A bifunctional enzyme, a bifunctional enzyme mutant and its application

Abstract

The present invention discloses a bifunctional enzyme, a bifunctional enzyme mutant and its application. The amino acid sequence of the bifunctional enzyme is shown in SEQ ID NO:1. The bifunctional enzyme is composed of a CKI enzyme and a CCT enzyme connected by a connecting peptide. The protein sequence of the CKI enzyme is shown in SEQ ID NO:2, the protein sequence of the CCT enzyme is shown in SEQ ID NO:3, and the amino acid sequence of the connecting peptide is shown in SEQ ID NO:4. The present invention has the following beneficial effects: (1) The bifunctional enzyme or mutant can replace the CKI enzyme and the CCT enzyme, so that the two-step reaction is combined into a one-step reaction. Since the intermediate product and the final product have low feedback inhibition on it, the enzyme activity is higher, which is more than 160% of the activity of the two enzymes, and is more suitable for in vitro enzymatic reactions. (2) When using the bifunctional enzyme for enzymatic reactions, only a single enzyme needs to be added to the reaction system, which reduces the production cost of the enzyme. (3) There is no need to consider the ratio of the enzymes in the reaction system, which makes the catalytic process simpler and more repeatable.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a bifunctional enzyme, a bifunctional enzyme mutant and applications thereof. Background Art

[0002] Citicoline, also known as nicotin, diphosphocholine, etc., is a key auxiliary factor in the biosynthesis of lecithin and an important component in the construction of biological membranes. After central nervous system damage, Citicoline participates in repair and regeneration, plays a neuroprotective role; it also plays an important role in the transfer of neurotransmitters and the conduction of bioelectricity. It has obvious clinical therapeutic effects on acute stroke, nerve damage caused by surgery, impaired consciousness, Parkinson's syndrome, dementia, glaucoma, etc. At the same time, as a nucleic acid drug, it can also be used in combination with protease inhibitors for the treatment of pancreatitis. With the continuous increase in the aging of my country's population, the market demand for drugs for Parkinson's syndrome, cerebrovascular diseases and other diseases continues to increase.

[0003] There are three main methods for synthesizing CDPC: biological fermentation, chemical synthesis and enzymatic synthesis. The biological fermentation method has problems such as long time cycle, low yield, and complicated subsequent purification steps; the chemical synthesis method has complicated steps, many intermediates, and will cause a certain degree of environmental pollution; the emerging enzymatic synthesis method uses enzymes to produce CDPC, that is, choline kinase (CKI, EC 2.7.1.32) catalyzes choline and adenosine triphosphate (ATP) to generate phosphocholine and adenosine diphosphate (ADP), and then uses phosphocholine dicytidine transferase (CCT, EC 2.7.7.15) to catalyze phosphocholine and cytidine triphosphate (CTP) to generate CDPC. It is a high-yield, green and environmentally friendly synthesis method that is in line with the development trend of the future industry.

[0004] The key to enzymatic synthesis lies in the use of enzymes. How to improve the activity, stability and manufacturing cost of catalytic enzymes directly affects its industrialization process. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a bifunctional enzyme, a bifunctional enzyme mutant and its application. The enzyme has the catalytic ability of two enzymes, choline kinase (CKI, EC 2.7.1.32) and choline phosphotransferase (CCT, EC 2.7.7.15), and has the advantages of high activity and low enzyme preparation cost, and is more suitable for industrial production.

[0006] The technical problem to be solved by the present invention is achieved through the following technical solutions:

[0007] A bifunctional enzyme, the amino acid sequence of the bifunctional enzyme is shown in SEQ ID NO: 1.

[0008] Preferably, the bifunctional enzyme is composed of a CKI enzyme and a CCT enzyme connected by a connecting peptide, the protein sequence of the CKI enzyme is shown in SEQ ID NO:2, the protein sequence of the CCT enzyme is shown in SEQ ID NO:3, and the amino acid sequence of the connecting peptide is shown in SEQ ID NO:4.

[0009] Preferably, the bifunctional enzyme is expressed by a gene sequence encoding the protein, the gene sequence is shown in SEQ ID NO: 10, and the construction method thereof is as follows:

[0010] (1) According to the protein sequences of choline kinase (CKI, EC 2.7.1.32) and choline cytidine diphosphate transferase (CCT, EC 2.7.7.15) of Streptococcus mitis, the sequences were optimized according to the codon preference of Escherichia coli DNA, and DNA sequences cki (SEQ ID NO: 5) and cct (SEQ ID NO: 6) were synthesized respectively;

[0011] (2) Designing amplification primers, which contain genes corresponding to the linker peptide and sequences homologous to the expression vector, and using the primers to perform polymerase chain reaction (PCR) on cki and cct to amplify the cki and cct fragments, respectively; at the same time, linearizing the expression vector by enzyme digestion, and connecting the two target DNA segments (cki and cct) to the vector using seamless cloning;

[0012] (3) The constructed plasmid (vector) is transferred into the expression strain for protein expression.

[0013] A bifunctional enzyme mutant is obtained by mutating the amino acid sequence of the connecting peptide in the bifunctional enzyme, wherein the mutation is a single point mutation or a combined mutation of the 352nd and 469th amino acids.

[0014] Preferably, the mutant SEQ ID NO:7 is that the amino acid at position 352 is changed from isoleucine (I) to threonine (T), the mutant SEQ ID NO:8 is that the amino acid at position 469 is changed from lysine (K) to glutamine (Q), and the mutant SEQ ID NO:9 is that the amino acid at position 352 is changed from isoleucine (I) to threonine (T) and the amino acid at position 469 is changed from lysine (K) to glutamine (Q).

[0015] Preferably, the mutation method is site-directed mutagenesis.

[0016] The invention discloses an application of a bifunctional enzyme, wherein the bifunctional enzyme is used for catalyzing the preparation of CDPC.

[0017] Preferably, the catalytic preparation of CDPC is prepared by using choline chloride, potassium chloride, magnesium chloride hexahydrate, potassium hydrogen phosphate dodecahydrate, ATP and CTP as the reaction system, adding a bifunctional enzyme to react, controlling the pH value to 7.5 and the temperature to 35°C.

[0018] An application of a bifunctional enzyme mutant for catalyzing the preparation of CDPC.

[0019] Preferably, the catalytic preparation of CDPC is prepared by using choline chloride, potassium chloride, magnesium chloride hexahydrate, potassium hydrogen phosphate dodecahydrate, ATP and CTP as the reaction system, adding a mutant of the bifunctional enzyme to react, controlling the pH value to 7.5 and the temperature to 35°C.

[0020] The above technical solution of the present invention has the following beneficial effects:

[0021] (1) The bifunctional enzyme (CCT-CKI) of the present application can completely replace the CKI enzyme and the CCT enzyme, so that the two-step reaction is combined into a one-step reaction. Since the intermediate and final products have low feedback inhibition on it, the enzyme activity is higher. Its activity is increased by more than 20% compared with the single enzyme combination, and it is more suitable for in vitro enzymatic reactions.

