FORM 2
THE PATENTS ACT 1970
(39 of 1970)
&
The Patents [Amendment] Rules, 2006
COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. TITLE OF THE INVENTION
A FERMENTATION MEDIA AND A PROCESS FOR THE PREPARATION OF CARBONIC
ANHYDRASE FOR CO2 CAPTURE
2. APPLICANT:
(a) NAME : Indian Oil Corporation Limited
(b) NATIONALITY : IN
(c) ADDRESS : IndianOil Bhavan, G-9, Ali Yavar Jung Marg, Bandra (East),
Mumbai - 400051, Maharashtra, India
3. PREAMBLE TO THE DESCRIPTION
COMPLETE
The following specification particularly describes the invention and the manner in which it is to be
performed:

FIELD OF THE INVENTION:
[001] The present invention relates to carbonic anhydrases and fermentation media for producing the carbonic anhydrases. Specifically, the present invention relates to a process for the preparation of carbonic anhydrases for CO2 capture. Wherein, the said process is able to produce two different variants of carbonic anhydrases wherein one variant catalyzes CO2 absorption, and another variant catalyzes CO2 desorption.
BACKGROUND OF THE INVENTION:
[002] Carbon capture seems to be a mandatory technology for the development of sustainable energy infrastructures considering global energy demand. Post-combustion capture is a mature and proven technology, but not economically attractive unless novel solvents and optimized processes are implemented. The use of carbonic anhydrase (CA), inspired by the CO2 metabolic process in cells, a natural fast biocatalyst, is a promising technique which can dramatically improve the implementation and economics of carbon capture under stringent environment demands.
[003] Carbonic anhydrases (CAs) catalyze the inter-conversion between carbon dioxide and bicarbonate. It is a ubiquitous natural catalyst responsible for metabolic CO2 transfer in mammals and plants and is also found widely distributed in microorganisms. The protein sequence and three-dimensional structure of CAs from different origins can vary widely, however, almost all CAs share in common a highly conserved active site structure that catalyzes CO2 hydration though a zinc-hydroxide mechanism.
[004] Carbonic anhydrase improves both CO2 absorption and low temperature CO2 desorption in amine-based process. Accordingly, advancement in carbonic anhydrase enzyme production process is required for the commercialization of enzyme catalyzed CO2 capture technology as enzyme accounts for ~30% of cost saving of overall CO2 capture process. Below is the prior art which discloses various method of preparation of carbonic anhydrases.
[005] However, there are major challenges for producing carbonic anhydrases for CO2 capture process such as low enzymes titer, low catalytic activity and non-specificity for CO2 absorption

and/or desorption, and high production cost. Low stabilization of enzyme in various amine solvents used in CO2 capture process. Low tolerance of enzyme to higher temperature during CO2 absorption and desorption process.
[006] Effendi et.al (Process Biochemistry 87 (2019): 55) have summarized CA production from various species such as Pseudomonas fragi, Sulfurihydrogenibium azorens, Mesorhizobium loti, Lactobacillus delbrueckii, Hahella chejuensis, Sulfurihydrogenibium azorens, Salmo trutta Labrax Coruhensis, Bacillus subtilis, Dunaliella sp., Neisseria gonorrhoeae, Hahella chejuensis, Persephonella marina, Thermovibrio ammonificans, Sulfurihydrogenibium yellowstonense, M. chthonoplastes, Citrobacter freundii, Bacillus sp., Thalassiosira weissflogii and Phaeodactylum tricornutum. However, in most of these processes genetic modifications have been done by conventional and recombinant techniques of the strains to produce enzymes. Mutant created after genetic manipulation by recombinant DNA technology required specific growth factors or media components to produce enzymes. Further, the stability of these carbonic anhydrase in amine solvents is not studied and their effectiveness and specificity for CO2 capture and high thermal stability is very low.
[007] US 7,892,814 B2 describes a method for the use of heat-stable carbonic anhydrase in CO2 extraction, e.g., from flue gas, natural gas or biogas. Furthermore, the document relates to isolated polypeptides having carbonic anhydrase activity at elevated temperatures and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides. However, the stability of enzyme in presence of amines was not studied. Moreover, the method of enzyme production is tedious, effect of impurities, and stability in presence of heavy metals were not provided. The enzyme produced herein is not specific for CO2 absorption and desorption.
[008] US 8,871,485 B2 disclosed a process for modified carbonic anhydrase enzymes, and a process of using same for the extraction, production and purification of carbon dioxide gas. More particularly, modified carbonic anhydrase enzymes are used for the production, purification of carbon dioxide and the products of the hydration reaction, hydrogen and bicarbonate ions. Also, this technology is used to enhance the production of carbon dioxide in

blood or in reverse osmosis desalination to remove carbon dioxide. Specifically, the prior art document relates to a modified carbonic anhydrase enzyme possessing improved activity and a process whereby immobilized modified carbonic anhydrase contained within a reactor device catalyzes the reversible hydration of carbon dioxide. However, enzyme used herein is not suitable for flue gas application. The stability at higher temperature, flue gas condition, impurities are not provided.
[009] US 2013/0149771 A1 relates to use of Persephonella carbonic anhydrase in CO2 extraction, e.g., from flue gas, natural gas, biogas or ambient air. The Persephonella carbonic anhydrases are especially well suited for these purposes due to their extreme thermo stability. However, the impact of enzyme in absorption, desorption, impurities are not studied. Moreover, the enzyme is not stable at flue gas condition. The enzyme produced herein is not specific for CO2 absorption and desorption.
[0010] WO2015056858A1 relates to carbonic anhydrase, a nucleic acid molecule coding the carbonic anhydrase, a recombinant vector comprising the nucleic acid molecule, a hose cell transformed with the recombinant vector, and a method for producing carbonic anhydrase using the host cell. The carbonic anhydrase of the present invention has high stability at high temperatures and exhibits carbon dioxide collecting activity even at high temperatures, and thus can be applied in a carbon dioxide collection process actually performed at high temperatures. In spite of the above advantages, the impact of enzyme in absorption, desorption, impurities are not studied. Moreover, the enzyme is not stable at flue gas condition. The enzyme produced herein is not specific for CO2 absorption and/or desorption.
[0011] 4763/MUM/2015 relates to method of carbon dioxide absorption in aqueous solution from mixture of gases and a method for desorption of carbon dioxide in gaseous form carbon dioxide enriched solution by enzyme linked polyhydral metal organic framework. Also, the document relates to composition of an enzyme linked polyhydral metal organic framework for carbon dioxide absorption in aqueous solution from mixture of the gases and/or carbon dioxide desorption in gaseous form in carbon dioxide enriched solution. An enzyme linked polyhydral metal organic framework is also described. The document also relates to method of preparing

