A PROCESS OF IMMOBILISING CDX-036 TRANSAMINASE ENZYME ON A SUBSTRATE

Abstract

ABSTRACT A PROCESS OF IMMOBILISING CDX-036 TRANSAMINASE ENZYME ON A SUBSTRATE The present invention relates to a process of immobilising CDX-036 Transaminase enzyme on a substrate. The invention provides a process of immobilisuing CDX-036, Transaminase, which is easy, feasible, economical and a scalable process of immobilization, for the synthesis of blockbuster Sitagliptin. The process is not only feasible and economical but has also been validated for its rate of conversions and recyclability with Sitagliptin synthesis and thus is direct utility and relevant to academia and Industry.

Specification

Description:FORM 2
THE PATENTS ACT, 1970
(39 OF 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)



Title: A PROCESS OF IMMOBILISING CDX-036 TRANSAMINASE ENZYME ON A SUBSTRATE


APPLICANT DETAILS:
(a) NAME: NATIONAL INSTITUTE OF PHARMACEUTICAL EDUCATION
AND RESEARCH (NIPER), HYDERABAD
(b) NATIONALITY: Indian
(c) ADDRESS: NH 9, Balanagar Main Rd, Kukatpally Industrial Estate,
Balanagar, Hyderabad, Telangana 500037, India







PREAMBLE TO THE DESCRIPTION:
The following specification particularly describes the nature of this invention and the manner in which it is to be performed.
A PROCESS OF IMMOBILISING CDX-036 TRANSAMINASE ENZYME ON A SUBSTRATE
FIELD OF INVENTION:
The present invention relates to a process of immobilising CDX-036 Transaminase enzyme on a substrate.

BACKGROUND OF THE INVENTION:
The following background discussion includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
Transaminases are important biocatalysts used for synthesizing chiral amines, and they can catalyze chemical reactions by transferring an amino group to a keto group in a regio- and stereoselective manner. Although the synthesis of chiral amines is a challenge, transaminase-based green processes for synthesizing chiral amines are highly efficient however the stability and reusability of these enzymes pose a challenge in chemical and pharmaceutical industries. Reports are available where they have immobilized Transaminases to increase their reusability; however, commercially utilizing those available immobilization techniques is not very feasible due to the use of expensive and artificial solid supports, use of non-commercial enzymes with limited substrate scope, difficulty in scaling up, decrease in stability and activity after immobilization and many others.
Chiral amines containing compounds are essential moieties used for the synthesis of almost 40% of pharmaceuticals and other biologically active compounds. Transaminases are important biocatalysts that are used for the synthesis of chiral amines in regio- and stereospecific manners with interesting substrate scope. Codexis reported that the manufacturing of sitagliptin using transaminase is an excellent illustration of the use of enzyme engineering for catalyzing non-native but highly significant industrial synthesis. Codexis picked up a wild-type enzyme from the Arthobacter species, which initially only accepted small molecules as substrates; however, using the substrate walking approach, they evolved the enzyme for 27 rounds of evolution for catalyzing the efficient conversion of pro-sitagliptin to sitagliptin in the presence of 50% DMSO as a co-solvent and reported the conversion of 200 g/L of pro-sitagliptin into the final product of sitagliptin in 24 hours with an ee.>99.5% . However, the process of doing this enzymatic conversion, although efficient, is very expensive and thus, to increase the economy of the reaction at an industrial scale, recycling of transaminase enzyme is an excellent approach.
Recyclability of enzymes can be achieved via enzyme immobilization on recoverable solid support. Several solid supports have been used for enzyme immobilization, such as carbon nanotubes (CNTs), graphene and graphene oxide, polymer nanofibers, mesoporous silica, magnetic nanoparticles , metal-organic framework compounds (MOFs), metal oxides, metal nanoparticles, metal hydroxides, natural biopolymers , biochar , zeolites, dextrin and dextran etc. Multiple reports of transaminase enzyme immobilization using different solid supports like epoxy and glutaraldehyde-based linkage on modified cellulose, cross-linking of enzyme aggregates (CLEA), polymeric resins like polystyrene, polyvinyl, polyacrylate, polypropylene, copper phosphate nanoflowers, silica nanoparticles, polylactic acid and others are available in the literature. However, there are several limitations. These methods have been developed non-uniformly with not so well characterized enzymes of unknown industrial importance from various bacterial species, poor enzyme loading, loss of enzymatic activity on immobilization, poor stability, loss of activity on multiple usages, expensive solid supports and sophisticated chemistry, issues with scale-up processes, use of uncharacterized cell lysate or unpurified enzyme and whole cell-based immobilization which can lead to reproducibility issues.
Hence, there is a need of an efficient Dextrin aldehyde-based process of preparation of recyclable CDX-036 Transaminase with high activity and stability.

