Production of transglutaminase in glutathione-producing recombinant Saccharomyces cerevisiae

Abstract

Transglutaminase (TG) catalyzes the formation of cross-links between proteins. TG from Streptoverticillium mobaraense (SmTG) is used widely in food, cosmetic, biomaterial and medical industries. SmTG is occasionally supplied as a mixture with the activator peptide glutathione. Currently, glutathione is industrially produced using a budding yeast, Saccharomyces cerevisiae, because of its intracellular high content of glutathione. In this study, active SmTG was produced together with glutathione in S. cerevisiae. SmTG extracted from S. cerevisiae expressing SmTG showed cross-linking activity when BSA and sodium caseinate were substrates. The cross-linking activity of SmTG increased proportionally as the concentration of added glutathione increased. Furthermore, SmTG was prepared by extracting SmTG from an engineered S. cerevisiae whose glutathione synthetic pathway was enhanced. The SmTG solution showed higher activity when compared with a SmTG solution prepared from a S. cerevisiae strain without enhanced glutathione production. This result indicates that a high content of intracellular glutathione further enhances active SmTG production in S. cerevisiae. S. cerevisiae co-producing SmTG and a higher content of glutathione has the potential to supply a ready-to-use industrial active TG solution.

Keywords: Coproduction; Glutathione; Saccharomyces cerevisiae; Streptoverticillium mobaraense; Transglutaminase; Yeast.

Fig. 1

Cross-linking assay using BSA as a substrate after incubation with a SmTG solution extracted from the BY4741-TG strain for several reaction times. Lane 1, molecular size marker; lane 2, BSA; lanes 3, 4, 5 and 6, 1×SmTG (+BSA) for 0, 30, 60 and 120 min, respectively; lane 7, 1×SmTG (−BSA)

Fig. 2

Effect of reduced glutathione (GSH) addition on polymerization of caseinate by SmTG. The concentration of proteins whose molecular weights were more than 100 kDa were detected after polymerization of caseinate by 0.2×SmTG extracted from the BY4741-TG strain in the presence of different GSH concentrations

Fig. 3

SmTG activity measured by the incorporation of MDC into N,N-dimethylcasein. 12×SmTG extracted from BY4741-TG and GCI-TG strains were used for cross-linking reactions. The values are means and the error bars show the SD (n = 5). Statistically significant differences are indicated by an asterisk (p = 0.023)

Fig. 4

Polymerization of caseinate by SmTG extracted from BY4741-TG and GCI-TG strains. Each sample volume loaded was 20 µL (a) and 5 µL (b). a Lane 1, protein molecular size marker; lane 2, caseinate control; lane 3, polymerized caseins by 12×SmTG extracted from the BY4741-TG strain; lane 4, polymerized caseins by 12×SmTG extracted from the GCI-TG strain; lane 5, BY4741-TG extract; lane 6, GCI-TG extract. b Expanded region of the SDS-PAGE in (a boxed region). Ratios of cross-linked α-casein were calculated from the intensity of each α-casein band and represented as relative values (%). c The concentration of proteins whose molecular weights were more than 100 kDa, were detected after polymerization of caseinate by 12×SmTG extracted from the BY4741-TG and GCI-TG strain