| The enzyme gradually penetrates into various industries,due to its mild reaction conditions,specificity of the substrate and high catalytic activity.However,the enzyme is susceptible to various factors such as strong acid,strong alkali,organic solvent and high temperature in the environment,and gradually deactivates or denatures the protein,loses the catalytic ability,and has a great influence on the separation and purification of the enzyme.Therefore,it is important to develop a suitable method to separate and purify high purity and high activity enzymes.As a composite separation technology,aqueous two-phase flotation has been gradually introduced into the separation and recovery of biological products because of its simple purification steps,mild operating environment and easy amplification.In this paper,bromelain and ?-glucosidase were used as examples to construct a novel ATPF method for separation and purification.The specific contents are as follows:(1)The thermosensitive polymer L64-IDA-Cu(?)was designed,synthesized and verified by FT-IR,atomic absorption spectrometry and transmittance experiments.The ionic liquid aqueous two-phase flotation(ILATPF)system of[Bmim]Br-K2HPO4 was constructed by investigating the effects of a series of ionic liquids(IL)and salts on the stability of enzymatic activity of bromelain.Then,bromelain was separated and purified by ILATPF using L64-IDA-Cu(?)as trapping agent.The experimental conditions were optimized by single factor experiments and response surface method:salt concentration,flotation time,L64-IDA-Cu(?)concentration,[Bmim]Br concentration and flotation flow rate were 0.50 g/mL,30 min,0.30 g/L,60%and 30 mL/min,and the best flotation effect was obtained:Ye and Kp values reached 97.43%and 3.45,respectively.For the mixture of L64-IDA-Cu(?)-bromelain complex and the upper phase[Bmim]Br obtained by flotation separation,the[Bmim]Br and L64-IDA-Cu(?)were removed by two-step precipitation using the thermosensitivity of L64-IDA-Cu(?)for further purification(Ye,Kp and PF values of the obtained bromelain were 96.90%,5.97 and 7.18,respectively).And compared with other purification methods,this experimental method has a good purification effect.Finally,SDS-PAGE analysis confirmed that the purity of purified bromelain was good Ultraviolet absorption spectroscopy(UV-vis),FT-IR and circular dichroism(CD)were used to verify that the purification process had no effect on the structure of bromelain(2)This experiment successfully expressed the fusion ?-glucosidase(Glu)containing GB and ELP tags:Glu-linker-ELP-GB(GLEGB).The ATPF system of PEG-(NH4)2S04 was constructed by investigating the effects of a series of polymers,salts and surfactants on the stability of Glu enzyme activity.GLEGB was then isolated and purified by the hydrophobic interaction of the GB tag on the fusion protein in combination with ATPF.The conditions of ATPF purified GLEGB were optimized by single factor experiment and response surface method(PEG 2000 concentration,BS-12 concentration,flotation time and flotation flow rate were 40.00%,1.50%,30 min and 30 mL/min,respectively)to obtain the best results(the Ye and PF values of purified GLEGB were respectively 172.86%and 9.58).Then,for the obtained mixture of GLEGB and PEG 2000,PEG 2000 was removed by using the inverse transition cycling(ITC)characteristic of the fused ELP tag to obtain a further purified GLEGB aqueous solution(Ye and PF values of purified GLEGB reached 124.92%and 24.26).).The ATPF-ITC method was also used to separate and purify other enzymes(Glu,Glu-linker-ELP(GLE),Glu-linker-GB(GLG),Glu-linker-ELP-6His(GLEH)),respectively.A good purification effect of the combination of GB and ELP labels was demonstrated.Finally,SDS-PAGE analysis confirmed that the purified GLEGB was of good purity.And the purchased Glu and purified GLEGB were compared by UV-vis,FT-IR and CD to verify that the purification process had no effect on the structure of GLEGB. |
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