OBJECTIVE OF THE PRESENT INVENTION:
An object of the present invention is to provide a Dextrin aldehyde-based preparation of recyclable CDX-036 Transaminase with high activity and stability.
Another object of the present invention is to provide a process of immobilising CDX-036, Transaminase, which is easy, feasible, and economical.
Another object of the present invention is to provide a scalable process of immobilization, for the synthesis of blockbuster Sitagliptin, which is not only feasible but also economical.

SUMMARY OF THE INVENTION:
In an embodiment, the invention provides a process of immobilising CDX-036 Transaminase enzyme on a substrate comprising dextrin, said steps comprises the steps of:
a) Converting Dextrin to dextrin aldehyde by oxidation;
b) Immobilizing CDX-036 Transaminase enzyme by the steps of:
i. Loading 10 mg of CDX-036 (TA) enzyme on dextrin aldehyde at 1:5, 1:10, 1:15 and 1:20 ratios using 50 mg, 100 mg, 150 mg, and 200 mg of dextrin, by dissolving CDX-036 (TA) enzyme and dextrin aldehyde in 5 ml of distilled water and stirring at 20oC for 16 hours;
ii. Adding 5 ml of 100 mM of chilled sodium bicarbonate and after 15 minutes, 5 mg of sodium borohydride was added, and stirring for 2 hours;
iii. Transferring the solution od step (ii) into a 10 Kda centricon, and then it was centrifuged for 15 min at 4300 rpm at 4oC to concentrate the immobilized enzyme followed by separating the immobilized enzyme by gel filtration chromatography.
The Invention also provides a process for the preparation of sitagliptin using immobilized CDX-036, which comprises treating 200 mg pro-sitagliptin substrate with 6 mg immobilized enzyme obtained from claim 1, 1 mg pyridoxal 5' phosphate (PLP) and 5 mM isopropyl amine in 2 mL of 25:75 (DMSO: water).

DETAILED DESCRIPTION OF DRAWING:
The accompanying drawings, which are incorporated in and constitute a part of this disclosure, illustrate exemplary embodiments and, together with the description, explain the disclosed principles. The reference numbers are used throughout the figures to describe the features and components. Some embodiments of system and/or methods in accordance with embodiments of the present subject matter are now described, by way of example only, and regarding the accompanying figures, in which:
Scheme 1a: illustrates scheme 1a, showing the steps involved in the present invention
Figure 1: illustrates UV-vis spectra of DNPH solution with the reaction of dextrin aldehyde (a) standard DNPH (b) 100 μL Dextrin aldehyde + 900 μL DNPH
Figure 2 (a) Gel Filtration chromatogram for purification of immobilized CDX-036 from free dextrin aldehyde and free enzyme present in the mixture (b) Bar graph depicting the best-optimized ratio of the enzyme to dextrin aldehyde for maximum enzyme loading.
Figure 3 illustrates Combined FTIR spectra of Dextrin, Dextrin Aldehyde (Dex. Ald.), and Immobilized Transaminase on Dextrin Aldehyde (Dex. Ald. Enz.)
Figure 4 illustrates (a) Scanning Electron Microscopy analysis at 2000x of (a) Dextrin polymer (b) dextrin aldehyde (c) immobilized transaminase enzyme on dextrin-aldehyde polymer
Figure 5 illustrates 1H NMR spectra of Dextrin in DMSO d6
Figure 6 illustrates 1H NMR spectra of Dextrin Aldehyde in D2O
Figure 7 illustrates Comparative graph of relative % conversion of Sitagliptin using immobilized and normal enzymes.