solid carbonate/ bicarbonate from carbon dioxide solution by enzyme linked polyhydral metal organic framework and a method for reactivating a carbon dioxide absorbing solution by using enzyme linked polyhydral metal organic framework.
[0012] The method of cultivating micro-organisms and production of carbonic anhydrase enzymes requires a complex regulation mechanism in a reactor. The efficiency of a microbial culture in terms of carbonic anhydrase enzyme production depends upon the medium, cultural parameters and operational conditions like temperature, pH, agitation rate and CO2/O2 concentration in reaction media etc. Hence, the formulation of suitable fermentation strategies plays a major role in deciding the fate of the microbial culture for its enzyme production and specificity.
[0013] However, there are major challenges in enzyme production for CO2 capture process such as low enzymes titer, low catalytic activity and non-specificity for CO2 absorption and/or desorption, and high production cost. Low stabilization of enzyme in various amine solvents used in CO2 capture process. Low tolerance of enzyme to higher temperature during CO2 absorption and desorption process.
OBJECTIVE OF THE PRESENT INVENTION:
[0014] The objective of the present invention is to develop a process for carbonic anhydrase
enzymes production from microbes using minimum media components and/or low-cost media
components.
[0015] The primary objective of the present invention is to provide a process for production of high titer of carbonic anhydrase enzymes from microbial culture using minimum media and/or low-cost media components.
[0016] The main objective of the present invention is to provide a process for production of two different variants of carbonic anhydrase wherein one variant catalyzes CO2 absorption and another variant catalyze CO2 desorption.

[0017] Another objective of the present invention is to provide a process that involves the chemical modification of bacterial carbonic anhydrase for its stabilization and improvement in catalytic activity.
[0018] Another objective of the present invention is to provide a process for production of efficient and low-cost enzyme for CO2 capture applications from flue gas, biogas or any waste gas stream containing CO2 from industrial sector.
SUMMARY OF THE INVENTION:
[0019] The present disclosure relates to a fermentation media for preparing carbonic anhydrases for CO2 absorption and desorption, wherein the fermentation media includes a carbon source, a nitrogen source, a chloride salt, KH2PO4, and limestone powder. The carbon source is in 2-5 % w/v, the nitrogen source is in 3-6% w/v, the chloride salt is in 0.1-4% w/v, KH2PO4 is in 0.1-0.6% w/v and the limestone powder is in 5-12% w/v. The fermentation media is used for preparing carbonic anhydrases for CO2 desorption and also used for preparing carbonic anhydrases for CO2 absorption. Specifically, the carbon source is in 2-5% w/v, the nitrogen source is in 3-6% w/v, the chloride salt is 0.1-4% w/v and KH2PO4 is in 0.1-0.6% w/v. The carbon source is selected from molasses, starch, cellulose, acid pre-treated lignocellulosic biomass or a combination thereof. The nitrogen source is selected from peptone, yeast extract, soya flour, de-oiled soya cake, or a combination thereof. The chloride salt is selected from sodium chloride, zinc chloride, magnesium chloride or a combination thereof.
[0020] Further, the present disclosure provides a process for preparing carbonic anhydrases for CO2 absorption from the fermentation media as mentioned above, wherein the process includes steps of a) sterilizing the fermentation media in a fermenter, b) inoculating 5 volume percent of a microbe culture into the fermentation media to prepare a culture media, c) maintaining a required pH of the culture media, wherein the required pH of the culture media is 4.5-5.5 pH, d) maintaining the temperature of culture media in the range of 45 to 55° C, passing 40-50 volume % of CO2 through the culture media for 16 hours, followed by passing 99.9 volume % of O2 through the culture media for 8 hour per day and total for 48 hours, e) collecting an enzyme

broth from the culture media followed by centrifugation to obtain carbonic anhydrases and precipitating the carbonic anhydrases as obtained after centrifugation, f) chemically modifying the carbonic anhydrases by adding enzyme modifiers. The step of sterilization is performed at 120°C for 20 minutes. The microbe for inoculation is selected from Bacillus thermoleovorans, Pseudomonas fragi, Bacillus stearothermophilus, Arthrobacter sp. or a combination thereof. The pH of the culture media is maintained by using 0.5 M acetic acid. The carbonic anhydrases which is obtained by the process, are further chemically modified by adding enzyme modifiers, wherein the enzyme modifiers are selected from acetic anhydride, hydroxyethyl-methylimidazolium, succinic anhydride, Hyaluronic acid, N-(iso-butoxymethyl) acrylamide, pyromellitic anhydride, pthalic anhydride, maleic anhydride, citraconic anhydride, succinic anhydride, benzoic anhydride, methoxypolyethylene glycol, methoxypolyethylene glycol succinimidyl succinate, monomethoxy polyethylene glycol activated with cyanuric chloride, tert-butyl methacrylate or a combination thereof. The enzyme modifiers are added at room temperature at a rate of 0.25-0.5ml/min to carbonic anhydrases followed by adding 1M sodium carbonate buffer in four aliquots over 3 hours.
[0021] Furthermore, the present disclosure provides a process for preparing carbonic anhydrases for CO2 desorption from the fermentation media as mentioned herein above. Wherein the process includes steps of a) sterilizing the fermentation media in a fermenter, b) inoculating 5 volume percent of a microbe culture into the fermentation media to prepare a culture media, c) maintaining a required pH of the culture media, wherein the required pH of the culture media is 8.5-10.5 pH, d) maintaining the temperature of culture media in the range of 45 to 55° C, passing 40-50 volume % of CO2 through the culture media for 16 hours, followed by passing 99.9 volume % of O2 through the culture media for 23 hour per day and for 48 hours, e) collecting an enzyme broth from the culture media followed by centrifugation to obtain carbonic anhydrases and precipitating the carbonic anhydrases as obtained after centrifugation, f) chemically modifying the carbonic anhydrases by adding enzyme modifiers. The sterilization step a) of the process is performed at 120°C for 20 minutes. The microbe for inoculation is selected from Bacillus thermoleovorans, Pseudomonas fragi, Bacillus stearothermophilus, Arthrobacter sp. or a combination thereof. The pH of the culture media is required to be maintained by using 0.5 M NaOH. The carbonic anhydrases which is obtained by the process, is chemically modified by enzyme modifiers wherein the enzyme modifiers are selected from acetic anhydride,

