| rAlcohol dehydrogenase?ADH,EC 1.1.1.1?is the key enzyme in alcohol metabolism.It has great application potential in disease diagnosis and medical analysis,food science and technology,biotechnology and enzyme industr etc.In human,the primary metabolism of alcohol occurred in stomach,during which Stomach alcohol dehydrogenase ??-ADH plays a major role.However,the activity of ??-ADH is most inhibited in acidic conditions,which limited its application area broading.In this paper,the ??-ADH was cloned,expressed and purified for characterization.In addition,the immobilization method of recombinant ??-ADH was explored for improving its alcohol tolerance and acidic stability.The adh7 gene which encoded ??-ADH was chemical synthesized,inserted into plasmid pET-32a?+?and transformed into E.coli BL21?DE3?,obtaining the engineered strain E-ADH.In the host E-ADH,the recombinant ??-ADH was expressed in the form of inactive inclusion body.The dissolution of inclusion body were optimized by orthogonal design as follows,urea concentration in buffer was 1 mol?L-1,pH was 8.0,and the ratio of inclusion body and solution was 160 mg?mL-1?w/v?.The recombinant ??-ADH was finally dissolved with an activity of 0.391 U?mL-1.Tyr fluorescence emission spectrum of the recombinant ??-ADH purified from the inclusion body solution showed it had part of the natural conformation.By affinity chromatography with 1mL HisTrapTM excel,the renatured ??-ADH was purified with an activity of 20.47 U?mg-1.Using ethanol as substrate,the recombinant ??-ADH showed Km of 16.9 mmol?L-1,kcat/Km of 77.5 L?mmol-1?min-1.The optimum reaction temperature of recombinant ??-ADH was 40°C.There was 61.6% activity remained after a 90 min incubation in 40°C.The optimum pH of recombinant ??-ADH was 9.0.There was nearly no activity loss when incubated in buffers with pH ranged from 7.0 to 11.0 for 30 min.However,there was no activity remained when pH was 3.0 or 4.0.When mixed with alcohol of 1000 mmol?L-1and incubated in 40°C for 40 min,there was 54.4% activity remained.In addition,the study tried to find an immobilization method to improve the activity of recombinant ??-ADH under acidic condition.The natural chitosan with positive charge group was chosen as immobilization medium.The immobilization reaction was carried out by mixing recombinan ??-ADH(0.3 mg?m L-1)with chitosan at the ratio of 1.5 mL?g-1in 2%glutaraldehyde?pH 8.0?at 40°C for 4 h.The unlinked groups in chitosan were finally blocked by 50 mg?mL-1 of chitosan oligosaccharides.The immobilized and post-modification ??-ADH exhibited Km of 21.9 mmol?L-1towards ethanol,displayed highest activity at pH 8.0,and showed activity of 0.094 ±0.021 U?g-1 carrier and 0.138±0.002 U?g-1 carrier at pH 3.0 and 4.0.When mixed with 1000 mmol?L-1alcohol and incubated in 40°C for 40 min,there was 78.6% remaining activity,suggested the higher alcohol tolerance and acidic stability of the enzyme after immobilization. |
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