F43Y及I354M,L358F定点突变对植酸酶热稳定性及酶活性的改善

对重组酵母PPNPm8的植酸酶phyAm基因进行PCR介导的定点突变,即将植酸酶43位的苯丙氨酸替换为酪氨酸(F43Y),将其354、358位的异亮氨酸、亮氨酸分别替换为甲硫氨酸和苯丙氨酸(I354M,L358F),得到了2个突变体PPNPm-1(F43Y)及PPNPm-2(I354M,L358F).含突变基因的重组表达载体pPIC9kphyAm-1,pPIC9kphyAm-2在毕赤酵母GS115中表达,对表达产物进行酶活性测定及热稳定性检测.结果表明:突变体PPNPm-1最适反应温度比未突变体PPNPm8上升了3℃,75℃处理10min,热稳定性提高15%,比活力提高11%;PPNPm-2最适反应温度未改变,热稳定性比PPNPm8仅提高3%,比活力降低6.5%.对突变前后的植酸酶空间结构进行比较预测,发现突变氨基酸Tyr43与空间位置相邻的Asn416之间形成氢键,增强了酶的热稳定性.

Site-directed Mutagenesis Improves the Thermostability and Specific-activity of Phytase Mutated by F43Y and I354M,L358F

The inability of currently available phytase to tolerate heat denaturation from feed processing remains one of the major practical concerns.To enhance the catalytic efficiency and thermostability of phytase expressed in Pichia pastoris,the mutants F43Y (PP-NP m-1) and I354M, L358F (PP-NP m-2) were constructed in vitro by site-directed mutagenesis with long-distance inverse PCR. The recombinants pPIC 9k-phyA m-1 and pPIC 9k-phyA m-2 were expressed in Pichia pastoris. The comparison experiments of mutant enzymes with natural phytase showed that the optimum temperature of PP-NP m-1 was increased by 3℃,and its thermostability was increased by 15%,when it was exposed for 10 min at 75℃. Its specific activity was increased by 11%. The optimum temperature of PP-NP m-2 was not increased, and its thermostability was only increased by 3%. However, the specific activity of PP-NP m-2 was decreased by 6.5%. By comparison between natural phytase and mutant phytase, it was found that hydrogen bond between Tyr43 and Asn416 promoted the thermostability.