Liu Aixia, Yu Xiao-Wei, Sha Chong, Xu Yan
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China.
Appl Biochem Biotechnol. 2015 Mar;175(6):3195-206. doi: 10.1007/s12010-015-1492-7. Epub 2015 Jan 25.
The phospholipase A2 (PLA2) from Streptomyces violaceoruber was successfully expressed in the methylotrophic yeast Pichia pastoris GS115 under the control of AOX1 promoter for the first time. The maximum activity of the recombinant PLA2 (rPLA2) reached 34.7 ± 0.2 U/mL, and specific activity was 170 ± 4 U/mg after purification. On the sodium dodecyl sulfate polyacrylamide gel electrophoresis of the culture supernatants, three bands of 21, 18, and 14.3 kDa were observed. The peptide mass fingerprinting analysis showed that all of these three bands were rPLA2 from S. violaceoruber. By the treatment with Endo H and PNGase F, it indicated that the rPLA2 occurred N-glycosylation. The enzymatic properties of this enzyme were determined. The rPLA2 exhibited a lower optimum pH (pH = 6.0) compared to the wild-type enzyme, which was a desirable property in the application of oil degumming. In the enzymatic degumming process, the phosphorus content decreased from 261.77 ± 3.51 mg/kg to 20.74 ± 0.23 mg/kg, which is very promising for the industrial application.
来自紫色链霉菌的磷脂酶A2（PLA2）首次在甲醇营养型酵母毕赤酵母GS115中，在AOX1启动子的控制下成功表达。重组PLA2（rPLA2）的最大活性达到34.7±0.2 U/mL，纯化后的比活性为170±4 U/mg。在培养上清液的十二烷基硫酸钠聚丙烯酰胺凝胶电泳中，观察到21、18和14.3 kDa的三条带。肽质量指纹图谱分析表明，这三条带均为来自紫色链霉菌的rPLA2。通过用内切糖苷酶H和肽-N-糖苷酶F处理，表明rPLA2发生了N-糖基化。测定了该酶的酶学性质。与野生型酶相比，rPLA2表现出较低的最适pH（pH = 6.0），这在油脂脱胶应用中是一个理想的特性。在酶法脱胶过程中，磷含量从261.77±3.51 mg/kg降至20.74±0.23 mg/kg，这对工业应用非常有前景。
