对磷脂酶D(phospholipase D,PLD)进行重组表达,并探究其在生物催化合成磷脂酰丝氨酸(phosphatidylserine, PS)中的应用。以大肠杆菌E. coli BL21 (DE3)作为PLD的异源表达宿主,构建重组菌E. coli BL21 (DE3)/pET-28a(+)-sspld,通过蛋白分析确定其分子量,并对培养条件进行优化,进一步探究重组菌的酶学性质。在有机相-水相双相反应体系中进行PS的制备,并对制备工艺进行系统优化。成功构建了重组菌株E. coli BL21 (DE3)/pET-28a(+)-sspld,通过蛋白分析确定其分子质量约为60 kDa。重组菌通过诱导条件优化,酶活最高可达38.58 U/mL,是优化前的2.26倍。PLD粗酶液的最适pH值为7.5,最适反应温度为60 ℃。制备工艺优化结果表明40 ℃条件下,在8 mL的乙酸乙酯中溶解64 mg的卵磷脂,在4 mL酶液中溶解160 mg的L-丝氨酸,得到的PS转化率最高,可达28%,产量为1.34 g/L。该PLD粗酶液催化性能良好,为酶法制备磷脂酰丝氨酸奠定了基础。
This study aimed to express recombinant phospholipase D (PLD) and explore its application in biocatalytic synthesis of phosphatidylserine (PS). The recombinant strain Escherichia coli BL21 (DE3)/pET-28a(+)-sspld was constructed, followed by optimizing its cultivation conditions to study its enzymatic properties. Besides, the preparation process of PS by the strain was also optimized. The results showed that a 60 kDa recombinant PLD was successfully expressed. The maximum activity of PLD produced reached 38.58 U/mL, which was 2.26-fold higher than that before optimization. Moreover, the optimum pH of PLD was 7.5 and its optimum reaction temperature was 60 ℃. Furthermore, the optimum reaction condition to prepare PS was as follows: reacted at 40 ℃ and dissolved 64 mg PC60 in 8 mL ethyl acetate and 160 mg L-ser in 4 mL PLD. Under this condition, the PS conversion rate was 28.0% and had the highest yield of 1.34 g/L. In conclusion, the recombinant strain exhibited favorable catalytic performance, which lays a foundation for enzymatic preparation of PS.
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