Description: FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents [Amendment] Rules, 2006

COMPLETE SPECIFICATION
(See Section 10 and Rule 13)
TITLE OF THE INVENTION: An enzyme and formulation thereof for reducing formation of acrylamide in food processing
APPLICANT:
NAME
NATIONALITY
ADDRESS : Krea Foods and Beverages Pvt Ltd
: Indian
: DL/40, VSS Nagar, Near Sainik School, Shahid Nagar, Bhubaneswar, India - 751007
NAME
NATIONALITY
ADDRESS : Indian Oil Corporation Limited
: Indian
: Indian Oil Bhavan, G-9, Ali Yavar Jung Marg, Bandra East, Mumbai, India - 400051
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 food science, particularly to food processing. More particularly, the invention relates to a reduction of acrylamide in thermally processed food.
BACKGROUND OF THE INVENTION
[002] In the past few decades, the risk of development of various lifestyle diseases and disorders have increased dramatically due to increased consumption of processed food. This is a consequence of various food toxins regularly consumed as a part of our daily diet. One such food toxin is the acrylamide. Acrylamide (acrylic amide), a chemical compound with the chemical formula C3H5NO is considered as carcinogenic for animals and humans. Acrylamide is often formed in high amounts through Maillard reaction which occurs during cooking, baking, frying & spray drying of starchy and carbohydrate rich foods. In the Maillard reaction, naturally present amino acid asparagine undergoes a conversion to form acrylamide, which is responsible for giving baked or fried foods their brown color, crust, and toasted flavor. The occurrence of acrylamide in a number of food and oven prepared foods was published (Tareke et al. Chem. Res. Toxicol. 13, 517-522. (2000)) and this resulted in world-wide concern. It has also been proved that considerable amounts of acrylamide are detectable in a variety of baked, fried and oven prepared common foods and it has been demonstrated that the occurrence of acrylamide in food was the result of baking process. Dietary acrylamide is present in thousands of food and beverage products consumed all over the world like potato chips, French fries, bakery and confectionery products, breakfast cereals, infant baby food, grain-based coffee and owing to 52% of our daily intake. Both cereal and potato-based food products that have been prepared by heating can have relatively high levels (30 - 7500 µg/kg) of acrylamide and account for a significant proportion of dietary exposure.
[003] Acrylamide is classified as a class 2A cancer causing chemical by WHO. It is also known to be mutagenic, neurotoxic and genotoxic. It presents a huge health risk to babies and children. Acrylamide in foods consumed by pregnant women may cause a reduction in their baby’s birth weight and head circumference. The point of higher concern is that younger children typically ingest each day twice as much. As acrylamide is formed from the reaction of L-asparagine (L-Asn) and reducing sugars contained in foods during heating processes, the presence of free asparagine is a limiting factor for acrylamide formation and the acrylamide formation in heated food products can be reduced by treating food materials with asparaginase enzyme before the heat treatment. Thus, acrylamide formation in heated food products can be reduced by treating food materials with asparaginase enzyme, which converts asparagine to aspartic acid, before thermal processing of food materials. As a food processing aid, asparaginases can reduce the formation of acrylamide in a range of starchy foods without changing taste and appearance of the end food product.
[004] Asparaginase can be obtained from various sources such as plants, animals and microorganisms. Asparaginase is the enzyme that hydrolyzes asparagine to aspartic acid, presents a potentially very effective means for reducing acrylamide formation in foods via removal of the precursor, asparagine, from the primary ingredients. Asparaginases derived from Aspergillus niger and Aspergillus oryzae has been used in food processing globally. However, these enzymes are not suitable for certain applications wherein the enzyme is required to exhibit asparaginase activity at higher temperature over a range pH conditions. Moreover, the production of these enzymes at larger scale is a complex, expensive, and that they exhibit weak kinetic properties. In search for a better solution, Escherichia coli asparaginase enzyme has been considered by virtue of its easy large-scale production. However, EcAsa-WT show low thermal stability, narrow pH range, higher glutaminase activity and low kinetic and enzyme specificity properties.
[005] Naturally occurring EcAsa-WT besides having affinity asparagine have also higher affinity for glutamine and thereby exhibiting an enhanced side glutaminase activity. This side activity leads to neurotoxicity and thrombo-embolism and thereby reduces the efficiency of acrylamide reduction in a food matrix, since both the amino acid substrates (asparagine and glutamine) generally co-exist. Owing to side activity by asparaginase due to lower specificity for asparagine, the efficiency of acrylamide reduction is compromised.
