DESC:FORM 2

THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003

COMPLETE SPECIFICATION
(See section 10 and rule 13)

“IMMOBILIZED WHOLE CELL NITRILASES FOR BIOCATALYSIS OF GABAPENTIN INTERMEDIATE”

GRANULES INDIA LIMITED
My Home Hub, 2nd Floor, 3rd Block, Madhapur, Hyderabad,
Telangana, INDIA - 500 081

The following specification particularly describes the invention and the manner in which it is to be performed.
IMMOBILIZED WHOLE CELL NITRILASES FOR BIOCATALYSIS OF GABAPENTIN INTERMEDIATE

FIELD OF INVENTION:
The present invention relates to immobilization of whole cell having nitrilase activity by cross-linked cell aggregates (CLCAs), and the use of such immobilized whole cell nitrilase enzymes for nitrile biocatalysis of gabapentin intermediate.

BACKGROUND OF THE INVENTION:
1-Cyanocyclohexaneacetic acid serves as a key precursor for the preparation of gabapentin, which is a useful drug in the treatment of seizures and neuropathic pain. Although several routes have been developed for the synthesis of cyanocarboxylic acid, most of them afford low yield and low purity of the object product because of their low regioselectivity.
Nitrilases are enzymes that can directly hydrolyze nitriles (containing —CN) to the corresponding carboxylic acids. While using nitrilase-mediated biotransformation, its stability and reusability are relatively poor. These obstacles can be circumvented by the immobilization of enzymes.
Recently, carrier-free immobilized enzymes, such as cross-linked enzyme aggregates (CLEAs) and cross-linked enzyme crystals (CLECs), have attracted increasing attention owing to the exclusion of additional carriers and the high volumetric productivity of immobilized enzyme. Cross-linked cell aggregates (CLCAs) as a carrier-free whole-cell immobilization method have a great potential for industrial application. Moreover, the lack of necessity for cell lysis and purification step reduce the cost of immobilization and simplify the manufacturing process.
The availability of immobilized biocatalysts being recycled can greatly reduce the operational cost in the production process. Compared with the free catalysts, separating the immobilized biocatalysts from the bioconversion reaction mixture is easier, making the repeated batch operations possible and simple. Moreover, immobilization improves the operational stability. Hence, the inventors of the present invention provide a Cross-linked cell aggregates (CLCAs) as a whole-cell immobilization method for application of Gabapentin intermediate.

OBJECTIVES OF THE INVENTION:
It is an object of the present invention to provide an immobilization of whole cell having nitrilase activity by cross-linked cell aggregates (CLCAs).

It is another object of the present invention to provide use of an immobilization of whole cell having nitrilase activity by cross-linked cell aggregates (CLCAs), in nitrile biocatalysis of gabapentin intermediate.

It is yet another object of the present invention to provide an immobilized whole cell nitrilases to give improved catalytic activity and recyclability of enzymes, which is simple and cost-effective.

SUMMARY OF THE INVENTION:
According to an aspect of the present invention, there is provided an immobilized whole cell having nitrilase activity.

According to another aspect of the present invention, there is provided an immobilization of whole cell having nitrilase activity by cross-linked cell aggregates (CLCAs).

According to yet another aspect of the present invention, there is provided use of an immobilization of whole cell having nitrilase activity by cross-linked cell aggregates (CLCAs), in nitrile biocatalysis of gabapentin intermediate.

According to a further aspect of the present invention, there is provided a process for the preparation of immobilized whole cell having nitrilase activity, which comprises mixing the whole cell with a polymer and cross-linking agent.

According to another aspect of the present invention, there is provided a process for the preparation of 1-Cyanocyclohexaneacetic acid from 1-Cyanocyclohexaneacetonitrile comprising the steps of:
a. contacting 1-Cyanocyclohexaneacetonitrile with an immobilized whole cell having nitrilase activity; and
b. optionally, isolating 1-Cyanocyclohexaneacetic acid from the reaction mixture.

According to another aspect of the present invention, there is provided a process for the preparation of gabapentin comprising reducing 1-Cyanocyclohexaneacetic acid obtained according to the process of the present invention.