[0022] (2) The bifunctional enzyme mutant of the present application has a significantly improved enzyme activity compared to the bifunctional enzyme alone, and an increase of more than 60% compared to the single enzyme combination.

[0023] (3) The present application uses a bifunctional enzyme to carry out enzymatic reaction, and only needs to add a single enzyme to the reaction system, which reduces the production cost of the enzyme and has stronger industrial adaptability.

[0024] (4) In the reaction involving the bifunctional enzyme or its mutants of the present application, there is no need to consider the ratio of the enzymes in the reaction system, making the catalytic process simpler and more reproducible. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the agarose electrophoresis diagram of the gene fragments cki and cct in Example 1.

[0026] Figure 2 This is the SDS-PAGE protein electrophoresis diagram of the three enzymes CKI, CCT and M47 in Example 1.

[0027] Figure 3 This is the agarose electrophoresis diagram of the gene fragments cki-cct and cct-cki in Example 2.

[0028] Figure 4 This is a diagram of the vector construction described in Example 2, wherein Figure A is pET22b-cct-cki and Figure B is pET22b-cki-cct.

[0029] Figure 5 The SDS-PAGE protein electrophoresis diagram of the two enzymes CKI-CCT and CCT-CKI in Example 2, wherein lane 1 in Figure A is a protein marker 20-100 kDa; lane 2 is a bifunctional enzyme CCT-CKI, about 65 kDa; lane 1 in Figure B is a protein marker 20-100 kDa; lane 2 is a bifunctional enzyme CKI-CCT, about 65 kDa. DETAILED DESCRIPTION

[0030] The specific embodiments of the present invention are described in detail below to facilitate a further understanding of the present invention. All experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained through commercial channels.

[0031] Example 1 Expression of CKI and CCT enzymes

[0032] According to the protein sequence of choline kinase (CKI, EC2.7.1.32) of Streptococcus mitis in GenBank (SEQ ID NO: 2), the sequence was optimized according to the codon preference of Escherichia coli DNA to synthesize the DNA sequence cki (SEQ ID NO: 5).

[0033] According to the protein sequence of Streptococcus mitis (Streptococcus mitis) cytidine phosphocholine transferase (CCT, EC 2.7.7.15) in GenBank (SEQ ID NO: 3), the sequence was optimized according to the codon preference of Escherichia coli DNA to synthesize the DNA sequence cct (SEQ ID NO: 6).

[0034] Design primers. The primer sequences are shown in Table 1.

[0035] Table 1. PCR amplification primers Table 1

[0036]

[0037] SEQ ID NO: 5 was used as a template, and the sense primer cki-F (SEQ ID NO: 11) and the antisense primer cki-R (SEQ ID NO: 12) were used for PCR amplification. The specific conditions were: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 1 minute, and the number of cycles was 30; 72°C for 10 minutes. The cki amplified fragment was obtained.

[0038] SEQ ID NO: 6 was used as a template, and the sense primer cct-F (SEQ ID NO: 13) and the antisense primer cct-R (SEQ ID NO: 14) were used for PCR amplification. The specific conditions were: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 1 minute, and the number of cycles was 30; 72°C for 10 minutes. The cct amplified fragment was obtained.

[0039] Figure 1 It is a 1% agarose electrophoresis diagram, as shown in the figure: lane 1 is a DNA marker 100-2000bp; lane 2 is a cct fragment, about 690bp; lane 3 is a cki fragment, about 870bp.

[0040] The pET22b vector was double-digested with BamH I and Nde I, and the cki and cct amplified fragments were ligated to the pET22b vector using a seamless cloning kit to construct pET22b-cki and pET22b-cct plasmids.

[0041] After sequencing verification, pET22b-cki and pET22b-cct plasmids were transferred into E. coli BL21 (DE3) strains. The transformed E. coli BL21 (DE3) monoclonal clones were inoculated into LB medium, cultured to the logarithmic phase, induced by adding 1mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 5 hours, and the cells were collected. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to screen high-expression strains. The LB medium contains 1% peptone, 0.5% yeast powder, and 1% NaCl.

[0042] Figure 2 It is the SDS-PAGE diagram of the expressed protein, as shown in the figure: lane 1 is the protein marker 20-100kDa; lane 2 is the CKI enzyme, about 34kDa; lane 3 is the CCT enzyme, about 27kDa.

[0043] Example 2 Construction of pET22b-cct-cki and pET22b-cki-cct plasmids

[0044] Gene modification was performed from the upstream so that the two proteins were connected by 8 glycine residues (SEQ ID NO: 4).

[0045] Design amplification primers. The primer sequences are shown in Table 2:

[0046] Table 2. PCR amplification primer list sequence list

[0047]

[0048] PCR amplification was performed using SEQ ID NO:5 as a template, sense primer cki-F (SEQ ID NO:11) and antisense primer linker-R1 (SEQ ID NO:16) as primers; PCR amplification was performed using SEQ ID NO:6 as a template, sense primer linker-F1 (SEQ ID NO:15) and antisense primer cct-R (SEQ ID NO:14) as primers, and the specific conditions were: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 2 minutes, and the number of cycles was 30; 72°C for 10 minutes. Amplified fragments cki-1 and cct-1 were obtained respectively.

[0049] The pET22b vector was double-digested with BamH I and Nde I, and the cki-1 and cct-1 amplified fragments were simultaneously connected to the pET22b vector using a seamless cloning kit to construct the pET22b-cki-cct plasmid.

[0050] Similarly, PCR amplification was performed using SEQ ID NO:6 as a template, sense primer cct-F (SEQ ID NO:13) and antisense primer linker-R2 (SEQ ID NO:18) as primers; and PCR amplification was performed using SEQ ID NO:5 as a template, sense primer linker-F2 (SEQ ID NO:17) and antisense primer cki-R (SEQ ID NO:12) as primers, with the specific conditions as follows: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 2 minutes, and the number of cycles was 30; 72°C for 10 minutes. Amplified fragments cki-2 and cct-2 were obtained respectively.

[0051] The pET22b vector was double-digested with BamH I and Nde I, and the cki-2 and cct-2 amplified fragments were simultaneously connected to the pET 22b vector using a seamless cloning kit to construct the pET22b-cct-cki plasmid.

[0052] Figure 3 It is a 1% agarose electrophoresis diagram, as shown in the figure: lane 1 is the DNA marker 100-2000bp; lane 2 is the cct-cki amplification product, about 1600bp; lane 3 is the cki-cct amplification product, about 1600bp.

[0053] See the vector construction diagram for Figure 4 , Figure A shows pET22b-cct-cki, and Figure B shows pET22b-cki-cct.

[0054] After sequencing verification, the genes were transferred into E. coli BL21 (DE3) strains respectively. The strains were cultured and protein expression was induced according to the same steps as in Example 1.

[0055] Figure 5 It is the SDS-PAGE diagram of the expressed protein, as shown in the figure: Lane 1 in Figure A is the protein marker 20-100kDa; Lane 2 is the bifunctional enzyme CCT-CKI, about 65kDa; Lane 1 in Figure B is the protein marker 20-100kDa; Lane 2 is the bifunctional enzyme CKI-CCT, about 65kDa.