hydroxyethyl-methylimidazolium, succinic anhydride, Hyaluronic acid, N-(iso-butoxymethyl) acrylamide, pyromellitic anhydride, pthalic anhydride, maleic anhydride, citraconic anhydride, succinic anhydride benzoic anhydride, methoxypolyethylene glycol, methoxypolyethylene glycol succinimidyl succinate, monomethoxy polyethylene glycol activated with cyanuric chloride, tert-butyl methacrylate or a combination thereof. The enzyme modifiers are added at room temperature at a rate of 0.25-0.5ml/min, at room temperature to the carbonic anhydrases produced in step (e), followed by adding 1M sodium carbonate buffer in four aliquots over 3 hours.
DETAILED DESCRIPTION OF THE INVENTION:
[0022] For promoting an understanding of the principles of the present disclosure, reference will now be made to the specific embodiments of the present invention further illustrated in the drawings and specific language will be used to describe the same. The foregoing general description and the following detailed description are explanatory of the present disclosure and are not intended to be restrictive thereof. It will nevertheless be understood that no limitation of the scope of the present disclosure is thereby intended, such alterations and further modifications in the illustrated composition, and such further applications of the principles of the present disclosure as illustrated herein being contemplated as would normally occur to one skilled in the art to which the present disclosure relates. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinarily skilled in the art to which this present disclosure belongs. The process, and examples provided herein are illustrative only and not intended to be limiting.
[0023] The present invention provides a process for preparing carbonic anhydrase enzymes from microbes using minimum media components and/or low-cost media components. The present invention also provides a process for production of high titer of carbonic anhydrase enzymes from microbial culture. More specifically, the invention discloses a process for production of two different variants of carbonic anhydrases wherein one variant catalyzes CO2 absorption and another variant catalyzes CO2 desorption. The disclosure further provides a process that involves the chemical modification of bacterial carbonic anhydrase for its stabilization and improvement in catalytic activity.

[0024] Further, to induce stability in industrial flue gas condition and to improve catalytic properties in carbonic anhydrase enzymes, the chemical modification of carbonic anhydrase enzyme has been carried out using group-specific reagents. The group-specific reagents placed in contact with the carbonic anhydrase enzyme and binds covalently to amino acid side-chains in the carbonic anhydrase enzyme to alter its activity and stability.
[0025] Further, the present disclosure provides a process of production of carbonic anhydrase variants responsible for CO2 absorption and desorption at high titer from bacterial mutant strain in an economical way by using cheaper media components and its usage thereof. The disclosure further provides a process that involves the chemical modification of bacterial carbonic anhydrase for improving its catalytic activity.
[0026] Specifically, the present disclosure provides a fermentation media for preparing carbonic anhydrases for CO2 absorption and desorption, wherein the fermentation medium includes a carbon source, a nitrogen source, a chloride salt, KH2PO4, and limestone powder.
[0027] In a specific embodiment, the fermentation media includes 2-5 % w/v of a carbon source, 3-6% w/v of a nitrogen source, 0.1-4% w/v of a chloride salt, 0.1-0.6% w/v of KH2PO4, and 5-12% w/v of limestone powder, wherein the fermentation medium is used for preparing carbonic anhydrases for CO2 desorption.
[0028] Further, the present disclosure provides a process for preparing carbonic anhydrases for CO2 absorption from a fermentation media, wherein the process includes steps of sterilizing the fermentation media in a fermenter, wherein the sterilization is carried out at 120°C for 20 minutes. Then inoculating a microbe culture into the fermentation media to prepare a culture media. The microbe for inoculation is selected from Bacillus thermoleovorans IOC-S3 (MTCC 25023), Pseudomonas fragi IOC S2 (MTCC 25025), Bacillus stearothermophilus IOC S1 (MTCC 25030), Arthrobacter sp. IOC-SC-2 (MTCC 25028) or a combination thereof. The microbe(s) for inoculation into the fermentation media such as Bacillus thermoleovorans IOC-S3 (MTCC 25023), Pseudomonas fragi IOC S2 (MTCC 25025), Bacillus stearothermophilus IOC

S1 (MTCC 25030), Arthrobacter sp. IOC-SC-2 (MTCC 25028) are obtained from Indian Oil R&D Centre, Sector-13, Faridabad.
[0029] Maintaining a required pH of the culture media, wherein the required pH of the culture media is 4.5-5.5, wherein the required pH of the culture media is maintained by using 0.5 M acetic acid. Maintaining the temperature of culture media in the range of 45 to 55° C, passing 40-50 volume % of CO2 through the culture media for 16 hours, followed by passing 99.9 volume % of O2 through the culture media for 8 hour per day and for 48 hours.
[0030] Thereafter, collecting an enzyme broth from the culture media followed by centrifugation to obtain carbonic anhydrases and precipitating the carbonic anhydrases, then chemically modifying the carbonic anhydrases by enzyme modifiers. The enzyme modifiers are selected from acetic anhydride, hydroxyethyl-methylimidazolium, succinic anhydride, Hyaluronic acid, N-(iso-butoxymethyl) acrylamide, pyromellitic anhydride; pthalic anhydride; maleic anhydride; citraconic anhydride; succinic anhydride; benzoic anhydride; methoxypolyethylene glycol; methoxypolyethylene glycol succinimidyl succinate; monomethoxy polyethylene glycol activated with cyanuric chloride; tert-butyl methacrylate or a combination thereof.
[0031] The enzyme modifiers are added at a rate of 0.25-0.5ml/min, at room temperature to the produced carbonic anhydrases.
[0032] The carbon source is selected from molasses, starch, cellulose, acid pre-treated lignocellulosic biomass or a combination thereof. The nitrogen source is selected from peptone, yeast extract, soya flour, de-oiled soya cake, or a combination thereof. The chloride salt is selected from sodium chloride, zinc chloride, magnesium chloride or a combination thereof.
[0033] In another specific embodiment, the present invention provides a fermentation medium having 2-5 % w/v of a carbon source, 3-6% w/v of a nitrogen source, 0.1-4% w/v of a chloride salt and 0.1-0.6% w/v of KH2PO4, wherein the fermentation medium is used for preparing carbonic anhydrases for CO2 absorption.

