Description:FIELD OF THE INVENTION
The present invention relates to a method for regeneration of a biocatalyst in a carbon dioxide capture process. More specifically, the present invention provides a method for regeneration of inactive and/or partially inactive immobilized carbonic anhydrase in an absorber/stripper unit during enzymatic CO2 capture. The method of the present invention helps in improving the shelf life of carbonic anhydrase enzyme and increases the CO2 capture efficiency and plant performance in both biogas purification and flue gas separation process.
BACKGROUND OF THE INVENTION
In order to mitigate climate change and to achieve net zero CO2 emissions, CO2 capture from large fixed-point sources are viable options. Various methods like pre combustion, post combustion and oxyfuel combustion are being used for CO2 capture. Among these, post-combustion capture by amine-based absorption is a mature and proven technology, but not economically attractive unless novel solvents and optimized processes are implemented. The major limitation of amine-based absorption technology includes sluggish kinetics, high solvent degradation, corrosively and energy intensive amine regeneration. The use of carbonic anhydrase, a natural fast biocatalyst, is a promising technique which can dramatically improve the implementation and economics of carbon capture under stringent environment demands. Carbonic anhydrases catalyze the inter-conversion between carbon dioxide and bicarbonate, improves the mass transfer, improves solvent CO2 loading capacity, and lowers the regeneration temperature. In enzymatic CO2 capture process, fixed beds of immobilized carbonic anhydrase are loaded in absorber and desorber column. The flue gas is fed to the bottom of the absorber where CO2 is removed through counter-current contact with solvent flowing from the top. The CO2-depleted treated gas exits from the top of the absorber. The CO2-rich amine, which exits the bottom of the absorber, passed through the top of desorber column. The desorber unit is then heated by an electric furnace to the desired temperature to regenerate the solvent. The regenerated lean amine was then re-circulated to the lean amine tank making the process continuous. In the whole process, the enzyme stability is a crucial factor to determine whether overall process will be commercially successful. However, after longer operation of enzymatic CO2 capture plant, the enzyme carbonic anhydrase loose part of its activity when the enzyme is (a) subjected to the action of heat, extreme pH; (b) interaction with heavy metals or sudden stress like high concentration of hydrocarbon, CO, SOx, NOx or H2S which may reduce the enzyme activity; and (c) interaction with reactive degraded amine components. The major causes of inactivation of carbonic anhydrase is due to aggregation, thiol-disulphide exchange, alterations in the primary structure, e.g., chemical modification of functional groups; cleavage of s-s bonds; dissociation of the prosthetic group from the active centre of the enzyme; dissociation of oligomeric proteins into subunits and conformational changes in the macromolecule.
Further, the replacement of enzyme from absorber and desorber column is cumbersome, cost intensive and also requires a long time shutdown of the equipment. Therefore, for an economical operation of enzymatic CO2 capture process; it is very important to develop a process for an effective in-situ regeneration of an immobilized biocatalyst/enzyme in the absorber and desorber column. Various studies are available on enzyme regeneration. For instance:
WO2013170384A1 describes a method for CO2 capture which includes operating a packed reactor comprising a reaction chamber containing packing including immobilized enzymes, by contacting a CO2 containing gas with a liquid solution in the reaction chamber to produce an ion-loaded solution and a CO2 depleted gas by an enzymatically catalyzed hydration reaction; monitoring enzyme activity of the immobilized enzymes; at a low enzyme activity threshold (i) stopping operation in the packed reactor, and (ii) replenishing the enzymatic activity by providing an enzyme replenishing solution into the packed reactor to contact the packing and provide a replenishing amount of the immobilized enzymes.
CN103055959B discloses a catalyst regeneration method and more particularly, to an active regeneration of carbon deposit catalysts during the cracking reaction of heavy and low-quality hydrocarbon oils. The method comprises: addition of a carbon deposition catalyst into a carbonic material, wherein the carbonic material is 1-30% of the mass percentage of the carbon deposition catalyst; enabling the carbon deposition catalyst in a fluidized bed regenerator at the temperature of 550-8000C to obtain a semi-regenerated catalyst; enabling the semi-regenerated catalyst to contact with the O2-containing gas for complete regeneration.
WO2013106932A1 discloses a method, process, apparatus, use and formulation for dual biocatalytic conversion of CO2 containing gas into carbon containing bio-products by enzymatic hydration of CO2 into bicarbonate ions in the presence of carbonic anhydrase.
Deanna M. D’Alessandro et al., 2010 highlights the CO2 capture technologies, namely postcombustion (predominantly CO2/N2 separation), precombustion (CO2/H2) capture, and natural gas sweetening (CO2/CH4). It discloses the effectiveness of CA as separator and CO2 absorbent.
