Cloning And Recombinant Expression Of AMP Deaminase Gene

AMP deaminase is widely used in industry which was first produced from mammaliancells, such as human erythrocytes, rat skeletal muscles etc. Now, it is usually produced bymicroorganism fermentation, including solid state fermentation and liquid fermentation. Butthis method has a lot of problems in many aspects, such as enzymatic activity and its stability.In order to solve these problems, we can express AMP deaminase gene from microorganismin particular host through recombinant DNA technology. So, this experiment is intend to cloneAMP deaminase gene from Aspergillus melleus, Aspergillus oryzae, Streptomyces murinusand express them in different hosts. Then the fermentation conditions of the recombinantstrain were optimized and the enzymatic properties of the recombinant enzyme were studied,including temperature, pH, ions etc.First, we got cDNA of Aspergillus melleus by RT-PCR. The conserved sequence wasamplified by PCR with degenerated primers. Then we amplified3′end and5′end sequence ofthis gene by RACE. A2082bp open reading frame was discovered by Vector NTI and Kozakrules. Finally, we amplified this gene named AMPDa by PCR and constructed therecombinant plasmid pEASY-T1-AMPDa. We amplified the AMPDb gene of Aspergillusoryzae and constructed the recombinant plasmid pEASY-T1-AMPDb. The mature protein geneAMPDc1and the complete structure gene AMPDc2were cloned from Streptomyces murinusby PCR. The recombinant plasmid pMD19-T-AMPDc1and pMD19-T-AMPDc2wereconstructed.The expression plasmid pET28a-AMPDa, pET28a-AMPDb and pET28a-AMPDc1wereconstructed and transformed into E.coli DE3separately. The activity of recombinant E.coli(pET28a-AMPDa), E.coli (pET28a-AMPDb) and E.coli (pET28a-AMPDc1) were detected.The results showed that the activity of recombinant E.coli (pET28a-AMPDc1) was relativelyhigh, which was about150U·mL-1.The expression plasmid pHY-p43-AMPDc2, pGAP9K-AMPDc1and pKLAC1-AMPDc1were successfully constructed and transformed into Bacillus subtilis WB600, Pichia pastorisGS115and Kluyveromyces lactis GG799separately. The activity of recombinant B.subtilisWB600(pHY-p43-AMPDc2), P.pastoris GS115(pGAP9K-AMPDc1) and Kluyveromyceslactis GG799(pKLAC1-AMPDc1) were detected. The results showed that the activity ofrecombinant B.subtilis and Kluyveromyces lactis were very low, the recombinant P.pastorisGS115was about800U·mL-1. The fermentation conditions of the recombinant P.pastorisGS115strain were studied. The results indicated that the optimum fermentation medium ofthe recombinant strain contains2%glycerin,2%peptone,1%yeast extract,0.5%KH2PO4and0.05%MgSO4·7H2O (pH6.0). And the recombinant enzyme activity of fermentation supernatant was2230±60U·mL-1with3%of inoculation amount,24hours of seed time,200r·min-1for96hours at temperature of30℃.The target protein was purified by Ni-chelating-column and the enzymatic properties ofthe recombinant enzyme were studied. The results demonstrated that the recombinant enzymeperformed the maximal activity at60℃, pH6.0. And this enzyme had thermostabilitybetween30℃and60℃and pH stability from5.0to8.0.