Activation Mechanism And Physiological Function Of Transglutaminase From Streptomyces Hygroscopicus

Transglutaminase (TGase; Protein-glutamine-glutamyltransferase, EC 2.3.2.13) is an enzyme capable of introducing covalent cross-links between proteins as well as peptides. TGase is a very important enzyme in food industry and has great potential application in pharmaceutical and textile industries. Streptomyces TGase is the only commercial enzyme that could covalent proteins. It has been reported that TGase from Streptomyces mobaraensis was secreted as zymogen and could be activated by protease. However, no evidence explain the physiological function of Streptomyces TGase; and the mechanism of TGase activation was still unclear in Streptomyces. Recently, in our lab, a new strain capable of producing a high activity of TGase, classified as Streptomyces hygroscopicus, was isolated from soil. To provide theory support to enhance the fermentation of this strain, the activation mechanism and physiological function of TGase from S. hygroscopicus were studied in this dissertation. The main results are listed as following:1. Translation from Pro-TGase to TGase exists in liquid culure of S. hygroscopicus.TGase and Pro-TGase were secreted to the liquid media and could be purified by ethanol precipitation and Fractogel EMD SO3-. TGase and Pro-TGase exhibited molecular mass as 40 kDa and 44 kDa, respectively. N-terminal amino acids sequences of Pro-TGase were: ASGDDEEREG.During the liquid culture of S. hygroscopicus, TGase was secreted as a zymogen and was subsequently activated. Pro-TGase appeared at 24h and reached a maximum concentration at 36h. Subsequently, with the level of Pro-TGase decreasing, the level of mature TGase increased gradually accompanied by a significant increase in TGase activity. TGase activity reached the highest value of 2.4 U/mL at about 60 h, when Pro-TGase was entirely converted into mature TGase.2. TGase-activating protease is located in the cell-free fraction and is inhibited by a TGase-activating protease inhibitor.TGase activation was silenced in cell-removed system. Both Pro-TGase level and TGase activity in the cell-free fraction remained steady from 0 to 24 h. 10 mg/mL cetyltrimethyl ammonium bromide (CTAB) could waken TGase activation in cell-removed system. CTAB-treated supernatant showed that the TGase activation process resumed and the rate of activation (from 1.2 to 2.3 U/mL).3. Transglutaminase activaton is catalyzed by multi-proteases in S. hygroscopicus liquid culture.TAP preparation (0.03 mg/mL) was purified by DEAE-Sepharose F. F. TAP (10μL) was added to 100μL Pro-TGase (0.26 mg/mL). This mixture was incubated at 30°C for 30 min. Pro-TGase then was cuted into TGase, and Pro-TGase was readily converted to TGase by the TAP-rich preparation, and the TGase activity was clearly detected (0.92 U/mL). TAP was detected as serine protease. Serine protease and metalloprotease involve in the TGase activation in cell-removed system. Under the inhibition of EDTA (10 mmol/L), TGase activity in cell-removed system did not increased and concentration of TGase and Pro-TGase kept stady; while under the inhibition of PMSF (1 mmol/L), TGase activity and the convertion from Pro-TGase to TGase were absolutely delayed.4. Purification, indentification and characterization of TAPI.TAPI was purified by Fractogel EMD SO3- column and Phenyl Sepharose 6 F. F. column. Yeild of TAPI was about 6.36mg/mL. TAPI (0.4μg) could inhibite Pro-TGase (2μg) activation mediated by TAP (0.2μg).N-terminal acid sequences of TAPI were: GLYAATTALV. TAPI belongs to Streptomyces subtilisin inhibitor (SSI) family. TAPI has surface activity. TAPI (0.1 mg/mL) decreased the surface tension of water from 72 to 60 mJ/m2 within 5 min. Based on these findings, a model for TAPI-regulated TGase activation process was proposed.5. In S. hygroscopicus solid culture, TGase was secreted and activated during differentiation. TGase may be involved in differentiation of Streptomyces.TGase was sereted and activated during the emergence of aerial hyphae rather then during the vegetative growth. And the seretion and activation process stopped when spores were formed. When white aerial hyphae first appeared at 48 h, TGase activaty was detected from the extract liquid (0.51 U/plate); after spores appeared at 144 h, TGase activity could not be detected. Pro-TGase activity from bovine trypsin activation first appeared at 48 h (0.03 U/plate). Subsequently, this activity increased gradually till reach 0.65 U/plate.Cystamine could inhibite TGase activity. Cystamine at the concentration of 25 mmol/L inhibited 75 % activity of TGase (0.17 U/mL). Cystamine (25 mmol/L) remarkablely delayed differentiation of S. hygroscopicus.