Savile et.al., 2011 summarizes recent approaches to improve carbonic acid (CA), and processes employing this enzyme, to maximize the CO2 capture by this extremely fast biocatalyst. It shows CA can be inactivated followed by reactivation giving rise to large scale production of enzyme which can effectively entrap larger amount of CO2. Also, discloses immobilization of CA both stabilizes the enzyme and limits the exposure to denaturing conditions in CO2 capture and sequestration processes.
Yongqi Lu et al., 2011 describes a CO2 capture process wherein biocatalyst carbonic anhydrase is used for promoting the CO2 absorption under Integrated Vacuum Carbonate Absorption Process conditions.
Kageoka et al., 1981 discloses reactivation after denaturation in Gnd. HCI, reactivation of various concentrations of the normal and variant enzymes which had been denatured in 5.0 M, Gnd. HCI for 24 hr at 25°C. was initiated by rapid dilution to 0.5 M Gnd' HCI in 0.1 M Tris-SO, pH 7.5, buffer with and without dithiothreitol. Aliquots were removed at various times and assayed for esterase activity.
Romero et.al (Journal of Molecular Catalysis B: Enzymatic 74 (2012) 224–229) in the article entitled “Reactivation of penicillin acylase biocatalysts: Effect of the intensity of enzyme–support attachment and enzyme load” describes a method were immobilized penicillin G acylase (PGA) was recovered by filtration to remove the inactivation medium and re-incubated in aqueous medium to promote reactivation.
Zawalich et.al (Proceedings of the IEEE 24th Annual Northeast Bioengineering Conference (Cat. No. 98CH36210), pp. 85-87. IEEE, 1998) in an article entitled “In-situ regeneration of co-enzyme NADH with an electro-enzymatic biomimetic membrane” describes a method for regeneration of enzyme lactate dehydrogenase (LDH).
Although several methods are available in the art, however the method described in the prior arts only involves immobilization/adding fresh enzyme on the fixed bed. Further, addition of fresh enzyme will impact the overall cost of the overall process. Furthermore, it is unlikely that the spraying the enzyme and immobilizing matrix will uniformly cover the fixed bed, and thus, the method does not activate the earlier immobilized enzymes. Moreover, over the time when the inactive enzymes over occupy the surface of fixed bed, it is difficult to functionalize new enzyme. Therefore, there is no prior art available for regeneration of carbonic anhydrase used in CO2 capture process. Furthermore, the regeneration method of enzyme varies depending on the specific activity, molecular weight, protein sequence, active site and prosthetic group. Thus, the present invention addresses the above problems and describes a method for reactivation of inactive enzyme already attached to the immobilized materials without adding any new fresh enzyme. Particularly, the present invention is related with the development of a method to regenerate carbonic anhydrase enzyme which has become substantially deactivated.
SUMMARY OF THE INVENTION
In an aspect of the present invention, there is provided a method for in-situ regeneration of an immobilized enzyme in enzymatic carbon dioxide capture, the method comprising of:
(i) treating the immobilized enzyme in an absorber/stripper unit of a fixed bed reactor with a flush-buffer solution at a temperature of 80- 95o C for a duration of 10-30 minutes;
(ii) adding a solution of chaotropic agent at a rate of 3-5 ml/minute for a duration of 60-120 minutes;
(iii) treating the solution of step (ii) with a solution of a modulator for a duration of 60-120 minutes; and
(iv) re-treating the enzyme with the flush-buffer solution to obtain regenerated/active enzyme.
In an embodiment of the present invention, there is provided a method for in-situ regeneration of an immobilized enzyme in enzymatic carbon dioxide capture, wherein prior to treatment with flush buffer solution in step (i), flow of amine gas and flue gas is stopped/ceased in the absorber or stripper unit of the fixed bed reactor.
In another embodiment of the present invention, there is provided a method for in-situ regeneration of an immobilized enzyme in enzymatic carbon dioxide capture, wherein the flush-buffer solution comprises a buffer solution in the range of 10-50mm and a surfactant in the range of 10-50 ppm.
In an embodiment of the present invention, there is provided a method for in-situ regeneration of an immobilized enzyme in enzymatic carbon dioxide capture, wherein the buffer solution is selected from carbonate buffer, phosphate buffer, 3-(N-morpholino) propanesulfonic acid (MOPS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), or Tris buffer.
In another embodiment of the present invention, there is provided a method for in-situ regeneration of an immobilized enzyme in enzymatic carbon dioxide capture, wherein the surfactant is selected from CTAB, P-123, TRITON X- 100 and Tween 20, sodium dodecyl sulfate (SDS) or a combination thereof.
In an embodiment of the present invention, there is provided a method for in-situ regeneration of an immobilized enzyme in enzymatic carbon dioxide capture, wherein the solution of chaotropic agent comprises guanidine, thiourea or urea in the range of 1-5 M guanidine, a reducing agent in the range of 0.2-0.5M and a chelating agent in the range of 1-5 mg/ml.
In another embodiment of the present invention, there is provided a method for in-situ regeneration of an immobilized enzyme in enzymatic carbon dioxide capture, wherein the solution of modulator comprises a boron compound in the range of 0.5-5 mM, zinc salt in the range of 0.5-1 mM, magnesium salt in the range of 0.1-0.3 mM and a stabilizer agent in the range of 5-10 ppm.