[006] A variety of asparaginases have been identified and reported in the literature for decreasing or eliminating the formation of acrylamide during the thermal processing of food materials. WO2004/030468 discloses an asparaginase derived from Aspergillus niger and WO2004/032648 discloses peptide sequences of asparaginases derived from Aspergillus oryzae and Penicillium citrinum.
[007] The literature provides a couple of methods for reducing the formation of acrylamide vis-à-vis asparaginases (EP 2949748A1, US 7527815B2, US 8105815B2, US 7867529B2, US 2005/0064084A1, US 2010/0183765A1, WO 2014/161935 Al, WO2008/128974, WO2008/128975 and WO2011/134916. Although the known asparaginase enzymes can catalyze the conversion of asparagine to aspartic acid in thermal processing of food materials, these enzymes are not able to exhibit asparaginase activity at low substrate concentrations, as required in variety of food matrix conditions for certain applications. Various applications will place different demands on the conditions under which the enzymes have to operate. Physical and chemical parameters that may influence the rate of an enzymatic conversion of asparagine to aspartic acid are temperature (which has a negative effect on enzyme stability), moisture content, pH, salt concentration, structural integrity of the food matrix, presence of activators or inhibitors of the enzyme, concentration of the substrate and products, etc.
OBJECTIVES OF THE INVENTION
[008] The object of the present invention is to provide a novel asparaginase molecule EcAsa-DM from E. coli using rationale protein engineering which has improved functional properties that are useful in reduction of acrylamide content in thermally processed food products.
[009] Another object of the present invention is to provide a novel formulation of EcAsa-DM asparaginase along with other important acrylamide reducing enhancers and formulation stabilizers for reducing acrylamide content in thermally processed food.
SUMMARY OF THE INVENTION
[0010] The present invention provides a novel asparaginase molecule EcAsa-DM from E. coli.
[0011] The present invention relates to the field of food. It relates to a modified E. coli asparaginase gene sequence, which encodes for enzyme having improved characteristics related specificity, activity and stability. The modified E. coli asparaginase enzyme is particularly useful in reducing the production of acrylamide during different cooking processes.
[0012] The present invention provides a modified E. coli asparaginase encoding nucleotide sequence of SEQ. ID 1.
[0013] The present invention in one of the aspects provides a modified E. coli asparaginase encoding nucleotide sequence of SEQ. ID 1, wherein said sequence comprises of 526-528 TAC?TTC and 862-864 AAA?CAT substitution.
[0014] In another aspect, the present invention provides a modified E. coli asparaginase enzyme of amino acid sequence of SEQ. ID 2.
[0015] The present invention in one of the aspects provides a modified E. coli asparaginase enzyme of amino acid sequence of SEQ. ID 2, wherein said enzyme comprises of Y176F and K288H amino acid substitutions.
[0016] In one of the aspects, the present invention provides a modified E. coli asparaginase enzyme, wherein the said enzyme having a 2.3-fold higher unit activity/L as compared to E. coli L-asparaginase II or wild type E. coli asparaginase.
[0017] In one of the aspects, the present invention provides a modified E. coli asparaginase enzyme, wherein said enzyme have 6-fold lower Km.
[0018] In one of the aspects, the present invention provides a modified E. coli asparaginase enzyme, wherein said enzyme have ~6-fold higher affinity for asparagine as compared to unmodified wild type E. coli asparaginase enzyme.
[0019] In one of the aspects, the present invention provides a modified E. coli asparaginase enzyme, wherein said enzyme have an enhanced asparaginase activity at an asparagine concentration as low as 0.07 mM.
[0020] In one of the aspects, the present invention provides a modified E. coli asparaginase enzyme, wherein said enzyme lacks glutaminase activity.
[0021] In another aspect, the present invention provides a formulation for reducing the formation acrylamide during processing of the food products, wherein said formulation comprises of a) a modified E. coli asparaginase enzyme of sequence of SEQ. ID 2; and b) a food grade enhancers, stabilizers, and preservatives.
[0022] In one of the aspects, the present invention provides a formulation comprising of a) a modified E. coli asparaginase enzyme of sequence of SEQ. ID 2; and b) a food grade enhancers, stabilizers, and preservatives, wherein the modified E. coli asparaginase enzyme of SEQ. ID 2 is in 6% - 12%.
[0023] In one of the aspects, the present invention provides a formulation comprising of a) a modified E. coli asparaginase enzyme of sequence of SEQ. ID 2; and b) a food grade enhancers, stabilizers, and preservatives, wherein the food grade enhancers, stabilizers, and preservatives consist of 100 - 300mM Glycine, 100 - 500mM NaCl, 50 - 400mM Sucrose, 25 - 200mM Tris buffer pH:8.0.
[0024] In one of the aspects, the present invention provides a formulation comprising of a) a modified E. coli asparaginase enzyme of sequence of SEQ. ID 2; and b) a food grade enhancers, stabilizers, and preservatives, wherein the formulation is an aqueous formulation consisting of 200mM Glycine, 200mM NaCl, 200mM Sucrose, 30mM Tris buffer and 80U/ml of modified E. coli asparaginase enzyme of sequence of SEQ. ID 2.