BRIEF DESCRIPTION OF DRAWINGS:

Figure 1: Biocatalytic conversion of 1-Cyanocyclohexaneacetonitrile to 1-Cyanocyclohexaneacetic acid with an Immobilized whole cell Nitrilase.

DETAILED DESCRIPTION:
The following description is provided to assist in a comprehensive understanding of exemplary embodiments of the invention. It includes various specific details to assist in that understanding but these are to be regarded as merely exemplary.
Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the embodiments described herein can be made without departing from the scope of the invention. In addition, descriptions of well-known functions and constructions are omitted for clarity and conciseness.
The terms and words used in the following description and claims are not limited to the bibliographical meanings, but, are merely used by the inventor to enable a clear and consistent understanding of the invention. Accordingly, it should be apparent to those skilled in the art that the following description of exemplary embodiments of the present invention are provided for illustration purpose only and not for the purpose of limiting the scope of the invention as defined by the appended claims and their equivalents.
It is to be understood that the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.
Features that are described and/or illustrated with respect to one embodiment may be used in the same way or in a similar way in one or more other embodiments and/or in combination with or instead of the features of the other embodiments.
It should be emphasized that the term “comprises/comprising” when used in this specification is taken to specify the presence of stated features, steps or components but does not preclude the presence or addition of one or more other features, steps, components or groups thereof.

In one embodiment, the present invention provides an immobilized whole cell having nitrilase activity.

In another embodiment, the present invention provides a nitrilase gene which expressed in bacterial host cell like E. coli BL21(DE3) as the expression host and pET-28a as the cloning vector, and whole cell or isolated enzyme was immobilized, they can be used directly for the conversion of the nitrile substrate to product.

In another embodiment, the present invention provides an immobilized whole cell having nitrilase activity with a specific amino acid sequence ID and the amino acid sequence is as following:
SEQ ID NO: 1
1 5 10 15 20 25 30 35
M N H P K F K X A A V Q A A P V F L D L D K T I D K T I A L M E D A A R N
40 45 50 55 60 65 70
G A E L I A F P E T W L P G Y P W Y A W L D A P A M W L P Q Y T Q R Y F D
75 80 85 90 95 100 105 110
N S L E Y G T P Q A E R I S K A A K D N N I V V S M G L S E R S G G S L Y I S
115 120 125 130 135 140 145 150
Q W F I D R D G Q T I A Q R R K L K P T H V E R T I F G E G D G S D L A V W
155 160 165 170 175 180 185
D T R L G R V G G L C C W E H X Q P L S K Y A M Y A Q N E Q V H V A A W
190 195 200 205 210 215 220 225
P S F S X Y E A G A Y A L G P E V N T S A S R I Y A V E G Q C F V L A P C A
230 235 240 245 250 255 260
T V S Q E M V D E M C T T D L Q K A L L K T G G G Y A R I F G P D G X Q L
265 270 275 280 285 290 295 300
H E S L P A G E E G L I Y A E I D L G L I S V S K A V G D P A G H Y S R P D V
305 310 315 320 325 330 335
T Q L I H N K A P R R A V V D T A P P S D P A P A V E R P H G A D E P A L P
340
A M A E L

In another embodiment present invention provides the amino acid sequence of described nitrilase gene is classified as shown in SEQ ID NO: 1 and that includes the feature of residue corresponding to X8 is V, A, H or G; X167 is V, L, N or M; X192 is F, Y, L, or K and X260 is E, S, Q or R.
Wherein X represents the position of amino acid in SEQ ID NO: 1.
The abbreviations used for genetically encoded amino acids are conventional and are as follows:
Amino Acid One-Letter Code
Alanine A
Arginine R
Asparagine N
Aspartate D
Cysteine C
Glutamate E
Glutamine Q
Glycine G
Histidine H
Isoleucine I
Leucine L
Lysine K
Methionine M
Phenylalanine F
Proline P
Serine S
Threonine T
Tryptophan W
Tyrosine Y
Valine V

Accordingly, the present invention provides immobilized whole cell nitrilase enzymes capable of converting 1-Cyanocyclohexaneacetonitrile to 1-Cyanocyclohexaneacetic acid shown in Figure 1.

