米黑根毛霉来源l-天冬酰胺酶的分子改造及高效表达

Molecular modification and highly efficient expression of l-asparaginase from Rhizomucor miehei

摘要:

l-天冬酰胺酶能够水解l-天冬酰胺生成l-天冬氨酸和氨，广泛存在于微生物、植物和部分啮齿类动物的血清中，在医药和食品行业中都具有重要应用。然而无论是在医药还是在食品行业中，l-天冬酰胺酶依然存在一些问题，如催化效率低、热稳定性差、产量低等。文中通过理性设计及5′非翻译区 (5′ untranslated region, 5′ UTR)改造提高米黑根毛霉Rhizomucor miehei来源的l-天冬酰胺酶 (RmAsnase) 的酶活及蛋白表达量。结果显示，通过同源建模结合序列比对分析构建的6个突变菌株中，突变酶A344E比酶活较野生酶提高了1.5倍。继而构建食品安全菌株枯草芽孢杆菌Bacillus subtilis 168/pMA5-A344E，对其进行UTR改造，获得重组菌株B. subtilis 168/pMA5 UTR-A344E，其酶活较原始菌提高了7.2倍，对重组菌B. subtilis 168/pMA5 UTR-A344E进行5 L罐研究，最终产量为489.1 U/mL。该酶活提高的重组菌株对l-天冬酰胺酶的工业化应用具有重要价值。

Abstract:

l-asparaginase hydrolyzes l-asparagine to produce l-aspartic acid and ammonia. It is widely distributed in microorganisms, plants and serum of some rodents, and has important applications in the pharmaceutical and food industries. However, the poor thermal stability, low catalytic efficiency and low yield hampered the further application of l-asparaginase. In this paper, rational design and 5′ untranslated region (5′ UTR) design strategies were used to increase the specific enzyme activity and protein expression of l-asparaginase derived from Rhizomucor miehei (RmAsnase). The results showed that among the six mutants constructed through homology modeling combined with sequence alignment, the specific enzyme activity of the mutant A344E was 1.5 times higher than the wild type. Subsequently, a food-safe strain Bacillus subtilis 168/pMA5-A344E was constructed, and the UTR strategy was used for the construction of recombinant strain B. subtilis 168/pMA5 UTR-A344E. The enzyme activity of B. subtilis 168/pMA5 UTR-A344E was 7.2 times higher than that of B. subtilis 168/pMA5-A344E. The recombinant strain B. subtilis 168/pMA5 UTR-A344E was scaled up in 5 L fermenter, and the final yield of l-asparaginase was 489.1 U/mL, showing great potential for industrial application.