In an embodiment of the present invention, there is provided a method for in-situ regeneration of an immobilized enzyme in enzymatic carbon dioxide capture, wherein the boron compound is selected from boric acid or an alkali metal salt of boric acid, sodium and potassium ortho-, pyro-, and meta-borates, polyborates, or borax or a combination thereof.

In another embodiment of the present invention, there is provided a method for in-situ regeneration of an immobilized enzyme in enzymatic carbon dioxide capture, wherein the zinc salt is selected from zinc-chloride (ZnCl2) or Zn-acetate or a combination thereof.

In an embodiment of the present invention, there is provided a method for in-situ regeneration of an immobilized enzyme in enzymatic carbon dioxide capture, wherein the magnesium salt is selected from manganese chloride (MnCl2), Mn-acetate, or a combination thereof.

In another embodiment of the present invention, there is provided a method for in-situ regeneration of an immobilized enzyme in enzymatic carbon dioxide capture, wherein the stabilizer agent is selected from ethylene glycol, polyethylene glycol (PEG) and glycerol.

In an embodiment of the present invention, there is provided a method for in-situ regeneration of an immobilized enzyme in enzymatic carbon dioxide capture, wherein the immobilized enzyme is carbonic anhydrase.

In another embodiment of the present invention, there is provided a method for in-situ regeneration of an immobilized enzyme in enzymatic carbon dioxide capture, wherein the carbonic anhydrase is obtained from microbes selected from Bacillus thermoleovorans IOC-S3 (MTCC 25023), Pseudomonas fragi IOC S2 (MTCC 25025), Bacillus stearothermophilus IOC S1 (MTCC 25030) and Arthrobacter sp. IOC-SC-2 (MTCC 25028).

These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description. This summary is provided to introduce a selection of concepts in a simplified form.

BRIEF DESCRIPTION OF DRAWINGS
These and other features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings wherein:
Figure 1 illustrates overall process of in-situ regeneration of carbonic anhydrase.

DETAILED DESCRIPTION OF THE INVENTION
For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are collected here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

Definition:
For the purposes of this invention, the following terms will have the meaning as specified therein:
The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”.

Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.
The term “including” is used to mean “including but not limited to” “including” and “including but not limited to” are used interchangeably.

