Establishment of Screening Platform and Direct Evolution of Recombinant Carbonic Anhydrase in E coli

Abstract

Carbonic Anhydrase (CA) catalyzes the chemical reaction in which carbon dioxide react with water to form bicarbonate ions Due to its high catalytic efficiency and its eco-friendliness scientists look insight to apply CA for capturing carbon dioxide in the industrial process However there exist technical difficulties to industrialize this process such as high cost limited heat tolerance and poor in stability In this study we chose CA from Sulfurihydrogenibium yellowstonense YO3AOP1 (denoted as SyCA) as candidate for enzyme characterics analysis Furthermore based on the results MlCA which owns the highest activity but is low heat tolerance and hCAII which can be highly expressed by pET system in E coli but poor in stability are chosen as candidates for direct evolution We expected to create a heat tolerance and stability CA through direct evolution thus applied it into industrial Carbon Capture and Storage (CCS) process At first the SyCA is successfully heterologous expressed in E coli BL21(DE3) and the characterics including enzyme activity heat stability pH effect ion effect and kinetic parameters were further tested In direct evolution experiment two different types of screening platfom and three differen stategies of mutations have been established As a result total 576 candidates were screened in EP-PCR experiment on hCAII but no candidates showed imporovments The amount of diverse mutation candidates have to raise to the level of 105 ~ 107 in order to pick up the desirable candidates with significant improvement in characteristics We have setted up the screening platform to selected CA with higher activity but further investigation are needed in the future

Date of Award	2019
Original language	English
Supervisor	I-Son Ng (Supervisor)