DESCRIPTION OF THE ACCOMPANYING DRAWINGS
[0025] Figure 1A: First mutation at Position 526 to 528 TAC(Y) to TTC(F); Figure 1B: Second mutation at Position 862 to 864 AAA(K) to CAT(H).
[0026] Figure 2: Expression and purification of EcAsa-WT(A) and EcAsa-DM (B). A) Purification profile of EcAsa-WT. Lane 1-Biorad Precision Plus Dual Marker, Lane 2- 50mM imidazole wash fraction, Lane 3 to Lane 10 - 100mM & 200mM imidazole wash fractions; B) Purification of EcAsa-DM. Lane 1- Biorad Precision Plus Dual Marker, Lane 2- 50mM imidazole wash fraction, Lane 3 to Lane 10 - 100mM & 200mM imidazole wash fractions; C) Immunoblots of EcAsa by Anti-His. Lane 1-Biorad Precision Plus Dual Marker, Lane 2 EcAsa-WT, Lane 3 EcAsa-DM; D) Secondary structure of 6X His- Asparaginase CD spectra showing the graph of wavelength against mean residual ellipticity of EcAsa-WT (blue) and EcAsa-DM (red).
[0027] Figure 3: Determination of enzymatic activity of asparaginases on asparagine and glutamine: A) Catalytic activity of both asparaginase were tested over a range of asparagine concentration as mentioned in the figure. Activity of EcAsa-WT is shown in yellow and EcAsa-DM variant is in red. B) Comparison of glutaminase activity of EcAsa-WT and EcAsa-DM variant.
[0028] Figure 4: Effect of pH and temperature on catalytic activity of EcAsa-DM variant: A) Enzymatic activity of EcAsa-DM variant was tested over a range pH 4 - 10. Acetate buffer was used in acidic range (pH 4-6), Tris buffer used for pH range 6.5 - 8.5, Hepes buffer was used for pH range 7 - 9 and Glycine-NaOH buffer was used for range of 8 - 10. Stock enzyme was diluted in the appropriate buffer before the assay was performed; B) EcAsa-DM variant activity was checked over a temperature range of 0 - 80?.
DETAILED DESCRIPTION OF THE INVENTION
[0029] While the invention is susceptible to variations and modifications other than those specifically described herein by specific embodiments and examples. It is to be understood that the present disclosure includes all modifications, equivalents, and alternative falling within the spirit and the scope of the invention as defined by the appended claims. The figures and protocols have been represented for only showing the specific details that are pertinent to understanding of the embodiments of the present invention and not to obscure the disclosure with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the disclosure.
[0030] Source of Asparaginase: Asparaginase (isozyme II) was obtained from Escherichia coli is an important therapeutic enzyme used in the treatment of leukemia. E. coli belongs to a group of bacteria informally known as coliforms that are found in the gastrointestinal tract of warm-blooded animals. Only the gene of interest has been taken out from E. coli and then mutated to increase the stability and other functional properties of the expressed enzyme. Then the gene of interest has been cloned and expressed in E. coli strains like DH5-a and BL21 codon plus (manufactured for experimental purpose). After the large amount expression of the protein during up-scaling process, the enzyme is purified and used in different food matrices. During the food processing steps, very high temperature is used which denatures the enzyme used for the reduction of free asparagine present in the food.
[0031] The inventors of present invention have developed a novel asparaginase enzyme, with improved characteristics to help reducing formation of acrylamide in thermally processed food. To arrive at the aimed objective, inventors have created several mutants with point mutations to wild type E. coli asparaginase (EcAsa-WT), to enhance different properties by using site directed mutagenesis based on rationale of protein engineering. Upon analyzing in silico and experimental results, inventors of the present inventions have identified 2-point mutations, which particularly improve stability and substrate affinity of the EcAsa. Through site directed mutagenesis of EcAsa-WT, a double mutant of EcAsa-WT ‘EcAsa-DM’ is developed. EcAsa-DM exhibits improved enzymatic and functional properties compared to its parent asparaginase regarding applications with the food industry (Tabel-1). The modified enzyme EcAsa-DM with amino acid sequence as depicted by of SEQ. ID. 2, possesses significantly improved enzymatic properties as compared to asparaginases obtained from A. niger and A. oryzae (Table 1-2). Mutated gene of EcAsa-WT represented as of EcAsa-DM with 6 nucleic acid substitutions over the EcAsa-WT is depicted by SEQ. ID. 1.
[0032]
Table 1: Comparative analysis of enzymatic activity of EcAsa-DM & Asparaginase from A. oryzae
Type Matrix used Enzyme used Acrylamide reduction Reference
Asparaginase from Aspergillus oryzae Potato matrix for French fries 10500 units/lit ~60%-80% of reduction DOI:10.1021/jf900174q
Evaluating the Potential for Enzymatic Acrylamide Mitigation in a Range of Food Products Using an Asparaginase from Aspergillus oryzae
EcAsa-DM from E. coli Potato matrix for French fries 700 units/lit ~75%- 95% of reduction Current data