DESCRIPTION OF THE INVENTION:
In the present document, the word "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any embodiment or implementation of the present subject matter described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

While the disclosure is susceptible to various modifications and alternative forms, specific embodiment thereof has been shown by way of example in the drawings and will be described in detail below. It should be understood, however that it is not intended to limit the disclosure to the specific forms disclosed, but on the contrary, the disclosure is to cover all modifications, equivalents, and alternative falling within the spirit and the scope of the disclosure.
The invention provides a process of immobilising CDX-036, Transaminase, which is easy, feasible, economical and a scalable process of immobilization, for the synthesis of blockbuster Sitagliptin. The process is not only feasible and economical but has also been validated for its rate of conversions and recyclability with Sitagliptin synthesis and thus is direct utility and relevant to academia and Industry.
The invention comprises CDX-036, Transaminase for immobilizing on natural polymer (dextrin) to develop a green, easy, efficient, scalable and reusable process. Not only is the process developed easy and scalable with high efficiency, but the use of dextrin as a solid support has multiple advantages like better loading yield, low cost, and easy process of enzyme loading, amongst others. The process developed shows improvement in enzyme activity over soluble free enzyme which is also anticipated as immobilization increases the enzyme stability and increases the surface area available for the reaction. Also, it is easily useable for up to 5 cycles and more without any significant decrease in activity.
The dextrin was converted to dextrin aldehyde, and CDX-036 was immobilized with an 82 % loading yield. After immobilization, this enzyme was used further for the synthesis of sitagliptin using pro-sitagliptin as a substrate and pyridoxal 5' phosphate as a cofactor in 25 % DMSO at 50oC and 400 rpm for four hours. In the first cycle, the percentage conversion of sitagliptin with immobilized enzyme was observed at 92 % as compared to only 80 % with soluble enzyme under the same conditions, and the immobilized enzyme maintained an excellent conversion rate till the five cycles as tested by us with percentage conversion of 70 % at fifth cycle. The immobilized enzyme was also analyzed using SEM and FTIR and showed a good loading.
Reagents and Equipment used in the experiments performed for the present invention:
Dextrin, pyridoxal 5' phosphate, dimethyl sulphoxide, ethanol, and Bradford reagent were purchased from Himedia. sodium meta periodate (Finar), dialyzed membrane (Merck), sodium bicarbonate (SRL), sodium borohydride (Sigma-Aldrich), transaminase enzyme (Codexis), sephacryl resin (Cytiva), pro-sitagliptin (Ami Life Sciences), isopropyl amine (Sigma-Aldrich), Heptane (Finar), Diethylamine (SDFCL). HPLC Agilent 1260 infinity, Daicel chiralcel column (4.6 mmID x 250 mmL x 5μL), Japan. UV-vis spectrophotometer Eppendorf, Multimode Reader BioTek (Cytation 5), Centrifuge 5810R Eppendorf.
Enzyme Immobilization
Activation of dextrin to dextrin aldehyde
To activate dextrin, sodium periodate was used as an oxidizing agent. Three grams of dextrin was dissolved in 22.50 ml of distilled water in a 100 ml round bottom flask (RBF). Further, 6 g of sodium periodate (NaIO4) was dissolved separately in 52.50 mL of distilled water. After that, dextrin solution (RBF) was covered with aluminium foil to avoid light exposure. Then, the slow addition of NaIO4 was completed in 15 minutes with constant stirring, and the reaction was continued for 24 hours.
After that, oxidized dextrin solution was dialyzed using an activated dialyzed membrane. Oxidized dextrin solution was poured into a dialyzed membrane to remove unreacted particles of NaIO4 from the reaction mixture. The dialysis was completed in 24 hours. Further, oxidized dextrin was lyophilized to remove excess water from the reaction mixture. After lyophilisation, the dry powdered dextrin aldehyde was obtained, and then it was directly used for enzyme immobilization.
2,4-Dinitrophenylhydrazine (DNPH) based colorimetric Assay for determination of aldehyde content
To determine the content of oxidation of dextrin, a DNPH assay was performed. Firstly, a standard curve of DNPH was made using 0.2-1.2 mg concentrations of DNPH dissolved in sulphuric acid, ethanol, and water. For making the DNPH solution, 40mg of DNPH was dissolved in 300 μL of concentrated sulphuric acid. Then, 3 mL of ethanol was added to the above-prepared slurry with continuous stirring at RT. After homogenization, the above solution volume was made up to 10 ml using distilled water. Further, dilutions were made to make standard curve, and the absorbance was taken at 357 nm.
The above standard curve was used to measure the aldehyde content of the activated dextrin. For the same, the dextrin aldehyde solution of 3 mg/ml (100 μl) was prepared and added to the DNPH 0.4 mg/ml (900 μl) solution. Then, the reaction mixture was stirred at 200 rpm and 25oC for one hour. After that reaction, the mixture was centrifuged at 10000 rpm and 4oC for 10 min. The absorbance of unreacted DNPH solution was measured at 357 nm.
