The microbial transglutaminase (mTGase) from Streptomyces mobaraense is widely used in the food industry. However, recombinant production of mTGase is challenging because the mTGase is synthesized as an inactive zymogen, and needs to be activated by proteolytic processing. In this study, self-cleaving intein Ssp DnaB was applied to activate the mTGase in Corynebacterium glutamicum. Premature cleavage of intein Ssp DnaB also occurred, but instead of suppressing premature cleavage, this phenomenon was used to produce active mTGase in C. glutamicum. Both SDS-PAGE analysis and mTGase activity assays indicated that the premature cleavage of intein Ssp DnaB activated the mTGase intracellularly in C. glutamicum. The subsequent N-terminal amino acid sequencing and site-directed mutagenesis studies further showed that the premature cleavage activated the mTGase intracellularly, in a highly specific manner. Moreover, the growth performance of C. glutamicum was not noticeably affected by the intracellular expression of active mTGase. Finally, the mTGase was produced in a 2 L bioreactor, with activity up to 49 U/mL, the highest intracellular mTGase activity ever reported. Using premature cleavage of intein Ssp DnaB to activate mTGase in C. glutamicum, we produced high levels of intracellular active mTGase. Moreover, this approach did not require any further processing steps, such as protease treatment or lengthy incubation, greatly simplifying the production of active mTGase. This efficient and simple approach has great potential for the large-scale industrial production of active mTGase.

中文翻译：

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谷氨酸棒杆菌中内含肽介导的活性微生物转谷氨酰胺酶的细胞内产生

来自 Streptomyces mobaraense 的微生物转谷氨酰胺酶 (mTGase) 广泛用于食品工业。然而，mTGase 的重组生产具有挑战性，因为 mTGase 是作为无活性酶原合成的，需要通过蛋白水解加工激活。在这项研究中，自切割内含肽 Ssp DnaB 被用于激活谷氨酸棒杆菌中的 mTGase。也发生了内含肽 Ssp DnaB 的过早裂解，但不是抑制过早裂解，而是利用这种现象在谷氨酸棒杆菌中产生活性 mTGase。SDS-PAGE 分析和 mTGase 活性测定均表明内含肽 Ssp DnaB 的过早裂解在细胞内激活了谷氨酸棒杆菌中的 mTGase。随后的 N 端氨基酸测序和定点诱变研究进一步表明，过早切割以高度特异性的方式在细胞内激活了 mTGase。此外，谷氨酸棒杆菌的生长性能不受活性 mTGase 的细胞内表达的显着影响。最后，mTGase 在 2 L 生物反应器中生产，活性高达 49 U/mL，是有史以来报告的最高细胞内 mTGase 活性。使用内含肽 Ssp DnaB 的过早裂解来激活谷氨酸棒杆菌中的 mTGase，我们产生了高水平的细胞内活性 mTGase。此外，这种方法不需要任何进一步的加工步骤，例如蛋白酶处理或长时间的孵育，大大简化了活性 mTGase 的生产。