[0033] Experimental data in Table 1 demonstrates that EcAsa-DM shows approximately 15-fold higher asparagine removal efficiency as compared to asparaginase from A. oryzae, which means significantly lower amount of enzyme will be required to treat potato matrix for French fries.
[0034]
Table 2: Comparative analysis of enzymatic activity of EcAsa-DM & Asparaginase from A. niger

Type Matrix used Average Enzyme used Acrylamide reduced Workable pH range Reference
Asparaginase from
Aspergillus niger Potato based matrix 2000 units/kg of sample 80% of reduction 4 - 8 GRAS report GRN000214 (Prevent ASem of DSM)
http://dx.doi.org/10.1016/j.foodchem.2016.04.105
The use of asparaginase to reduce acrylamide levels in cooked food

EcAsa-DM from E. coli Potato based matrix 800 units/kg of sample ~75% - 95% of reduction 4.0 - 10 Current data

[0035] Experimental data given in Table 2 demonstrates that EcAsa-DM shows higher amount of acrylamide reduction using comparatively lower dose in a potato-based matrix than the asparagine from A. niger. The activity pH range of EcAsa-DM enzyme is much wider than the asparagine from A. niger, supporting the improvement in its enzymatic property. EcAsa-DM shows 2.5-3-fold higher asparagine removal efficiency compared to asparaginase from A. niger. Moreover A. niger data shows ~80% reduction, whereas EcAsa-DM shows >90% reduction on acrylamide.
[0036] EcAsa-DM was cloned in E. coli DH5-a and expressed the asparaginase enzyme through E. coli BL21 codon plus expression vector. The clones were sequenced to confirm the mutation. His-tagged EcAsa-WT and EcAsa-DM expression in E. coli BL21(DE3) cells were confirmed by immunoblotting with anti-6His antibody.
[0037] Further, a composition comprising EcAsa-DM (Acryl-aid) was formulated to mitigate reduction of acrylamide in thermally processed food products. Said composition comprises of various food grade ingredients along with a uniquely designed EcAsa-DM enzyme molecule. The EcAsa-DM enzyme molecule has improved efficiency and advanced enzymatic properties to remediate acrylamide formation in various food matrices without making any changes in the regular production process. In one of the preferred aspects the composition comprising EcAsa-DM (Acryl-aid) is a liquid formulation of EcAsa-DM enzyme along with acrylamide reducing enhancers, food grade stabilizers and food grade preservatives, which is a one stop enzymatic processing aid for reducing acrylamide formation during thermal processing of food. Acrylamide reducing enhancers, food grade stabilizers and food grade preservatives maybe as per given in table 3. All the components used in the formulation are common ingredients compatible with food applications.
[0038]
Table 3: Composition and function of constituents of Acryl-Aid liquid formulation
Composition Function Workable Range
Glycine acrylamide reducing enhancers and thermal stabilizer of enzyme 100mM-300mM
Sucrose food grade preservative and enzyme stabilizer 50mM-400mm
NaCl Enzyme stabilizer 100mM -500mM
Tris-buffer Maintains specific pH for optimal activity of enzyme 25mM-200mM
Enzyme Asparaginase activity 6% - 12%