The present invention relates to a method for regeneration of carbonic anhydrase enzyme. Particularly, the present invention provides a method for regeneration of partially inactive immobilized carbonic anhydrase in absorber/stripper units in a fixed bed reactor during enzymatic CO2 capture. The present invention also discloses composition of chaotropic agent and enzyme (CA) modulator as well as their method of application for rejuvenation inactive carbonic anhydrase. The method for rejuvenation of inactive enzyme in the absorber and stripper column involves the following sequential steps: Discontinuing the amine flow to the fixed bed of immobilized enzyme in the absorber and stripper; treating the inactive and/or partial inactive enzyme with a flush-buffer solution at 80-95oC; adding a solution containing desired concentration of chaotropic agent in step-2 for desired period; treating solution of modulator and maintaining desired contact time after step-3 resulting the activation of inactive and/or partially inactive enzyme and finally washing the enzyme by fresh flush-buffer solution as described in step-2.
Thus, in accordance with the present invention, there is provided a method for in-situ regeneration of an immobilized enzyme in enzymatic carbon dioxide capture, the method comprising of:
(i) treating the immobilized enzyme in an absorber/stripper unit of a packed/bed reactor with a flush-buffer solution at a temperature of 80- 95o C for a duration of 10-30 minutes;
(ii) adding a solution of chaotropic agent at a rate of 3-5 ml/minute for a duration of 60-120 minutes;
(iii) treating the solution of step (ii) with a solution of a modulator for a duration of 60-120 minutes; and
(iv) re-treating the enzyme with the flush-buffer solution to obtain a regenerated/active enzyme.
In an embodiment of the present invention, there is provided a method wherein prior to treatment with flush buffer solution in step (i), flow of amine gas and flue gas is stopped/ceased in the absorber or stripper unit. The amine flow and flue gas flow to be stopped in either absorber or stripper unit before starting the rejuvenation process and the temperature was kept between 30-50oC.
In another embodiment of the present invention, there is provided a method for in-situ regeneration of an immobilized enzyme for carbon dioxide capture, wherein the flush-buffer solution comprises a buffer solution in the range of 10-50mm and a surfactant in the range of 10-50 ppm. The flush-buffer solution was prepared by the following steps: (a) preparing 10-50mM buffer solution; (b) adding 10-50ppm surfactant to the solution prepared. The flush-buffer solution to be flowed at a rate of 1-10 ml/minutes of immobilized enzyme as fixed bed in the absorber and stripper column for enzyme activation. The total contact time between enzyme and flush-buffer should be maintained between 10 to 30 minutes and the volume of flush-buffer should be at least 3-5 times than that of the volume of the immobilized enzyme.

In another embodiment of the present invention, there is provided a method for in-situ regeneration of an immobilized enzyme for carbon dioxide capture, wherein the buffer solution is selected from carbonate buffer, phosphate buffer, 3-(N-morpholino) propanesulfonic acid (MOPS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), or Tris buffer. The concentration of the buffer in the solution is ranging from 10 mM to 50 mM. The pH is from about 7 to 8.5.

In an embodiment of the present invention, there is provided a method for in-situ regeneration of an immobilized enzyme for carbon dioxide capture, the surfactant can nonionic, anionic, cationic, or amphoteric. In another embodiment of the present invention, there is provided a method wherein the surfactant is selected from CTAB, P-123, TRITON X- 100 and Tween 20, sodium dodecyl sulfate (SDS) or a combination thereof.

In another embodiment of the present invention, there is provided a method for in-situ regeneration of an immobilized enzyme for carbon dioxide capture, wherein the solution of chaotropic agent comprises guanidine, thiourea or urea in the range of 1-5 M guanidine, a reducing agent in the range of 0.2-0.5M and a chelating agent in the range of 1-5 mg/ml. The chaotropic agent was added at a rate of 3-5 ml/minutes. The volume of chaotropic agent should be 2-3 times volume than that of immobilized enzyme volume. The total contact time between chaotropic agent and inactive enzyme should be maintained as 60-120 minutes.

In an embodiment of the present invention, there is provided a method for in-situ regeneration of an immobilized enzyme for carbon dioxide capture, wherein the solution of enzyme modulator comprises a boron compound in the range of 0.5-5mM, zinc salt in the range of 0.5-1mM, magnesium salt in the range of 0.1-0.3 mM and an enzyme stabilizer agent in the range of 5-10 ppm. The enzyme-modulator was prepared by following steps: (a) preparation of a 0.5-5 mM boron compound for stabilization of protein backbone of enzyme; addition of 0.5-1 mM Zn salts to the solution and adding 0.1-0.3mM Mg salt as structure stabilizing agent. The contact time between enzyme modulator and enzyme should be at least 60-120 minutes. The volume of modulator should be 2-3 times volume than that of volume of immobilized enzyme.

