MODIFIED GENE SEQUENCES ENCODING CHOLINE OXIDASE AND A METHOD FOR PREPARING BETAINE USNG THE SAME

Abstract

The present invention provides at least two modified gene sequences, Sequence 1 comprising of nucleotide sequence of SEQ. ID no.1, and Sequence 2 comprising of nucleotide sequence of SEQ. ID no.2, encoding the enzyme choline oxidase wherein the gene sequences have been obtained by modifying the codA gene (Accession no. X84895) encoding choline oxidase from Arthrobacter globiformis, and a method to enzymatically produce betaine using choline oxidases encoded by Sequence 1, and Sequence 2, wherein the enzymatically produced betaine has minimal undesired trimethylamine contamination.

Claims

1) A modified gene sequence of codA gene comprising a nucleotide sequence of SEQUENCE ID no.1 or SEQUENCE ID no. 2 for encoding choline oxidase; wherein the choline oxidase encoded by said nucleotide sequence has stability in the pH range 7.0 to 9.0; the choline oxidase encoded by said nucleotide sequence has stability at a temperature range of 10-50° C.; the choline oxidase encoded by said nucleotide sequence has stability at a temperature range of 10-50° C. for up to 120 minutes; and the choline oxidase thus encoded is useful to produce betaine from choline.

2) The modified gene sequence as claimed in claim 1, wherein, the modified gene sequence comprises of nucleotide sequence of SEQ. ID no.1 having 70-80% similarity with codA gene derived from Arthrobacter globiformis.

3) The modified gene sequence as claimed in claim 1, wherein, the modified gene sequence comprises of nucleotide sequence of SEQ. ID no.2 having 70-80% similarity with codA gene derived from Arthrobacter globiformis.

4) A method for producing betaine from choline comprising the steps of: a) making a reaction mixture comprising of choline aqueous solution, an enzyme and optionally hydrogen peroxide catalyzing agents; b) carrying out a reaction using the reaction mixture in a reactor; and c) on completion of the reaction, separating the enzyme from the mixture; wherein, the enzyme comprises choline oxidase encoded by nucleotide sequence of SEQUENCE ID no.1 or SEQUENCE ID no.2; and the method has choline to betaine conversion efficiency of 1:1 molar ratio.

5) The method as claimed in claim 4, wherein, the choline aqueous solution is 1-50% by weight, more preferably 15-30% by weight.

6) The method as claimed in claim 4, wherein, the hydrogen peroxide catalyzing agent is catalase or peroxidase.

7) The method as claimed in claim 4, wherein, the reaction of step b) is carried at a temperature range of 20-40° C. and a pH range of 6-9.

8) The method as claimed in claim 4, wherein, the reaction is carried in the presence of thermal stabilizers and/or cofactors.

9) The method as claimed in claim 8, wherein, the thermal stabilizers and/or cofactors are multivalent cations including but not limiting to KCl, and MgCl.sub.2.

10) The method as claimed in claim 4, wherein, betaine is produced enzymatically with minimal contamination of undesired trimethylamine.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0057] A complete understanding of the system and method of the present invention may be obtained by reference to the following drawings: FIG. 1 depicts a graph showing the activity of modified choline oxidase encoded by Sequence 1 comprising of nucleotide sequence of SEQ. ID no. 1, at various pH conditions;

[0058] FIG. 2 depicts a graph showing the activity of modified choline oxidase encoded by Sequence 2 comprising of nucleotide sequence of SEQ. ID no. 2, at various pH conditions;

[0059] FIG. 3 depicts a graph showing the activity of modified choline oxidase encoded by Sequence 1 comprising of nucleotide sequence of SEQ. ID no. 1, and Sequence 2 comprising of nucleotide sequence of SEQ. ID no. 2, at various temperatures after incubating at the respective temperatures for 20 mins (short-term exposure to different temperatures);

[0060] FIG. 4 depicts a graph showing the activity of modified choline oxidase encoded by Sequence 1 comprising of nucleotide sequence of SEQ. ID no.1, and Sequence 2 comprising of nucleotide sequence of SEQ. ID no. 2, at 37° C. after incubating at the respective temperatures for up to 120 mins (long-term exposure);

[0061] FIG. 5 depicts a graph comparing the activities of native choline oxidase derived from Arthrobacter globiformis and of modified choline oxidase encoded by Sequence 1 comprising of nucleotide sequence of SEQ. ID no. 1., at 45° C. when the enzymes were tagged with a ligand or a tag, more particularly His-tag; and

[0062] FIG. 6 depicts a graphical representation of conversion ratio of choline to betaine using modified choline oxidase encoded by Sequence 1 comprising of nucleotide sequence of SEQ. ID no.1, or Sequence 2 comprising of nucleotide sequence of SEQ. ID no.2.