Transaminase enzyme immobilization on dextrin aldehyde
In this process, the transaminase enzyme was immobilized on the surface of dextrin aldehyde, and further, it was purified and used in chemical reactions. To optimize the best enzyme-to-support ratio for maximum enzyme loading 10 mg of CDX-036 (TA) enzyme was loaded on dextrin aldehyde at 1:5, 1:10, 1:15 and 1:20 ratios using 50 mg, 100 mg, 150 mg, and 200 mg of dextrin. For enzyme loading, a given amount of TA and dextrin aldehyde were taken together and dissolved in 5 ml of distilled water and stirred at 20oC for 16 hours. Then, 5 ml of 100 mM of chilled sodium bicarbonate was added to the same solution. After 15 minutes, 5 mg of sodium borohydride was added, and stirring continued for 2 hours. Further, the above solution was transferred into a 10 Kda centricon, and then it was centrifuged for 15 min at 4300 rpm at 4oC to concentrate the immobilized enzyme. Then, immobilized enzyme was separated using gel filtration chromatography using sephacryl-200. For this, the reaction was loaded on a sephracryl-200 column and eluted with 100 mM phosphate buffer (pH 7.5). One ml fractions were collected, and their absorbance was measured at 280 to determine the elution of the protein. All the collected samples were also tested using Bradford Assay. The eluted immobilized enzyme was further concentrated and stored at 4oC.
FTIR, NMR and SEM analysis
Activated dextrin aldehyde and immobilized transaminase enzyme were characterized by FTIR and NMR spectroscopy. The FTIR spectra of the synthesized samples were measured using Attenuated Total Reflection (ATR) on Agilent Cary 630 FTIR spectrophotometer. The 1H NMR spectra were measured on a Bruker AscendTM 500 MHz FT-NMR spectrometer in CDCl3 solvent, and TMS was used as the internal reference. Surface analysis of dextrin, activated dextrin aldehyde and immobilized transaminase enzyme on dextrin aldehyde was measured using a Scanning electron microscope (FESEM) of a JEOL JIB4700F (FIB-SEM) and utilized secondary electron beam and lower electron detectors. Due to the sensitive nature of the specimens, SEM images were captured at a relatively low operating voltage of 5V and a current of 10 mA. Before imaging, the specimens were carefully drop-cast onto a silica substrate and then dried at 40oC. At the tabletop, a gold sputtering instrument was used to coat the samples with a thin conductive film of gold, approximately a few nanometers thick, for 45 seconds to prevent charging effects and to obtain high-quality SEM images of the biological samples.
Activity and Recyclability of Immobilized CDX-036
Immobilized enzyme was taken and used to convert pro-sitagliptin to sitagliptin. For this, 200 mg pro-sitagliptin substrate was taken along with 6 mg immobilized enzyme, 1 mg pyridoxal 5' phosphate (PLP), and 5 mM isopropyl amine in 2 mL of 25:75 (DMSO: water). Simultaneously, the same experiment was performed with a standard free soluble enzyme (without immobilization), and both reactions were monitored under the same conditions. The reaction was monitored using Agilent 1260 infinity HPLC analysis, 0.7 mL/min, mobile phase 40:60:1:1 (heptane: ethanol: diethylamine: D.H2O) at 270 nm.
The results of the experiments are mentioned below:
Result:
Activation of dextrin to dextrin aldehyde
This investigation uses dextrin as a solid support for enzyme immobilization. Dextrin is a very economical, abundantly available, and stable polymer, so it is an important solid support for enzyme immobilization at an industrial scale. Activation of dextrin is done by converting dextrin to dextrin aldehyde using sodium periodate, as can be seen in Scheme 1. After complete oxidation, the obtained dextrin aldehyde is dried using lyophilization and is stored at 4oC. The final yield of the obtained dextrin aldehyde was 94%. Further, the percentage conversion of dextrin to dextrin aldehyde is determined using the DNPH assay. DNPH assay is the most commonly used assay for the measurement of aldehydes and ketones. The nucleophilic addition of NH2 of Dinitrophenyl Hydrazine (DNPH) on the aldehyde and keto group leads to the formation of a yellow-coloured Hydrazone precipitate (Scheme S1). The amount of DNPH consumed can be directly related to the concentration of aldehyde and ketone present in the unknown samples. Dextrin aldehyde solution of 3mg/ml (100 μl) was prepared and added to the DNPH 0.4 mg/ml (900 μL) solution. Then, the reaction mixture was stirred at 200 rpm and 25oC for one hour. Different concentrations of Formaldehyde were used as a control for this assay (Figure S2). After that reaction, the mixture was centrifuged at 10000 rpm and 4oC for 10 min. The absorbance of unreacted DNPH solution was measured at 375 nm (Figure 1). The amount of unreacted DNPH was calculated using the DNPH assay standard curve (Figure S1), and aldehyde content was calculated using the following formula (REF). The final aldehyde content of the obtained dextrin aldehyde was found to be 366.40 mmol/g.