In another embodiment, the present invention provides an immobilization of whole cell having nitrilase activity by cross-linked cell aggregates (CLCAs).

In another embodiment, the present invention provides use of an immobilization of whole cell having nitrilase activity by cross-linked cell aggregates (CLCAs), in nitrile biocatalysis of gabapentin intermediate.

In another embodiment, the present invention provides a process for the preparation of immobilized whole cell having nitrilase activity, which comprises mixing the whole cell with a polymer and cross-linking agent by cross-linking method and isolating the immobilized cell.
The aforementioned process involves mixing whole cell with a carrier selected from celite, diatomite, perlite or gac and the like; preferably celite, stirred for a sufficient period of time, then add polymer selected from polyethyleneimine, polypropylenimine, polyallylamine, polyvinylamine and the like; preferably polyethyleneimine; and cross-linking agent is selected from glutaraldehyde, cyanuric chloride, hexamethylene diisocyanate or toluene di-isothiocyanate and the like; preferably glutaraldehyde, again stirred for an hour and isolating the immobilized cell carried out by any conventional techniques known in the art, for example filtration or decantation of solution followed by drying to get immobilized whole cell nitrilase enzyme. Wherein the whole cell nitrilase enzyme comprises, the amino acid sequence of nitrilase gene is classified as SEQ ID NO: 1.

In another aspect, the biological materials that can be immobilized by the method of this invention include enzymes, cell mass (intact or disrupted cells, viable or non-viable), antibodies and coenzymes.

In another embodiment, the present invention provides, the biocatalytic conversion of nitrile substrate into carboxylic acid product in the presence of immobilized whole cell nitrilase enzyme,

wherein R is,
C3–7 cycloalkyl which represents a cyclic alkyl radical having 3 to 7 carbon atoms being unsubstituted or substituted with one to more substituents selected from cyano, carboxy, nitro, amino, alkyl amino, and halogen; or
C1-8 alkyl which represents a straight-chain or branched alkyl radical having 1 to 8 carbon atoms being unsubstituted or substituted with one to more substituents selected from aryl, substituted aryl, heteroaryl, substituted heteroaryl, and alkyl amine.

In another embodiment, the immobilized whole cell nitrilase can be used in the preparation of key intermediates of active pharmaceutical ingredients like Gabapentin, Gabapentin enacarbil, Pregabalin, Baclofen, Levetiracetam, and Brivaracetam, etc.

In another embodiment of the present invention provides a process for the preparation of 1-Cyanocyclohexaneacetic acid from 1-Cyanocyclohexaneacetonitrile comprising the steps of:
a. contacting 1-Cyanocyclohexaneacetonitrile with immobilized whole cell having nitrilase activity; and
b. optionally, isolating 1-Cyanocyclohexaneacetic acid from the reaction mixture.

The aforementioned process involves addition of 1-Cyanocyclohexaneacetonitrile into the buffer solution such as phosphate or acetate buffer under stirring at room temperature for a sufficient period of time, followed by addition of immobilized whole cell having nitrilase activity and stirring at room temperature for a sufficient period of time for completion of reaction. The immobilized whole cell is separated and reused for subsequent cycle. The reaction mass is directly used for next step without isolation of 1-Cyanocyclohexaneacetic acid. Optionally, the reaction mass is acidified with an acid selected from hydrochloric acid, acetic acid, citric acid, tartaric acid, sulfuric acid, phosphoric acid and the like; preferably hydrochloric acid and the product 1-Cyanocyclohexaneacetic acid is isolated through filtration followed by drying.

In another aspect, the present invention provides a process for the preparation of gabapentin comprising reducing 1-Cyanocyclohexaneacetic acid obtained according to the process of the present invention.

The above process involves the reduction of 1-Cyanocyclohexaneacetic acid into gabapentin, which can be carried out by the methods known in the art or as per the process described herein by the present invention.

