Study on Catalytic Synthesis of Triolein by Recombinant Rice Bran Thermostable Lipase RBL-LC6

Enzymatic synthesis of triolein (TAG) has become an important approach for green development in the fine chemical industry due to its high product purity, low pollution, and simple downstream separation. However, the industrial application of enzymatic TAG synthesis is limited by insufficient thermal stability and low catalytic efficiency of lipases at high temperatures. Therefore, developing lipases with excellent thermostability and high catalytic activity is the key to technological breakthroughs. In this study, recombinant rice bran thermostable lipase (RBL-LC6) derived from rice bran was used as the catalyst to investigate the optimal conditions for the enzymatic esterification of oleic acid and glycerol for TAG production at high temperatures. The effect of temperature on product distribution was first explored. Then single-factor experiments were carried out to examine the effects of reaction time, temperature, water mass fraction, enzyme mass fraction, and substrate molar ratio on TAG synthesis. The enzymatic reaction kinetics was also analyzed. The results showed that as the temperature increased from 40 ℃ to 90 ℃, RBL-LC6 exhibited better catalytic performance for TAG synthesis at 80 ℃, with a TAG yield of 48.04%. After optimization, the highest TAG yield reached 55.13% under the optimal conditions: reaction time 6 h, temperature 80 ℃, water mass fraction 1.0%, enzyme mass fraction 0.2%, and substrate glycerol/oleic acid molar ratio 1∶3.5. Kinetic analysis indicated that the esterification catalyzed by RBL-LC6 followed the Ping-Pong Bi-Bi mechanism and the enzyme exbibited a stronger affinity for glycerol than for oleic acid. This study aimed to provide theoretical and technical support for the green enzymatic synthesis of TAG under high-temperature conditions.