipase in the Enzymatic Synthesis of the Structured Lipid 1,3-Dioleoyl-2-Palmitoylglycerol

Abstract

The enzymatic synthesis of 1,3-dioleoyl-2-palmitoylglycerol (OPO), one of the main components of human milk fats, has been hindered by the relatively high cost of sn-1,3-specific lipases and the deficiency in biocatalyst stability. The sn-1,3-specific lipase from Aspergillus oryzae (AOL) is highly and efficiently immobilized with the polystyrene-based hydrophobic resin D3520, with a significant 49.54-fold increase in specific lipase activity compared with the AOL powder in catalyzing the synthesis of OPO through the acidolysis between palm stearin and oleic acid (OA). The optimal immobilization conditions were investigated, including time course, initial protein concentration and solution pH. The sn-1,3 specificity of lipases under different immobilization conditions was evaluated and identified as positively associated with the lipase activity, and the pH of the immobilization solution influenced the regiospecificity and synthetic activity of these lipases. Immobilized AOL D3520, as the biocatalyst, was used for the enzymatic synthesis of the structured lipid OPO through the acidolysis between palm stearin and OA. The following conditions were optimized for the synthesis of structured lipid OPO: 65 °C temperature; 1:8 substrate molar ratio between palm stearin and OA; 8% (w/w) enzyme load; 3.5% water content of the immobilized lipase; and 1 h reaction time. Under these conditions, highly efficient C52 production (45.65%) was achieved, with a tripalmitin content of 2.75% and a sn-2 palmitic acid (PA) proportion of 55.08% in the system.

Aspergillus oryzae Lipase‐Catalyzed Synthesis of Dioleoyl; Palmitoyl‐Rich Triacylglycerols in Two Reactors

“…The adsorption method described by Cai et al [8] was followed to immobilize the AOL on the macroporous adsorption resin D3520. Approximately 0.3 g of AOL powder was dissolved in 5 ml of pH 9.5, 0.1 M of Gly-NaOH buffer solution.…”

“…The FA in all of the TAG products and the sn-2 MAG were converted into the corresponding fatty acid methyl esters (FAME) using a 2 mol/L KOH-methanol solution. The FAME were analyzed by a 7890A GC platform (Agilent, Santa Clara, USA) equipped with an FID and a DB-23 column (30 m × 0.25 mm × 0.25 µm, Agilent, Santa Clara, USA) as described previously [8]. The temperature of the column oven was initially held at 50 °C for 2 min, increased to 180 °C at a constant rate of 10 °C/min, and then held at 180 °C for 5 min.…”

“…Response surface methodology were used to optimize the reaction conditions of the AOL-catalyzed acidolysis is calcium loss through the feces [4][5][6]. Therefore, most researchers have imitated this special structure in the production of OPO-rich TAG [5,7,8].…”

“…The relatively high cost of commercial immobilized lipases hinders the production of OPO-rich TAG in industry. We developed a novel Aspergillus oryzae lipase (AOL) which had been characterized as sn-1,3 regiospecific [8,9]. We immobilized AOL on the polystyrene-based hydrophobic resin D3520 as a feasible non-commercial catalyst for the production of OPO-rich TAG.…”

“…Our results showed that the regiospecific lipase activity of immobilized AOL increased 49.54-fold compared with the AOL powder catalyzed synthesis of OPO-rich TAG from palm stearin and commercial oleic acid. The optimum conditions optimized for the synthesis of OPO-rich TAG were: temperature, 65 °C; substrate molar ratio between palm stearin and OA, 1:8; enzyme load, 8 %(w/w); water content of the immobilized lipase, 3.5 %; and reaction time 1 h. Under these conditions, the C52 content was 45.65 %, and a sn-2 palmitic acid (PA) proportion of 55.08 % in the system [8].…”