Another embodiment of the present invention provides, an immobilized whole cell catalysis having different types of activity for example but not limited to nitrilase, transaminase, keto-reductase, alkene-reductase and imine-reductase activity which used for the biocatalytic conversion of nitrile substrate into carboxylic acid product, keto substrate into amine product, keto substrate into alcohol product, alkene substrate into alkane product and imine substrate into amine product.

In another embodiment, the present invention provides, the use of an immobilized whole cell catalysis having different types of activity for example but not limited to nitrilase, transaminase, keto-reductase, alkene-reductase and imine-reductase activity, in the preparation of key intermediates of active pharmaceutical ingredients like Gabapentin, Gabapentin enacarbil, Pregabalin, Baclofen, Levetiracetam, Empagliflozin, Dexmethylphenidate, Nicotine, Brivaracetam, Ravuconazole, Rosuvastatin, Atorvastatin, Sitagliptin, Ticagrelor, Migalastat, Sacubitril, Cinacalcet, Apremilast, Lopinavir, Tenofovir, Ursodeoxycholic acid, Dapoxetine, Droxidopa, Sertraline, Duloxetine, Luliconazole, Vibegron, Mirabegron, Montelukast, Atazanavir, Chenodeoxycholic acid and Tofacitinib etc.

ADVANTAGES OF THE PRESENT INVENTION:
1. Immobilization of cell increases the stability and allows storage of cell for longer duration at higher temperature like 2-8 oC, which otherwise requires temperature as low as -20 oC to -78 oC in case of free cell.
2. Immobilization of cell facilitates recycling biocatalyst for multiple times without losing considerable activity.
3. Immobilization of cell prevents damage of cell membrane thereby minimizing contamination of product by proteins or other cell related materials. This contributes in improving quality of product.
4. In biocatalysis, removal of biocatalyst is one difficult task. Immobilization of cell eases up removal of biocatalyst after reaction by simple filtration.
5. Immobilization of cell by cross linked cell aggregate requires very low quantities of cross linker than carrier based immobilization.

In the foregoing section, embodiments are described by way of examples to illustrate the processes of invention. However, these are not intended in any way to limit the scope of the present invention. Variants of the examples that would be evident to a person ordinarily skilled in the art are within the scope of the present invention.

EXAMPLES:
The process details of the invention are provided in the examples given below, which are provided by way of illustration only and therefore should not be construed to limit the scope of the invention.

Example 1:
Preparation of immobilized whole cell nitrilase enzyme
To a clean and dry RBF (Round-bottom flask), 370 mL of phosphate buffer, 10 g of whole cell expressing nitrilase enzyme and 2 g of celite were added, stirred for 30 min, followed by addition of 11 mL of 5% polyethyleneimine and 3.7 mL of 30% glutaraldehyde solution and stirred for an hour. The reaction mass of immobilized cell was separated by filtration and washed with phosphate buffer. The solid obtained was dried under suction to obtain 25 g of immobilized nitrilase enzyme.

Example 2:
Preparation of immobilized whole cell nitrilase enzyme
To a clean and dry RBF, 2.5 L of phosphate buffer, 70 g of whole cell expressing nitrilase enzyme and 14 g of celite were added, stirred for 30 min, followed by addition of 77 mL of 5% polyethyleneimine and 25.9 mL of 30% glutaraldehyde solution and stirred for an hour. The reaction mass of immobilized cell was separated by filtration and washed with phosphate buffer. The solid obtained was dried under suction to obtain 175 g of immobilized nitrilase enzyme.

Example 3:
Preparation of immobilized whole cell nitrilase enzyme
To a clean and dry RBF, 2 L of phosphate buffer, 100 g of whole cell expressing nitrilase enzyme and 20 g of celite were added, stirred for 30 min, followed by addition of 110 mL of 5% polyethyleneimine and 259 mL of 30% glutaraldehyde solution and stirred for an hour. The reaction mass of immobilized cell was separated by filtration and washed with phosphate buffer. The solid obtained was dried under suction to obtain 250 g of immobilized nitrilase enzyme.

