Cloning Characterization and Application of Recombinant Carbonic Anhydrase in Escherichia coli

  • 何 太皓

Student thesis: Doctoral Thesis

Abstract

In this study we use IDT g-block synthesis to obtain 2 CA gene from Helicobacter pylori (HpCA) and human beings (hCAII) Through genetic engineering in E coli the activity of HpCA and hCAII was achieved up to 93 87 WAU/mg 15743 WAU/mg in pET28a system with highest expression level For hCAII the thermostability was very poor The pH stability was between pH6 0 and pH8 0 whereas the activity maintained more than 80% In salinity effect the activity kept at 70% in the salinity of 3% to 4% Agar has been used for immobilization of hCAII The residual activity is 500 4 WAU/mg when encapsulating whole cells in 2% (w/v) agar It can be reused 5 times with the final activity of 62 16% The immobilized CA kept 100% activity after 3 weeks of preservation Surface display of hCAII mediated by ice nucleation protein (INP) on cell has been designed to enhance the utilized efficiency of whole cell The results showed that INP-hCAII can be overexpressed on the outer membrane of E coli through trypsin digestion The kinetic parameters of hCAII were also calculated and the diffusion coefficient of CO2 in E coli was determined to be De = 157 μm2/s and its diffusion coefficient in agar was De = 0 413 μm2/s
Date of Award2019
Original languageEnglish
SupervisorI-Son Ng (Supervisor)

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