| Transglutaminases (TGases, protein-glutamine: amine γ-glutamyltransferase, EC2.3.2.13) are a family of enzymes that catalyze transfer reaction between a γ-carboxamide group of the glutamyl residue (amine acceptor) and a ε-amino group of a lysine residue or a primary amine group in a polyamine (amine donor). The results of this activity introduces the modification of the protein conformation and cross-linking reaction either intra or intermolecular in the proteins, leading to the improvement of functional properties. TGases are widely distributed in nature, but the research on plant TGase was confined by the difficult isolation and purification. Heterologous expression of plant TGase is an effective a way to solve this problem. The aim of this article is to express active TGZ in Escherichia coli and Pichia pastoris expression system. In order to achieve large-scale and soluble protein production, the coding sequence of tgz gene was optimized according to the codon bias of P. pastoris and synthesized using SOEing-PCR. Meanwhile, the functional properties of purified TGZ and MTG were compared.In order to study the effect of chloroplast import peptide for the protein expression and production, tgz and ptgz were expressed in ArcticExpress E. coli respectively. The result of expression showed that this import peptide did not affect the expression level and the solubility of recombinant protein. But the recombinant TGases were mainly presented as inclusion bodies and activated by a complicated refolding process. The expression conditions of recombinant strain were optimized, and the maximal expression of recombinant TGZ was achieved by culturing recombinant strain ArcticExpress E. coli to OD600of0.4at LB medium at37°C, and induced by0.2mmol/L IPTG for16h at16°C when a predicted chloroplast import peptide was used. The inclusion bodies were solubilized in8mol/L urea, and then purified by an affinity method. Under optimal expression condition, the specific activity and production of refolded TGZ were0.34U/mg and1.41mg/L.Because the TGZ expressed in E. coli was mainly present in inclusion bodies, we expressed this gene in P. pastoris to achieve a soluble expression. TGZ gene was synthesized according to the P. pastoris preferred codons. In order to generate the optimized DNA sequence,14B-type repeat regions were eliminated and the full length of TGZ gene was divided into three fragments, A, B and C. The optimized coding sequence was synthesized by SOEing-PCR, and a six-histidine tag was added for the purification. Compared with tgz gene, tgzo gene was changed289nucleotides, involving239amino acids, and the content of G+C decreased from53.1%to40.3%, which consistent with the P. pastoris. tgz and tgzo were ligated into Eco R I-Not I digested pPIC9K to create recombinant vector pPIC9K-tgz and pPIC9K-tgzo. These two recombinant plasmid were linearized with restriction enzyme, and transformed into P. pastoris GS115competent cells using electroporation. The His+Mut+multi-copy transformants G-tgz and G-tgzo were selected by phenotypes, G418resistance and colony PCR. The secreted TGZ was initially separated by Superdex200resin and further purified on a cation exchange resin. The activity of TGZ following purification was0.32U/mg protein and the production was1.44mg/L. The secreted TGZo was purified by affinity method, its specific activity was0.89U/mg, and the production was4.4mg/L. After codon optimization, the activity of TGZo was2.6times of TGZ expressed in E. coli and2.8times of TGZ expressed in P. pastoris, the production was3.1times of TGZ expressed in these two expression system. These result indicated that the activity and yield of TGZo was successfully improved through codon optimization.In order to improve the expression of G-tgzo, induction medium, final methanol concentration, induction time, loading volume, initial pH, biomass before induction, oleic acid and PMT1were optimized. And then P-B design and response surface methodology was applied to improve the production of the TGZo. The final composition of the optimized culture conditions were obtained as follows: the recombinant strain was cultured to OD600of2in BMGY medium, and then the strain was transferred to a500mL flask containing37mL optimized medium (2%peptone,1%yeast extract,100mmol/L phosphate buffer pH6.5,1.31%methanol,0.006%metal ions,0.07%oleic acid,1.34%YNB and4×10-5%biotin) induced for96h at28°C.1.31%methanol,0.006%PMT1and0.07%oleic acid were added every24h. Under optimal expression condition, the TGZo activity increased to1078mU/mL in the shake flask system and the production of TGZo was enhanced to7.6mg/L after purification using affinity method. The activity was80%and the production was73%higher than that the production before optimization. Thus, this recombinant strain G-tgzo may have highly potential for the plant TGase production. Characterization of different TGase was studied by the fluorometric method. The result showed that recombinant TGZ has less binding capacity to casein than that MTG. The optimal temperature of TGZo was37°C, less than the45°C of TGZe and MTG, but the heat stability of recombinant TGZ were4~60°C that higher than the MTG. The optimal pH of TGZo and TGZe were8.0, and they had a better activity and stability under alkaline condition. Metal ions have different impact on the activity of recombinant enzyme. Among these metal ions, low concentration of Ca2+has a strongest promote to the activity of TGZe and TGZo. The cross-linking reaction of TGZo and MTG to the different protein substrates were studied, the results showed that two enzymes had a significant polymerization effect on casein and soy protein isolate. And the reaction catalyzed by TGZo to the soy protein isolate was higher than that of MTG, but there was no cross-linking effect of TGZo on whey protein. Application of recombinant TGZo into milk yoghurt showed that it could improve the textural properties and apparent viscosity of yoghurt, meanwhile reduce the whey separation, but the improvement of yoghurt treated with TGZo was less than MTG because of the TGZo properties. In addition, recombinant TGZo was used to improve the functional properties of different content fat yoghurt. The result showed that fat has no effect on the enzyme activity. These results will further lay the foundation for the application of plant TGase in food industry. |
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