[0056] The harvested bacteria are broken by ultrasound, and the supernatant is collected by centrifugation. Purified enzymes can be obtained through ammonium sulfate precipitation, hydrophobic chromatography, ion exchange chromatography and desalting steps.

[0057] Example 3 Enzyme activity determination

[0058] Prepare a standard reaction system: the system contains 10mM choline, 10mM CTP (cytidine triphosphate), 10mM ATP (adenosine triphosphate), 50mM potassium chloride, 20mM magnesium chloride and 50mM Tris. Set the detection temperature to 35°C and pH to 8.0. Add a certain amount of enzyme to react, and use high performance liquid chromatography to detect the amount of CDPC generated, with a detection wavelength of 276nm.

[0059] The enzyme activity unit is defined as: 1U is the amount of enzyme that produces 1μmol CDPC in 1 minute at 35°C.

[0060] The enzyme activity was determined using the above enzyme activity determination method using the two enzymes in Example 1 and the enzyme described in Example 2, respectively.

[0061] The results show that:

[0062] In Example 1, the molar ratio of the two enzymes CKI:CCT is 1:1, and the total concentration of the two enzymes is the same as the enzyme concentration in Example 2. After activity measurement, the activity of CCT-CKI is significantly higher than the combination of the two enzymes, about 120% of the combination of the two enzymes; while the activity of enzyme CKI-CCT is about 40% of the combination of the two enzymes.

[0063] Therefore, CCT-CKI can be used as a bifunctional enzyme with better activity, and the protein sequence is SEQ ID NO: 1.

[0064] Example 4 Selection of bifunctional enzyme linker

[0065] According to the method described in Example 2, the connecting primers were designed, and the CCT and CKI proteins were connected using different linkers, and the cct sequence was located before the cki. Table 3 shows some connecting peptide sequences and the corresponding enzyme activities. The enzyme activity detection method was the same as in Example 3, and the CCT-CKI enzyme activity in Example 2 was taken as 100% as a control.

[0066] Table 3. Enzyme activities of different linker peptide sequences

[0067] Serial number Connector peptide sequence Bifunctional enzyme activity 1 GGGGGGGG 100％ 2 GGGGGG 89％ 3 GGGGSGGGGS 93％ 4 GGGSGSGG 68％ 5 GSAGSAAGSGEF 53％ 6 EAAAKEAAAK 73％

[0068] The results showed that the new enzyme CCT-CKI constructed with sequence number 1 using 8 glycine residues as a connecting peptide (SEQ ID NO: 4) had higher activity, and the protein sequence was SEQ ID NO: 1.

[0069] Example 5

[0070] Through multiple sequence alignment on the NCBI website and analysis of the protein high-level structure using online modeling software, it was determined that some sites may have a greater impact on the activity of the protein, and single-point mutations were performed on some sites of SEQ ID NO: 17. The primers and mutation sites are shown in Table 4.

[0071] The mutant sequence was connected to the pET22b vector (the method was as in Example 2), and after the mutant sequence was sequenced and verified to be correct, it was respectively transferred into the E. coli BL21 (DE3) strain. The strain was cultured and protein expression was induced according to the same steps as in Example 1. The expression activity of each protein was detected according to the activity detection method described in Example 3, and the CCT-CKI enzyme activity in SEQ ID NO: 1 in Example 4 was 100% as a control. The results are shown in Table 4.

[0072] Table 4. Comparison of primers and enzyme activities for single point mutation

[0073]

[0074] In the above Table 4, except for the I352T and K469Q mutant enzymes (M4 and M7) which have a certain improvement in enzyme activity, the other mutations have little effect. Therefore, two positions, M4 (SEQ ID NO: 7) and M7 (SEQ ID NO: 8), were selected for mutation.

[0075] Example 6

[0076] According to the method of Example 5, the mutant M47 was prepared using the primers M4 and M7 in Table 4, and the expression activity of each protein was detected according to the activity detection method of Example 3. Compared with the CCT-CKI enzyme, the enzyme activity was increased by 32%. Compared with the single enzyme combination, it was increased by about 60%. The protein sequence of the M47 enzyme is shown in SEQ ID NO: 9. The SDS-PAGE image of the M47 enzyme is shown in Figure 2As shown in the figure: Lane 1 is a protein marker 20-100kDa; Lane 4 is M47 enzyme, about 65kDa; Lane 2 is a control, CKI enzyme, about 34kDa; Lane 3 is a control, CCT enzyme, about 27kDa.

[0077] Example 7 Preparation of CDPC by Bifunctional Enzyme CCT-CKI

[0078] The reaction solution was prepared according to the following formula: 10 g / L choline chloride, 3.8 g / L potassium chloride, 10.2 g / L magnesium chloride hexahydrate, 18 g / L potassium hydrogen phosphate dodecahydrate, 35 g / L ATP and 35 g / L CTP were contained in a 1 L reaction system, the pH value was adjusted to 7.5, and 6000 U of the bifunctional enzyme CCT-CKI (SEQ ID NO: 1) of Example 3 was added to start the reaction. During the reaction, the pH value was controlled to be 7.5 and the temperature was 35° C.

[0079] After 9 hours of reaction, the amount of CDPC produced was 19 g / L as determined by HPLC.

[0080] Example 8 Preparation of CDPC by Bifunctional Enzyme Mutants

[0081] The reaction solution was prepared according to the conditions of Example 7, and 6000 U of the bifunctional enzymes M4, M7 and M47 of Examples 5 and 6 were added to start the reaction. During the reaction, the pH value was controlled to be 7.5 and the temperature was controlled to be 35°C.

[0082] After 9 hours of reaction, the CDPC production amounts detected by HPLC were 21 g / L, 20 g / L and 25 g / L respectively, and the M47 enzyme had the best effect.

[0083] Example 9 Preparation of CDPC by Bifunctional Enzyme Mutant M47

[0084] The reaction solution was prepared according to the following formula: 10 g / L choline chloride, 3.8 g / L potassium chloride, 10.2 g / L magnesium chloride hexahydrate, 18 g / L potassium hydrogen phosphate dodecahydrate, 35 g / L ATP and 35 g / L CTP were contained in a 1 L reaction system, and the pH value was adjusted to 7.5.

[0085] In a constant temperature stirring reaction tank, the bifunctional enzyme M47 in Example 6 was mixed with the LX1000HA wet carrier, and the immobilized carrier and enzyme mass ratio was 20:1. Glutaraldehyde was added according to 10% of the amount of enzyme, and stirred at 100 rpm for 16 hours. After filtering and removing the fixative, the immobilized liquid was washed twice with 0.02M potassium phosphate buffer (pH 8.0) to obtain the immobilized bifunctional enzyme. The immobilized enzyme was loaded into the reaction column equipment, and the enzyme reaction column was obtained after exhausting the bubbles.

[0086] The reaction solution was slowly passed through the enzyme reaction column using a constant flow pump, and the temperature during the reaction was controlled at 35° C. After 10 hours of reaction, the reaction solution was collected, and the amount of CDPC produced was about 24 g / L as determined by high performance liquid chromatography (HPLC).

