Liu Song, Rao Shengqi, Chen Xiao, Li Jianghua
Science Center for Future Foods, Jiangnan University, Wuxi, Jiangsu, China.
National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, Jiangsu, China.
Front Bioeng Biotechnol. 2022 Sep 7;10:996138. doi: 10.3389/fbioe.2022.996138. eCollection 2022.
Glutaminase (EC 3.5.1.2) can catalyze the deamidation of glutamine, which has been used to improve umami taste in oriental fermented foods. However, a high salt concentration is still a fundamental challenge for glutaminase application, especially in soy sauce production. To improve the salt tolerance of glutaminase, the self-assembling amphiphilic peptides EAK16 and ELK16 were fused to the N-terminus of a mutant (E3C/E55F/D213T) derived from glutaminase, yielding the fusion enzymes EAK16-E3C/E55F/D213T and ELK16-E3C/E55F/D213T, respectively. As ELK16-E3C/E55F/D213T was expressed as insoluble active inclusion bodies, only the purified EAK16-E3C/E55F/D213T was subjected to further analyses. After the incubation with 18% (w/v) NaCl for 200 min, the residual activities of EAK16-E3C/E55F/D213T in a NaCl-free solution reached 43.6%, while E3C/E55F/D213T was completely inactivated. When the enzyme reaction was conducted in the presence of 20% NaCl, the relative activity of EAK16-E3C/E55F/D213T was 0.47-fold higher than that of E3C/E55F/D213T. As protein surface hydrophobicity and protein particle size analysis suggested, oligomerization may play an important role in the salt-tolerance enhancement of the fusions. Furthermore, EAK16-E3C/E55F/D213T achieved a 0.88-fold increase in the titer of glutamic acid in a model system of soy sauce fermentation compared to E3C/E55F/D213T. Therefore, the fusion with self-assembling amphiphilic peptides is an efficient strategy to improve the salt-tolerance of glutaminase.
谷氨酰胺酶（EC 3.5.1.2）可催化谷氨酰胺的脱酰胺反应，该反应已被用于改善东方发酵食品的鲜味。然而，高盐浓度仍然是谷氨酰胺酶应用面临的一个基本挑战，尤其是在酱油生产中。为了提高谷氨酰胺酶的耐盐性，将自组装两亲性肽EAK16和ELK16融合到源自谷氨酰胺酶的突变体（E3C/E55F/D213T）的N端，分别产生融合酶EAK16-E3C/E55F/D213T和ELK16-E3C/E55F/D213T。由于ELK16-E3C/E55F/D213T表达为不溶性活性包涵体，因此仅对纯化的EAK16-E3C/E55F/D213T进行进一步分析。在18%（w/v）NaCl中孵育200分钟后，EAK16-E3C/E55F/D213T在无NaCl溶液中的残余活性达到43.6%，而E3C/E55F/D213T完全失活。当酶反应在20%NaCl存在下进行时，EAK16-E3C/E55F/D213T的相对活性比E3C/E55F/D213T高0.47倍。蛋白质表面疏水性和蛋白质粒径分析表明，寡聚化可能在融合体耐盐性增强中起重要作用。此外，与E3C/E55F/D213T相比，EAK16-E3C/E55F/D213T在酱油发酵模型系统中的谷氨酸产量提高了0.88倍。因此，与自组装两亲性肽融合是提高谷氨酰胺酶耐盐性的有效策略。
