Research On The Regulation Of Bromelain Activity Based On Surfactants

The application of enzyme has attracted widespread attention in the fields of medicine,food and chemical industry,and thus,how to maintain its activity and stability is an important issue.In this dissertation,the effects of surfactant on the activity and thermal stability of bromelain from fruit pineapple was studied,and casein was used as the substrate.The surfactants used included anionic surfactant(sodium dodecyl sulfate(SDS)),conventional cationic surfactants(cetyl trimethyl ammonium bromide(CTAB)and dodecyl trimethyl ammonium bromide(DTAB)),gemini surfactants(pentane-1,5-bis(dimethyldodecyl ammonium bromide)(12-5-12),octamethylene-1,5-bis(dimethyldodecyl ammonium bromide)(12-8-12),dodecane-1,12-bis(dimethyldodecylammonium bromide)(12-12-12),pentane-1,5-bis(dimethylcetyl ammonium bromide)(16-5-16)and octamethylene-1,8-bis(dimethylcetyl ammonium bromide)(16-8-16)),and nonionic surfactant(octylphenyl polyoxyethylene ether(Tx-100)).The interaction between surfactant and bromelain was further studied by fluorescence spectroscopy and ~1H NMR.And then,the surfactant reverse micelles were used to extract bromelain.The results showed that cationic and non-ionic surfactants could promote bromelain activity,and anionic surfactants could inhibit bromelain activity.When the concentration of DTAB reached 2 mM and that of CTAB reached 0.3 mM,the activity of crude bromelain enzyme in the fruit pineapple could be increased by about 20%-30%,and the commercial bromelain activity could be increased by 70%and 50%,respectively.In the presence of 12-s-12(s=5,8,12)or 16-s-16(s=5,8),crude bromelain activity could be increased by 50%or 20%and commercial bromelain activity could be increased by 80%or 60%once the concentration of 12-s-12 reached 0.1 mM or that of 16-s-16 reached 20 μM.The shorter the hydrophobic chain of the surfactant,the stronger the resistance of bromelain to temperature,pH,and ionic strength.In Tx-100/16-5-16 mixed system,the activity of bromelain crude enzyme could be further enhanced,indicating a synergistic effect of non-ionic/cationic surfactants.Moreover,anionic surfactants reduced the fluorescence intensity of bromelain,and cationic surfactants increased the fluorescence intensity of bromelain.The reduction in the fluorescence intensity of bromelain corresponded to the attenuation of bromelain activity,and the increase in the fluorescence intensity of bromelain corresponds to an increase in the activity of bromelain.The study of ~1H NMR spectra showed that with the increase of surfactant concentration,the chemical shifts of hydrogen on the hydrophobic chain of the surfactant migrated to the low field,and those of the polar head of the cationic surfactants shifted to the low field,with the widening of the half peak width.The chemical shift of the signal of the anionic surfactant head group shifted to a high field with the narrowing of the half peak width.Bromelain could make the chemical shifts of all the protons in the surfactants moved to high field.Surfactant could also change the chemical shift of bromelain.When the cationic surfactant was present,the chemical shift of bromelain shifted to a low field and the half-peak width became broadened.When the anionic surfactant was present,the chemical shift of bromelain moved toward the high field and the half-peak width became narrowed.What’s more,the chemical shifts of cationic surfactants and bromelain were also related to temperature.With the increase of incubation temperature,the chemical shifts of the surfactant and bromelain moved to a higher field.Therefore,the electrostatic interaction and hydrophobic interaction between the cationic surfactant and bromelain made the bromelain conformation more stretched.In addition,gemini surfactant reverse micelles also exhibited excellent performance in bromelain extraction.Gemini surfactant with a longer spacer could not only extract bromelain over a wider range of surfactant concentrations,but also showed a better extraction efficiency.Among the surfactants used in the dissertation,12-12-12 reverse micelles showed the best extraction efficiency for bromelain;the activity recovery of bromelain was 160%,the purification factor was 3,and the extraction efficiency was 60%.