Example 4:
Preparation of 1-Cyanocyclohexaneacetic acid using immobilized whole cell nitrilase enzyme
To a clean and dry RBF, 200 mL of 0.007M phosphate buffer (pH 8.0) and 15 g of 1-Cyanocyclohexaneacetonitrile were added, stirred for 15 min at room temperature, then add 12.5 g of immobilized whole cell enzyme and stirred for 16 hours at same temperature. After complete conversion, the immobilized enzyme was separated by filtration, washed with 2x 15 mL of phosphate buffer and used for second cycle. Combined filtrate was cooled to 0-5 oC and acidified with 15% hydrochloric acid to adjust pH 3. The precipitate was isolated by filtration and washed with 3x 15 mL of water. The solid obtained was dried to get 1-Cyanocyclohexaneacetic acid (14.1g, 83% yield).
Second Cycle: The enzyme separated from first cycle was reused for second cycle. To a round bottomed flask, 200 mL of 0.007M phosphate buffer (pH 8.0) and 15 g of 1-Cyanocyclohexaneacetonitrile were added, stirred for 15 min at room temperature, then add the immobilized whole cell recovered from the first cycle and stirred for 16h at room temperature. After complete conversion, the immobilized enzyme was separated by filtration, washed with 2x 15 mL of phosphate buffer and used for subsequent cycle. Combined filtrate was cooled to 0-5 oC and acidified with 15% hydrochloric acid to adjust pH 3. The precipitate was isolated by filtration and washed with 3x 15 mL of water. The solid obtained was dried to get 1-Cyanocyclohexaneacetic acid (14.3 g, 85%).

Example 5:
Preparation of 1-Cyanocyclohexaneacetic acid using immobilized whole cell nitrilase enzyme
To a clean and dry RBF, 520 mL of 0.007M phosphate buffer (pH 8.0) and 40 g of 1-Cyanocyclohexaneacetonitrile were added, stirred for 15 min at 25oC, then add 25 g of immobilized whole cell enzyme and stirred for 24 h at 25oC. After complete conversion, the immobilized enzyme was separated by filtration, washed with 2x 40 mL of phosphate buffer and used for second cycle. Combined filtrate was cooled to 0-5 oC and acidified with 15% hydrochloric acid to adjust pH 3. The precipitate was isolated by filtration and washed with 3x 40 mL of water. The solid obtained was dried to get 1-Cyanocyclohexaneacetic acid (38.8g, 86% yield).
Second Cycle: The enzyme separated from first cycle was reused for second cycle. To a round bottomed flask, 200 mL of 0.007M phosphate buffer (pH 8.0) and 40 g of (1-cyanocyclohexyl) acetonitrile were added, stirred for 15 min at 25oC, then add the immobilized whole cell recovered from the first cycle and stirred for 24h at room temperature. After complete conversion, the immobilized enzyme was separated by filtration, washed with 2x 40 mL of phosphate buffer and used for subsequent cycle. Combined filtrate was cooled to 0-5 oC and acidified with 15% hydrochloric acid to adjust pH 3. The precipitate was isolated by filtration and washed with 3x 40 mL of water. The solid obtained was dried to get 1-Cyanocyclohexaneacetic acid (38.8g, 86% yield).