[0039] In food products, asparagine is the limiting substrate in food matrix and is the major precursor for formation of acrylamide in thermally processed starchy food products. Therefore, an asparaginase enzyme with a higher specificity and affinity towards asparagine at low substrate concentration would be highly desirable and the novel variant of E. coli asparaginase enzyme “EcAsa-DM” described herein have demonstrated this enhanced affinity towards asparagine, which contributes to higher reduction of acrylamide in thermally processed starchy foods.
[0040] Another drawback of naturally occurring EcAsa-WT regarding with higher affinity for glutamine and thereby exhibiting an enhanced side glutaminase activity is also addressed by the novel variant of E. coli asparaginase enzyme ‘EcAsa-DM’. This side activity of naturally occurring EcAsa-WT enzyme leads to neurotoxicity and thrombo-embolism and thereby reduces the efficiency of acrylamide reduction in a food matrix, since both the amino acid substrates (asparagine and glutamine) generally co-exist. Thereby, the asparaginase activity becomes less specific in existing state and decreases efficiency of acrylamide reduction. However, EcAsa-DM have negligible glutaminase activity, which translates into a higher specificity and enhanced asparaginase activity of EcAsa-DM.
[0041] At higher temperatures and at a wide range of pH, the naturally occurring EcAsa-WT enzyme is not suitable to exhibit efficient asparaginase activity. Thereby, this property hinders its usage in a variety of food applications and food matrices. The novel EcAsa-DM in contrast showcases both enhanced thermal stability and has shown activity at wide range of pH (pH 4 - pH 10). This enhanced functional property would enhance the use ability of EcAsa-DM across a variety of food processing applications.
Preparation of clone and expression:
[0042] Preparation of Insert DNA (EcAsa DM gene): Asparaginase gene was extracted out from E. coli K-12 strain and subjected to site directed mutagenesis was done as per standard methods known in the art, on two residue sites namely Y176 and K288 of the extracted asparaginase gene to synthesize novel variant asparaginase gene (EcAsa-DM) to enhance the functional properties of the asparaginase enzyme.
[0043] Preparation of Cloning vector: pET28a cloning vector is prepared as per standard protocol of the vector known in the art.
[0044] Insertion of cloned sequence into a vector: Cloned DNA was inserted in to pET system.
[0045] Transformation: Then the expression host (BL21 CODON plus) is transformed with the pET vector with the inserted gene.
[0046] Optimization: Induction and optimization was done for the expression of the target protein in lab scale.
[0047] Scale up and purification: Scaling up was done for expressing the protein of interest at higher amount and process for purification of the novel EcAsa-DM was optimized. Purified proteins were then used in different food matrices for acrylamide reduction study.
EXAMPLES
EXAMPLE 1
[0048] Cloning of double mutant of E. coli asparaginase gene (EcAsaY176F-K288H/ EcAsa-DM): E. coli asparaginase (EcAsa) is an enzyme used to treat leukaemia for a long time, however it has not been tested for food application. Several point mutations were created on naturally occurring asparaginase to enhance its different properties. Based on the analysis of in-silico studies and experiments, substitution of 2 residues Y176F and K288H have been identified to improve stability of the protein and substrate affinity. Wild type asparaginase (EcAsa-WT) was cloned in the E. coli using pET28a expression vector. Through site directed mutagenesis of EcAsa-WT, a double mutant named “EcAsa-DM” was created where Y176 and K288 residues were mutated to F and H respectively (Figure 1A and 1B). All the clones were sequenced to confirm the mutation. His-tagged EcAsa-WT enzyme and EcAsa-DM enzyme expression in E. coli BL21(DE3) cells were confirmed by immunoblotting with anti-6His antibody (Figure 2C).
EXAMPLE 2
[0049] Expression and purification of EcAsa-DM: Initially EcAsa-WT enzyme and EcAsa-DM enzyme were expressed as intracellular soluble protein in E. coli BL21(DE3) as shake flask culture to optimize the protein yield. A range of expression conditions is tested by varying IPTG concentration, temperature and time. Both proteins were expressed at optimal level at 30°C upon 4 hours induction with 0.3mM IPTG. The same conditions were identified to be optimal for expression in 5L and 10L batch cultures using bench top bioreactor. The E. coli biomass produced can be stored at -80°C till used for purification. 6xHis-EcAsa-WT enzyme and 6xHis-EcAsa-DM enzyme was purified by Ni-NTA agarose affinity chromatography. Both proteins were purified to 85-90% homogeneity for subsequent studies and applications. CD spectra of purified proteins showed that both the proteins are well folded with very little unstructured regions. EcAsa-DM is more structured than its wild type counterpart and hence it may be more stable compared to EcAsa-WT. Earlier Khushoo et.al. (Protein Expr Purif. 2004 Nov;38(1):29-36) expressed E. coli asparaginase as extracellular protein and purified it from culture supernatant. Our data showed EcAsa-DM has 1.5 times yield and 2.3-fold higher unit activity per liter (Table 4). This clearly showed that expression conditions are better suited to EcAsa-DM production.
[0050]
Table 4: Comparative analysis of E. coli Asparaginase expression
Source Reference Yield of Purified protein (mg/L) Unit Activity/ L of purified protein
E. coli L-asparaginase II wild type (Extracellular) Khushoo et. al, 95mg/L 20950 UI/L
Novel EcAsa-DM (intracellular) Current data 149mg/L 48448 UI/L

EXAMPLE 3
[0051] Enzyme activity of Asparaginase: EcAsa activity assay was routinely performed as per standard methods using asparagine as substrate. The reaction was stopped by addition of trichloroacetic acid and amount of ammonia (NH3) was measured by reaction with 8- hydroxyquinoline at alkaline pH (Nessler’s assay). Both enzymes were found to be active over a broad range of asparagine concentration (0.156 mM to 160 mM). However, EcAsa-DM showed better activity compared to its wild type counterpart at lower substrate concentrations (Figure 3A). EcAsa-DM did not show any glutaminase activity, whereas wildtype enzyme had significant glutaminase activity (Figure 3B). This showed that double mutant is more specific to asparaginase activity as compared to wildtype.
[0052] Enzyme kinetics analysis showed EcAsa-DM variant has 6-fold lower Km and thus have ~6-fold higher affinity for asparagine as compared to EcAsa-WT (Table 5). EcAsa-DM variant showed activity at asparagine concentration as low as 0.03 mM, whereas naturally occurring asparaginase enzyme did not show any detectable activity below 0.4 mM. High substrate affinity of EcAsa-DM will ensure effective removal of low amount of asparagine present in the food samples.
[0053]
Table 5: Comparative analysis of enzyme kinetics parameters
EcAsa-WT EcAsa-DM variant
Km (mM) 0.5 ± 0.10 0.085 ± 0.015
Vmax 22.47 ±2.2 16.22±1.8
Min. detectable [asparagine] mM 0.4mM-0.6mM 0.07mM-0.1mM