[0087] Comparative Example 1

[0088] The reaction solution was prepared according to Example 7, the pH value was adjusted to 7.5, and the CCK and CKI enzymes described in Example 1 were added to start the reaction (molar ratio 1:1). During the reaction, the pH value was controlled to be 7.5 and the temperature was 35°C.

[0089] After 9 hours of reaction, the amount of CDPC produced was less than 16 g / L according to HPLC.

[0090] The above-mentioned Examples 7 to 9 are all examples of using the bifunctional enzyme or bifunctional enzyme mutant of the present application to prepare CDPC. Comparative Example 1 uses a traditional method to prepare CDPC. The results are shown in Table 5:

[0091] Table 5. Comparison of the preparation methods of Examples 7-9 and Comparative Example 1

[0092]

[0093] From the results, it can be seen that under the same reaction solution, the yield of CDPC prepared using the bifunctional enzyme of the present application (19) is much higher than that of comparative example 1 (<16); and the yield of CDPC prepared using the bifunctional enzyme mutant of the present application (Examples 8 and 9) is higher than that of CDPC prepared using the bifunctional enzyme (Example 7), and the effect of multi-point mutation is better; and the results of preparing CDPC (CCT-CKI M47) using the two-point mutant enzyme also show that the difference between the results of fixed enzyme and free enzyme production is not large and can be ignored.

[0094] Therefore, in the present application, since the intermediate product or the final product has low feedback inhibition on the bifunctional enzyme, the application of the bifunctional enzyme or the bifunctional enzyme mutant of the present invention in the enzymatic reaction is more effective than using two separate enzymes.

