Engineering robust carbonic anhydrase immobilized

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Activity of immobilized (Covalent bond) BCA. Activity of immobilized (physical adsorption) BCA. ➢ Enzyme immobilisation via covalent bonding was irreversible ...

Engineering robust carbonic anhydrase immobilized on silica nanoparticles for carbon dioxide capture Pradeep

1 G.C. ,

Zhengyang

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

Introduction

6 5

4 3

Covalent bond

Physical adsorption

2 1 Mr 1 2 3 4

6 6 5

5

Physical adsorption

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.

3a

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+

H2CO3

carbon dioxide + water

carbonic acid

bicarbonate + hydrogen ion

Before reaction

 Compare the reusability of immobilized enzyme.

After reaction

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.

Enzyme purification

Activity of immobilized (physical adsorption) BCA

Silica particle 100

2a

100 100

98.5

98.49

100

Relative activity (%)

2b 80

60

39.07

40

20

2.05

0

0

Fig.2: SEM image of porous (2a) and non porous (2b) silica nanoparticles

OH R

CH

Silica particles

-H2O

3

4

Reuse of immobilized BCA (times)

Conclusions

Enzyme immobilization H 2N Enzyme CH2

Epoxy group

2

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

O

1

 Enzyme immobilisation via covalent bonding was irreversible compared with enzyme physical adsorption

H 2N O

H2N

H 2N

Silica particles functionalized with epoxy group

Enzyme Enzyme immobilized on silica nano particles (physical adsorption)

HN OH

H 2N

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.

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