DESCRIPTION OF THE INVENTION

[0063] The present invention will now be described more fully hereinafter with reference to the accompanying drawings in which a preferred embodiment of the invention is shown. This invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiment set forth herein. Rather, the embodiment is provided so that this disclosure will be thorough, and will fully convey the scope of the invention to those skilled in the art.

[0064] The present invention provides a modified gene sequence, Sequence 1 comprising of nucleotide sequence of SEQ. ID no.1, encoding the enzyme choline oxidase wherein the gene sequence has been obtained by modifying the codA gene, i.e. Sequence ID no. 3, (Accession no. X84895) encoding choline oxidase from Arthrobacter globiformis, and a method to enzymatically produce betaine using choline oxidases encoded by Sequence 1.

[0065] The present invention further provides an alternative modified gene Sequence 1 i.e. Sequence 2, comprising of nucleotide sequence of SEQ. ID no.2, encoding the enzyme choline oxidase wherein the gene sequence has been obtained by modifying the codA gene, i.e. Sequence ID no. 3, (Accession no. X84895) encoding choline oxidase from Arthrobacter globiformis, and a method to enzymatically produce betaine using choline oxidases encoded by Sequence 2.

[0066] In the preferred embodiment the invention provides at least two gene sequences, Sequence 1 comprising of nucleotide sequence of SEQ. ID no.1, and Sequence 2 comprising of nucleotide sequence of SEQ. ID no.2, encoding the enzyme choline oxidase wherein the gene sequences have been obtained by modifying the codA gene, i.e. Sequence ID no. 3, (Accession no. X84895) encoding choline oxidase from Arthrobacter globiformis.

[0067] In another embodiment of the present invention, the invention relates to at least two recombinant vectors carrying at least one modified gene Sequence 1 comprising of nucleotide sequence of SEQ. ID no.1, or Sequence 2 comprising of nucleotide sequence of SEQ. ID no.2, encoding the enzyme choline oxidase; wherein the recombinant vector comprises of a nucleic acid sequence of the present invention, a promoter, transcriptional and translational stop signals, and optionally of nucleic acid sequence encoding a tag, particularly His-tag, on either the N′ or C′ terminal of the encoded polypeptide sequence of choline oxidase of present invention. More specifically, the present invention relates to expression of nucleotide sequence encoding the enzyme choline oxidase derived from Arthrobacter globiformis in a T7 expression system, wherein the choline oxidase polypeptide is tagged on either its N′ or C′ terminal with an appropriate tag molecule or ligand, more particularly His-tag. The recombinant vector system can be any vector (linear or circular), a plasmid or a virus, into which the nucleotide sequence of the present invention can be inserted using recombinant DNA technology.

[0068] In yet another embodiment of the present invention, the invention relates to at least two recombinant microorganisms transformed with at least one recombinant vector carrying at least one modified gene Sequence 1 comprising of nucleotide sequence of SEQ. ID no.1, or Sequence 2 comprising of nucleotide sequence of SEQ. ID no.2, encoding the enzyme choline oxidase; wherein the recombinant microorganism is preferably Escherichia coli but not limited thereto.

[0069] In yet another embodiment the invention provides a method for producing modified choline oxidase encoded by either Sequence 1 comprising of nucleotide sequence of SEQ. ID no.1, or Sequence 2 comprising of nucleotide sequence of SEQ. ID no.2, using recombinant microorganisms transformed with recombinant vectors carrying at least one modified gene

[0070] Sequence 1 comprising of nucleotide sequence of SEQ. ID no.1, and Sequence 2 comprising of nucleotide sequence of SEQ. ID no.2, encoding the enzyme choline oxidase. The method comprises the following steps of: [0071] 1) culturing recombinant microorganisms transformed with recombinant vectors carrying modified choline oxidase encoded by either Sequence 1 comprising of nucleotide sequence of SEQ. ID no.1, or Sequence 2 comprising of nucleotide sequence of SEQ. ID no.2, in an appropriate nutrient media either complex or synthetic for 20-48 hrs, preferably for 30 hrs, at temperature range of 20-42° C., preferably at 25° C., in a medium of pH range of 6-8.5, preferably 6.5-7.5; [0072] 2) inducing the recombinant cells to produce choline oxidase enzyme by treating them with isopropyl β-D-1-thiogalactopyranoside (IPTG) or any other inducer based on the T7 expression system carrying the modified gene encoding choline oxidase (the enzyme generally accumulates as soluble fraction and/or in inclusion bodies inside the microbes depending upon the fermentation process parameters); [0073] 3) separating the microorganisms from the nutrient media by either centrifugation or tangential flow filtration to obtain a harvest of microorganisms; [0074] 4) grinding or lysing or using digestive enzymes for breaking the harvested microorganism cells open to release the inner contents including inclusion bodies containing the enzyme, choline oxidase; and [0075] 5) isolating and purifying the enzyme choline oxidase by a combination of methods such as salting-out with ammonium sulfate, precipitation with an organic solvent such as ethanol, etc., ion-exchange chromatography, gel filtration, affinity chromatography, and the like.