[0095] Although the present invention has been disclosed as above by the embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various choices and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention is limited by the claims and their equivalents. Sequence Listing <110> Beijing Tiankai Yida Biotechnology Co., Ltd. <120> A bifunctional enzyme, a bifunctional enzyme mutant and its application <130> 20210824 <160> 18 <170> SIPOSequenceListing 1.0 <210> 1 <211> 526 <212> PRT <213> Artificial amino acid sequence <400> 1 Met Lys Ala Ile Ile Leu Ala Ala Gly Leu Gly Thr Arg Leu Arg Pro 1 5 10 15 Met Thr Glu Asn Thr Pro Lys Ala Leu Ile Gln Val Asn Gln Lys Pro 20 25 30 Leu Ile Glu Tyr Gln Ile Glu Phe Leu Lys Glu Lys Gly Ile Asn Asp 35 40 45 Ile Ile Ile Ile Val Gly Tyr Leu Lys Glu Gln Phe Asp Tyr Leu Lys 50 55 60 Glu Lys Tyr Gly Val Arg Leu Val Phe Asn Asp Lys Tyr Ala Asp Tyr 65 70 75 80 Asn Asn Phe Tyr Ser Leu Tyr Leu Val Lys Glu Glu Leu Ala Asn Ser 85 90 95 Tyr Val Ile Asp Ala Asp Asn Tyr Leu Phe Lys Asn Met Phe Arg Asn 100 105 110 Asp Leu Thr Arg Ser Thr Tyr Phe Ser Val Tyr Arg Glu Asp Cys Thr 115 120 125 Asn Glu Trp Phe Leu Val Tyr Gly Asp Asp Tyr Lys Val Gln Asp Ile 130 135 140 Ile Val Asp Ser Lys Ala Gly Arg Ile Leu Ser Gly Val Ser Phe Trp 145 150 155 160 Asp Ala Pro Thr Ala Glu Lys Ile Val Ser Phe Ile Asp Lys Ala Tyr 165 170 175 Ala Ser Gly Glu Phe Val Asp Leu Tyr Trp Asp Asn Met Val Lys Asp 180 185 190 Asn Ile Lys Glu Leu Asp Val Tyr Val Glu Glu Leu Glu Gly Asn Ser 195 200 205 Ile Tyr Glu Ile Asp Ser Val Gln Asp Tyr His Lys Leu Glu Glu Ile 210 215 220 Leu Lys Asn Glu Asn Gly Gly Gly Gly Gly Gly Gly Gly Met Glu Lys 225 230 235 240 Ile Ile Lys Glu Lys Ile Ser Ser Leu Leu Ser Gln Glu Glu Glu Val 245 250 255 Leu Ser Val Glu Gln Leu Gly Gly Met Thr Asn Gln Asn Tyr Leu Val 260 265 270 Lys Thr Thr Asn Lys Gln Tyr Ile Val Lys Phe Phe Gly Lys Gly Thr 275 280 285 Glu Lys Leu Ile Asn Arg Gln Asp Glu Lys Tyr Asn Leu Glu Leu Leu 290 295 300 Lys Asp Leu Asp Leu Asp Val Lys Asn Tyr Leu Phe Asp Ile Glu Path 305 310 315 320 Gly Ile Lys Val Asn Glu Tyr Ile Glu Ser Ala Ile Thr Leu Asp Ser 325 330 335 Thr Ser Ile Lys Thr Lys Phe Asp Lys Ile Ala Pro Ile Leu Gln Ile 340 345 350 In His Ala Ser Gly Lys Glu Leu Arg Gly Glu Phe Ala Pro Phe Glu 355 360 365 Glu Ile Lys Lys Tyr Glu Ser Leu Ile Glu Glu Lys Ile Pro Tyr Ala 370 375 380 Asn Tyr Glu Ala Val Arg Glu Glu Val Phe Ser Leu Glu Lys Arg Leu 385 390 395 400 Pathway Asp Leu Gly Val Asp Arg Lys Ser Cys His Ile Asp Leu Val Pro 405 410 415 Glu Asn Phe Ile Glu Ser Pro Gln Gly Arg Leu Tyr Leu Ile Asp Trp 420 425 430 Glu Tyr Ser Ser Met Asn Asp Pro Met Trp Asp Leu Path Path Leu Phe 435 440 445 Leu Glu Ser Glu Phe Thr Pro Gln Glu Glu Glu Val Phe Leu Ser His 450 455 460 Tyr Glu Ser Asp Lys Thr Pro Val Ser Arg Glu Lys Ile Thr Ile Tyr 465 470 475 480 Lys Ile Leu Gln Asp Thr Ile Trp Ser Leu Trp Thr Val Tyr Lys Glu 485 490 495 Glu Gln Gly Ala Asp Phe Gly Asp Tyr Gly Val Ser Arg Tyr Gln Arg 500 505 510 Ala Val Lys Gly Leu Ala Tyr Tyr Gly Gly Ser Asp Glu Lys 515 520 525 <210> 2 <211> 289 <212> PRT <213> Streptococcus mitis CKI enzyme protein sequence <400> 2 Met Glu Lys Ile Ile Lys Glu Lys Ile Ser Ser Leu Leu Ser Gln Glu 1 5 10 15 Glu Glu Val Leu Ser Val Glu Gln Leu Gly Gly Met Thr Asn Gln Asn 20 25 30 Tyr Leu Val Lys Thr Thr Asn Lys Gln Tyr Ile Val Lys Phe Phe Gly 35 40 45 Lys Gly Thr Glu Lys Leu Ile Asn Arg Gln Asp Glu Lys Tyr Asn Leu 50 55 60 Glu Leu Leu Lys Asp Leu Asp Leu Asp Val Lys Asn Tyr Leu Phe Asp 65 70 75 80 Ile Glu Ala Gly Ile Lys Val Asn Glu Tyr Ile Glu Ser Ala Ile Thr 85 90 95 Leu Asp Ser Thr Ser Ile Lys Thr Lys Phe Asp Lys Ile Ala Pro Ile 100 105 110 Leu Gln Ile Ile His Ala Ser Gly Lys Glu Leu Arg Gly Glu Phe Ala 115 120 125 Pro Phe Glu Glu Ile Lys Lys Tyr Glu Ser Leu Ile Glu Glu Lys Ile 130 135 140 Pro Tyr Ala Asn Tyr Glu Ala Val Arg Glu Glu Val Phe Ser Leu Glu 145 150 155 160 Lys Arg Leu Ala Asp Leu Gly Val Asp Arg Lys Ser Cys His Ile Asp 165 170 175 Leu Val Pro Glu Asn Phe Ile Glu Ser Pro Gln Gly Arg Leu Tyr Leu 180 185 190 Ile Asp Trp Glu Tyr Ser Ser Met Asn Asp Pro Met Trp Asp Leu Ala 195 200 205 Ala Leu Phe Leu Glu Ser Glu Phe Thr Pro Gln Glu Glu Glu Val Phe 210 215 220 Leu Ser His Tyr Glu Ser Asp Lys Thr Pro Val Ser Arg Glu Lys Ile 225 230 235 240 Thr Ile Tyr Lys Ile Leu Gln Asp Thr Ile Trp Ser Leu Trp Thr Val 245 250 255 Tyr Lys Glu Glu Gln Gly Ala Asp Phe Gly Asp Tyr Gly Val Ser Arg 260 265 270 Tyr Gln Arg Ala Val Lys Gly Leu Ala Tyr Tyr Gly Gly Ser Asp Glu 275 280 285 Lys <210> 3 <211> 229 <212> PRT <213> Streptococcus mitis CCT enzyme protein sequence <400> 3 Met Lys Ala Ile Ile Leu Ala Ala Gly Leu Gly Thr Arg Leu Arg Pro 1 5 10 15 Met Thr Glu Asn Thr Pro Lys Ala Leu Ile Gln Val Asn Gln Lys Pro 20 25 30 Leu Ile Glu Tyr Gln Ile Glu Phe Leu Lys Glu Lys Gly Ile Asn Asp 35 40 45 Ile Ile Ile Ile Val Gly Tyr Leu Lys Glu Gln Phe Asp Tyr Leu Lys 50 55 60 Glu Lys Tyr Gly Val Arg Leu Val Phe Asn Asp Lys Tyr Ala Asp Tyr 65 70 75 80 Asn Asn Phe Tyr Ser Leu Tyr Leu Val Lys Glu Glu Leu Ala Asn Ser 85 90 95 Tyr Val Ile Asp Ala Asp Asn Tyr Leu Phe Lys Asn Met Phe Arg Asn 100 105 110 Asp Leu Thr Arg Ser Thr Tyr Phe Ser Val Tyr Arg Glu Asp Cys Thr 115 120 125 Asn Glu Trp Phe Leu Val Tyr Gly Asp Asp Tyr Lys Val Gln Asp Ile 130 135 140 Ile Val Asp Ser Lys Ala Gly Arg Ile Leu Ser Gly Val Ser Phe Trp 145 150 155 160 Asp Ala Pro Thr Ala Glu Lys Ile Val Ser Phe Ile Asp Lys Ala Tyr 165 170 175 Ala Ser Gly Glu Phe Val Asp Leu Tyr Trp Asp Asn Met Val Lys Asp 180 185 190 Asn Ile Lys Glu Leu Asp Val Tyr Val Glu Glu Leu Glu Gly Asn Ser 195 200 205 Ile Tyr Glu Ile Asp Ser Val Gln Asp Tyr His Lys Leu Glu Glu Ile 210 215 220 Leu Lys Asn Glu Asn 225 <210> 4 <211> 8 <212> PRT <213> Linker peptide <400> 4 Gly Gly Gly Gly Gly Gly Gly Gly 1 5 <210> 5 <211> 870 <212> DNA <213> Optimized synthetic CKI enzyme coding gene <400> 5 atggaaaaaa tcatcaaaga aaaaatctct tctctgctgt ctcaggaaga agaagttctg 60 tctgttgaac agctgggtgg tatgaccaac cagaactacc tggttaaaac caccaacaaa 120 cagtacatcg ttaaattctt cggtaaaggt accgaaaaac tgatcaaccg tcaggacgaa 180 aaatacaacc tggaactgct gaaagacctg gacctggacg ttaaaaacta cctgttcgac 240 atcgaagctg gtatcaaagt taacgaatac atcgaatctg ctatcaccct ggactctacc 