Example 6:
Preparation of 1-Cyanocyclohexaneacetic acid using immobilized whole cell nitrilase enzyme
To a clean and dry RBF, 650 mL of 0.007M phosphate buffer (pH 8.0) and 50 g of 1-Cyanocyclohexaneacetonitrile were added, stirred for 15 min at 25oC, then add 25 g of immobilized whole cell enzyme and stirred for 24 h at 25oC. After complete conversion, the immobilized enzyme was separated by filtration, washed with 2x 50 mL of phosphate buffer and used for second cycle. Combined filtrate was cooled to 0-5 oC and acidified with 15% hydrochloric acid to adjust pH 3. The precipitate was isolated by filtration and washed with 3x 50 mL of water. The solid obtained was dried to get 1-Cyanocyclohexaneacetic acid (50.2g, 89% yield).
Second Cycle: The enzyme separated from first cycle was reused for second cycle. To a round bottomed flask, 650 mL of 0.007M phosphate buffer (pH 8.0) and 40 g of 1-Cyanocyclohexaneacetonitrile were added, stirred for 15 min at 25oC, then add the immobilized whole cell recovered from the first cycle and stirred for 27h at room temperature. After complete conversion, the immobilized enzyme was separated by filtration, washed with 2x 50 mL of phosphate buffer and used for subsequent cycle. Combined filtrate was cooled to 0-5 oC and acidified with 15% hydrochloric acid to adjust pH 3. The precipitate was isolated by filtration and washed with 3x 40 mL of water. The solid obtained was dried to get 1-Cyanocyclohexaneacetic acid (49.9 g, 88% yield).

Example 7:
Preparation of 1-Cyanocyclohexaneacetic acid using immobilized whole cell nitrilase enzyme
To a clean and dry RBF, 200 mL of 0.007M phosphate buffer (pH 8.0) and 30 g of 1-Cyanocyclohexaneacetonitrile were added, stirred for 15 min at 35oC, then add 25 g of immobilized whole cell and stirred for 12h at 35oC. After complete conversion, the immobilized enzyme was separated by filtration, washed with 2x 30 mL of phosphate buffer and used for second cycle. Combined filtrate was cooled to 0-5 oC and acidified with 15% hydrochloric acid to adjust pH 3. The precipitate was isolated by filtration and washed with 3x 30 mL of water. The solid obtained was dried to get 1-Cyanocyclohexaneacetic acid (29.8g, 88% yield).
Second Cycle: The enzyme separated from first cycle was reused for second cycle. To a round bottomed flask, 200 mL of 0.007M phosphate buffer (pH 8.0) and 30 g of 1-Cyanocyclohexaneacetonitrile were added, stirred for 15 min at 35oC, then add immobilized whole cell recovered from the first cycle and stirred for 12h at 35oC. After complete conversion, the immobilized enzyme was separated by filtration, washed with 2x 30 mL of phosphate buffer and used for subsequent cycle. Combined filtrate was cooled to 0-5 oC and acidified with 15% hydrochloric acid to adjust pH 3. The precipitate was isolated by filtration and washed with 3x 30 mL of water. The solid obtained was dried to get 1-Cyanocyclohexaneacetic acid (29.8g, 88% yield).
Example 8:
Preparation of 1-Cyanocyclohexaneacetic acid using immobilized whole cell nitrilase enzyme
To a clean and dry RBF, 2000 mL of 0.007M phosphate buffer (pH 8.0) and 100 g of 1-Cyanocyclohexaneacetonitrile were added, stirred for 15 min at 45oC, then added 25 g of immobilized whole cell and stirred for 16h at 45oC. After complete conversion, the immobilized enzyme was separated by filtration, washed with 2x 50 mL of phosphate buffer and used for second cycle. Combined filtrate was cooled to 0-5 oC and acidified with 15% hydrochloric acid to adjust pH 3. The precipitate was isolated by filtration and washed with 3x 100 mL of water. The solid obtained was dried to get 1-Cyanocyclohexaneacetic acid (102.6g, 91% yield).

Example 9:
Preparation of 1-Cyanocyclohexaneacetic acid using immobilized whole cell nitrilase enzyme
To a clean and dry RBF, 600 mL of 0.007M ammonium acetate buffer (pH 8.0), 50g of immobilized whole cell and 100g of 1-cyanocyclohexaneacetonitrile were added and stirred at 35oC for 18h. After complete conversion, the immobilized enzyme was separated by filtration and washed with 100 mL of ammonium acetate buffer. Combined filtrate was washed with 2x 150 mL of toluene. The aqueous layer was carried over to next step without isolation of the product. The recovered enzyme was used for subsequent cycle of reaction.

Using the above procedures, the immobilized whole cell enzyme was reused for multiple cycles.

