以2-巯基-1-甲基咪唑(MMI)为混合模式配基,人血免疫球蛋白G(IgG)为模型蛋白,探讨混合模式配基双水相系统分离抗体的新方法,主要包括MMI-聚乙二醇(PEG)的制备和IgG分配研究。首先优化了PEG4000的环氧氯丙烷活化反应条件,环氧氯丙烷和PEG比例为3.5:1,三氟化硼乙醚溶液加量为0.05 mL g 1PEG,开环反应时间为8 h;40%氢氧化钠溶液加量为0.15 mL g-1PEG,闭环反应时间为4 h,环氧基团接入量可达到460μmol g-1PEG,偶联配基MMI,得到MMI-PEG混合模式配基。进一步考察了IgG在PEG4000/(NH4)2SO4双水相系统中的分配,发现添加MMI-PEG配基有效提高了IgG分配系数,加入适量NaCl,分配系数达到了17.1。结果表明,混合模式配基双水相萃取可以实现IgG的上相富集,为抗体分离提供了一个新途径。
内生拟盘多毛孢菌J63(Pestalotiopsis sp.J63)是从东海的海滩滩涂中新分离出的一株真菌。研究考察了无机盐对该菌株产漆酶响,以及对漆酶催化底物和脱色偶氮类染料甲基橙的影响。结果表明添加H3BO3、SrCl2.6H2ONaF、KBr、MnCl2.4H2O和ZnSO4.7H2O对漆酶的产生有明显的促进作用,其中H3BO3的作用最明显,可使酶活增加221.8%。在漆酶催化底物反应时,除1 mmol L 1FeSO4.7H2O能够抑制漆酶酶活,其余无机盐均对酶活有促进作用。14 mmol L 1KAl(SO4)2对酶活的促进作用明显,使相对酶活达到124.9%。3 mmol L 1KBr使相对酶活达到116.7%,与其他菌株所产漆酶受到KBr抑制的特性相比具有优势。添加14 mmol L 1KAl(SO4)2和3 mmol L 1KBr后相应的脱色率分别达到了97.3%和75.6%,高于对照组64%的脱色率。同时还研究了适宜该菌株产漆酶的温度和pH,当温度为26oC,pH为6.0时酶活最高,达到3280.6 U L 1。
Disulfide bond formation protein A (DsbA) is one of the important helper proteins for folding in protein synthesis in vivo. In this study, purification of recombinant DsbA was investigated by examining four important factors with Box-Behnken design method, a statistic-based design of experiments. The optimal operation conditions were obtained by adopting the effectiveness coefficient method on the multi-objective problem, which takes the protein recovery, purification efficiency and throughput of ion-exchange chromatography into account. After the optimization, protein recovery of 96.8% and purity higher than 95% DsbA was achieved, and the productivity was (377.9±1.7)mg soluble DsbA per liter broth. The purified protein was identified by peptide mass fingerprinting matching the record of gi|2624856, a mutant of DsbA. The DsbA was preliminarily applied to the refolding of denatured lysozyme in vitro.
