Surfactant Protein of the Streptomyces Subtilisin Inhibitor Family Inhibits Transglutaminase Activation in Streptomyces hygroscopicus

Abstract

Transglutaminase (TGase) is widely used in the food industry for improving protein properties by catalyzing the cross-linking of proteins. In Streptomyces, TGase is secreted as a zymogen, and an activation process has been observed in liquid culture. However, the activation mechanism remains unclear. In the present study, the TGase activation process in Streptomyces hygroscopicus was investigated by biochemical approaches. In a liquid culture, Pro-TGase was secreted and gradually was converted into active TGase during the growth period; however, in a cell-free system in which cells were removed from the liquid culture, TGase activation stalled unexpectedly. Subsequently, the TGase activation process was found to be inhibited by a TGase-activating protease inhibitor (TAPI). N-Terminal amino acid sequencing and a homology search of the purified TAPI revealed that it is a member of the Streptomyces subtilisin inhibitor (SSI) family. Furthermore, it was found that TAPI (0.1 mg/mL) decreased the surface tension of water from 72 to 60 mJ/m2 within 5 min, suggesting that it possesses surface activity. This is the first report that an SSI member functions as a surfactant protein. On the basis of these findings, a model for TAPI-regulated TGase activation process was proposed. This study provides novel insights into the TGase activation process in Streptomyces.

The order of expression is a key factor in the production of active transglutaminase in Escherichia coli by co-expression with its pro-peptide

“…The purification of TGase from S. hygroscopicus culture was performed as previously described [28]. The purification of the recombinant enzyme (pro-TGase and TGase) was conducted using nickel affinity chromatography (HisTrap™ FF crude, GE Healthcare) and gel filtration (Superdex™75 10/300 GL, GE Healthcare).…”

“…Dispase-mediated activation of pro-TGase was performed as previously described [13]. Protein content and SDS-PAGE analysis were conducted as previously described [28]. TGase activity was determined using N-CBZ-Gln-Gly at pH 6.0 and 37°C [28].…”

“…Protein content and SDS-PAGE analysis were conducted as previously described [28]. TGase activity was determined using N-CBZ-Gln-Gly at pH 6.0 and 37°C [28]. Amino acid sequencing of TGase N-terminal region was performed by Shanghai Gene Core Biotechnologies Co., Ltd.…”

Prokaryote-derived protein inhibitors of peptidases: A sketchy occurrence and mostly unknown function

“…The SSI coding gene is present in 31 out of the 33 species with fully sequenced genomes. Apparently, one way of wielding the physiological effect by SSI is the control of the proteolytic activation of transglutaminase, an extracellular enzyme that cross-links proteins to high molecular weight aggregates, by endogenous proteases [58–60]. …”

Discovery of two transglutaminases derived from same zymogen for the Streptomyces hygroscopicus and analysis of their formation processes

“…In a previous study a high‐yield TGase production strain was isolated from Streptomyces hygroscopicus . TGase activation in S. hygroscopicus was catalysed by various proteases and required at least a serine protease and a metalloprotease . The TGase‐activating protease is inhibited by a protein of the Streptomyces subtilisin inhibitor (SSI) family, has surface activity and is precipitated by cetyltrimethylammonium bromide …”