Example 10:
Preparation of Gabapentin from isolated 1-Cyanocyclohexaneacetic acid
To a clean and dry RBF, 300 mL of aqueous ammonia and 11 g of ammonium acetate and 50g of 1-cyanocyclohexaneacetic acid were added and stirred to get clear solution. The solution was transferred to an autoclave, then 15 g of Raney nickel was added and hydrogenated under 15 kg/cm2 pressure at 50-60oC. After completion of reaction, Raney nickel was separated by filtration through celite. Water was removed by distillation. The crude product was purified by recrystallization from a mixture of water and isopropanol. The solid obtained was filtered and dried to get the title compound. Yield: 75%.

Example 11:
Preparation of Gabapentin from non-isolated 1-Cyanocyclohexaneacetic acid
To 1-Cyanocyclohexaneacetic acid (obtained from example 9 reaction mass), 23g of ammonium acetate was added in to a clean and dry RBF and stirred to get clear solution. The solution was transferred to an autoclave and 18 g of Raney Nickel was added and hydrogenated under 15 kg/cm2 pressure at 60oC. After completion of reaction, Raney nickel was separated by filtration. Water was removed by distillation followed by addition of acetone to recrystallize the product. The solid obtained was filtered and dried to get the title compound. Yield: 75%.

Dated this: 19th day of August, 2024.
Signature:
Name: Mr. Rama Rao Javvaji
Patent Agent Reg. No.: IN/PA-1669
GRANULES INDIA LIMITED
My Home Hub, 2nd Floor, 3rd Block,
Madhapur, Hyderabad, Telangana, INDIA-500 081
,CLAIMS:We Claim:
1. An immobilized whole cell having nitrilase activity.

2. An immobilized whole cell having nitrilase activity as claimed in claim 1, wherein the enzyme is immobilized by cross-linked cell aggregates (CLCAs).

3. An immobilized whole cell having nitrilase activity as claimed in claim 1, wherein the whole cell nitrilase enzyme comprises the amino acid sequence of SEQ ID NO: 1.

4. A process for the preparation of immobilized whole cell having nitrilase activity, which comprises mixing the whole cell with a polymer and cross-linking agent by cross-linking method and isolating the immobilized cell.

5. The process as claimed in claim 4, wherein the polymer is selected from polyethyleneimine, polypropylenimine, polyallylamine and polyvinylamine; and the cross-linking agent is selected from glutaraldehyde, cyanuric chloride, hexamethylene diisocyanate and toluene di-isothiocyanate.

6. A process for the preparation of 1-Cyanocyclohexaneacetic acid from 1-Cyanocyclohexaneacetonitrile comprising the steps of:
a. contacting 1-Cyanocyclohexaneacetonitrile with immobilized whole cell having nitrilase activity; and
b. optionally, isolating 1-Cyanocyclohexaneacetic acid from the reaction mixture.

7. A process for preparation of gabapentin comprising reducing 1-Cyanocyclohexaneacetic acid obtained according to claim 6.

8. A biocatalytic conversion of nitrile substrate into carboxylic acid product in the presence of immobilized whole cell nitrilase enzyme,

wherein R is,
C3–7 cycloalkyl which represents a cyclic alkyl radical having 3 to 7 carbon atoms being unsubstituted or substituted with one to more substituents selected from cyano, carboxy, nitro, amino, alkyl amino, and halogen; or
C1-8 alkyl which represents a straight-chain or branched alkyl radical having 1 to 8 carbon atoms being unsubstituted or substituted with one to more substituents selected from aryl, substituted aryl, heteroaryl, substituted heteroaryl, and alkyl amine.

9. Use of an immobilized whole cell having nitrilase activity by cross-linked cell aggregates (CLCAs), in the preparation of key intermediates of active pharmaceutical ingredients.

Dated this: 19th day of August, 2024.
Signature:
Name: Mr. Rama Rao Javvaji
Patent Agent Reg. No.: IN/PA-1669
GRANULES INDIA LIMITED
My Home Hub, 2nd Floor, 3rd Block,
Madhapur, Hyderabad, Telangana, INDIA-500 081