Study On Diversity Of Soil Bacteria And Activity Of Carbonic Anhydrase And Its Gene Cloning In Typical Karst Ecosystem

Karst ecosystem is one of important terrestrial carbon sinks.The karstification has asignificant effect on atmospheric CO₂sequestration. The studies have shown thatmicroorganisms and their CA have a significant role in promoting the karstification.Carbonic anhydrase （CA, EC4.2.1.1） is a zinc metalloenzyme that catalyzes theinterconversion of CO₂and HCO₃^-. In this paper, the spatial and temporal characteristicsof soil CA activity and bacterial diversity in the typical karst ecosystem were investigated,exemplified by the Karst Experimental Site in Guilin and taking Silai clastic rock area inMaocun as the control area. The relationship between bacterial diversity and soil CAactivity was also preliminarily explored. Moreover, the cloning and expression of γ-CAgene from a typical bacteria producting extracellular CA was studied. The results of thispaper provides a scientific base for in-depth study on the role of microorganisms and theirCA in the the karst carbon sinks, and lay a foudation for the future study on theenhancement of carbon fixation and sequestration by a large amount of microbial CAobtained using the technology of fermentation engineering and enzyme engineering. Themain results in this pater are as follows.（1） The CA activities in the soils of the Karst Experimental Site were generally higherthan those of Silai clastic rock area. There were obvious differences in soil CA activityamong different geomorphological positions in the Karst Experimental Site. The CAactivity generally decreased with the increase in the soil depth. The seasonal variation ofsoil CA activity was characterized by rising in spring, reaching the highest level insummer, and then decreasing in autumn and winter.（2） The soil bacterial diversity index of each sampling site was between2.57and3.91.UPGMA cluster analysis showed that soil bacterial diversity was influenced by thegeomorphological position and the season. The soil bacterial community composition wasthe most similar in the same karst geomorphological position of the Karst ExperimentalSite.（3） The soil bacterial diversity had a certain correlation to the soil CA activity, whichwas affected by the season.（4） The γ-CA gene from a typical bacteria which could produce extracellular CA was cloned. The fragment length of this gene was513bp. The molecular weight was18.84kDa.The isoelectric point was6.42. The molecule structure model predicted that this enzymewas located at the extracellular, and was a trimer enzyme with a left-handed parallel βhelical domain（LBH）. Constructing the recombinant plasmid of PET-32a （+） RZγCA, theactivity of γ-CA by the prokaryotic expression was21.08U/g fresh cell.