Establishment And Application Of The Analytical Method For Cross-Linking Sites Of Transglutaminase

Transglutaminase(TGase)is a kind of acyltransferase which exists widely in animal,plant and microbial tissues.It can catalyze the covalent cross-linking reaction between glutamine and lysine in protein and form a complex spatial network structure.It has important physiological functions in organisms.TGase can give protein products better texture properties,so it is widely used as an industrial enzyme in soybean products,dairy products and other food protein processing industries.Liquid chromatography-mass spectrometry(LC-MS),as a common tool for proteomic analysis,can be used to analyze the cross-linking of disulfide bonds and other proteins.However,at present,there is little research and development of LC-MS methods to analyze TGase cross-linking sites,and there is no research on the selection tendency and dynamic changes of cross-linking sites in the process of food protein TGase cross-linking.In this study,a LC-MS method for analyzing TGase cross-linking sites was established and validated.On this basis,the dynamic process of TGase cross-linked soybean protein molecules was analyzed,and the endogenous protein substrates and cross-linking sites of TGase enzyme during Bacillus licheniformis sporulation were identified.Through the cross-linking of standard peptides and standard proteins,a LC-MS method for analyzing TGase cross-linking sites was established and verified.The process of this method is as follows: after the cross-linked samples were denatured and digested with trypsin,the cross-linked peptides were analyzed by high performance liquid chromatography-electrospray time-of-flight mass spectrometry(HPLC-ESI/Q-TOF),then the cross-linked peptides and sites were identified by the modified PLink software,and the cross-linked peptides were quantified by Masshunter software.The synthetic peptides are cross-linked and analyzed,and the synthetic peptides are hidden in the huge protein database.This method can still accurately identify the cross-linked peptides,showing strong reliability.Bovine serum albumin(BSA)was cross-linked and analyzed.The method successfully revealed the dynamic changes of its cross-linked peptides and cross-linking sites,and showed that TGase(MTGase)from Streptococcus cerevisiae preferred K(548),K(455),Q(603),Q(440),K(28)and Q(549)for BSA cross-linking sites.TGase cross-linking treatment can enhance the gel properties,water-holding capacity and stability of food proteins,but there are no studies on site selection and dynamic changes of TGase cross-linked food proteins at the molecular level.Soybean protein isolate(SPI)was used as the object.Using the established cross-linking analysis method,the crosslinking process of MTGase was analyzed,and it was found that MTGase was easy to cross-link SPI at the following sites: Q(117)of protein component Glycinin G5,K(87)of Sucrose-binding protein,K(208)of Seed linoleate 9S-lipoxygenase-2,Q(298)and Q(304)of Glycinin G1,and K(405)of Glycinin G4.TGase is thought to be involved in the process of spore formation in Bacillus,but there is a lack of systematic and comprehensive research on the type and quantity of protein substrates played by TGase in this process.In this study,the spore form of Bacillus licheniformis was taken as the object.Through the comparison between wild-type strains and TGase gene(tgl)deficient strains,the types of protein cross-linking substrates and their cross-linking sites of TGase were revealed by cross-linking analysis.The heat tolerance evaluation experiment showed that the heat tolerance of Bacillus licheniformis spores was enhanced after tgl gene knockout.Gene expression analysis showed that the transcription and expression of tgl gene occurred in the stable and senescent stages of cells,which was associated with sporulation.The types of substrates affected by TGase during spore formation were successfully identified by using the established cross-linking site analysis technique.In addition to the proteins GerQ and SafA reported in the literature,spore proteins such as CotO,CotJC,GerKA,GerKB,YutH,GerKC,CotG,GerB3,GerD,GerPE,YsxE and GerPC were also found to be cross-linked by TGase.This study is not only the direct biochemical data about the protein cross-linking in spore formation,but also the direct evidence of the highly ordered and resistant structure of the spore coat.In this study,a method for analyzing TGase cross-linking sites was established and successfully applied to the analysis of food protein cross-linking process and bacterial sporulation.This work is of great significance for revealing the molecular mechanism of TGase processed food proteins and elucidating the physiological function of TGase in vivo.