In another embodiment of the present invention, there is provided a method for in-situ regeneration of an immobilized enzyme for carbon dioxide capture, wherein the boron compound is selected from boric acid or an alkali metal salt of boric acid, sodium and potassium ortho-, pyro-, and meta-borates, polyborates, or borax or a combination thereof. For enzyme having a 10-20% loss in activity, the concentration of boron compound should be 0.5-2 mM; whereas for enzyme having activity 20-50% loss in activity the concentration of boron compound should be 2-5mM.

In an embodiment of the present invention, there is provided a method for in-situ regeneration of an immobilized enzyme for carbon dioxide capture, wherein the zinc salt is selected from zinc-chloride (ZnCl2) or Zn-acetate or a combination thereof.

In another embodiment of the present invention, there is provided a method for in-situ regeneration of an immobilized enzyme for carbon dioxide capture, wherein the magnesium salt is selected from manganese chloride (MnCl2), Mn-acetate, or a combination thereof.

In an embodiment of the present invention, there is provided a method for in-situ regeneration of an immobilized enzyme for carbon dioxide capture, wherein the enzyme stabilizer agent is selected from ethylene glycol, polyethylene glycol (PEG) and glycerol.

In another embodiment of the present invention, there is provided a method for in-situ regeneration of an immobilized enzyme for carbon dioxide capture, wherein the immobilized enzyme is carbonic anhydrase.

In an embodiment of the present invention, the carbonic anhydrase enzyme is obtained from Bacillus thermoleovorans IOC-S3 (MTCC 25023) and/or Pseudomonas fragi IOC S2 (MTCC 25025), and/or Bacillus stearothermophilus IOC S1 (MTCC 25030) and/or Arthrobacter sp. IOC-SC-2 (MTCC 25028).

The activity of the rejuvenated/activated enzyme obtained in the present invention is tested by p-NPA hydrolysis or Wilbur–Anderson assay. The carbon dioxide (CO2) absorption and desorption performance of rejuvenated/activated enzyme in presence of amines were tested. The amines used for enzyme stability test may include primary, secondary, tertiary or poly amines at temperature up to 95oC. Accordingly, the method of the present invention helps in in-situ regeneration of enzyme in absorber and stripper column and improves the life of enzyme by keeping the CO2 capture efficiency intact. Further, the method also prevents frequent change in the enzyme and helps in regeneration of carbonic anhydrase enzyme which has become substantially deactivated.

EXAMPLES
The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.

Example 1: In-situ regeneration of inactive carbonic anhydrase in CO2 capture process using active mixed amine system:
(a) Assessing initial biocatalyst/enzyme performance in absorber and stripper column and obtaining inactive and/or partially inactive immobilized carbonic anhydrase.
• The CO2 capture was performed in a 5kg CO2 capture/day pilot plant. The synthetic flue gas having composition (20-35% CO2, 1.5-2.0% O2 and balance N2) was prepared and passed to the absorber column with various flow rates (20-100 SLPH). The fixed bed in the absorber column has a volume of 30CC and was loaded with 4.3g enzyme (Varient1) immobilized-Al2O3 support (2-3 mm). CO2 absorption from the mixed gas occurs by countercurrent contact with solvent having 15%AMP+13.0%TAA+7.0% PEHA+5.0% Pz+0.4% K2CO3 in the column; where solvent is fed on the top and gas enter at the bottom of the column. The CO2 captured amine (Rich amine) is stored in
the rich amine tank. The rich amine was routed to the absorber (stripper) where it was heated to regenerate the solvent and pure CO2. The stripper was loaded with 4.3g enzyme (Varient-2) immobilized-Al2O3 support (2-3 mm). The regenerated amine (Lean amine) is again used for next cycle CO2 capture.
• Carbonic anhydrase was obtained from Pseudomonas fragi IOC S2 (MTCC 25025).
• The initial activity of carbonic anhydrase was found as 984 WAU/g of immobilized Al2O3 support in the absorber and 876 WAU/g of immobilized-Al2O3 in the desorber.
• In order to obtain the inactive enzyme, both the absorber and desorber after 4000h of continuous operation were subjected to high temperature up to 120oC for 8hour and thereafter the amine flow was stopped and an acetic acid solution of pH~3.5 was recirculated in the absorber and stripper for 24hours. Thereafter, the final enzyme obtained with activity was obtained as 531 WAU/g of support and 446 WAU/g respectively for both absorber and desorber column respectively.

