Separation of two microbial transglutaminase isomers from Streptomyces mobaraensis using pH-mediated cation exchange chromatography and their characterization

Abstract

Microbial transglutaminase (MTGase) derived from Streptomyces mobaraensis has been widely used in the food, biotechnology and medicine fields. The lot-to-lot consistency and product stability of MTGase must be ensured. The structure and charge variants of MTGase can influence its bioactivity. In this study, MTGase isomers (MTG I1 and MTG I2) were found during the separation of MTGase by pH-mediated cation-exchange chromatography. MTG I1 and MTG I2 had the same molecular weight and N-terminal amino acid sequences, but they showed charge heterogeneity. The affinity of MTG I2 for substrates was higher than that of MTG I1, and the thermal stability and the acid-base tolerance of MTG I1 were significantly higher than that of MTG I2. Therefore, the ratio of MTG I1/MTG I2 was positively correlated with the stability of MTGase. The buffer pH and the ionic strength of the eluent had significant effects on the separation of MTG I1 and MTG I2, and the elution gradient steepness and column load showed little effect on the separation of the MTG I1 and MTG I2 peaks. We built a stable and repeatable separation method for MTG I1 and MTG I2. MTG I1 could transform into MTG I2, but MTG I2 was unable to transform into MTG I1, making the transformation of MTG I1 to MTG I2 was irreversible. When MTG I2 was removed from the MTGase, a portion of the MTG I1 could transform into MTG I2. Therefore, one way to increase the stability of MTGase was to reduce the transformation of MTG I1 to MTG I2.