嗜热蓝细菌碳酸酐酶基因的异源表达及酶学性质

IPTG-induced, heterologous expression and characterization of carbonic anhydrase from Thermosynechococcus elongatus PKUAC-SCTE542

摘要:

【背景】碳酸酐酶(carbonic anhydrase，CAH)因其高效催化CO2转化为HCO3–的能力成为当今碳减排工艺中的研究热点，但因工厂烟道气的温度较高，因此寻求热稳定性高的嗜热碳酸酐酶是碳酸酐酶仿生学固碳的关键所在。【目的】克隆嗜热蓝细菌Thermosynechococcus elongatus PKUAC-SCTE542和Synechococcus lividus PCC6715的碳酸酐酶基因Ecah、Pcah，实现其在大肠杆菌细胞中异源高效表达，并进行初步酶学性质研究。【方法】利用PCR技术获得碳酸酐酶基因cah，构建重组基因工程菌BL21-_pETM11_CAH，利用IPTG诱导方法高效表达蛋白，表达产物(CAH)经Ni-Agarose亲和层析柱纯化后，进行酶学性质研究。【结果】从E542、PCC6715中克隆得到大小均为534 bp的碳酸酐酶基因，以CO2为底物，酶催化CO2水合的活性分别为42.6 WAU/mg-protein、47.6 WAU/mg-protein。碳酸酐酶ECAH 50 °C处理30 min后，酶活提高了8%，而PCAH却下降了10%。Zn2+、磺胺对两种碳酸酐酶有显著抑制作用，Ca2+对ECAH有轻微激活作用，对PCAH无显著抑制作用。【结论】E542嗜热蓝细菌表达的碳酸酐酶比PCC6715的热稳定性强，符合处理工业高温点源烟道气的CO2的基本要求，丰富了碳减排的嗜热碳酸酐酶基因库。

Abstract:

[Background] Carbonic anhydrase has become the hotspot in carbon reduction research due to its ability to efficiently convert CO2 to HCO3－. Because of the flue gas high temperature, searching for thermostable, the thermophilic carbonic anhydrase is the key to achieve the biomimetic capture of CO2 in industrial flue gas. [Objective] The carbonic anhydrase (CAH) gene of Thermosynechococcus elongatus PKUAC-SCTE542 (Ecah) and Synechococcus lividus PCC6715 (Pcah) were cloned and expressed in Escherichia coli, and the enzymatic properties were characterized. [Methods] The two genes of the CAH were amplified by PCR. The recombinant plasmid pETM11-ECAH, pETM11-PCAH was overexpressed in BL21(DE3) pLysS by IPTG induced. The recombinant carbonic anhydrase was purified with Ni-Agarose His-tagged affinity chromatography and the enzymatic properties were further checked. [Results] Two length of 534 bp carbonic anhydrase were both obtained from E542 and PCC6715. The CO2 hydration activity of the ECAH and the PCAH was 42.6 WAU/mg-protein, 47.6 WAU/mg-protein, respectively. After 50 °C incubation for 30 min, the ECAH activity was increased by 8%, but the PCAH was decreased by 10%. The ECAH activity was increased to about 108% after treated by Ca2+ for 30 min, but no significant inhibition of PCAH activity was observed. Both the ECAH and PCAH activities were significantly inhibited by Zn2+ and sulphanilamide. [Conclusion] The ECAH of the Thermosynechococcus elongatus PKUAC-SCTE542 showes favorable thermostability than PCAH of the Synechococcus lividus PCC6715. EACH meets the basic requirements for CO2 treatment of industrial high-temperature point source flue gas, and enriches the thermophilic carbonic anhydrase gene pool.