拉达克轮丝菌TGase在大肠杆菌中的分子改造

【背景】谷氨酰胺转氨酶是一种能够催化酰基转移反应的酶,催化各种蛋白质分子之间或之内发生交联反应,在食品、化妆品、医药等领域具有重要的潜在价值。【目的】克隆来自拉达克轮丝菌(Streptoverticillium ladakanum) B1的谷氨酰胺转氨酶(TGase)基因并对其进行分子改造,使其在大肠杆菌中获得高效异源表达。【方法】分别克隆来自拉达克轮丝菌谷氨酰胺转氨酶的自身前导肽(pro)和除前导肽以外的成熟谷氨酰胺转氨酶(TGase)基因,以pET-22b为表达载体构建pro、TGase共表达和融合表达两种表达模式,在这两种表达模式的基础上进一步用定点突变的方法对成熟TGaseN端前4个氨基酸进行改造,检测不同表达模式以及突变对酶活的影响。【结果】当采用前导肽与TGase共表达时,可以直接得到活性形式的TGase,比酶活达到37.71 U/mg。在融合表达的基础上,将TGaseN端前3个氨基酸DSD突变为AAA,比酶活达到14.04U/mg,相较于原始表达模式提高了14.05%。【结论】前导肽与TGase共表达可以直接产生活性TGase,对于融合表达模式,合适位点的突变有利于提高TGase酶活。

Molecule evolution of TGase from Streptoverticillium ladakanum in Escherichia coli

[Background] Transglutaminase (TGase) is an enzyme that catalyzes the acyl transfer reaction, which can catalyze cross-linking reactions between or within various protein molecules, and has important potential value in the food, cosmetics, medicine and other fields. [Objective] Molecular engineering and site-directed mutagenesis were carried out by cloning glutamyl transferase from Streptoverticillium ladakanum B1 to obtain efficient heterologous expression in E. coli. [Methods] The gene of pro and TG from Streptoverticillium ladakanum were cloned into pET-22b to generate co-expression and fusion expression, respectively. Based on these two expression patterns, the first four amino acids of the mature TGase N-terminal were modified by site-directed mutagenesis to detect the effect of different expression patterns and mutation on enzyme activity. [Results] When co-expression of the pro-peptide with TGase, the active form of TGase can be obtained directly, and the specific activity reaches 37.71 U/mg. Based on the fusion expression, the first three amino acid DSD of the N-terminal of TGase were mutated to AAA, and the specific enzyme activity reached 14.04 U/mg, which was 14.05% higher than the original expression pattern. [Conclusion] The co-expression of pro-peptide with TGase can directly produce the active form of TGase. For the fusion expression pattern, the mutation of the appropriate site is beneficial to increase the TGase activity.