The Expression And Enzymology Properties Of Thermostable Recombinant Carbonic Anhydrase

Carbonic anhydrase (CA) is a zinc metalloenzyme which can effectively catalyze the interconversion of carbon dioxide and bicarbonate. It is widely distributed in animal、plant and microorganism. The enzyme play an important role in various physiological functions, which include facilitated diffusion of CO2, photosynthesis, pH homeostasis, and ion transport. At present, carbonic anhydrase is applied to biosensors, natural actives screening, biological detection and CO2capture. The purpose of this research is to develop a kind of thermostable carbonic anhydrase, and apply it to an CO2capturing process-IVCAP. In this paper, we cloned the gene of carbonic anhydrase(β-class) which comes from thermophiles Methanocella conradii HZ254and expressed it in E. coli, then studied the property of the recombinant enzyme.Primers were designed according to the encoding gene of carbonic anhydrase (mtc) which comes from Methanocella conradii HZ254in the NCBI. The mtc gene sequence with Ndel and BamHI sites on either end was amplified through PCR employing DNA of mtc as template. Both the PCR product and pET-24a(+) vector were digested with NdeI and BamHl and ligated to each other to yield recombinant plasmid. Competent E. coli JM109cells were transformed with recombinant plasmid. Transformants with recombinant plasmids were screened on the plate containing kanamycin, and were identified by colony PCR and double digestion of restriction enzymes. The result showed that the target gene fragments were successfully inserted into the recombinant plasmids. The DNA sequencing further confirmed it. The engineering bacteria was named JM109-pET24a-mtc. The molecular mass of induced expression product was29kDa estimated by SDS-PAGE and it was nearly identical to the expected. The expression product have the carbonic anhydrase activity, but no esterase activity.The induced expression conditions of JM109-pET24a-mtc were optimized. The expression amount reached to the maximum when the engineering bacteria was induced with1mM IPTG, which was added after the4h growth at37℃, for10h at30℃. This condition considered the stability of recombinant enzymes、inducing efficiency and economic cost.The properties of the recombinant carbonic anhydrase were studied. The results showed that the enzyme activity increased dramatically after incubation at55℃for30min. The optimal pH on the stability of recombinant enzyme was in the range of pH6.00-8.00. Its catalytic activity was enhanced in the presence of1mM Fe2+、Mg2+、Mn2+and Ca2+, but inhibited by1mM Cu2+and Zn2+. F-has no effect on the activity, but1mM of sulfanilamide、I-、Br-、HCO3-、Cl-、NO3-and SO42-inhibited it at the different levels.