(b) Regeneration of enzyme activity in absorber and desorber
• Preparation of flush buffer and its treatment with inactive enzyme: The 50mM of phosphate buffer having pH 8 was prepared and 30ppm of CTAB was added to it at room temperature. The medium was stirred for 1hour at room temperature to obtain a clear solution. The solution was stored at room temperature prior to its application. In the treatment method, firstly, the amine flow was stopped to the fixed bed enzyme chamber. The flush buffer solution was passed through the catalyst at a rate of 4ml/min for a period of 30 minutes.

Preparation of chaotropic agent and its treatment
Aqueous solution of 5M Urea was prepared in a beaker and to the solution percaptoethanol solution was added so that its concentration in the final mixture percaptoethanol was 0.5M at a rate of 1 ml/min. Finally, 5mg/ml of EDTA was added to the solution. The whole solution was stirred for 1h at room temperature at 500 rpm and the obtained solution was stored at room temperature. During the treatment, 1000 ppm of the above solution was added at a rate of 5ml/min for 2hours.

Preparation of CA-modulator and its treatment
5mM aqueous solution of borax solution was prepared followed by addition of 1mM Zn acetate. The mixture was stirred at 500 rpm for 30 minutes followed by addition of 5ppm of PEG. The solution was again stirred for another 30minutes to obtain the final CA-Modulator. 100 ppm of CA-modulator was added to the inactive immobilized at a rate of 5ml/min for 2hours. After, all the treatment, the immobilized enzyme was rewashed with freshly prepared flush buffer solution at a rate of 4 ml/min to obtain the final rejuvenated immobilized enzyme.

(c) Activity analysis of regenerated enzyme
The activity of immobilized enzyme was studied by Wilbur–Anderson assay using previously reported protocols:

Table-1: Activity of Carbonic anhydrase before and after regeneration.
Enzyme Initial Activity (WAU/g) Activity of partial inactive enzyme % activity of partial inactive enzyme relative to initial activity Activity of enzyme after rejuvenation % activity of rejuvenated enzyme relative to initial activity
CA-Variant-1 984±5 531±5 53.9 889±5 90.3
CA-Variant-2 876±5 447±5 51.0 821±5 93.7

Example-2: Role of various components for the in-situ regeneration of inactive carbonic anhydrase in CO2 capture.
To evaluate the synergistic role of various components in the regeneration of carbonic anhydrase various different experiments were performed. The enzyme assay was performed by using a previously used protocol. Typically using the electrometric method of Wilbur and Anderson in which the time required (in seconds) for a saturated CO2 solution to lower the pH of 0.012M Tris·HCl buffer from 8.3 to 6.3 at 0°C is determined. The time without enzyme is recorded at T0; with enzyme, T.6.0 ml of chilled 0.02 M Tris·HCl buffer, pH 8.0 added to a 20ml beaker. The temperature was maintained at 0-4°C and pH was recorded. 0.1g of immobilized enzyme was added
and 4 ml of CO2 saturated water and the time required for the pH to drop from 8.3 to 6.3 was recorded. WAU was measured by the following equation.
WAU/g = 2(T0-T)/T*g of enzyme

Table.2: Role of various components for the in-situ regeneration of inactive carbonic anhydrase
Sl. No Treatment method Initial activity (WAU/g)
Activity of partial inactive CA (WAU/g)
Activity of regenerated CA (WAU/g) after treatment
A Treatment of flush buffer 984±5 531±5 550±5
B Treatment of chaotropic agent 984±5 531±5 356±5
C Treatment of CA-modulator 984±5 531±5 552±5
A followed by B 984±5 531±5 441±5
A followed by C 984±5 531±5 556±5
B followed by C 984±5 531±5 623±5
C followed by B 984±5 531±5 524±5
C followed by A 984±5 531±5 601±5
A followed by C followed by B 984±5 531±5 619±5
A followed by B followed by C 984±5 531±5 889±5