EXAMPLE 4
[0054] Effect of pH and temperature on activity of noble asparaginase variant EcAsa-DM: Enzyme activity of EcAsa-DM variant was evaluated for pH in the range of 4-10 (Figure 4). Table 6 shows that EcAsa-DM variant showed highest activity in the range of pH 6.5-8 and is active in a pH range of 4.0-10. As the asparaginase variant EcAsa-DM shows highest activity at pH 7.0 (Figure 4A) its activity is highly retained in water. Table 7 shows that highest activity of EcAsa-DM variant is over a temperature range of 40-60°C (Figure 4B). Taken together EcAsa-DM variant showed highest activity between 50? to 60? and neutral pH. This temperature and pH ranges are optimum for the usage in food processing since a processing of food happens at such temperature range and the addition of the enzyme during pre-processing of the food matrix in lukewarm water.
[0055]
Table 6: Activity of EcAsa-DM at a range of pH
pH Unit activity(µmole/min/µg) % of retained activity
4 2.496146435 24.96146435
5 2.893063584 28.93063584
6 3.132947977 31.32947977
7 10 100
7.5 7.255298651 72.55298651
8 6.801541426 68.01541426
8.5 5.674373796 56.74373796
9 6.445086705 64.45086705
10 3.615606936 36.15606936

[0056]
Table 7: Activity study of EcAsa-DM at different reaction temperature
Reaction temperature (?) Unit activity(µmole/min/µg) % of retained activity
0 0.897880539 14.02938343
4 1.529222864 23.89410726
15 2.230571612 34.85268144
25 3.664739884 57.26156069
30 4.204238921 65.69123314
40 4.795761079 74.93376686
50 6.428387925 100.4435613
60 5.950545922 92.97728003
70 1.217726397 19.02697495
80 1.037893385 16.21708414

EXAMPLE 5
[0057] Acryl-Aid Formulation stability studies: Different combinations of stabilizing agents have been explored to prepare formulation comprising EcAsa-DM (Acryl-Aid formulation). All the additives and stabilizing agents are food compatible and routinely used in food preparations. Table 8 shows an accelerated stability studies that were conducted for different EcAsa-DM formulations over a period of 30 days. EcAsa-DM formulation comprising (80U/ml EcAsa-DM with 200mM Glycine, 200mM NaCl, 200mM Sucrose, 30mM Tris buffer pH:8.0 and water) is stable at 4°C over a period of 30 days without any substantial loss of enzyme activity.
[0058]
Table 8: Activity study of Acryl-aid at 4? for 28 days
Day Unit activity(µmole/min/µg) % of retained unit activity
0 3.1 100
7 3.03 97.74193548
14 2.9 93.5483871
21 3.09 91.67741935
28 2.79 90

EXAMPLE 6
[0059] Acrylamide reduction activity of EcAsa-DM variant in carbohydrate-based food matrix: A potato matrix is used to test the acrylamide reduction activity of Acryl-aid formulation containing EcAsa-DM variant using the standard protocol as described in Hendriksen et al (2009, Journal of Agricultural and Food Chemistry, 57(10), 4168–4176) with modifications. Acryl-aid formulation showed 85-90% reduction in acrylamide content (Table 11). Moreover, significantly lower dosage of enzyme was used as compared to L-asparaginase from Bacillus subtilis.
[0060]
Table 9: Comparative analysis of specific activity of EcAsa-DM & Asparaginase from B. subtilis

Organism Specific activity (unit activity/mg of enzyme) Reference
L-asparaginase from Bacillus subtilis 45.4 units/mg Effective treatment for suppression of acrylamide formation in fried potato chips using L-asparaginase from Bacillus subtilis
EcAsa-DM 400 units/mg Current data

EXAMPLE 7
Analytical Method for Acrylamide study:
[0061] Treatment method:
(i) Potatoes were washed and peeled off and Equal size of potato strips (1cm) were cut
(ii) Removal of starch from potato was done by vigorous water washing;
(iii) To increase the efficacy of the enzyme the blanching of the potatoes was done at warm water (85?) for 5 mins as a pre-treatment step;
(iv) Blanched potatoes were then dried and then were treated with novel EcAsa-DM asparaginase variant for 45 mins;
(v) Excess moisture of the potato was soaked using tissue paper;
(vi) Potatoes were deep fried at 180? for 5mins; and
(vii) As a control sample, potatoes were kept at only distilled water then are subjected to frying.
[0062] Extraction of sample for measuring acrylamide:
(i) 5g of homogenized sample was weighed approximately in 50 ml Tarsons tube;
(ii) 10 ml of Mili-Q water was added to sample Vortex for 60 seconds;
(iii) 10 ml of Acidified Acetonitrile was added and vortexed 5mins for defatting;
(iv) 4 g of MgSO4 dried and 1.66 g of Sodium acetate trihydrate were added;
(v) It was vortexed for 3 min followed by centrifugation for 5 min at 4000 rpm;
(vi) 2 ml of upper layer was taken for Cleanup in Dis Q tube;
(vii) Then it was vortexed and centrifuged for 5 min at 10000 rpm;
(viii) 1 ml of sample was then taken for evaporation; and
(ix) After evaporation, it was reconstituted with 1 ml of Mili-Q water to dilute to 5 times in Mili-Q water and was collected in injection vial for running to LC-MS/MS.
[0063] Comparative analysis of acrylamide reduction: Table 10 demonstrates that there is 5-fold more reduction of acrylamide in the final food product in case of EcAsa-DM treated food than that of EcAsa-WT treated food product.
[0064]
Table 10: Comparative acrylamide reduction by EcAsa-DM & EcAsa-WT in potato-based matrix
Sample Dosage of enzyme Amount of acrylamide (µg/kg)
Blank Not treated 2009
EcAsa-WT 0.8U/gm 170
EcAsa-DM 0.8U/gm 31

