Studies On High Density Culture Of Transglutaminase By Recombinant Escherichia Coli For Transglutaminase Production

Transglutaminase （protein-glutamine: amine γ-glutamyltransferase, EC2.3.2.13;TGase） is a family of enzymes that catalyze the formation of amide bond betweenγ-carboxamide groups of peptide-bound glutamine residues and primary amino groupsof either protein-bound lysine residues or free amines, resulting in the formation ofprotein/protein cross-links or protein/amine conjugates. In the absence of aminesubstrates, reactive glutamine residues are deamidated into glutamic acid residues. Dueto its great potential to improve various functional proprties of proteins, TGase ismainly used to enhance texture and stability.In order to improve cell concentration, we optimized the fermentation conditionsand medium constituents for the production of recombinant MTG by E.coli Rosetta（DE3）. With the same of the synthesized medium, high cell density in10L fermentorwere studied. We also studied a series of denaturation, solubilization, purification andrefolding, and finally gained the functional protins.In order to increase the cell concentration, we studied the growth of E.coliRosetta（DE3） and the influence of production of MTG. The optimal fermentationconditions were as follows:25mL operational volume in250mL shake flask,inoculation size5%（V/V）, initial pH7.0, the optima temperature was37℃, the bestinduction was started when OD600reached to0.6⁰.8（growth for4h）by adding0.8mmol/L IPTG and induction time was4h.Based on the optimum fermentation conditions, we finially fined the optimalcomposition of the culture medium by single factor experiment and Orthogonal: glucose4g/L, Yeast extract3g/L, NH₄Cl4.0g/L, Na₂HPO₄2.0g/L, KH₂PO₄1.0g/L,MgSO₄·7H₂O1.0g/L, NaCl3.0g/L, trace elements solution2.0mL/L, Vitamin B11.0mg/L, VitaminB20.5mg/L, Vitamin B50.5mg/L, Vitamin B60.2mg/L. When thestrain was cultivated in shake flask fermentation by using this synthetic medium, thefinal OD increased to5.8from1.8, the amount of recombinant proteins up to36.2%.The cell growth rate and cell concentration has been greatly improved.On the basis of the above studies, the high cell-density fermentation ofrecombinant E.coli Rosetta（DE3） in10Liter fermentor was carried out. Therecombinant MTG-expressing bacteria grew to OD600of51.5and the finial cell densityreach112g wet weight per liter by adopting the dissolved oxygen feed-back fed-batch. But mostly recombinant protein was inactive inclusion body. After renaturation, thespecific activity and the protein concentration of MTG were8.1U/mg and4mg/L.