MgCl₂Stress Regulation Biosynthesis Of Transglutaminase Of Streptomyces Mobaraensis And Enzyme Application Properties

Transglutaminase （shorten as TGase, EC2.3.2.13, protein-glutamine: amineγ-glutamyltransferase） is a family of enzymes that can catalyze the cross-linking ofproteins to high molecular weight aggregates by transfer reaction betweenappropriate glutamine and lysine residues. It attacts a great interest, since it modifysthe functional properties of protein such as solubility, hydration ability, rheologicaland emulsifying properties as well as heat stability in model systems. However,scarcely sources, low yield and expensive price of TGase limit its application in thefood industry. Based on the theory that Streptomyces cell growth in disadvantageconditions are benefit to the synthesis of secondary metabolites, this paper aimed atimproving TGase production by stress methods. The most suitable condition wasfound. Furthermore, mechanism of enhancement of TGase production was studied.Meanwhile, TGase from MgCl₂media was purified and characterized, and appliedinto yogurt.Effects of heat treatment, supplementation alcohol or NaCl on the TGaseproduction and cell growth of Streptomyces mobaraensis showed that stress methodsimproved the TGase production in varying extent. Response of surface design ofheat treatment, addition alcohol and/or NaCl to media was used to estimate theirinteraction effects on TGase production. Results showed that the interaction effectwas not significant and salt stress was the best way to improve TGase production. Inorder to find better condition for TGase production, eight kinds of different neutralsalts （NaCl, Na₂SO₄, C₆H₅O₇Na₃, MgCl₂, CaCl₂, CH3COONa, KCl and Na₃PO₄）were added into the media. Results showed that the optimal salt for TGaseproduction was MgCl₂. In order to study the enhancement of TGase production byMgCl₂stress, effects of MgCl₂concentration on cell growth, period of TGasebiosynthesis and proteases production were studied. The highest yield of TGase wasobserved at96h of incubation （4.3U/ml）, when a basic medium was supplementedof0.1mol/l MgCl₂, compared with the basic media （2.1U/ml） by120h.Western-blot analysis showed faster transformation of pro-TGase into the matureenzyme in MgCl₂medium vs. the control medium. Total protease, metalloproteaseand serine protease were also biosynthesized in a fast rate in the media containingMgCl₂. The results demonstrated that the MgCl₂stress improved the production of key proteases involved in the activation of Pro-TGase. Furthermore, in order todetermine the key enzyme for Pro-TGase activation, the inhibitors formetalloprotease （EDTA） and serine protease （PMSF） were added into two kinds ofmedia separately, and TGase activities were determined during fermentation. Resultsshowed that the key enzyme for TGase production was metalloprotease.Strain viability, morphological differentiation and DNA fragment of S.mobaraensis during fermentation in the control media and MgCl₂media werecompared. MgCl₂inhibited cell growth and the formation of strain sphere. With theextension of the fermentation time, MgCl₂promoted the bacterial programmed celldeath. According to bacterial morphological differentiation in both kinds of media, itwas found that S. mobaraensis in MgCl₂media appeared bent, branched and amyloidearlier than in the control media; as well as, fimbriae and spores appeared in MgCl₂media, not in the control. These results suggested that the MgCl₂promotedmycelium differentiation. Comparing S. mobaraensis DNA changes in two kinds ofmedia, it was found that MgCl₂promote bacterial DNA cleavage, which indicatedthat MgCl₂stimulated morphological differentiation and programmed cell death,thus shortened the fermentation period.Comparing two dimensional gel electrophoresis of the bacterial cell protein incontrol medium and MgCl₂medium cultured for24h by PDQuest, fifty-threedifferentially expressed proteins were found and50proteins was identifiedsuccessfully by mass spectrometry. Their physiological functions were analyzedaccording to KEGG data. Results showed that proteins involved in primarymetabolism, such as sugar metabolism, amino acid metabolism, nucleotidemetabolism were inhibited by MgCl₂stress, while those involved in secondarymetabolism, pressure stress protein and energy metabolism-related enzymes wereincreased. These results showed that MgCl₂stress inhibited the primary metabolismof Streptomyces, decreased bacterial cell growth rates. However, it promotedproteins involved in secondary metabolism or synthesis of secondary metabolitesprecursors. Thus, TGase was biosynthesized earlier with high yields and myceliaprogrammed cell death was promoted.In order to purify TGase from high-salt media, gel filtration and ion exchangechromatography method were combined after concentrating fermentation liquid byultrafiltration. Using liquid chromatography/tandem mass spectrometryidentification and the protein database comparison, the purified enzyme showed high homology with AAN01353from S. mobaraensis. Characterization of purifiedenzyme showed higher heat and pH stability. Application of the pure enzyme intoskim and whole milk yogurt showed that it could improve the functional propertiesof yogurt, but the excessive cross-linking caused the oversize of yogurt gel particlesand the course texture. In addition, milk fat affected the protein cross-linking and thetexture of the yogurt. Furthermore, TGase was used to cross-link yak yogurt protein.Results showed that the texture of cross-linking yak yogurt was more delicate andacceptable. It provided the foundation for the development of yak milk products.