The Study On The Molecular Modification Of Streptomyces Hygroscopicus Transglutaminase For Enhanced Catalytic Properties

Transglutaminase （EC2.3.2.13, TGase） catalyzes an acyl transfer reaction between aγ-carboxyamide group of glutamine and ε-amino group of lysine or other primary amine,resulting in the formation of ε-（γ-glutamyl）-lysine peptide chain bridges. Because of thiscapability, TGase has been used in many industrial areas, such as food engineering, textile andleather processing, materials engineering, and biomedical engineering. Among TGaseenzymes, that from Streptomyces is the main source in commercialization because of itsCa2+-independence and broad substrate specificity for acyl donors. However, the low catalyticactivity and poor thermal stability of TGase limite its industrial applications. Therefore, moreinterest has been focused on the development of higher activity and more thermal stableTGase domestically as well as globally.In our lab, a new strain （S. hygroscopicus WSH03-13） capable of producing a highactivity of TGase was isolated from soil previously. Streptomyces TGase is naturallysynthesized as a zymogen （pro-TGase）, which is then processed through the removal of itsN-terminal pro-peptide to produce active TGase. In this study, the recombinant Escherichiacoli was used to express pro-TGase and its mutants. Several strategies were adopted toimprove the catalytic activity and thermal stability, such as pro-peptide engineering,N-terminal residues saturation mutagenesis, and fusing stabilization tag. Furthermore, thestable mechanism of TGase was analyzed and discussed. The main results are listed asfollowing:（1） Analyzing the function of pro-peptide in S. hygroscopicus TGaseThe structure of S. hygroscopicus pro-TGase was simulated and analyzed. Thepro-peptide globally consisted of-helix^L12-N30,-helix^R37-S42and loop^L43-P57. Seven hydrogenbonds were predicted between pro-peptide （Y12, N27, N30, and R32） and TGase. When thefour amino acid residues were mutated into A, the mutated pro-TGase （Y12A） was expressedas inclusion bodies, and the secretion of other mutants were partially inhibited. Deletion of theC-terminal loop^L43-A52in pro-peptide increased the secretion of TGase by approximately70%as compared with wild type enzyme （WT）. Deletion of the C-terminal α-helixL37-A42had noincrease in TGase secretion, but increased the specific activity from15.4U/mg to17.3U/mg.To analyze the roles of α-helixL37-A42, it was substituted with AAA and GGG, respectively.The corresponding mutants exhibted22.2%and24.8%higher specific activity than WT, andthe efficiency of pro-peptide cleavage was2-fold higher than that of WT. These resultssuggest that the pro-peptide is important for TGase secretion and activity.（2） Enhancement of TGase activity and thermal stability by introducing tags into theC-terminus of pro-peptideThe sites for introducing tags located before the cleavage site （L53） of dispase. Five tagsincluding GG, GGG, GGGG, GGGGS, and PTPPTTPT were chosen to introduce into theC-terminus of pro-peptide. The mutants with last two tags exhibited26.1%and35.3%higheractivities than that of WT. To further improve the properties of TGase, eleven importantpeptides from pro-TGase were chosen as tags. Among these tags, the mutants with tag2 （SPARPGESW） and tag4（KTIWTHANH） exhibited40%higher specific activity, especiallyfor the later one, also exhibted90%longer half-life at50℃than WT.（3） Deletion combined with saturation mutagenesis of N-terminal residues in TGaseresults in enhanced activity and thermal stabilityThe mutated TGase missing the first four residues （Del1-4） showed an increase inspecific activity of32.9%. However, the mutants missing five or more than five residuesexhibited reduced activity. Then, the fifth residue E62in mutant Del1-4was selected forsubstitution with the nineteen other amino acids. The mutant replacing the fifth residue withan aspartic acid （Del1-4/E62D） exhibited a85%higher specific activity and a1.7-fold longerhalf-life at50°C when compared with WT. The melting temperature of the mutated TGaseincreased from68.9℃to79.1℃by circular dichroism spectroscopy （CD） analysis. Analyzingthe structure of Del1-4/E62D, it was found that the distance, hydrophobic interaction, chargedistributions bewteen N-terminal region and loop^335-346are very important for TGase activityand thermal stability.（4） Analyzing the function of the C-terminal residues and enhancing TGase thermalstability through fusion a stabilization tag into its C-terminusWhen deletion of the C-terminal residue S389, there was no influence in TGase activity.However, deletion of the C-terminal residue W388, the mutated TGase lost activity.Analyzing the structure of C-terminal region, W388could form hydrogen bonds withN-terminal residues D76, A77, and Y78. To analyze the roles of hydrogen bonds, the W388was substituted with A. The activity and thermal stability of mutant W388A decreased by50%, suggesting that the hydrogen bonds were important for TGase activity and thermalstability. Furthermore, a tag （IGCIILT） from the C-terminus of RNase HI was chosen asstabilization tag to fuse into the C-terminus of TGase. The half-life of the correspondingmutant （TG-tag1） at50°C increased from3.7min to9.1min. To analyze the reasons ofincreased thermal stability, the structure of TG-tag1was investigated through CD andfluorescence spectroscopy. The secondary structure of TG-tag1had no significant differencewith WT. However, the hydrophobicity of TG-tag1was increased through fluorescencespectroscopy and ANS binding analysis.（5） High cell density culture of recombinant E. coli via fed-batch for over-production ofpro-TGaseTo improve the properties of TGase, the benefical mutagenesis were combined. Thespecific activity and half-life of mutant Del1-4/E62D-tag1were increased to26.8U/mg and11.2min. Then, the mutant E62D-tag1was cultured with batch and fed-batch fermentation.The highest extracellular activity of TGase in batch culture was14.8U/mL. To achieve highcell density fed-batch culture of recombinant E. coli producing pro-TGase, a two-stagefeeding strategy was proposed and applied and cell concentration （OD600） could reach120.The highest extracellular activity was32.5U/mL after optimizing the induction cellconcentration. Based on the culture condition, the addition times of glycine and Ca2+wasoptimized. The highest extracellular activity reached47.4U/mL.