The Study On The Thermostability And Substrate Specificity Modification Of Microbial Transglutaminase

Transglutaminase（EC 2.3.2.13,TGase）catalyzes the reaction of theγ-carboxamido group of a glutamine residue with the primary amine of an acyl acceptor to formε-（γ-glutamyl）covalent bond.TGases are widely distributed in various organisms,and microbial-derived TGases are commonly used for industrial processing due to they are calcium-independent during catalysis and easy to be produced.Streptomyces mobaraenesis derived TGase（sm TG）is commercial available.However,sm TG showed weak thermostability and low activity towards certain substrates which restricted its utility horizon.Previously,Escherichia coli used for heterologous expression of sm TG was convenient,but the yield of active sm TG was low,and expression of sm TG as zymogen is tedious for subsequent enzyme purification and property characterization.In this study,a reported high activity and thermostability sm TG variant S2P-S23V-Y24N-S199A-K294L（TGm1）was used as research target.Its recombinant active expression and enzyme property modification were systematically investigated,and the main findings were as follows.（1）Construction of E.coli for active expression of microbial transglutaminasesm TG was natively expressed in zymogen and its pro-peptide was removed by meltalloprotease（TAMEP）to activate sm TG.In this study,the pro-peptide of sm TG was replaced with these pro-peptides from the other Streptomyces TGases.Fusing with the pro-peptide of Streptomyces hygroscopicus TGase resulted in intracellular TGase activity reached6.78 U/m L,which was 64%higher than using the original pro-peptide.Thioredoxin A was fused to TGm1 zymogen,and the intracellular TGase activity further reached 9.8 U/m L.TGm1zymogen was co-expressed with TAMEP which obtained intracellular TGase activity of 0.79U/m L.Through co-expression with molecular chaperones Dna K/Dna J/Grp E,the resulted intracellular TGase activity reached 9.5 U/m L.The 3L bioreactor was used for the fermentation,which resulted in 15.4 U/m L of intracellular TGase activity.（2）Construction of E.coli for secretory expression of microbial transglutaminaseThe pel B signal peptide was redesigned,and those pel B variants with the lowest and highest m RNA folding free energy were fused to TGm1.TGm1 zymogen can be activated by TAMEP in the periplasmic space before releasing to the extracellular space of E.coli.These pel B variant with the lowest m RNA folding free energy significantly enhanced the secretion of TGm1,and the best variant resulted in detected extracellular TGase activity of 1.5 U/m L,which was 57.9%higher than using native pel B.Knockdown of outter membrane protein gene lpp has further promoted the extracellular TGase activity which finally reached to 2 U/m L.TGm1 fused with the pro-peptide of S.hygroscopicus and the original pro-peptide left a residual FRAPD and FRAP tag in its N-terminus after TAMEP cleavage,respectively.FRAP-TGm1 and FRAPD-TGm1 displayed 11.31 and 22 min half-life(t_1/2)at 60°C,and their specific activity were 50 and 49.1 U/mg,respectively.（3）Rational design the thermostability and specific activity of microbial transglutaminaseRosetta Cartesian＿ddg was adopted to carry out whole-protein proline scan based on FRAPD-TGm1.The single mutation A287P and A265P improved the thermostability of FRAPD-TGm1,that the melting temperature（T_m）of FRAPD-TGm1-A265P and FRAPD-TGm1-A287P increased by 0.69 and 2.13℃,respectively.The T_m of FRAPD-TGm1-A287P reached 67.3℃.Molecular docking and energy prediction were carried out using Discovery Studio,the mutations that can enhance the binding affinity between FRAPD-TGm1 and its substrate were experimentally validated,and obtained single mutation E28T can significantly enhance the specific activity of TGm1,which reached 61.8 U/mg.Desigining surface charge was carried out based on the combinatory mutant FRAPD-TGm1-E28T-A265P-A287P（FRAPD-TGm2）using Rosetta Supercharge.The resulted variant FRAPD-TGm2-N96E-S144E-N163D-R183E-R208E-K325E（FRAPD-TGm3）displayed t_1/2（60°C）of 122.9 min and the specific activity reached 83.7 U/mg,which were 4.6-fold and 70.5%higher than that of FRAPD-TGm1,respectively.（4）Investigating substrate binding sites and modification of the specific activity of microbial transglutaminaseDeep learning framework DUnet was built based on convolutional neural network.DUnet was trained using protein-ligand dataset（sc PDB）for predicting of protein-ligand binding sties.DUnet predicted 29 substrate binding sites for sm TG,and 10 sites were experimentally validated which consist of acyl receptor binding area by alanine scan.Previous study showed sm TG displayed weak catalytic activity towards the acyl donor of peptide GGGGQR.In this study,a rational design protocol was built based on Rosetta script,which can resolve molecular docking and virtual mutagenesis issue.Through engineering the key residues within the catalytic pocket,the specific activity against GGGGQR of FRAPD-TGm2-G250H and FRAPD-TGm2-Y278E were dramatically improved.FRAPD-TGm2 displayed a 7.3 U/mg activity against GGGGQR which was 1.13-fold higher than FRAPD-TGm2.