| Transglutaminase (Transglutaminase, R-glutaminyl peptide: amine y-glutaminyl transferase, EC 2.3.2.13) are thiol enzymes that improve protein functions, reform structures and increase nutrition value through catalyzing protein reticulation by introducing isopeptide bonds (ε - (γ-glutaminyl) lysine bonds). These eukaryotic transglutaminase, Ca~2+- dependent, are involved in numerous biological functions. In prokaryotes, TGase activity has been found only in actinomycetes, Ca~2+- independent.Although the physiological role of this bacterial transglutaminase is still unknown, the enzyme has been pay more attention in fields of food application and protein modification. But till now, TGase has not been used widely because the low total recovery of activity and the low stability against long-time store. Ajinomoto Co. (Japan) has incubated Streptoverticillium mobaraense to obtain TGase and researched for the stability of TGase in presence of partial protein hydrolysate. Meanwhile the total recovery of activity of TGase produced by domestic company is as 1/10 times as that of Ajinomoto Co. So this research purposes looking for the way to enhance the total recovery of activity and the stability during production and storage.Methods: (1) TGase activity is measured by Grossowicz colorimetry. Proteins are determined by the Folin hydroxybenzene dye-bingding method. (2) Calculate the remained enzyme activity after heated to compare the effect on stability of TGase in presence of different addivites. (3) The thermodynamic data (temperature of inactivation point - Tm, enthalpy - AH, loss weight) of TGase in presence of addivites are measured by TG/DSC.Results: (1) After incubation, Streptoverticillium mobaraense forms a kind of pellet and is removed by filtration. Then an ultrafiltration membrane which block off those molecular weight larger than 10000 is used to concentrate the enzyme for 2.5 to 3 times. Before add 1.5 times ethanol as the volume of TGase, modify pH of TGase to 7.5 to 8.0. The total recovery of activity of the process is about 85 %. (2) The remained enzyme activity of TGase in presence of trehalose is increased 35 %, meanwhile that of TGase in presence of sugar is increased 30 %. The remained enzyme activity of TGase in presence of glucose is only increased 25 %. Compare in presence of skim milk with whey powder, the remained enzyme activity of TGase is increased 10 % more. (3) Tm of pure TGase is 123 ℃. After add protectants into TGase , the highest Tm is 155 ℃. AH of pure TGase is 17.9 J/g. After add protectants into TGase, the highest AH is 58.4 J/g. Loss weight of pure TGase is 35 %. After add protectants into TGase, the lowest loss weight is 28 %.Conclusion: (1) Times of concentrated filtrate affects time, enzyme activity and protein concentration during ultrafiltration. The pH of TGase modified before adding ethanol and volume of ethanol influence the total recovery of activity and appearance of concentrated TGase. (2) As a precipitator, ethanol for harmless, obtainable and volatilizable is fit for be used during deposition. (3) The effect of saccharide as additives increasing stability of TGase is trehalose > sugar > glucose. (4) The effect of whey protein and casein is more than that of trehalose. Whey powder equals to whey protein and skim milk powder equals to casein when comparing their effect on stablility of TGase. (5) More the effect of additives on stability of TGase, more increased Tm and AH of TGase while more depressed loss weight. (6) Tm and AH of TGase in presence of trehalose is higer than in presence of sugar and glucose, so the effect of trehalose on stability of TGase is the best in them. (7) The thermodynamic data of TGase in presence of whey protein (casein) corresponds with whey powder (skim milk powder), so their effect on stability of trehalose is similar.
|
PDF Full Text Request