[0034] Further, the present disclosure provides a process for preparing carbonic anhydrases for CO2 desorption from a fermentation media, wherein the process includes steps of sterilizing the fermentation media in a fermenter, wherein the sterilization is carried out at 120°C for 20 minutes. Then inoculating a microbe culture into the fermentation media to prepare a culture media. The microbe for inoculation is selected from Bacillus thermoleovorans IOC-S3 (MTCC 25023), Pseudomonas fragi IOC S2 (MTCC 25025), Bacillus stearothermophilus IOC S1 (MTCC 25030), Arthrobacter sp. IOC-SC-2 (MTCC 25028) or a combination thereof.
[0035] Maintaining a required pH of the culture media, wherein the required pH of the culture media is 8.5-10.5, wherein the required pH of the culture media is maintained by using 0.5 M NaOH. Maintaining the temperature of culture media in the range of 45 to 55° C, passing 40-50 volume % of CO2 through the culture media for 16 hours, followed by passing 99.9 volume % of O2 through the culture media for 23 hour per day and for 48 hours.
[0036] Thereafter, collecting an enzyme broth from the culture media followed by centrifugation to obtain carbonic anhydrases and precipitating the carbonic anhydrases, then chemically modifying the carbonic anhydrases by enzyme modifiers. The enzyme modifiers are selected from acetic anhydride, hydroxyethyl-methylimidazolium, succinic anhydride, Hyaluronic acid, N-(iso-butoxymethyl) acrylamide, pyromellitic anhydride; pthalic anhydride, maleic anhydride, citraconic anhydride, succinic anhydride, benzoic anhydride, methoxypolyethylene glycol, methoxypolyethylene glycol succinimidyl succinate, monomethoxy polyethylene glycol activated with cyanuric chloride, tert-butyl methacrylate or a combination thereof.
[0037] The enzyme modifiers are added at a rate of 0.25-0.5ml/min, at room temperature to the produced carbonic anhydrases.
[0038] The carbon source is selected from molasses, starch, cellulose, acid pre-treated lignocellulosic biomass or a combination thereof. The nitrogen source is selected from peptone,

yeast extract, soya flour, de-oiled soya cake, or a combination thereof. The chloride salt is selected from sodium chloride, zinc chloride, magnesium chloride or a combination thereof.
Carbonic anhydrase enzyme production from microbes using minimum media and/or low-cost media components.
[0039] Below is the step-by-step process description for preparation of fermentation media for carbonic anhydrase production responsible for CO2 absorption:
a) About 2-5 % w/v carbon source as molasses, starch, cellulose, acid pre-treated lignocellulosic biomass or a combination thereof.
b) Addition of 3-6% w/v peptone, yeast extract, soya flour or defatted soya flour or de-oiled soya cake as N source.
c) About 0.5-1 %w/v of NaCl added to the mixture described in step a) and b).
d) About 0.1-4 % w/v of zinc chloride and about 0.1-0.6 % w/v KH2PO4 added to the media described in step- c.
e) Autoclaving the media components.
f) Adding 1-5% (V) of the seed culture to the above media.
g) Maintaining the pH of the media at 4.5-5.5 using 0.5 M acetic acid.
h) Passing CO2 (40-50 v %) to the culture media for 16 h followed by O2 (99.9%) for 8 h per
day for 48h. i) Maintaining the temperature of fermentation media in the range of 45 to 55° C. j) Collecting the enzyme broth and centrifuging to obtain enzyme and precipitating the
enzyme.
[0040] Below is the step-by-step process description for preparation of fermentation media for carbonic anhydrase production responsible for CO2 desorption:
a) About 2-5 % w/v carbon source as molasses, starch, cellulose, acid pre-treated lignocellulosic biomass or a combination thereof.
b) Addition of 3-6% w/v peptone, yeast extract, soya flour or defatted soya flour or de-oiled soya cake as N source.
c) About 0.5-1 %w/v of NaCl added to the mixture described in step a) and b).

d) About 0.1-4 % w/v of magnesium chloride and about 0.1-0.6 % w/v KH2PO4 added to the
media described in step-c.
e) Adding 5-12% w/v of limestone powder to the fermentation broth.
f) Autoclaving the media components.
g) Adding 1-5% (V) of the seed culture to the above media.
h) Maintaining the pH of the media at 8.5-10.5 using 0.5 M NaOH.
i) Passing CO2 (40-50 v %) to the culture media for 1 h followed by O2 (99.9%) for 23 h per
day for 48h.Maintaining the temperature of fermentation media in the range of 45 to 55° C. j) Collecting the enzyme broth and centrifuging to obtain enzyme and precipitating the
enzyme.
Chemical modification of carbonic anhydrase
[0041] The enzymes obtained from the above disclosed process steps j) and j) were subjected for chemical modification. The chemical modification for the obtained enzyme involves the following steps:
a) The enzyme solution (3 mg/ml) was prepared in 1 M sodium carbonate buffer (pH 8.5)
b) In step a), group-specific enzyme modifiers were added in appropriate ratio.
c) The solution obtained through step b) placed in a shaker at 50-55° C for 20-24 h.
d) The excess reagents were separated by centrifugation and the modified enzymes were separated.
Activity evaluation of the enzyme
[0042] The evaluation of activity of enzyme by following tests:
a) The thermal stability, activity towards impurities, heavy metals, was tested.
b) The stability of the enzymes in presence of amines was tested.
c) CO2 absorption and desorption in presence of amines were tested.
[0043] Further, the low cost carbon source in the media includes 2-5% w/v molasses, starch, cellulose, acid pre-treated lignocelluloses biomass, corn sugar, or a combination thereof.

