The Enzymological Characteristics,Cloning And Expression Analysis Of Ornithine Carbamoyltransferase From Citrullus Lanatus

Citrullus lanatus is an important economic crops and fruits,rich in nutrients;as citrulline is an effective scavenger of oxygen free radicals in watermelon which plays an important role in resistant metabolism of Citrullus lanatus.Citrulline is an intermediate metabolite involved in the urea cycle,regulating body functions in the human body.Ornithine carbamoyltransferase(OCT)is a key enzyme in the synthesis of citrulline metabolism,it catalyzes the condensation of ornithine and carbamyl phosphate to form citrulline and phosphate,and also involved in the formation of pyrimidine and polyamines in higher plants.Study on OCT separation and purification will contribute to having a better understanding of its function on metabolism and regulation under abiotic stresses in the synthesis of citrulline,which will provide reference for the improvement of watermelon varieties and breeding new varieties.Cloning,expression and purification of OCT will lay the foundation for researches of gene function and catalytic reaction mechanism.In this study,we optimized the extraction condition OCT from Citrullus lanatus,isolated and purified Citrullus lanatus OCT enzyme by heating treatment,salting out and dialysis,and investigated some of the enzymatic properties of the OCT.At the same time we analyzed the OCT gene with the method of molecular biology,bulit the expression vector of recombinant OCT,with expression,purification,refolding and activity analysis on it.The results are as follows:1.The optimal conditions for the extraction of OCT were Tris-HCl buffer pH 8.5,solid-liquid ratio 1:5 and extraction time 4 h.The purification factor and recovery rate of this enzyme are 1.69 and 8.53%respectively by heating treatment,salting out and dialysis;Enzymatic characterization demonstrated that its optimum temperature and pH was 30 ℃and 8.0,the Km for carbamoyl phosphate and ornithine value was 3.73 and 8.34 mmol/L,Vmax was 0.32 and 0.22 μmol/(mL·min)respectively.Metal ions Ca2+,Mg2+ and Mn2+ promoted the enzyme activity,whereas Fe3+,Cu2+,phosphoric acid,arginine and EDTA could inhibit its activity.2.The ORF of OCT gene is 1137bp,which encodes 378 amino acids with a molecular weight of about 41.8kD and an isoelectric point of 8.61;it contains two binding domains,Carbamoyl-P binding domain and Ornithine binding domain,respectively.The secondary structure of OCT protein is containing 32.8%a-helix,32.8%the random coil,25.4%extended strand and 8.99%β-folding.The N-terminal of OCT protein is predominant in hydrophilic amino acids,and the C-terminal hydrophobic amino acids.The protein is not a transmembrane protein without signal peptide.The OCT from Citrullus lanatus is close affinity to Cucumis sativus and Cucumis melo,the similarity is 92%and 93%,respectively.3.The optimal induced expression conditions of recombinant OCT inclusion body proteins in E.coli expression system is the initial induction of OD600 0.6,IPTG concentration 0.6 mmol/L,the induction time 10 h and induced temperature of 32 ℃.The refolding rate of the purified inclusion body protein is 42.55%,and it is activity.