Activity of immobilized (Covalent bond) BCA. Activity of immobilized (physical adsorption) BCA. â¢ Enzyme immobilisation via covalent bonding was irreversible ...
Engineering robust carbonic anhydrase immobilized on silica nanoparticles for carbon dioxide capture Pradeep
1 G.C. ,
1 Zhao ,
1 Kasargod ,
2 Dumsday ,
1 Haritos ,
Bhuvana Geoff Victoria 1* Lizhong He 1Department of Chemical Engineering, Monash University, Clayton, VIC3800, Australia. 2CSIRO, Clayton South, VIC 3169, Australia. Covalent bond
2 1 Mr 1 2 3 4
6 6 5
4 3 2 1 Mr 1 2 3 4 5 6
Carbon dioxide (CO2) is the major anthropogenic green house gas leading to global warming. Several approaches has been adopted to reduce carbon significantly from atmosphere. Using an enzymatic approach (bovine carbonic anhydrase) offers the best alternative options because they are highly specific in reactions, work in mild conditions, and environmentally friendly. The industrial and broad applications of enzyme are limited due to its poor stability and reusability. Immobilization of enzyme makes them more stable under storage and operational conditions thus, become less sensitive towards the working environment. Additionally, the immobilized enzymes can be reused.
3b Fig 3: SDS-PAGE of bovine carbonic anhydrase immobilized on porous silica particles (3a) and non porous particles (3b); Mr: protein marker, 1: original protein solution, 2: proteins in SDS sample buffer eluted from particles 3: supernatant after immobilization, 4: wash solution (PBS and 1 M NaCl), 5: ethanolamine wash, 6: wash solution (PBS buffer)
Enzyme Activity (Wilbur-Anderson method) Carbonic anhydrase
Research objectives Engineer robust carbonic anhydrase by immobilizing on silica nanoparticles
CO2 + H2O
HCO3- + H+
carbon dioxide + water
bicarbonate + hydrogen ion
Compare the reusability of immobilized enzyme.
Methodology and results Methods Bovine carbonic anhydrase (BCA) was expressed on E. coli BL21 (DE3) cells, produced, and purified using Ni-affinity column chromatography. The enzymes were immobilized on porous and non-porous silica nano particles1.
Fig 4: BCA activity measured by Wilbur-Anderson method. The change in colour was monitored by Bromothymol blue dye when the pH 8.0 changes to 7.5. Here, 10: Control (without enzyme), 11: Free purified enzyme (BCA), 12: Immobilized enzyme (BCA) on silica particles
Multiple testing (Wilbur-Anderson method) Activity of immobilized (Covalent bond) BCA
The immobilization methods were compared and subsequently enzyme activities were tested for carbon dioxide conversion by Wilbur-Anderson method2.
Activity of immobilized (physical adsorption) BCA
Silica particle 100
Relative activity (%)
Fig.2: SEM image of porous (2a) and non porous (2b) silica nanoparticles
Reuse of immobilized BCA (times)
Enzyme immobilization H 2N Enzyme CH2
Fig.5: Bar graph showing the reusability of immobilized BCA.
Fig.1: SDS-PAGE of BCA. Here, Mr: protein marker, Crude: before chromatography; Pure: purified BCA after chromatography
Enzyme immobilisation via covalent bonding was irreversible compared with enzyme physical adsorption
H 2N O
Silica particles functionalized with epoxy group
Enzyme Enzyme immobilized on silica nano particles (physical adsorption)
Enzyme immobilized on silica nanoparticles (covalent bond)
Immobilised enzyme was active and was reused four times with no loss of activity as determined by the Wilbur-Anderson method
References 1. Zhengyang, Z., Junfei, T., Zhangxiong, W., Jian, L., Dongyuan, Z., Wei, S., and Lizhong, H. (2013) J. Mater. Chem. B,1, 4719-4722 2. Wilbur, K.M. and Anderson, N.G. (1948) J. Biol. Chem., 176, 147-154.