[0076] In yet another embodiment the invention provides a method for producing betaine enzymatically by converting choline to betaine using the modified choline oxidases encoded by either Sequence 1 comprising of nucleotide sequence of SEQ. ID no.1, and Sequence 2 comprising of nucleotide sequence of SEQ. ID no.2.;wherein the enzyme, modified choline oxidase, is immobilized over a solid support. The choline was treated with choline oxidase using either a batch orcontinuous process. In either case, it was preferred that the enzyme, choline oxidase, be immobilized by a water-insoluble support, usually a polymer, either by physical absorption, covalent bonding, or by entrapment. In the first two methods of immobilization, a choline oxidase/water-insoluble polymer conjugate was formed which makes retention and recycling of the enzyme possible. Synthetic supports which are suitable for use with the present invention include polymers based upon acrylamide, maleic anhydride, methacrylic acid, and styrene; natural supports include agarose, cellulose, dextran and starch; and commonly used adsorbents which have been used to immobilize enzymes include affinity resins, calcium carbonate, cellulose, clays, collagen, diatomaceous earth, hydroxylapatite and the like. Another suitable method for the immobilization of the choline oxidase is by use of a hollow fiber reactor. This type of system is preferable where contamination of active chemical residues needs to be avoided, as for example in the production of food stuffs.

[0077] The method for producing betaine enzymatically by converting choline to betaine using the modified choline oxidases comprising the steps of: [0078] 1) making a reaction mixture comprising of: [0079] a) choline aqueous solution (1-50% by weight, more preferably 15-30% by weight) 0.1-0.5 M, more preferably 0.14 M; [0080] b) Catalase or Peroxidase 2-100 units/ml [0081] 2) carrying out the reaction using the reaction mixture in a reactor at a temperature of 20-40° C. and pH 6-9, more preferably at a temperature of from about 30-37° C. and at a pH of 7.5-8.5; and [0082] 3) on completion of the reaction, separating the enzyme from the mixture and initiating a fresh set of reaction by contacting the recovered enzyme with a fresh reaction mixture;

[0083] wherein,

[0084] hydrogen peroxide catalyzing agents may be added to the solution to inhibit the inactivation of the choline oxidase due to hydrogen peroxide levels to improve the efficiency of the process;

[0085] multivalent cations such as KCl or MgCl.sub.2are commonly used as thermal stabilizers and/or cofactors; and

[0086] choline to betaine conversion is 1:1 at molar level.

[0087] The present invention is more fully described hereinafter and with reference to illustrative examples. It is to be understood, however, that these examples are presented in order to more fully describe the present invention, and are correspondingly not intended to be construed to limit the present invention.

EXAMPLE 1

Synthesis and Extraction of Modified Choline Oxidase

[0088] Modified choline oxidase enzyme was synthesized using fermentation process by recombinant Escherichia coli transformed with recombinant vectors carrying at least one gene coding for choline oxidase, either Sequence 1 comprising of nucleotide sequence of SEQ. ID no. 1, or Sequence 2 comprising of nucleotide sequence of SEQ. ID no.2.

[0089] The medium required for the production of choline oxidase can either be synthetic or complex medium which comprises of a carbon source, nitrogen source, inorganic substance and other nutrients. Examples of carbon source include glucose, sodium gluconate, glycerol and the like; examples of nitrogen source include tryptone, peptone, casein digest, yeast extract, and the like; and examples of inorganic substance include sodium, potassium, calcium, manganese, magnesium, cobalt, and the like which are usually contained in a normal medium. Examples of preferred medium is 1% tryptone, 0.5% yeast extract, 2-3% glucose, 1.0% K.sub.2HPO.sub.4, 0.1% NaH.sub.2PO.sub.4, 0.05% MgSO.sub.4.Math.7H.sub.2O, 0.01% CaCl.sub.2.Math.2H.sub.2O and 0.01% vitamin mixture and 0.02% trace metal mixture, in which tryptone and yeast extract is used as the sole nitrogen source, and glucose as the carbon source.

