| Most proteins come from the fermentation broth of organisms(such as yeast,Aspergillus niger,Pseudomonas aeruginosa,etc.)or the extract of natural products(such as apples,pineapples,eggs,etc.),and the large-scale application of protein products depends on the extraction and purification technology.At present,the reverse micelle liquid-liquid extraction technology is still in the laboratory research stage,and the mechanism remains to be clarified.There are some problems need to be dealed,such as high concentration of surfactant,low loading capacity of protein,easy emulsification in the extraction process,and low activity recovery of protein.In this thesis,the efficiency of reverse micelles in protein extraction was studied systematically.The surfactants used included single-chained quaternary ammonium surfactants(dodecyl trimethyl ammonium bromide(DTAB)?cetyl trimethyl ammonium bromide(CTAB)),and gemini quaternary ammonium surfactants(pentane-1,5-bis(dimethyldodecyl ammonium bromide)(C12-C5-C12),octamethylene-1,5-bis(dimethyldodecyl ammonium bromide)(C12-C8-C12),dodecane-1,12-bis(dimethyldodecylammonium bromide)(C12-C12-C12),pentane-1,5-bis(dimethylcetyl ammonium bromide)(C16-Cs-C16)and octamethylene-1,8-bis(dimethylcetyl ammonium bromide)(C16-C8-C16)).Bromelain(BM)squeezed from pineapple pulp was taken as the target protein,extraction efficiency by quaternary ammonium surfactant reverse micelle and the effect on protein activity were discussed from the aspects of pH,ionic strength,structure and concentration of surfactant,dilution multiple of crude enzyme,solvent,cosolvent and so on.Then,the target protein was changed.Based on the properties of protein,the structure of quaternary ammonium surfactant and the composition of the reverse micelles were designed and optimized,by doing so,the extraction efficiency of the reverse micelles and the activity recovery was improved.The main contents of this work are as follows:1.Reverse micelles prepared from single-chained quaternary ammonium surfactant/isooctane/butanol/hexanol were used toextract bromelain from pineapple pulp.It was usful to increase extraction efficiency by decreasing the polarity of tryptophan via increasing the concentration of surfactant or pH value,adding appropriate amount of NaCl during forward extraction and KBr during backward extraction.When DTAB reverse micelle was used,the extraction efficiency(EF),activity recovery(AR)and purification multiple(PF) of bromelain were 52%,54%and 1.0,respectively,under optimal condition.With CTAB reverse micelle,the optimal values of EF,AR and PF of bromelain were 54%,82%and 1.5,respectively.Therefore,increasing the hydrophobic chain length can improve the extraction efficiency of single-chained quaternary ammonium surfactant reverse micelle.2.With the increase of the spacer length of Gemini quaternary ammonium surfactant(C12-C5-C12?C12-C8-C12,C16-C5-C16?C16-C8-C16),the extraction efficiency of reverse micelles for bromelain increased,but decreased when the spacer length increased to 12(C12-C12-C12).With the increase of hydrophobic chain length(C12-C5-C12?C16-C5-C16,C12-C8-C12?C16-C8-C16),the reverse micelle extraction efficiency decreased.For C12-Cs-C12(s=5,8,12)surfactant reverse micelles,C12-C8-C12 reverse micelles exibited the highest extraction efficiency(AR:107 %,PF:1.6);for C16-Cs-C16(s=5,8)surfactant reverse micelles,C16-C8-C16 reverse micelles had the higher extraction efficiency(AR:98 %,PF:1.8).3.The extraction efficiency of mixed reverse micelles for bromelain depended on the composition of reverse micelles.With 2030 mg·mL-1 C12-C8-C12/1014 mg·mL-1 DTAB mixed reverse micelles,the extraction efficiency for bromelain was better than that with C12-C8-C12 or DTAB reverse micelles.With 1.5·40 mg·ML-1 C16-C8-C16/1030 mg·mL-1 CTAB mixed reverse micelles,the extraction efficiency for bromelain was lower than C16-C8-C16 or CTAB reverse micelles.The 20 mg·mL-1 C12-C8-C12/10 mg·mL-1 DTAB mixed reverse micelles showed the best extraction efficiency,AR,PF and EF were 98%,1.4 and 68%,respectively.4.The transfer of bromelain between two phases became more convenient by using isooctane as solvent,and n-butanol and n-hexanol as the auxiliary solvent.The separation effiency was improved with the increase of the spacer length of Gemini surfactant.For DTAB,CTAB,C12-C5-C12,C16-C5-C16 and C16-C8-C16,the reverse micelles prepared by isooctane,15 %n-butanol and 5 % n-hexanol,the extraction efficiency was higher than others.C12-C8-C12/hexane/10 %hexanol/water reverse micelle and C12-C12-C12/hexane/10 %hexanol/water reverse micelle can achieve the high-efficiency purification of bromelain in a wider pH range.5.Bovine serum albumin(BSA),ovalbumin(OVA)and polyphenol oxidase(PPO)were extracted with quaternary ammonium surfactant reverse micelles to investigate the selectivity of proteins.For the exraction of BSA and bromelain,C12-C12-C12 or C16-C8-C16 was the best surfactant for the purification;C12-C5-C12 or C16-C8-C16 is more suitable for the extraction of hydrophobic OVA.However,all these quaternary ammonium surfactant reverse micelles were not suitable for extracting PPO from apple juice. |
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