300 tctatcaaaa ccaaattcga caaaatcgct ccgatcctgc agatcatcca cgcttctggt 360 aaagaactgc gtggtgaatt cgctccgttc gaagaaatca aaaaatacga atctctgatc 420 gaagaaaaaa tcccgtacgc taactacgaa gctgttcgtg aagaagtttt ctctctggaa 480 aaacgtctgg ctgacctggg tgttgaccgt aaatcttgcc acatcgacct ggttccggaa 540 aacttcatcg aatctccgca gggtcgtctg tacctgatcg actgggaata ctcttctatg 600 aacgacccga tgtgggacct ggctgctctg ttcctggaat ctgaattcac cccgcaggaa 660 gaagaagttt tcctgtctca ctacgaatct gacaaaaccc cggtttctcg tgaaaaaatc 720 accatctaca aaatcctgca ggacaccatc tggtctctgt ggaccgttta caaagaagaa 780 cagggtgctg acttcggtga ctacggtgtt tctcgttacc agcgtgctgt taaaggtctg 840 gcttactacg gtggttctga cgaaaaataa 870 <210> 6 <211> 690 <212> DNA <213> Optimized synthetic gene encoding CCT enzyme <400> 6 atgaaagcta tcatcctggc tgctggtctg ggtacccgtc tgcgtccgat gaccgaaaac 60 accccgaaag ctctgatcca ggttaaccag aaaccgctga tcgaatacca gatcgaattc 120 ctgaaagaaa aaggtatcaa cgacatcatc atcatcgttg gttacctgaa agaacagttc 180 gactacctga aagaaaaata cggtgttcgt ctggttttca acgacaaata cgctgactac 240 aacaacttct actctctgta cctggttaaa gaagaactgg ctaactctta cgttatcgac 300 gctgacaact acctgttcaa aaacatgttc cgtaacgacc tgacccgttc tacctacttc 360 tctgtttacc gtgaagactg caccaacgaa tggttcctgg tttacggtga cgactacaaa 420 gttcaggaca tcatcgttga ctctaaagct ggtcgtatcc tgtctggtgt ttctttctgg 480 gacgctccga ccgctgaaaa aatcgtttct ttcatcgaca aagcttacgc ttctggtgaa 540 ttcgttgacc tgtactggga caacatggtt aaagacaaca tcaaagaact ggacgtttac 600 gttgaagaac tggaaggtaa ctctatctac gaaatcgact ctgttcagga ctaccacaaa 660 ctggaagaaa tcctgaaaa cgaaaactaa 690 <210> 7 <211> 526 <212> PRT <213> I want to make sure that I have the right to do so. <400> 7 Met Lys Ala Ile Ile Leu Ala Ala Gly Leu Gly Thr Arg Leu Arg Pro 1 5 10 15 Met Thr Glu Asn Thr Pro Lys Ala Leu Ile Gln Val Asn Gln Lys Pro 20 25 30 Leu Ile Glu Tyr Gln Ile Glu Phe Leu Lys Glu Lys Gly Ile Asn Asp 35 40 45 Ile Ile Ile Val Gly Tyr Leu Lys Glu Gln Phe Asp Tyr Leu Lys 50 55 60 Glu Lys Tyr Gly Val Arg Leu Val Phe Asp Lys Tyr Ala Asp Tyr 65 70 75 80 Asn Phe Tyr Ser Leu Tyr Leu Val Lys Glu Glu Leu Ala Asn Ser 85 90 95 Tyr Val Ile Asp Ala Asp Asn Tyr Leu Phe Lys Asn Met Phe Arg Asn 100 105 110 Asp Leu Thr Arg Ser Thr Tyr Phe Ser Val Tyr Arg Glu Asp Cys Thr 115 120 125 Asn Glu Trp Phe Leu Val Tyr Gly Asp Asp Tyr Lys Val Gln Asp Ile 130 135 140 Ile Val Asp Ser Lys Ala Gly Arg Ile Leu Ser Gly Val Ser Phe Trp 145 150 155 160 Asp With Pro Thr With Glu Lys With Val Ser Phe With Asp With Lys With Tyr 165 170 175 Ala Ser Gly Glu Phe Val Asp Leu Tyr Trp Asp Asn Met Val Lys Asp 180 185 190 Asn Ile Lys Leu Glu Asp Val Tyr Val Glu Leu Glu Gly Asn Ser 195 200 205 Ile Tyr Glu Ile Asp Ser Val Gln Asp Tyr His Lys Leu Glu Glu Ile 210 215 220 Leu Lys Asn Glu Asn Gly Gly Gly Gly Gly Gly Gly Gly Met Glu Lys 225 230 235 240 Ile Ile Lys Glu Lys Ile Ser Ser Leu Leu Ser Gln Glu Glu Glu Val 245 250 255 Leu Ser Val Glu Gln Leu Gly Gly Met Thr Asn Gln Asn Tyr Leu Val 260 265 270 Lys Thr Thr Asn Lys Gln Tyr Ile Val Lys Phe Phe Gly Lys Gly Thr 275 280 285 Glu Lys Leu Ile Asn Arg Gln Asp Glu Lys Tyr Asn Leu Glu Leu Leu 290 295 300 Lys Asp Leu Asp Leu Asp Val Lys Asn Tyr Leu Phe Asp Ile Glu Ala 305 310 315 320 Gly Ile Lys Val Asn Glu Tyr Ile Glu Ser Ala Ile Thr Leu Asp Ser 325 330 335 Thr Ser Ile Lys Thr Lys Phe Asp Lys Ile Ala Pro Ile Leu Gln Thr 340 345 350 Ile His Ala Ser Gly Lys Glu Leu Arg Gly Glu Phe Ala Pro Phe Glu 355 360 365 Glu Ile Lys Lys Tyr Glu Ser Leu Ile Glu Glu Lys Ile Pro Tyr Ala 370 375 380 Asn Tyr Glu Ala Val Arg Glu Glu Val Phe Ser Leu Glu Lys Arg Leu 385 390 395 400 Ala Asp Leu Gly Val Asp Arg Lys Ser Cys His Ile Asp Leu Val Pro 405 410 415 Glu Asn Phe Ile Glu Ser Pro Gln Gly Arg Leu Tyr Leu Ile Asp Trp 420 425 430 Glu Tyr Ser Ser Met Asn Asp Pro Met Trp Asp Leu Ala Ala Leu Phe 435 440 445 Leu Glu Ser Glu Phe Thr Pro Gln Glu Glu Glu Val Phe Leu Ser His 450 455 460 Tyr Glu Ser Asp Lys Thr Pro Val Ser Arg Glu Lys Ile Thr Ile Tyr 465 470 475 480 Lys Ile Leu Gln Asp Thr Ile Trp Ser Leu Trp Thr Val Tyr Lys Glu 485 490 495 Glu Gln Gly Ala Asp Phe Gly Asp Tyr Gly Val Ser Arg Tyr Gln Arg 500 505 510 Ala Val Lys Gly Leu Ala Tyr Tyr Gly Gly Ser Asp Glu Lys 515 520 525 <210> 8 <211> 526 <212> PRT <213> Amino acid sequence of artificially synthesized bifunctional enzyme mutant <400> 8 Met Lys Ala Ile Ile Leu Ala Ala Gly Leu Gly Thr Arg Leu Arg Pro 1 5 10 15 Met Thr Glu Asn Thr Pro Lys Ala Leu Ile Gln Val Asn Gln Lys Pro 20 25 30 Leu Ile Glu Tyr Gln Ile Glu Phe Leu Lys Glu Lys Gly Ile Asn Asp 35 40 45 Ile Ile Ile Ile Val Gly Tyr Leu Lys Glu Gln Phe Asp Tyr Leu Lys 50 55 60 Glu Lys Tyr Gly Val Arg Leu Val Phe Asn Asp Lys Tyr Ala Asp Tyr 65 70 75 80 Asn Asn Phe Tyr Ser Leu Tyr Leu Val Lys Glu Glu Leu Ala Asn Ser 85 90 95 Tyr Val Ile Asp Ala Asp Asn Tyr Leu Phe Lys Asn Met Phe Arg Asn 100 105 110 Asp Leu Thr Arg Ser Thr Tyr Phe Ser Val Tyr Arg Glu Asp Cys Thr 115 120 125 Asn Glu Trp Phe Leu Val Tyr Gly Asp Asp Tyr Lys Val Gln Asp Ile 130 135 140 Ile Val Asp Ser Lys Ala Gly Arg Ile Leu Ser Gly Val Ser Phe Trp 145 150 155 160 Asp Ala Pro Thr Ala Glu Lys Ile Val Ser Phe Ile Asp Lys Ala Tyr 165 170 175 Ala Ser Gly Glu Phe Val Asp Leu Tyr Trp Asp Asn Met Val Lys Asp 180 185 190 Asn Ile Lys Glu Leu Asp Val Tyr Val Glu Glu Leu Glu Gly Asn Ser 195 200 205 Ile Tyr Glu Ile Asp Ser Val Gln Asp Tyr His Lys Leu Glu Glu Ile 210 215 220 Leu Lys Asn Glu Asn Gly Gly Gly Gly Gly Gly Gly Gly Met Glu Lys 225 230 235 240 Ile Ile Lys Glu Lys Ile Ser Ser Leu Leu Ser Gln Glu Glu Glu Val 245 250 255 Leu Ser Val Glu Gln Leu Gly Gly Met Thr Asn Gln Asn Tyr Leu Val 260 265 270 Lys Thr Thr Asn Lys Gln Tyr Ile Val Lys Phe Phe Gly Lys Gly Thr 275 280 285 Glu Lys Leu Ile Asn Arg Gln Asp Glu Lys Tyr Asn Leu Glu Leu Leu 290 295 300 Lys Asp Leu Asp Leu Asp Val Lys Asn Tyr Leu Phe Asp Ile Glu Path 305 310 315 320 Gly Ile Lys Val Asn Glu Tyr Ile Glu Ser Ala Ile Thr Leu Asp Ser 325 330 335 Thr Ser Ile Lys Thr Lys Phe Asp Lys Ile Ala Pro Ile Leu Gln Ile 340 345 350 In His Ala Ser Gly Lys Glu Leu Arg Gly Glu Phe Ala Pro Phe Glu 355 360 365 Glu Ile Lys Lys Tyr Glu Ser Leu Ile Glu Glu Lys Ile Pro Tyr Ala 370 375 380 Asn Tyr Glu Ala Val Arg Glu Glu Val Phe Ser Leu Glu Lys Arg Leu 385 390 395 400 Pathway Asp