[0090] The cultivation of recombinant E. coli was normally done at temperature range of 20-42° C., preferably at 25° C., in a medium of pH range of 6-8.5, preferably 6.5-7.5 and the cells were cultured for 20-48 hrs, more preferably for 30 hrs. However, the culturing conditions may vary depending on various factors such as conditions of microorganism and should not be limited to those described here. Synthesis of choline oxidase is induced to the highest extent using IPTG or lactose with varied concentration for protein resulting in inclusion body form and/or as soluble fraction.

[0091] Choline oxidase got accumulated inside the recombinant cells when cultured in the above mentioned method. The resultant fermentation broth was then treated to separate the cells from the broth using centrifugation method and or tangential flow filtration employing hollow fiber or filtration cassettes. The harvested cells were either ground or lysed to extract the enzyme. The grinding of cells can be carried out in a conventional manner, for example, by means of mechanical grinding, auto-digestion with a solvent, freezing, ultrasonic treatment, pressurization, or the like. Then the enzyme was isolated and purified either by conducted by combining known methods such as salting-out with ammonium sulfate, precipitation with an organic solvent such as ethanol, etc., ion-exchange chromatography, gel filtration, affinity chromatography, and the like.

[0092] For example, bacterial cells were harvested by subjecting the resultant culture to centrifugation or tangential filtration, washed, suspended in 0.1 M Tris-HCl buffer (pH 8.0), ground with sonicator and centrifuged. The supernatant as cell—free extract was then purified using, for example, fractionation with ammonium sulfate or using affinity chromatography Ni-NTA chromatography.

[0093] Molecular weight of the modified choline oxidase encoded by either Sequence 1 comprising of nucleotide sequence of SEQ. ID no.1, or Sequence 2 comprising of nucleotide sequence of SEQ. ID no. 2., when determined by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) was about 70,000 Da (70.0 kDa).

EXAMPLE 2

Enzyme Activity Assay of Modified Choline Oxidase

[0094] Choline oxidase activity was measured by two step enzymatic reaction. Choline oxidase catalyses the production of betaine, followed by production of quinoneimine through the hydrogen produced in the first enzymatic reaction.

[0095] Reaction 1 was catalyzed by choline oxidase:

##STR00001##

[0096] Reaction 2 was catalyzed by peroxidase:

##STR00002##

[0097] Each 100 ml of reaction mixture comprised of Tris buffer (97 mM), Choline chloride (0.14 M), EDTA (33 μM), KCl (2.2 mM), 4-Aminoantipyrine (48 mM), Phenol (2.1 mM), and Peroxidase (5 U/ml).

[0098] Enzymes were assayed by pipetting 3.0 ml of reaction mixture into a cuvette (d=1.0cm) and equilibrate at 37° C. for about 5 minutes followed by addition of 0.05 ml of the enzyme solution* and mix by gentle inversion. The increase in optical density at 500 nm against the working solution for 3 to 4 minutes in a spectrophotometer thermostated at 37° C., was recorded and the LOD per minute was calculated from the initial linear portion of the curve.

[0099] Calculation

[0100] Activity can be calculated by using the following formula:

[00001]$\mathrm{Volume}\mathrm{activity}\left(U/\mathrm{ml}\right)=\frac{\Delta \mathrm{OD}/\min \times \mathrm{Vt}\times \mathrm{df}}{12.0\times 1/2\times 1.0\times \mathrm{Vs}}=\Delta \mathrm{OD}/\min \times 10.17\times \mathrm{df}$
Weight activity (U/mg)=(U/ml)×1/C [0101] Vt: Total volume (3.05 ml) [0102] Vs: Sample volume (0.05 ml) [0103] 12.0: Millimolar extinction coefficient of quinoneimine dye under the assay conditions (F/micromole) [0104] ½: Factor based on the fact that one mole of H.sub.2O.sub.2 produces half a mole of quinoneimine dye [0105] 1.0: Light path length (cm) [0106] Df: Dilution factor [0107] C: Enzyme concentration in final sample dilution (mg/ml)

[0108] Specific activity shall be defined as micromole H.sub.2O.sub.2 which is formed in 1 min by 1 mg enzyme [U/mg].

EXAMPLE 3

Determination of Optimal Ph of Choline Oxidase Activity

[0109] The optimal pH for activity of choline oxidase was determined by adding choline oxidase to a mixture of 100 mM Tris-HCl buffer, potassium phosphate buffer, water (pH 5.0 to 10.0), incubating for 10 minutes at 25° C., and measuring the activity of choline oxidase as detailed under Example 2 (37° C., pH 8.0).