[0065] Effects of number of enzyme units of EcAsa-DM used on percentage of Acrylamide reduction: Table 11 demonstrates reduction in percentage of acrylamide in French fries upon using 0.8U of EcAsa-DM/gm.
[0066]
Table 11: Percentage Acrylamide reduction by EcAsa-DM
Sl. No Units/gm of potato Acrylamide content in
Treated Sample (ppb) Acrylamide content in
Untreated Sample (ppb) Food Matrix Acrylamide Reduction (%) Enzyme
Matrix
1 0.8U EcAsa-DM crude Enzyme 552 1205 French fries 54.3 Buffer
2 0.8U EcAsa-DM purified Enzyme BLQ 223.5 French fries Acrylamide Not detected Water
3 0.8U EcAsa-DM purified Enzyme 31 2009 French fries 98 Water
4 0.8U EcAsa-DM
purified Enzyme 298 2946 French fries 89.8 Water
5 0.8U EcAsa-DM purified Enzyme 695 2939 French fries 76.5 Water

[0067] Effect of blanching in acrylamide reduction study in potato-based food products: Table 12 demonstrates that the addition of pre-processing steps of blanching in potato-based food matrix aids the process of reduction of acrylamide. Due to the addition of the blanching step for 5 mins at 85?, the reduction of acrylamide increases up to 5%-10%.
[0068]
Table 12: Effect of blanching in acrylamide reduction study in potato-based food products

Sample
(French fries) Blanched (at 85? for 5mins) + EcAsa-DM treated Only EcAsa-DM treated
acrylamide(µg/kg) present in final food product % Reduction acrylamide(µg/kg) present in final food product % Reduction
Set 1 300.15 85% 408.20 79.6%
Set 2 458.30 78.3% 636.6 70%
Set 3 180.81 91% 251.12 87.5%
Set 4 348.59 82.7% 523.9 74%

EXAMPLE 8
[0069] Acrylamide reduction in banana chips: Unripe banana was peeled and weighed properly using weighing balance and were cut into round slices of equal sizes. Round slices of banana were soaked in water in a particular dosage of Acryl-aid where water:banana ratio is 2:1 for 15-25 minutes. The water was further drained to remove excess starch. The round slice of banana is fried at 150 - 170 °C for 2-3 minutes until turned to a yellowish brown colour. Salt solution is added to the frying process and completed till the banana is crisp in nature and the excess oil is drained and the chips are allowed to cool for packaging purposes.
[0070] Acrylamide Extraction Procedure and analysis by LC-MS/MS chromatography: Weigh homogenized sample 5 g approximately in 50 ml Tarsons tube and added 10 ml of Mili-Q water to sample. Vortex for 60 seconds and add 10 ml of Acidified Acetonitrile and vortexed for 5 minutes. To this is added 4 g of Mgso4 dried + 1.66 g of Sodium acetate trihydrate and vortexed for 3 minutes. Centrifuge the sample for 5 minutes at 4000rpm. Collect 2 ml of upper layer for Cleanup in Dis Q tube and vortex and centrifuge for 5 minutes at 10000 rpm. Take 1 ml for evaporation and after evaporation, reconstitute with 1 ml of Mili-Q water and dilute to 5 times in Mili-Q water and collect injection vial and use for injecting to LC-MS/MS.
Mobile phase: A) Water, B) 0.1 % MeOH
Column: HSS T3 Column 100 mm 1.7 µ
MRM: 72.1> 55, 72.1>27.1
[0071]
Table 13: Acrylamide analysis Result
S. No Units/gm
of sample Acrylamide content in treated
Sample (ppb) Acrylamide content
in untreated
sample (ppb) Food Matrix Acrylamide Reduction (%)
1 1.2 U Acryl-aid BLQ 870.97 Banana Chips Not detectable
2 0.8 U Acryl-aid 493.95 1922 Banana Chips 74.3 %
3 0.8 U Acryl-aid 258.06 1265 Banana Chips 79.6%
4 1.2 U Acryl-aid 269.62 1456 Banana Chips 82.1 %
5 1.2 U Acryl-aid 274.32 1793 Banana Chips 84.7 %