[0044] Additionally, the N2 source includes 3-6% w/v soya flour or defatted soya flour or de-oiled soya cake, Gelatin, Polypeptide, Yeast Extract.
[0045] Specifically, the carbon source in the fermentation media is cellulose and acid pre-treated lignocellulosic biomass in ratios selected from 1:2 or 1:3.
[0046] The lignocellulosic biomass for the fermentation is selected from rice straw, wheat straw, corn stover, and cotton stalk or sugarcane bagasse, wherein the acid pre-treatment of lignocellulosic biomass is done by concentrated sulphuric acid.
[0047] Particularly, the soya flour for the fermentation media is selected from defatted soya flour or plain soya flour.
[0048] In the media components, about 5-12% w/v of limestone powder is added, for the production of carbonic anhydrase responsible for CO2 desorption.
[0049] In accordance with present invention, the enzyme compositions contain CA (carbonic anhydrase) 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).
[0050] The seed culture of about 1-5 v% was added to the fermenter broth.
[0051] The pH was regulated during the fermentation process for the production of carbonic anhydrase enzyme.
[0052] The pH was maintained at 4.5-5.5 using 0.5-1 M acetic acid for production of carbonic anhydrase responsible for CO2 absorption activity.
[0053] Additionally, CO2 (40-50 v %) to the culture media for 16 h followed by O2 (99.9%) for 8 h per day for 48h for production of carbonic anhydrase responsible for CO2 absorption activity.
[0054] Furtherance, CO2 (40-50 v %) to the culture media for 1 h followed by O2 (99.9%) for 23 h per day for 48h for production of carbonic anhydrase responsible for CO2 desorption activity.

[0055] The CO2 or O2 is sparged using nano or micro-bubbler to the fermentation media.
[0056] Prior to fermentation, the fermentation media is sterilized by autoclaving at about 120° C for about 20 minutes.
[0057] The fermentation media is grown for 48 h at 30-37°C for the growth of the culture media.
[0058] The enzyme is separated from the reaction mixture by centrifugation of cell debris.
[0059] The supernatant was separated and precipitated using precipitating agent ammonium chloride, ethanol or acetone.
[0060] The chemical modification of enzyme was carried out in presence of group-specific enzyme modifiers.
[0061] The group-specific enzyme modifiers includes acetic anhydride, hydroxyethyl-methylimidazolium, succinic anhydride, Hyaluronic acid, N-(iso-butoxymethyl) acrylamide, pyromellitic anhydride, pthalic anhydride, maleic anhydride, citraconic anhydride, succinic anhydride, benzoic anhydride, methoxypolyethylene glycol, methoxypolyethylene glycol succinimidyl succinate, monomethoxy polyethylene glycol activated with cyanuric chloride, tert-butyl methacrylate or a combination thereof.
[0062] The enzyme was added with 3-5 mM chemical modifiers in presence of enzyme concentration 3-10 mg/L.
[0063] The group-specific enzyme modifiers were added in a rate of 0.25-0.5 ml/min to the enzymatic solution.
[0064] The modified enzyme in reaction medium were placed in a shaker at 50-55°C for 20-24 h.
[0065] The resulting modified enzyme was subsequently lyophilized or precipitated prior to use.

[0066] The resulting enzyme was inserted to amine solvents for 30 days and the activity was monitored. The activity of enzyme was measured by p-NPA hydrolysis or Wilbur–Anderson assay.
[0067] The amines were used for enzyme stability test may include primary, secondary, tertiary or poly amines at temperature up to 95°C.
[0068] The enzyme stability was studied at 130°C.
[0069] The effect of CA (carbonic anhydrase) stability was measured in presence of SO2, NO/NO2, and H2S.The enzyme activity also measured in presence of heavy metals Hg and Cd.
[0070] The CO2 absorption and desorption capacity were measured in presence of enzyme and amine in pilot plant after immobilizing on solid support.
Table-1. Media composition for production of carbonic anhydrase in fermentation broth.

Media composition Carbonic anhydrase promoting CO2 absorption Carbonic anhydrase promoting CO2 desorption
2-5 % w/v carbon source as molasses, starch, cellulose, acid pre-treated lignocellulosic biomass or a combination. 2-5 % w/v carbon source as molasses, starch, cellulose, acid pre-treated lignocellulosic biomass or a combination.
3-6% w/v peptone, yeast extract, soya flour or defatted soya flour or de-oiled soya cake as N source. 3-6% w/v peptone, yeast extract, soya flour or defatted soya flour or de-oiled soya cake as N source.
0.5-1 % w/v of NaCl 0.5-1 % w/v of NaCl
0.1-4 % w/v of zinc chloride 0.1-4 % w/v of magnesium chloride

0.1-0.6 % w/v KH2PO4 0.1-0.6 % w/v KH2PO4
- 5-12% w/v of limestone powder
De-ionized water is added to make up the volume. De-ionized water is added to make up the volume.
Fermentation condition pH of the media at 4.5-5.5 using 0.5 M acetic acid Maintaining the pH of the media at 8.5-10.5 using 0.5 M NaOH
Passing CO2 (40-50 v %) to the culture media for 16 h followed by O2 (99.9%) for 8 h per day for 48h Passing CO2 (40-50 v %) to the culture media for 16 h followed by O2 (99.9%) for 8 h per day for 48h
Maintaining the temperature of fermentation media in the range of 45 to 55° C. Maintaining the temperature of fermentation media in the range of 45 to 55° C.
Example-1: Production of carbonic anhydrase enzyme in fermentation media
a) Production of carbonic anhydrase variants from microbes promoting CO2 absorption
[0071] The production of carbonic anhydrase was carried out in a 20 L glass jacketed vessel. The media components of fermentation media used to give in Table 2.
Table-2: The media components of fermentation media

Code Media composition g/L
A Molasses 20
B Peptone 30
C NaCl 5
D ZnCl2 1
E KH2PO4 1
[0072] The fermenter containing 15 L medium was sterilized at 120° C. for 20 minutes. After cooling, the temperature was kept at 30° C, pH adjusted to 5.0 using 0.5 M acetic acid. 5 V% of Bacillus thermoleovorans IOC-S3 (MTCC 25023) was inoculated to the fermentation medium. CO2 (40-50 v %) was passed to the culture media for 16 h followed by O2 (99.9%) for 8 h per

day for 48h. The pH of the fermentation process was regulated and maintained at 5.0 using acetic acid (0.5M). After 48 hours of fermentation, the enzyme broth was collected, centrifuged and analysis of clear enzyme broth was carried out. The concentration of carbonic anhydrase was analyzed by UV-Vis spectrometer at 280nm and enzyme activities were analyzed by Wilbur– Anderson assay. 43.4 g/L of enzyme was obtained with activity of 1051 WAU/ml of enzymatic solution. Effect of media composition on enzyme titer and enzyme activity was given in Table-3.
Table-3: Effect of media composition on enzyme titer and enzyme activity

