| Transglutaminase (EC2.3.2.13, TGase) is a family of enzymes that catalyzecross-linking between the γ-carboxyamide group in glutamine residues (acyl donors) and avariety of primary amines (acyl acceptors), including the amino group of lysine. TGase cancrosslink proteins and improve the property of proteins, which attracts more and moreresearchers’ attention.In this study, TGase from Streptomyces hygroscopicus was modified in two ways tomake it more suitable for industrial application:1. Microbial TGase (MTG) from S. hygroscopicus was secreted as pro-MTG, and thenactivated by an additional protease, which has the potential to hydrolyze MTG and itssubstrates. In addition, the cost of MTG purification also increased. In order to solve theabove problems, a pH-dependent intein (Synechocystis sp. strain PCC6803DnaBmini-intein，SDB) was inserted between pro-region and MTG to mediate the activationprocess of pro-MTG by controlling the environmental pH. The results showed that therecombinant protein (pro-SDB-MTG) was successfully expressed in Escherichia coli andconverted to MTG in vitro within24h, at pH7.0,25℃. The MTG activity was0.93U·mL-1.The specific activity and Kmof recombinant MTG were14.3U·mg-1and69.4mmol·L-1,respectively. These results were nearly the same as wild type MTG. Meanwhile, the circulardichroism (CD) spectra of wild type MTG and recombinant MTG was also the same. All ofthese results proved that the insertion of SDB between pro-region and MTG did not affect thestructure and function of recombinant MTG. In addition, a high flexible region was found inpro-MTG by homology modeling and molecular simulation dynamics. This flexible regionwas comprised by the C-terminal of pro-region and N-terminal of MTG, which explained thatthe insertion of SDB between pro-region and MTG did not affect the structure and function ofMTG. Next, we investigated the effect of MTG first residue on SDB cleavage activity andMTG activity. We constructed two variants: pro-SDB-MTG(D1S) and pro-SDB-MTG(D1Δ).The two variants were successfully expressed in E. coli, and the cleavage time ofpro-SDB-MTG(D1S) and pro-SDB-MTG(ΔD1) was6h and36h, respectively. Meanwhile,the MTG activity of pro-SDB-MTG(D1S) and pro-SDB-MTG(ΔD1) was1.28U·mL-1and0.94U·mL-1, respectively. The specific activity of MTG(D1S) and MTG(ΔD1) was10.5U·mg-1and12.6U·mg-1, respectively.2. The improvement of MTG thermostability was helpful to its industrial application. Wewant to improve MTG thermostability by engineering a disulfide bond in its N-terminalregion. First, a loop region, comprised by the residues from3to11, was selected by proteinsequence aligment and structure analysis. Next, we use the software, disulfide by design(Dbd), to analysis the loop (3-11). The results showed two residues had the probability toform disulfide bonds with the residues near them: D4C and G284C, T8C and E59C. Weconstructs two mutants: pro-MTG(4-284) and pro-MTG(8-59), and purified MTG(4-284) andMTG(8-59) after activation. The formation of disulfide bond in MTG(4-284) and MTG(8-59)was detected by the DTNB method. When the recombinant MTG and its variants were treated at55℃for10min, MTG(4-284) and MTG(8-59) still had95%and65%activity,respectively. But the recombinant MTG showed only15%activity in10min at55℃. TheT50of recombinant MTG, MTG(4-284) and MTG(8-59) was58,65and63℃, respectively.Meanwhile, the resistance of trypsin-proteolysis of MTG(4-284) and MTG(8-59) was alsoimproved. The specific activity of recombinant MTG, MTG(4-284) and MTG(8-59) was13.3U·mg-1，12.5U·mg-1，5.2U·mg-1, respectively.In this study, we modified S. hygroscopicus MTG in two aspects. On one hand, theactivation process of pro-MTG was mediated by intein, instead of the additional protease. Onthe other hand, the MTG thermostability was improved by engineering a disulfide bond in theN-terminal region of MTG. All the results in this study might be helpful for the industryapplication of MTG. |
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