[0072] Table 13 demonstrates that multiple dosage of a various concentration of the enzyme has been applied to the banana-based food matrix as a preliminary step of food processing and reduction of acrylamide in banana-based products (chips). Two sample sets treated with 0.8U/gm of Acryl-aid; and three sets of samples treated with 1.2U/gm of purified enzyme have been processed to yield data for reduction of acrylamide in banana chips. 0.8U/gm of sample shows the partial reduction whereas a higher % of reduction has been noticed in 1.2 U/gm of sample treated banana chips. The reduction % of acrylamide has been in the range of 70%-85% for the two dosage steps.
[0073] Important technical characteristics of EcAsa-DM: EcAsa-DM and formulation comprising EcAsa-DM possess specific advantageous characteristics as list herein below over the asparaginase known in the art:
(i) Substrate specific affinity towards asparagine is increased substantially for the EcAsa-DM. This enhanced functional property of the enzyme contributes towards better conversion of limiting substrate and acrylamide precursor molecule (asparagine) to the aspartic acid in starchy food matrix products.
(ii) EcAsa-DM asparaginase can be produced in a E. coli BL21(DE3) cells as an Intracellular/Periplasmic enzyme in a quantity, which is more than 2-times of the quantity of wild type Escherichia coli L-asparaginase II produced.
(iii) Specific activity of EcAsa-DM is 8-times higher as compared to other bacterial asparaginases namely Bacillus sp.
(iv) Formulation comprising EcAsa-DM asparaginase along with an acrylamide reducing enhancer (Glycine) and other formulation stabilizers have shown enhanced stability up to 4-5 weeks at 4 ?.
(v) Formulation comprising EcAsa-DM asparaginase showcases 75% - 95% reduction in acrylamide in potato-based food products subjected with pre-processing steps of blanching of the potato-based food matrix.
(vi) Formulation comprising EcAsa-DM asparaginase exhibits 5-times enhanced reduction of acrylamide in potato-based food products in comparison to naturally occurring E. coli asparaginases under same environmental and food processing conditions.
(vii) Enhanced thermal stability & wider pH working range (4 – 10) of EcAsa-DM asparaginase increases its use ability in a wide variety of food matrices and food processing applications.
[0074] Advantages:
(i) EcAsa-DM has significantly increased affinity for asparagine, which helps in removal of low amount of asparagine from starchy food matrix products with a lower dosages of EcAsa-DM.
(ii) EcAsa-DM has no glutaminase activity, thereby having higher specificity and enhanced asparaginase activity.
(iii) EcAsa-DM has highest asparaginase activity at 40 – 60 °C and neutral pH.
(iv) EcAsa-DM has enhanced thermal stability & wider pH working range
(v) EcAsa-DM can be produced in significantly higher quantities as compared to wild type asparaginase from E. coli.
(vi) Formulation comprising EcAsa-DM have enhanced stability up to 4 - 5 weeks at 4 °C.
(vii) Formulation comprising EcAsa-DM is required to reduces acrylamide production.
(viii) Formulation comprising EcAsa-DM reduces acrylamide production up to 75% - 95% in potato-based food products.
(ix) EcAsa-DM is developed from a Generally Recognized as Safe (GRAS) organism: K-12 strain of E. coli.
(x) EcAsa-DM has significantly increased specificity and asparaginase activity as compared to wild type asparaginase from E. coli.
(xi) Formulation comprising EcAsa-DM asparaginase is non-toxic, non- mutagenic and non-cytotoxic.
, Claims: We Claim:
1. A modified E. coli asparaginase encoding nucleotide sequence of SEQ. ID 1.
2. The modified E. coli asparaginase gene sequence as claimed in claim 1, wherein said gene sequence comprises of 526-528 TAC?TTC and 862-864 AAA?CAT substitution.
3. A modified E. coli asparaginase enzyme of amino acid sequence of SEQ. ID 2.
4. The modified E. coli asparaginase enzyme as claimed in claim 3, wherein said enzyme comprises of Y176F and K288H amino acid substitutions.
5. The modified E. coli asparaginase enzyme as claimed in claim 3, wherein the said enzyme having a 2.3-fold higher unit activity/L as compared to E. coli L-asparaginase II.
6. The modified E. coli asparaginase enzyme as claimed in claim 3, wherein said enzyme have 6-fold lower Km and have ~6-fold higher affinity for asparagine as compared to unmodified wild type E. coli asparaginase enzyme, and wherein said enzyme have an enhanced asparaginase activity at an asparagine concentration as low as 0.03 mM, and wherein said enzyme lacks glutaminase activity.
7. A formulation for reducing the formation acrylamide during processing of the food products, wherein said formulation comprises of a) a modified E. coli asparaginase enzyme of sequence of SEQ. ID 2; and b) a food grade enhancers, stabilizers, and preservatives.
8. The formulation as claimed in claim 7, wherein the modified E. coli asparaginase enzyme of SEQ. ID 2 is in 6% - 12%.
9. The formulation as claimed in claim 7, wherein the food grade enhancers, stabilizers, and preservatives consist of 100 - 300mM Glycine, 100 - 500mM NaCl, 50 - 400mM Sucrose, 25 - 200mM Tris buffer pH:8.0.
10. The formulation as claimed in claim 7, wherein the formulation is an aqueous formulation consisting of 200mM Glycine, 200mM NaCl, 200mM Sucrose, 30mM Tris buffer and 80U/ml of modified E. coli asparaginase enzyme of sequence of SEQ. ID 2.