Media composition Enzyme titer (g/L) Enzyme activity (WAU/ml)
A+B 4.2 354
C+D 1.5 57
A+B+C 10.3 153
A+B+D 12.3 523
A+B+C+D 16.7 533
B+C+D+E 18.8 563
A+B+C+D+E 43.4 1051
Variation of fermentation condition was given in Table-4(a) and 4(b) Table-4(a): Variation of fermentation condition

Process condition Enzyme titer (g/L) Enzyme activity (WAU/ml)
pH=3 in the fermentation broth 0.5 12
pH=8 in the fermentation broth 18.7 126
Table- 4(b): Variation of fermentation condition

Process condition Enzyme titer (g/L) Enzyme activity (WAU/ml)
No CO2 sparging 9.8 115
CO2 sparging for 2h at a rate of 0.5 VVM 18.3 522
CO2 sparging for 4h at a rate of 0.5 VVM 20.2 662
CO2 sparging for 6h at a rate of 0.5 VVM CO2 sparging for 6h at a rate of 0.5 VVM 24.5 668

1051
CO2 sparging for 8h at a rate 43.4
of 0.5 VVM CO2 sparging
for 8h at a rate of 0.5 VVM
CO2 sparging for 10h at a 43.6 1053
rate of 0.5 VVM CO2
sparging for 8h at a rate of
0.5 VVM
b) Production of carbonic anhydrase variants from microbes promoting CO2 desorption
[0073] The production of carbonic anhydrase was carried out in a 20 L glass jacketed vessel. The components of fermentation media used given in table-5.
Table-5: Components of fermentation media

Code Media composition g/L
A Molasses 20
B Peptone 30
C NaCl 5
D MgCl2 1
E KH2PO4 1
F Limestone powder 5
[0074] The fermenter containing 15 L medium was sterilized at 120° C. for 20 minutes. After cooling, the temperature was kept at 30° C, pH adjusted to 8.5 using 0.5 M NaOH. 5 V% of Bacillus thermoleovorans IOC-S3 (MTCC 25023) was inoculated to the fermentation medium. CO2 (40-50 v %) was passed to the culture media for 1 h followed by O2 (99.9%) for 23 h per day for 48h. The pH of the fermentation process was regulated and maintained at 8.5 if required using 0.5 M NaOH. After 48 hours of fermentation, the enzyme broth was collected, centrifuged and analysis of clear enzyme broth was carried out. The concentration of carbonic anhydrase was analyzed by UV-Vis spectrometer at 280 nm and enzyme activities were analyzed by Wilbur– Anderson assay. 54.3 g/L of enzyme was obtained with activity of 1321 WAU/ml of enzymatic solution.
[0075] Effect of media composition on enzyme titer and enzyme activity is given in Table-6

Table-6: Media composition on enzyme titer and enzyme activity

Media composition Enzyme titer (g/L) Enzyme activity (WAU/ml)
A+B 8.2 234
C+D 2.8 85
A+B+C 16.4 198
A+B+D 15.7 256
A+B+C+D 18.3 512
B+C+D+E 20.3 560
A+B+C+D+E 21.4 578
A+B+C+D+E+F 54.3 1321
Variation of fermentation condition was given in Table-7
Table-7: Variation of fermentation condition

Process condition Enzyme titer (g/L) Enzyme activity (WAU/ml)
pH=3 in the fermentation broth 0.3 22
pH=12 in the fermentation broth 18.7 256

Process condition Enzyme titer (g/L) Enzyme activity (WAU/ml)
Limestone powder (0g/L) 21.4 578
Limestone powder (2g/L) 22.3 625
Limestone powder (5g/L) 54.3 1321
Limestone powder (8g/L) 55.2 1323
Limestone powder (10g/L) 55.8 1327
Example 2. Chemical modification of carbonic anhydrase
[0076] Chemically modified carbonic anhydrase was prepared by adding succinic anhydride (3 mM) in a rate of 0.25 ml/min to the enzyme variants produced in example1a and 1b (3 mg/mL, determined by Bradford assay) in 1 M sodium carbonate buffer (pH 8.5) in four aliquots over 3 h at room temperature. The final molar ratio of succinic anhydride to number of enzyme containing primary amines was approximately 30:1. Following modification, excess reagents

were separated by centrifugal filtration using a polyethersulfone membrane with a 10-kDa cut off at 4C. The resulting modified enzyme cocktail was subsequently lyophilized prior to use.
The activities of enzyme before and after chemical modification were given in table 8.
Table- 8: Activities of enzyme before and after chemical modification

Enzyme Activity of unmodified Activity of chemically
enzyme (WAU/ml) modified enzyme (WAU/ml)
Carbonic anhydrase 1051 2103
promoting CO2
absorption
Carbonic anhydrase 1321 2314
promoting CO2
desorption
Example 3. Stability of modified enzymes in presence of amines.
[0077] The test of enzyme stability were carried out using DGPCR where Solvent and CA (carbonic anhydrase) were kept at 95 degree C (it is soluble in free form) for 800h and subsequently activities ere compared with control. Table-9 represents activity retention of modified and unmodified enzyme compared to initial activity.
Table-9: Activity retention of modified and unmodified enzyme compared to initial activity

Amine solvent Variant of Variant of Variant of Variant of
Carbonic Carbonic Carbonic Carbonic
anhydrase anhydrase anhydrase anhydrase
promoting CO2 promoting CO2 promoting promoting CO2
absorption absorption CO2 desorption
(Chemically modified) desorption (Chemically modified)
Diglycolamine 37 91 37 87
Diethanolamine 28 97 24 94
Methyldiethanolamine 49 99 46 104
Aminomethylpropanol 33 98 25 98
2-piperidineethanol 62 95 53 98
Piperazine 51 97 45 98

Hydroxyethylpiperazine 55 98 56 99
Methyl piperazine 67 99 62 96
Aminoethylpiperazine 68 98 69 91
Aminoethylethanolamine 41 89 39 88
Triethanolamine 67 98 64 114
Pentaethylenehexamine 34 89 22 81
Diisopropanolamine 35 99 56 97
Table. 10: Impact of impurities on the activity of chemically modified

Time (h) Relative activity with impurity (NOx:5000 ppm, SOX: 5000ppm, H2S:
10,000ppm)
Amine Variant of Variant of Variant of Variant of
solvent Carbonic Carbonic carbonic carbonic
anhydrase anhydrase anhydrase anhydrase
promoting CO2 promoting CO2 promoting CO2 promoting CO2
absorption absorption
(Chemically
modified) desorption desorption
(chemically
modified)
0 100 100 100 100
20 45 99.8 47 99.2
800 21 98.2 18 98.5
3000 7 98.1 12 97.9
Table. 11: Impact of impurities on the activity of chemically modified.

