Background

Error-prone PCR

Error-prone PCR technology has a wide range of applications in vitro random mutation . It is currently the most extensively used in vitro directed evolution of proteins and plays an important role in improving the catalytic properties of enzyme molecules^[1-3]. Error-prone PCR has been applied to improve the activity of glucanase, glycosyl trehalose synthase, starch hydrolase, lipase, laccase β, cellulase etal. What’s more, it is also very vital in guiding the study of the key amino acid residues that affect the catalytic properties of enzymes^[4-9].

A Biref Introduction of MICP

Microbial induced calcium carbonate precipitation (MICP) is a natural phenomenon that microorganisms form calcium carbonate deposits in their surrounding microenvironment through metabolism. As early as 1973, Boquet et al.^[10] first discovered the phenomenon of soil microbial induced calcium carbonate precipitation (MICP). Most microorganisms in nature can generate cementation substances through their own metabolic activities, causing cementation between particles^[11], thus improving the strength of soil and reducing its water permeability. Microbial cement has the characteristics of environmental protection, economy and efficiency, and has become a research hotspot in the fields of biology, civil engineering and environment. There are two key enzymes for biomineralization, namely urease and carbonic anhydrase^[12].

Fig.1 How to create more efficient enzymes

Reaction Principle

Urease hydrolyzes urea to increase cell pH and carbonate concentration, forming alkaline conditions for calcium carbonate deposition. When Ca²⁺ adsorbed on the cell surface meets carbonate ions, it forms calcium carbonate precipitation and eventually biomineralization. Carbonic anhydrases (CA) are zinc containing metalloenzymes that can efficiently catalyze the reversible reactions between carbon dioxide, water, carbonic acid and H⁺. Carbonic anhydrase can effectively promote biomineralization. In natural conditions, the hydration rate of CO₂ is quite low, which greatly limits the generation of calcium carbonate precipitation. However, the CO₂ hydration rate increases rapidly when the reaction above is catalyzed by carbonic anhydrase, reaching 10⁸ times that in natural conditions.

Therefore, the rapid deposition of biologically induced calcium carbonate mineralization requires urease to decompose urea to increase the pH value of the environment, and carbonic anhydrase to promote the rapid hydration of CO₂ to form a large amount of CO₃^2–, thus forming calcium carbonate deposition with Ca²⁺ in the environment^[12]. The mineralization and deposition cost of carbonic anhydrase mineralizing bacteria is low, there is no secondary pollution to the environment, and it can effectively store carbon dioxide, which has great potential in the research of environmental engineering fields such as mitigating global warming and other engineering fields^[12]. At present, Sporosarcina pasteurii is the strain with the best biomineralization efficiency among known natural bacteria.

Fig.2 A brief description of MICP induced by urease and carbonic anhydrase

Our Goal

MICP research has been focused on screening strains with higher urease activity^[13], in order to quickly produce CO₃^2– by accelerating the decomposition of urea, but the activity of carbonic anhydrase of the strain has been ignored. This study attempts to improve the activity of carbonic anhydrase by means of error-prone PCR to explore the effect of the increase of carbonic anhydrase activity on the mineralization rate of microbial cement.

References

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