Leu Gly Val Asp Arg Lys Ser Cys His Ile Asp Leu Val Pro 405 410 415 Glu Asn Phe Ile Glu Ser Pro Gln Gly Arg Leu Tyr Leu Ile Asp Trp 420 425 430 Glu Tyr Ser Ser Met Asn Asp Pro Met Trp Asp Leu Path Path Leu Phe 435 440 445 Leu Glu Ser Glu Phe Thr Pro Gln Glu Glu Glu Val Phe Leu Ser His 450 455 460 Tyr Glu Ser Asp Gln Thr Pro Val Ser Arg Glu Lys Ile Thr Ile Tyr 465 470 475 480 Lys Ile Leu Gln Asp Thr Ile Trp Ser Leu Trp Thr Val Tyr Lys Glu 485 490 495 Glu Gln Gly Ala Asp Phe Gly Asp Tyr Gly Val Ser Arg Tyr Gln Arg 500 505 510 Ala Val Lys Gly Leu Ala Tyr Tyr Gly Gly Ser Asp Glu Lys 515 520 525 <210> 9 <211> 526 <212> PRT <213> Amino acid sequence of artificially synthesized bifunctional enzyme mutant <400> 9 Met Lys Ala Ile Ile Leu Ala Ala Gly Leu Gly Thr Arg Leu Arg Pro 1 5 10 15 Met Thr Glu Asn Thr Pro Lys Ala Leu Ile Gln Val Asn Gln Lys Pro 20 25 30 Leu Ile Glu Tyr Gln Ile Glu Phe Leu Lys Glu Lys Gly Ile Asn Asp 35 40 45 Ile Ile Ile Val Gly Tyr Leu Lys Glu Gln Phe Asp Tyr Leu Lys 50 55 60 Glu Lys Tyr Gly Val Arg Leu Val Phe Asp Lys Tyr Ala Asp Tyr 65 70 75 80 Asn Phe Tyr Ser Leu Tyr Leu Val Lys Glu Glu Leu Ala Asn Ser 85 90 95 Tyr Val Ile Asp Ala Asp Asn Tyr Leu Phe Lys Asn Met Phe Arg Asn 100 105 110 Asp Leu Thr Arg Ser Thr Tyr Phe Ser Val Tyr Arg Glu Asp Cys Thr 115 120 125 Asn Glu Trp Phe Leu Val Tyr Gly Asp Asp Tyr Lys Val Gln Asp Ile 130 135 140 Ile Val Asp Ser Lys Ala Gly Arg Ile Leu Ser Gly Val Ser Phe Trp 145 150 155 160 Asp With Pro Thr With Glu Lys With Val Ser Phe With Asp With Lys With Tyr 165 170 175 Ala Ser Gly Glu Phe Val Asp Leu Tyr Trp Asp Asn Met Val Lys Asp 180 185 190 Asn Ile Lys Leu Glu Asp Val Tyr Val Glu Leu Glu Gly Asn Ser 195 200 205 Ile Tyr Glu Ile Asp Ser Val Gln Asp Tyr His Lys Leu Glu Glu Ile 210 215 220 Leu Lys Asn Glu Asn Gly Gly Gly Gly Gly Gly Gly Gly Met Glu Lys 225 230 235 240 Ile Ile Lys Glu Lys Ile Ser Ser Leu Leu Ser Gln Glu Glu Glu Val 245 250 255 Leu Ser Val Glu Gln Leu Gly Gly Met Thr Asn Gln Asn Tyr Leu Val 260 265 270 Lys Thr Thr Asn Lys Gln Tyr Ile Val Lys Phe Phe Gly Lys Gly Thr 275 280 285 Glu Lys Leu Ile Asn Arg Gln Asp Glu Lys Tyr Asn Leu Glu Leu Leu 290 295 300 Lys Asp Leu Asp Leu Asp Val Lys Asn Tyr Leu Phe Asp Ile Glu Ala 305 310 315 320 Gly Ile Lys Val Asn Glu Tyr Ile Glu Ser Ala Ile Thr Leu Asp Ser 325 330 335 Thr Ser Ile Lys Thr Lys Phe Asp Lys Ile Ala Pro Ile Leu Gln Thr 340 345 350 Ile His Ala Ser Gly Lys Glu Leu Arg Gly Glu Phe Ala Pro Phe Glu 355 360 365 Glu Ile Lys Lys Tyr Glu Ser Leu Ile Glu Glu Lys Ile Pro Tyr Ala 370 375 380 Asn Tyr Glu Ala Val Arg Glu Glu Val Phe Ser Leu Glu Lys Arg Leu 385 390 395 400 Ala Asp Leu Gly Val Asp Arg Lys Ser Cys His Ile Asp Leu Val Pro 405 410 415 Glu Asn Phe Ile Glu Ser Pro Gln Gly Arg Leu Tyr Leu Ile Asp Trp 420 425 430 Glu Tyr Ser Ser Met Asn Asp Pro Met Trp Asp Leu Ala Ala Leu Phe 435 440 445 Leu Glu Ser Glu Phe Thr Pro Gln Glu Glu Glu Val Phe Leu Ser His 450 455 460 Tyr Glu Ser Asp Gln Thr Pro Val Ser Arg Glu Lys Ile Thr Ile Tyr 465 470 475 480 Lys Ile Leu Gln Asp Thr Ile Trp Ser Leu Trp Thr Val Tyr Lys Glu 485 490 495 Glu Gln Gly Ala Asp Phe Gly Asp Tyr Gly Val Ser Arg Tyr Gln Arg 500 505 510 Ala Val Lys Gly Leu Ala Tyr Tyr Gly Gly Ser Asp Glu Lys 515 520 525 <210> 10 <211> 1581 <212> DNA <213> Artificially synthesized bifunctional enzyme mutant gene sequence <400> 10 atgaaagcta tcatcctggc tgctggtctg ggtacccgtc tgcgtccgat gaccgaaaac 60 accccgaaag ctctgatcca ggttaaccag aaaccgctga tcgaatacca gatcgaattc 120 ctgaaagaaa aaggtatcaa cgacatcatc atcatcgttg gttacctgaa agaacagttc 180 gactacctga aagaaaaata cggtgttcgt ctggttttca acgacaaata cgctgactac 240 aacaacttct actctctgta cctggttaaa gaagaactgg ctaactctta cgttatcgac 300 gctgacaact acctgttcaa aaacatgttc cgtaacgacc tgacccgttc tacctacttc 360 tctgtttacc gtgaagactg caccaacgaa tggttcctgg tttacggtga cgactacaaa 420 gttcaggaca tcatcgttga ctctaaagct ggtcgtatcc tgtctggtgt ttctttctgg 480 gacgctccga ccgctgaaaa aatcgtttct ttcatcgaca aagcttacgc ttctggtgaa 540 ttcgttgacc tgtactggga caacatggtt aaagacaaca tcaaagaact ggacgtttac 600 gttgaagaac tggaaggtaa ctctatctac gaaatcgact ctgttcagga ctaccacaaa 660 ctggagaaa tcctgaaaaa cgaaaacggt ggtggtggtg gtggtggtgg tatggaaaaa 720 atcatcaaag aaaaaatctc ttctctgctg tctcaggaag aagaagttct gtctgttgaa 780 cagctgggtg gtatgaccaa ccagaactac ctggttaaaa ccaccaacaa acagtacatc 840 gttaaattct tcggtaaagg taccgaaaaa ctgatcaacc gtcaggacga aaaatacaac 900 ctggaactgc tgaaagacct ggacctggac gttaaaaact acctgttcga catcgaagct 960 ggtatcaaag ttaacgaata catcgaatct gctatcaccc tggactctac ctctatcaaa 1020 accaaattcg acaaaatcgc tccgatcctg cagatcatcc acgcttctgg taaagaactg 1080 cgtggtgaat tcgctccgtt cgaagaaatc aaaaaatacg aatctctgat cgaagaaaaa 1140 atcccgtacg ctaactacga agctgttcgt gaagaagttt tctctctgga aaaacgtctg 1200 gctgacctgg gtgttgaccg taaatcttgc cacatcgacc tggttccgga aaacttcatc 1260 gaatctccgc agggtcgtct gtacctgatc gactgggaat actcttctat gaacgacccg 1320 atgtgggacc tggctgctct gttcctggaa tctgaattca ccccgcagga agaagaagtt 1380 ttcctgtctc actacgaatc tgacaaaacc ccggtttctc gtgaaaaaat caccatctac 1440 aaaatcctgc aggacaccat ctggtctctg tggaccgttt acaaagaaga acagggtgct 1500 gacttcggtg actacggtgt ttctcgttac cagcgtgctg ttaaaggtct ggcttactac 1560 ggtggttctg acgaaaaata a 1581 <210> 11 <211> 36 <212> DNA <213> Primer DNA sequence <400> 11 aagaaggaga tatacatatg gaaaaaatca tcaaag 36 <210> 12 <211> 43 <212> DNA <213> Primer DNA sequence <400> 12 gacggagctc gaattcggat ccttattttt cgtcagaacc acc 43 <210> 13 <211> 37 <212> DNA <213> Primer DNA sequence <400> 13 aagaaggaga tatacatatg aaagctatca tcctggc 37 <210> 14 <211> 43 <212> DNA <213> Primer DNA sequence <400> 14 gacggagctc gaattcggat ccttagtttt cgtttttcag gat 43 <210> 15 <211> 64 <212> DNA <213> Primer DNA sequence <400> 15 acggtggttc tgacgaaaaa ggtggtggtg gtggtggtgg tggtatgaaa gctatcatcc 60 tggc 64 <210> 16 <211> 64 <212> DNA <213> Primer DNA sequence <400> 16 gccaggatga tagctttcat accaccacca ccaccaccac cacctttttc gtcagaacca 60 ccgt 64 <210> 17 <211> 64 <212> DNA <213> Primer DNA sequence <400> 17 aaatcctgaa aaacgaaaac ggtggtggtg gtggtggtgg tggtatggaa aaaatcatca 60 aaga 64 <210> 18 <211> 64 <212> DNA <213> Primer DNA sequence <400> 18 tctttgatga ttttttccat accaccacca ccaccaccac caccgttttc gtttttcagg 60 attt 64