Time (h) Relative activity with impurity (PbCl2=50 ppm, CdCl2 = 50ppm,
HgCl2 = 50ppm)
Variant of Variant of Variant of Variant of
Carbonic Carbonic carbonic carbonic
anhydrase anhydrase anhydrase anhydrase
promoting CO2 promoting CO2 promoting CO2 promoting CO2
absorption absorption
(Chemically
modified) desorption desorption
(chemically
modified)
0 100 100 100 100
20 89 99.3 86 99.2
800 59 98.7 63 98.5
3000 41 98.6 55 97.9

Example 4. CO2 absorption and desorption using chemically modified carbonic anhydrase enzyme variants.
[0078] The CO2 capture efficiency of modified enzymes were studied 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 (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 30 CC and was loaded with 4.3 g enzyme (Variant of carbonic anhydrase responsible for CO2 absorption) 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.
[0079] The rich amine was routed to the desorber (stripper) where it was heated at 90°C to regenerate the solvent and pure CO2. The stripper was loaded with 4.3 g enzyme (Variant of carbonic anhydrase responsible for CO2 desorption) immobilized-Al2O3 support (2-3 mm). The regenerated amine (Lean amine) is again used for next cycle CO2 capture. Table.12 represents the CO2 absorption and desorption capacity of chemically modified carbonic anhydrase enzyme

Enzyme variants Methyldiethanolamine Aminomethylpropanol
CO2 CO2 CO2 CO2 desorption
Absorption desorption Absorption (%) at 90°C
(Mol/L) (%) at 90°C (Mol/L)
Variant of Carbonic 2.7 - 2.2 -
anhydrase promoting
absorption CO2
Variant of Carbonic 4.4 - 4.1 -
anhydrase promoting
CO2 absorption
(Chemically modified)

Variant of Carbonic - 62 53
anhydrase promoting
CO2 desorption
(Chemically modified)
Variant of Carbonic - 96 91
anhydrase promoting
CO2 desorption
(Chemically modified)
Technical advantages of the invention:
[0080] In enzymatic CO2 capture, the enzyme carbonic anhydrase was used to improve CO2 absorption and lower the regeneration temperature. In the present process, the carbonic anhydrase having two different functions were involved. One variant of carbonic anhydrase catalyses the forward reaction of converting CO2 to carbonates and another variant catalyses the reverse reaction of the conversion of bicarbonates to CO2.
[0081] Production of carbonic anhydrase by conventional process is tedious and involves costly media components. Further, it is difficult to separate the enzyme by conventional process and provides enzymes having lower activity.
[0082] Additionally, the carbonic anhydrase obtained by the conventional process is less stable in amine solvents, impurities in flue gas and heavy metals.
[0083] In order to overcome the above limitations, the present invention relates to a process of carbonic anhydrase enzymes production from microbes using minimum media components and/or low-cost media components. The present invention also provides a process for production of high titer of carbonic anhydrase enzymes from microbial culture using minimum media and/or low-cost media components. More specifically, the invention discloses a process for production of two different variants of carbonic anhydrase wherein one variant catalyzes CO2 absorption and another variant catalyze CO2 desorption. The disclosure further provides a process that involves the chemical modification of bacterial carbonic anhydrase for its stabilization and improvement in catalytic activity.

[0084] The present invention involves the following major steps:
(Step 1): Production of Carbonic anhydrase variants from microbes using minimum media and/or
low-cost media components.
(Step-2): Chemical modification of carbonic anhydrase.
(Step-3): Activity and stability evaluation of carbonic anhydrase variants
[0085] The process of cultivating micro-organisms and production of carbonic anhydrase enzymes requires a complex regulation mechanism in a reactor. The present invention involves high titer of carbonic anhydrase enzymes using minimum media components and/or low-cost media components like cellulose or pre-treated lignocellulosic biomass as carbon source, soya flour or defatted soya flour or de-oiled soya cake as nitrogen source, limestone powder as carbonate source and gaseous CO2/O2 in presence of microbial culture. The composition of fermentation broth and conditions were also varied to obtain two variants of carbonic anhydrase enzyme responsible for CO2 absorption and desorption, respectively.
[0086] The present invention describes a process where the efficiency of a microbial culture in terms of carbonic anhydrase enzyme production depends upon the medium, cultural parameters and operational conditions like temperature, pH, agitation rate and CO2 concentration in reaction media etc. Hence, the formulation of suitable fermentation strategies plays a major role in deciding the fate of the microbial culture for its enzyme production ability.
[0087] The second step further involves chemical modification of as produced carbonic anhydrase to induce stability using group-specific reagents.
[0088] Native carbonic anhydrase has limitations over optimum utilization due to low thermal stability, and poor activity in harsh condition, like in the presence of high concentration of organic amines and trace contaminants (heavy metal, sulfur, oxide of sulfur, and nitrogen oxide). Chemical modification of enzyme is one of the process for making CA (carbonic anhydrase) more industrially viable.

