Enhanced carbonic anhydrase activity and stability by chaperone GroELS for applications in biomineralization

Abstract

High activity and stability of CA are essential and urgent to propose due to extreme and harsh environment of carbon capture and sequestration Three CA species as human CA II (hCAII) Mesorhizobium loti (MlCA) and Sulfurihydrogenibium yellowstonense (SyCA) have been optimized by recombinant engineering in E coli under the inducible pET system However there are still difficulties in the over-production of heterologous protein Here the use of a novel fusion partner and molecule chaperone are the strategies for improving the protein expression levels which refers to activity and capability The TrxA and GroELS have been chosen to assist the protein folding of CA TrxA fusion expression revealed a forceful function in enhancing protein production in which the CA activity was approximately improved to 50-180% Unfortunately MlCA was mostly accumulated as inclusion bodies Therefore the second strategy of using chaperonin GroELS was attempted; strong and weaken GroELS Interestingly the weaken GroELS was highly assisted soluble protein folding for those CAs in this study Improvement of soluble protein expression levels elevated the CA activity over 100% where MlCA showed a huge improvement about 30 times Despite this weaken GroELS was not powerful enough to assist the thermal capability of MlCA as strong GroELS which strong GroELS successfully shifted the optimum temperature to 50oC Furthermore we designed CA under a constitutive promoter to optimize the system and balance with GroELS Unexpectedly the results possessed lower CA activity than those under the inducible system Yet the weaken GroELS has dramatically intensified the thermostability of SyCA We considered that weaken GroELS was promoted only for CA which initially has thermostability properties Moreover an in vitro assay was also conducted to prove the GroELS assistance in the activity of CA Subsequently the soluble protein of CA was applied in a two-column system to verify the function of CA as biocatalyst for CO2 fixation and conversion The yield of calcium carbonate acquired was consistent with CA activity Finally SyCA has been chosen to covalently bond on novel polyacrylonitrile (PAN) and polyethylene terephthalate (PET) nanofibers (PAN-PET-PAN donated as AEA) that was first fabricated by electrospinning The resulting composite materials further crosslinked by the glutaraldehyde which significantly increased thermostability up to 89 8% and 18 0% after heating at 60oC for 1 hour for immobilized crude and pure SyCA respectively After four repetitive attempts in the demonstration of CO2 sequestration immobilized crude and pure SyCA on AEA also effectively improved the total CaCO3 yields to be 5 8 folds and 2 2 folds compared to free enzyme Furthermore the endurance of immobilized crude was investigated on flue gasses which was retained its activity up to 57% on 50 mM NOx and 61% on 50 mM SOx presence This study presents efficient strategies engineering to improve the productivity and stability immobilization techniques towards an industrial operating system and also conduct to prospective area in the CAs

Date of Award	2020
Original language	English
Supervisor	I-Son Ng (Supervisor)