[0110] As depicted in FIG. 1 and FIG. 2, modified choline oxidases encoded by SEQ. ID no.1 and SEQ. ID no.2 are stable in the pH range of about 6.0 to 10 .0,preferably pH 7.0 to 9.0. The optimal pH is about pH 8.0.

EXAMPLE 4

Determination of Optimal Temperatue of Choline Oxidase Activity

[0111] The optimal temperature for activity of choline oxidase was determined by adding choline oxidase to a mixture of 10 mM Tris-HCl buffer (pH 8.0) at temperature ranging from 10 to 65° C., incubating the mixture under the same condition for 0-120 minutes and measuring the activity of choline oxidase as detailed under Example 2(37° C., pH 8.0).

[0112] Short-Term Thermal Stability

[0113] The modified choline oxidases encoded by SEQ. ID no.1 and SEQ. ID no. 2were tested for activity at various temperatures after incubating at the respective temperatures for 20 mins As depicted in FIG. 3, both the modified choline oxidases showed stability at a temperature range of 10-50° C,preferably 25-45° C., more preferably at about 37° C.

[0114] Long-Term Thermal Stability

[0115] The modified choline oxidases encoded by SEQ. ID no.1 and SEQ. ID no. 2 were tested for activity at 37° C. over a period of 120 mins As depicted in FIG. 4, both the modified choline oxidases enzyme reaction proceeds efficiently till up to 120 mins

EXAMPLE 5

Thermal Stability of Modified Choline Oxidase Tagged with A Ligand or A Tag

[0116] The native gene of choline oxidase derived from Arthrobacter globiformis and the modified gene of choline oxidase derived from Arthrobacter globiformis, Sequence 1 comprising of nucleotide sequence of SEQ. ID no.1, was expressed using T7 expression system encoding either the native enzyme or the modified enzyme, choline oxidase, along with a tag, more particularly His-tag. The activity of choline oxidase was determined by adding choline oxidase to a mixture of 10 mM Tris-HCl buffer (pH 8.0) at 45° C., incubating the mixture under the same condition for 0-120 minutes and measuring the activity of choline oxidase every 30 minutes as detailed under Example 2(37° C., pH 8.0).

[0117] FIG. 5 depicts a graph comparing the activities of native choline oxidase derived from Arthrobacter globiformis and of modified choline oxidase encoded by Sequence 1 comprising of nucleotide sequence of SEQ. ID no.1., when the enzymes were tagged with a ligand or a tag, more particularly His-tag.

[0118] The result clearly shows that His-tagged modified choline oxidase was significantly more stable than the His-tagged native choline oxidase. The modified enzyme was stable at 45° C. for more than 60 minutes, more appropriately for more than 30 minutes, whereas the His-tagged native enzyme lost around 80% of the activity within less than 30 minutes.

EXAMPLE 6

Betaine Production

[0119] The reaction mixture for the production of betaine contained: [0120] a) 20 mM Tris-HCl buffer (pH 8), 0.129 mole of choline, 275 units of peroxidase, and 1373 units of choline oxidase in a final volume of 142 ml. [0121] b) 20 mM Tris-HCl buffer (pH 8), 2.486 mole of choline, 6300 units of peroxidase, and 31012 units of choline oxidase in a final volume of 150 ml.

[0122] The reaction was carried at 37° C. and samples were withdrawn intermittently to analyse the conversion of choline to betaine. The enzymes source was purified choline oxidase either from Seql or the Seq 2, described above.

[0123] The amount of betaine formed was determined by the liquid chromatography-mass spectrometry (LC-MS). Similarly, the reactions were setup with varied concentration of substrate and enzyme and conversion were estimated using LC-MS. As depicted in FIG. 6, choline is converted to betaine with high efficiency.

TABLE-US-00001 TABLE 1 Estimation of the reaction products of choline oxidation with choline oxidase either Seq ID 1 or Seq ID 2 Compound Amount (Mole) A Choline added 0.129 Betaine formed 0.124 B Choline added 2.486 Betaine formed 2.467

[0124] Tri-Methyl Amine estimated in enzyme reactor:

[0125] During the above enzyme reactor setup for choline to betaine conversion the TMA was also estimated using LCMS method.

TABLE-US-00002 TABLE 2 Provides estimated trimethylamine (TMA) in the reaction Compound Amount (Mole) TMA - (enzyme reactor - initial sample) 0.010 TMA - (enzyme reactor - termination 0.000 sample)

[0126] As it is evident from Table 2, the presence of TMA is minimal and hence betaine produced is of high quality.