[0089] The group-specific reagents used for modification carbonic anhydrase placed in contact with the enzyme and binds covalently to amino acid side-chains in the enzyme. Binding group-specific reagents are responsible for the primary and secondary hydrophobic face modification resulting better stability. The coordination geometry of Zn with CO2 depends on factors like π-stacking, and σ type H-bonding interactions. Carbonic anhydrase modification by involving a change in pol ype pti de c h a in, provide a sc op e to i mprove catalyti c a cti vity a nd sta bil ity.
[0090] Finally, the modified enzymes were evaluated for their stability, and catalytic activity under flue gas condition. The possible advantages of the present inventions, but not limited to, are: (i) Production of carbonic anhydrase enzyme at high titer from bacterial strain in an economical way by using cheaper media components, (ii) Easy separation of enzyme form fermentation broth, (iii) Easy separation of enzyme form fermentation broth, (iv) Easy separation of enzyme form fermentation broth, (v) Enzyme variants obtained showed high thermal tolerance up to 110°C and pH 4-14 as the enzyme obtained showed high stability to heavy metals, impurities like SO2, NO2, and H2S, (vi) Enzyme variants show long term stability in amine solutions, (vii) Enzyme variants show long term stability in a mi ne solutio ns.

We Claim:
1. A fermentation media for preparing carbonic anhydrases for CO2 absorption and desorption, wherein the fermentation media comprises a carbon source, a nitrogen source, a chloride salt, KH2PO4, and limestone powder.
2. The fermentation media as claimed in claim 1, wherein the carbon source is 2-5 % w/v, the nitrogen source is 3-6% w/v, the chloride salt is 0.1-4% w/v, KH2PO4 is 0.1-0.6% w/v, and the limestone powder is 5-12% w/v, wherein the fermentation media is used for preparing carbonic anhydrases for CO2 desorption.
3. The fermentation media as claimed in claim 1, wherein the carbon source is 2-5 % w/v, the nitrogen source is 3-6% w/v, the chloride salt is 0.1-4% w/v and KH2PO4 is 0.1-0.6% w/v, wherein the fermentation media is used for preparing carbonic anhydrases for CO2 absorption.
4. The fermentation media as claimed in claim 1-3, wherein the carbon source is selected from molasses, starch, cellulose, acid pre-treated lignocellulosic biomass or a combination thereof.
5. The fermentation media as claimed in claim 1-3, wherein the nitrogen source is selected from peptone, yeast extract, soya flour, de-oiled soya cake, or a combination thereof.
6. The fermentation media as claimed in claim 1-3, wherein the chloride salt is selected from sodium chloride, zinc chloride, magnesium chloride or a combination thereof.
7. A process for preparing carbonic anhydrases for CO2 absorption from the fermentation media as claimed in claim 1 and 3, wherein the process comprising steps of:

a) sterilizing the fermentation media in a fermenter;
b) inoculating 5 volume percent of a microbe culture into the fermentation media to prepare a culture media;

c) maintaining a required pH of the culture media, wherein the required pH of the culture media is 4.5-5.5 pH;
d) maintaining the temperature of culture media in the range of 45 to 55° C, passing 40-50 volume % of CO2 through the culture media for 16 hours, followed by passing 99.9 volume % of O2 through the culture media for 8 hour per day and total for 48 hours;
e) collecting an enzyme broth from the culture media followed by centrifugation to obtain carbonic anhydrases and precipitating the carbonic anhydrases as obtained after centrifugation;
f) chemically modifying the carbonic anhydrases by adding enzyme modifiers.

8. The process as claimed in claim 7, wherein the sterilization step a) is performed at 120°C for 20 minutes.
9. The process as claimed in claim 7, wherein the microbe for inoculation is selected from Bacillus thermoleovorans, Pseudomonas fragi, Bacillus stearothermophilus, Arthrobacter sp. or a combination thereof.
10. The process as claimed in claim 7, wherein the required pH of the culture media is maintained by using 0.5 M acetic acid.
11. The process as claimed in claim 7, wherein the enzyme modifiers are selected from acetic anhydride, hydroxyethyl-methylimidazolium, succinic anhydride, Hyaluronic acid, N-(iso-butoxymethyl) acrylamide, pyromellitic anhydride, pthalic anhydride, maleic anhydride, citraconic anhydride, succinic anhydride, benzoic anhydride, methoxypolyethylene glycol, methoxypolyethylene glycol succinimidyl succinate, monomethoxy polyethylene glycol activated with cyanuric chloride, tert-butyl methacrylate or a combination thereof.
12. The process as claimed in claims 7-11, wherein, the enzyme modifiers are added at room temperature at a rate of 0.25-0.5ml/min to the carbonic anhydrases followed by adding 1 M sodium carbonate buffer in four aliquots over 3 hours.

13. A process for preparing carbonic anhydrases for CO2 desorption from the fermentation
media as claimed in claim 1 and 2, wherein the process comprising steps of:
a) sterilizing the fermentation media in a fermenter;
b) inoculating 5 volume percent of a microbe culture into the fermentation media to prepare a culture media;
c) maintaining a required pH of the culture media, wherein the required pH of the culture media is 8.5-10.5 pH;
d) maintaining the temperature of culture media in the range of 45 to 55° C, passing 40-50 volume % of CO2 through the culture media for 16 hours, followed by passing 99.9 volume % of O2 through the culture media for 23 hour per day and for 48 hours;
e) collecting an enzyme broth from the culture media followed by centrifugation to obtain carbonic anhydrases and precipitating the carbonic anhydrases as obtained after centrifugation;
f) chemically modifying the carbonic anhydrases by adding enzyme modifiers.

14. The process as claimed in claim 13, wherein the sterilization step a) is performed at 120°C for 20 minutes.
15. The process as claimed in claim 13, wherein the microbe for inoculation is selected from Bacillus thermoleovorans, Pseudomonas fragi, Bacillus stearothermophilus, Arthrobacter sp. or a combination thereof.
16. The process as claimed in claim 13, wherein the required pH of the culture media is maintained by using 0.5 M NaOH.
17. The process as claimed in claim 13, wherein the enzyme modifiers are selected from acetic anhydride, hydroxyethyl-methylimidazolium, succinic anhydride, Hyaluronic acid, N-(iso-butoxymethyl) acrylamide, pyromellitic anhydride, pthalic anhydride, maleic anhydride, citraconic anhydride, succinic anhydride, benzoic anhydride, methoxypolyethylene glycol, methoxypolyethylene glycol succinimidyl succinate,

monomethoxy polyethylene glycol activated with cyanuric chloride, tert-butyl methacrylate or a combination thereof.
18. The process as claimed in claims 13-17, wherein, the enzyme modifiers are added at room temperature at a rate of 0.25-0.5ml/min, at room temperature to the carbonic anhydrases produced in step (e), followed by adding 1M sodium carbonate buffer in four aliquots over 3 hours.