Scheme 1. Schematic representation of activation of dextrin from dextrin-to-dextrin aldehyde using NaIO4.
Figure 1 illustrates UV-vis spectra of DNPH solution with the reaction of dextrin aldehyde (a) standard DNPH (b) 100 μL Dextrin aldehyde + 900 μL DNPH
Transaminase enzyme immobilization on dextrin aldehyde
For obtaining the maximum enzyme loading, enzyme and solid supports were mixed at 1:5, 1:10, 1:15 and 1:20. For this, 10 mg of enzyme was loaded on 50, 100, 150 and 200 mg of the dextrin aldehyde. After loading, the immobilized enzyme was separated from the free enzyme and free dextrin aldehyde by gel filtration chromatography using Sephacryl-200 resin, as mentioned in detail in the methods. The fractions were collected and separated by estimating their protein content by measuring their absorbance at 280 nm and by doing a Bradford assay (Figure 2a). As can be seen in Figure 2a, as intended, three different peaks were observed corresponding to enzyme immobilized on dextrin (peak a), free dextrin (peak b, not visible with Bradford) and free enzyme (peak c), respectively. The fractions of peak a were collected, concentrated, and stored in potassium phosphate buffer (pH-7.5) at 4oC. On analysis, it was found that enzyme loading of 82% was maximum at 1:5 (enzyme to dextrin aldehyde ratio), followed by 78 % at 1:10, 68 % at 1:15 and 63 % at 1:20 ratio (Figure 2b). Thus, a 1:5 ratio was maintained for further enzyme loading at a large scale.
Figure 2 illustrates (a) shows Gel Filtration chromatogram for purification of immobilized CDX-036 from free dextrin aldehyde and free enzyme present in the mixture (b) Bar graph depicting the best-optimized ratio of the enzyme to dextrin aldehyde for maximum enzyme loading.
Fourier-transform infrared (FTIR) Analysis:
FTIR spectra of dextrin, dextrin aldehyde and transaminase enzyme immobilized on dextrin aldehyde were measured on attenuated total reflectance (ATR)-FTIR of Agilent carry 630 at scanning range between 650 cm−1 and 4000 cm−1 (Figure 3). A broadband appeared at 3277 cm-1 due to O-H, stretching vibration of alcohol bond in dextrin (48). Other peaks at 2931 cm−1 and 2889 cm−1 belonged to the CH bond stretching vibrations of dextrin.
The absorption band was reported at 1643 cm-1 due to the bending vibration of a water molecule (H-OH) (49,50).
Other peaks at 1337 cm-1 correspond to the bending vibration of the CH3 and CH2 group and peak at 1145 cm−1 due to the stretching vibration of the C-O bond (51,52). The peak observed at 993 cm−1 is attributed to ring bond vibration, and the peak at 847 and 768 cm−1 is due to the bending vibration of the outside plane of the CH group.
The band at 3277 cm-1 is present in dextrin; however, it is not seen in the case of the lower band in the form of noise) in dextrin aldehyde, it may be due to the conversion of the OH group of dextrin into the CHO group of dextrin aldehyde. One more peak was also observed in dextrin aldehyde at 1735 cm-1 of C=O stretching of the aldehyde group present in dextrin aldehyde, and aldehyde content was also checked by DNPH assay, which is explained in section 2.2.2. C-H stretching of the aldehyde group is also present in the dextrin aldehyde sample at 2889 cm-1. A secondary amine peak appears at the near absorption of 3420 cm-1, but with hydrogen bond with concentrated liquids, the absorbance of this band was shifted towards lower frequencies of about 100 cm-1.