Claims

1. A bifunctional enzyme, characterized in that: The amino acid sequence of the bifunctional enzyme is shown in SEQ ID NO:1; the bifunctional enzyme is composed of a CKI enzyme and a CCT enzyme connected by a connecting peptide, the amino acid sequence of the CKI enzyme is shown in SEQ ID NO:2, the amino acid sequence of the CCT enzyme is shown in SEQ ID NO:3, and the amino acid sequence of the connecting peptide is shown in SEQ ID NO:4; the nucleotide sequence encoding the bifunctional enzyme is shown in SEQ ID NO:10, and the CKI enzyme and the CCT enzyme are derived from Streptococcus mitis ( Streptococcus mitis ).

2. Use of a bifunctional enzyme according to claim 1 in catalytic preparation of citicoline (CDPC).

3. The use according to claim 2, characterized in that: The catalytic preparation of cytidine diphosphocholine (CDPC) uses choline chloride, potassium chloride, magnesium chloride hexahydrate, potassium hydrogen phosphate dodecahydrate, ATP and cytidine triphosphate (CTP) as the reaction system, adds a bifunctional enzyme for reaction, controls the pH value to be 7.5, and the temperature to be 35°C.

4. The bifunctional enzyme mutant according to claim 1, characterized in that: The mutant is obtained by subjecting the amino acid sequence of the bifunctional enzyme shown in SEQ ID NO:1 to I352T and / or K469Q mutations, and the amino acid sequences of the mutant are shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:

9.

5. A bifunctional enzyme mutant according to claim 4, characterized in that: The mutation method was site-directed mutagenesis.

6. Use of a mutant of a bifunctional enzyme according to any one of claims 4 to 5 in catalytic preparation of cytidine diphosphocholine (CDPC).

7. The use according to claim 6, characterized in that the catalytic preparation of cytidine diphosphocholine (CDPC) is based on choline chloride, potassium chloride, magnesium chloride hexahydrate, potassium hydrogen phosphate dodecahydrate, ATP and cytidine triphosphate (CTP) as the reaction system, the mutant of the bifunctional enzyme is added to react, the pH value is controlled to 7.5, and the temperature is 35°C.

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