Example-3: CO2 capture performance in amine solution after in-situ regeneration of the carbonic anhydrase.
The CO2 capture efficiency of rejuvenated enzyme was further studied using amine composition (15% AMP+13.0% TAA+7.0% PEHA+5.0% Pz+0.4% K2CO3) in enzymatic CO2 capture pilot plant having 5 kg/day CO2 capture capacity. In this pilot plant, the enzymes were kept in fixed bed in both absorber and desorber column. The synthetic flue gas having composition (35% CO2, 2.0% O2 and balance N2) was prepared and passed to the absorber column with various flow rates (55 SLPH). The fixed bed in the absorber column has a volume of 30CC and was loaded with 4.3g reactivated enzyme (CA-Variant-1) immobilized-Al2O3 support (2-3 mm). CO2 absorption from the mixed gas occurs by counter-current contact with amine solvent in the column; where solvent is fed on the top and gas enter at the bottom of the column. The CO2 captured amine (Rich amine) is stored in the rich amine tank. The rich amine was routed to the desorber (stripper) where it was heated at 94oC to regenerate the solvent and pure CO2. The stripper was loaded with 4.3g enzyme (CA-Variant-2) immobilized-Al2O3 support (2-3 mm). The regenerated amine (Lean amine) is again used for next cycle CO2 capture.

Table. 3: CO2 capture efficiency of CA before and after regeneration
Enzyme variants Initial activity CO2 capture/desorption efficiency
CO2 capture/desorption efficiency with partial inactive CA (Mol/L) CO2 capture efficiency Activity of regenerated CA (WAU/g) after treatment
CA-Variant-1 CO2 uptake (5.2 Mol/L) CO2 uptake (2.1 Mol/L) CO2 uptake (4.9 Mol/L)
CA-Variant-2 CO2 desorption (96%) at 94oC CO2 desorption (53 %) at 94oC CO2 desorption (92%) at 94oC , Claims:1. A method for in-situ regeneration of an immobilized enzyme in enzymatic carbon dioxide capture, the method comprising of:
i. treating the immobilized enzyme in an absorber/stripper unit of a fixed bed reactor with a flush-buffer solution at a temperature of 80- 95o C for a duration of 10-30 minutes;
ii. adding a solution of chaotropic agent at a rate of 3-5 ml/minute for a duration of 60-120 minutes;
iii. treating the solution of step (ii) with a solution of a modulator for a duration of 60-120 minutes; and
iv. re-treating the enzyme with the flush-buffer solution to obtain regenerated/active enzyme.
2. The method as claimed in claim 1, wherein prior to treatment with flush buffer solution in step (i), flow of amine gas and flue gas is stopped/ceased in the absorber or stripper unit of the fixed bed reactor.

3. The method as claimed in claim 2, wherein the flush-buffer solution comprises a buffer solution in the range of 10-50mm and a surfactant in the range of 10-50 ppm.

4. The method as claimed in claim 3, wherein the buffer solution is selected from carbonate buffer, phosphate buffer, 3-(N-morpholino) propanesulfonic acid (MOPS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), or Tris buffer.

5. The method as claimed in claim 3, wherein the surfactant is selected from CTAB, P-123, TRITON X- 100 and Tween 20, sodium dodecyl sulfate (SDS) or a combination thereof.

6. The method as claimed in claim 1, wherein the solution of chaotropic agent comprises guanidine, thiourea or urea in the range of 1-5 M guanidine, a reducing agent in the range of 0.2-0.5M and a chelating agent in the range of 1-5 mg/ml.

7. The method as claimed in claim 1, wherein the solution of modulator comprises a boron compound in the range of 0.5-5 mM, zinc salt in the range of 0.5-1 mM, magnesium salt in the range of 0.1-0.3 mM and a stabilizer agent in the range of 5-10 ppm.

8. The method as claimed in claim 7, wherein the boron compound is selected from boric acid or an alkali metal salt of boric acid, sodium and potassium ortho-, pyro-, and meta-borates, polyborates, or borax or a combination thereof.

9. The method as claimed in claim 7, wherein the zinc salt is selected from zinc-chloride (ZnCl2) or Zn-acetate or a combination thereof.

10. The method as claimed in claim 7, wherein the magnesium salt is selected from manganese chloride (MnCl2), Mn-acetate, or a combination thereof.

11. The method as claimed in claim 1, wherein the stabilizer agent is selected from ethylene glycol, polyethylene glycol (PEG) and glycerol.

12. The method as claimed in claim 1, wherein the immobilized enzyme is carbonic anhydrase.

13. The method as claimed in claim 12, wherein the carbonic anhydrase is obtained from microbes selected from Bacillus thermoleovorans IOC-S3 (MTCC 25023), Pseudomonas fragi IOC S2 (MTCC 25025), Bacillus stearothermophilus IOC S1 (MTCC 25030) and Arthrobacter sp. IOC-SC-2 (MTCC 25028).