According to the above explanation, we obtained a peak at 3274 cm-1, which is almost lower than 100 cm-1 of 3420 cm-1, which indicates the bond formation between the transaminase enzyme NH2 group and the dextrin aldehyde C=O group. The absorption band at 1639 cm-1 is due to imino stretching vibrations of C=N, which confirms the formation of Schiff bases and the medium absorption band at 1018 cm-1 for skeletal vibration (53). The formation of the C=N bond between dextrin aldehyde and transaminase enzyme clearly indicates the transaminase enzyme immobilization on the solid support of dextrin aldehyde.
Figure 3 illustrates Combined FTIR spectra of Dextrin, Dextrin Aldehyde (Dex. Ald.), and Immobilized Transaminase on Dextrin Aldehyde (Dex. Ald. Enz.)
Scanning Electron Microscopy (SEM) Analysis
Surface morphology of dextrin, activated dextrin (dextrin aldehyde) and immobilized transaminase on dextrin aldehyde were shown in Figures 4 (a), (b) and (c) at different magnifications. The shape and size of dextrin, dextrin aldehyde and immobilized transaminase on dextrin aldehyde granules were significantly different. The dextrin shape and size are granular spherical shape and smooth at 2000x magnifications. Dextrin aldehyde's surface (embedded smooth surface) was different compared to dextrin, which may be due to activation of the dextrin surface (dextrin to dextrin aldehyde) at 2000x magnifications. The surface morphology of the immobilized transaminase enzyme surface was smooth and cracked, and it was different compared to dextrin and activated dextrin aldehyde. These results indicate enzyme immobilization on the surface of dextrin aldehyde. In Figure 4 (c), magnification at 2000x particles was observed on the embedded smooth surface. It may be due to transaminase enzyme immobilization on the surface of dextrin aldehyde.
Figure 4 illustrates (a) Scanning Electron Microscopy analysis at 2000x of (a) Dextrin polymer (b) dextrin aldehyde (c) immobilized transaminase enzyme on dextrin-aldehyde polymer
NMR analysis:
The 1H NMR spectrum of dextrin (Fig. 5, 500 MHz, DMSO-d6, ppm), the peaks at δ = 3.33-3.64, 4.56, 4.99, are for to H2-H6, anomeric protons (H1). The peaks at δ = 4.56, 4.99-5.09 and 5.09, 3.64 are assigned to -OH (2) and -OH (3, 4 or 6) protons, respectively (54,55). Figure 5 illustrates 1H NMR spectra of Dextrin in DMSO d6. In the 1H NMR spectrum of dextrin aldehyde (Fig. 6, 500 MHz, D2O, ppm), the peaks at δ = 8.35 are for the aldehyde group, which indicates the activation of the dextrin surface. Other peaks are the same as in dextrin, but all are merged, so the peaks integration is not possible for each separate peak. Figure 6 illustrates 1H NMR spectra of Dextrin Aldehyde in D2O.
Activity and Recyclability of Immobilized CDX-036
Immobilized transaminase enzyme activity was performed towards the well-known reaction synthesis of pro-sitagliptin to sitagliptin in Scheme 2. To know the enzyme activity, an experiment was conducted using 200 mg pro-sitagliptin substrate, 6 mg immobilized enzyme, 1 mg pyridoxal 5' phosphate (PLP) and 5 mM isopropyl amine in 2 mL of 25:75 (DMSO: water). Simultaneously, the same experiment was performed with a standard free-soluble enzyme, and both reactions were monitored under the same conditions. The reaction was monitored using Agilent 1260 infinity HPLC analysis, 0.7 mL/min, mobile phase 40:60:1:1 (heptane: ethanol: diethylamine: D.H2O) at 270 nm. The reaction was monitored for 4 hours and recycled five times Table 1 (Supplementary File).

