诱导温度调控华根霉(Rhizopus chinensis)前导肽脂肪酶的表达及稳定性研究

比较了7 L发酵罐中不同诱导温度对华根霉前导肽脂肪酶在巴斯德毕赤酵母(Pichia pastoris)中表达水平和稳定性的影响。通过实验表明在28℃条件下,最大酶活可以达到1412.5 U/mL,是30℃条件下的2.3倍,25℃条件下的1.3倍;而其产率可以达到12811 U/L·h~(-1),比产率达到82.6 U/g_(DCW)·h~(-1)。同时,在实验过程中发现诱导温度对目的蛋白的降解和聚合有重要的影响。通过对蛋白酶活性的测定和还原SDS-PAGE证实随着诱导温度的提高,蛋白酶活性急剧上升,从而导致由前导肽(37 kD)降解为成熟肽(30 kD)的降解作用的加剧。此现象在30℃条件下最为明显,在诱导84 h时前导肽已经全部降解为成熟肽。另外,通过非还原SDS-PAGE和分析表明随着诱导温度的提高也导致了二聚体的含量也逐渐增加。

Expression of Rhizopus chinensis lipase induced by different temperatures and stability of recombinant protein

Different induction temperatures were compared for the production of Rhizopus chinensis lipase （RCL） from Pichia pastoris in 7 L fermentor. The comparison showed that the optimum induction temperature was 28 ℃, at which the maximum enzyme activity was 1410.2 U/mL which was 2.3-fold and 1.3-fold higher than that at 30℃ and 25℃. And the productivity also reached 12811 U/L·h^-1 with a value of 82.6 U/gDCW·h^-1 specific productivity. It could be observed that proteolysis degradation had an important effect on RCL. Through determination of proteolysis activity and SDS-PAGE the results showed that proteases degraded proRCL （37 kD） to mRCL （30 kD） seriously, especially at 30℃. Under this condition the proRCL was completely degraded at 84 h. Besides, the concentration of dimer increased with the rise of temperature.