Scheme 2 Schematic representation of pro-sitagliptin to sitagliptin using transaminase enzyme with PLP as a cofactor.
Immobilized enzyme and normal enzyme activity were performed with pro-sitagliptin substrate in five cycles, as shown in Figure 7. In the 1st cycle, the relative % conversion of sitagliptin with immobilized enzyme was observed at 92.02, and with free soluble enzyme, the conversion was 80.66. In the 2nd cycle, the conversion of sitagliptin was 62.00 and 60.00, for 3rd cycle it was 76.50 and 72.00, in the fourth cycle, % the conversion was 90.00 and 76.00 and in the fifth cycle, it decreased a little bit to 60.00 and 78.00 with immobilized and free soluble enzymes, respectively. Thus, the immobilized enzyme is highly stable, have equivalent or better conversion rates than the free soluble enzyme and can be easily recovered and used for multiple cycles.
Figure 7 illustrates Comparative graph of relative % conversion of Sitagliptin using immobilized and normal enzymes.
Enzyme immobilization offers excellent support for increasing the accessibility of enzymes to the substrates with a larger turnover for a long time. For immobilization, 1: 5 ratio of TA enzyme: Dextrin aldehyde showed good immobilization out of 1: 10, 1: 15, and 1:20 ratios. Immobilized enzyme was purified via Sephacryl-200 gel filtration column and the immobilized enzyme was collected as a clear peak and concentrated to get the final concentrations of around 8-9 mg/ml. Further the enzymatic activity of purified immobilized enzyme (IE) activity was measured using highly desirable industrial substrate which is pro-sitagliptin (200 mg) along with 6 mg of immobilized enzyme, 1 mg pyridoxal 5' phosphate (PLP) and 5 mM isopropyl amine in 2 ml of 25:75 (DMSO: water). Free soluble CDX-036 was used as control under same conditions. Immobilized enzyme and free soluble enzyme activity were measured upto 5 cycles with a conclusion that the immobilized enzyme has better or equal enzymatic activity (92% conversion by immobilized enzyme in 1st cycle as compared to 80 % by free soluble enzyme) when compared to free soluble CDX-036, it is easily recoverable and as tested by us, is easily reusable upto 5 cycles without significant loss in the activity.
, Claims:We Claim:
1. A process of immobilising CDX-036 Transaminase enzyme on a substrate comprising dextrin, said steps comprises the steps of:
a) Converting Dextrin to dextrin aldehyde by oxidation;
b) Immobilizing CDX-036 Transaminase enzyme by the steps of:
i. Loading 10 mg of CDX-036 (TA) enzyme on dextrin aldehyde at 1:5, 1:10, 1:15 and 1:20 ratios using 50 mg, 100 mg, 150 mg, and 200 mg of dextrin, by dissolving CDX-036 (TA) enzyme and dextrin aldehyde in 5 ml of distilled water and stirring at 20oC for 16 hours;
ii. Adding 5 ml of 100 mM of chilled sodium bicarbonate and after 15 minutes, 5 mg of sodium borohydride was added, and stirring for 2 hours;
iii. Transferring the solution od step (ii) into a 10 Kda centricon, and then it was centrifuged for 15 min at 4300 rpm at 4oC to concentrate the immobilized enzyme followed by separating the immobilized enzyme by gel filtration chromatography.
2. The process as claimed in claim 1, wherein Dextrin to dextrin aldehyde is converted by the steps of:
a) dissolving 3 grams of dextrin in 22.50 ml of distilled water in a 100 ml round bottom flask (RBF) followed by adding 6 g of oxidising agent comprising sodium periodate (NaIO4) solution, said solution is prepared by dissolving NaIO4 separately in 52.50 mL of distilled water; said NaIO4 was added slowly with constant stirring, and the reaction was continued for 24 hours;
b) dialyzing the oxidized dextrin solution for 24 hours by an activated dialyzed membrane, wherein the oxidized dextrin solution was poured into a dialyzed membrane to remove unreacted particles of NaIO4 from the reaction mixture;
c) lyophilizing the oxidized dextrin to remove excess water from the reaction mixture.
3. The process as claimed in claim 1, wherein the gel filtration chromatography comprises loading the reaction on a sephracryl-200 column, eluting with 100 mM phosphate buffer (pH 7.5) followed by concentrating the eluted immobilized enzyme.
4. An economic process for the preparation of sitagliptin using dextrin-immobilized CDX-036, comprising treating 200 mg pro-sitagliptin substrate with 6 mg immobilized enzyme obtained from claim 1, 1 mg pyridoxal 5' phosphate (PLP) and 5 mM isopropyl amine in 2 mL of 25:75 (DMSO: water).

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