阅读说明：本技术 L-塔格糖的合成方法 (Synthetic method of L-tagatose ) 是由 张新帅 朱闰婕 李咏欣 于 2021-09-17 设计创作，主要内容包括：本发明公开了L-塔格糖的合成方法,将半乳糖醇在氧化酶GatDH以及激酶TgK联合作用下转化成L-塔格糖-6-磷酸,然后利用磷酸水解酶APase去磷酸得到L-塔格糖；或者D-半乳糖通过还原酶aldR还原成半乳糖醇,然后半乳糖醇通过氧化酶GatDH可转化成L-塔格糖。本发明采用廉价大宗的D-半乳糖或半乳糖醇作为原料,利用多个酶连续转化得到昂贵的L-塔格糖。该制备方案具有多方面的工业应用优势,使用原料便宜、制备路线短、反应条件简单且废物排放低；以上这些特点都很好的符合了当今绿色工业生产的基本要求。(The invention discloses a synthesis method of L-tagatose, which comprises the steps of converting galactitol into L-tagatose-6-phosphate under the combined action of oxidase GatDH and kinase TgK, and then dephosphorizing by using phosphohydrolase APase to obtain L-tagatose; or D-galactose is reduced to galactitol by reductase aldR, and then the galactitol is converted to L-tagatose by oxidase GatDH. The invention adopts cheap bulk D-galactose or galactitol as raw material, and obtains expensive L-tagatose by continuous conversion of a plurality of enzymes. The preparation scheme has the advantages of various industrial applications, cheap used raw materials, short preparation route, simple reaction conditions and low waste discharge; the characteristics well meet the basic requirements of the current green industrial production.)

A method for synthesizing L-tagatose, characterized by comprising the steps of:

(1) mixing galactitol, adenosine disodium triphosphate ATP, nicotinamide adenine dinucleotide monosodium salt, magnesium salt and potassium salt, adjusting the pH value of a system to 7.0-8.5, adding galactitol dehydrogenase (GatDH) and ketohexokinase (TgK), reacting at room temperature for 4-10 hours, then adding barium oxalate, stirring for dissolving, adding absolute ethyl alcohol, mixing and precipitating L-tagatose-6-phosphoric acid, and centrifuging to collect white solid precipitate; re-dissolving white solid precipitate to remove Ba²⁺Ionizing and further purifying to obtain L-tagatose-6-phosphoric acid;

(2) taking L-tagatose-6-phosphate, adding phosphohydrolase (APase), and reacting at 20-30 ℃ for 2-5 hours to generate the L-tagatose.

2. The method of synthesis according to claim 1, characterized in that: and (2) simultaneously adding Lactate Dehydrogenase (LDH), acetate kinase (AcK), sodium pyruvate and acetic acid phosphate solution into the step (1).

3. The method of synthesis according to claim 1, characterized in that: re-dissolving the white solid precipitate in the step (1) to remove Ba²⁺Dissolving the white solid precipitate in Tris buffer solution with pH value of 1.0, and adding anhydrous sodium sulfate and Ba²⁺Ion reaction to produce white barium sulfate precipitate, filtering and eliminating.

4. The method of synthesis according to claim 1, characterized in that: further purification as described in step (1) is to remove Ba²⁺Adjusting the pH value of the supernatant after ionization to be neutral, loading the supernatant into an anion resin exchange column, and eluting and separating by using an ammonia water solution with gradient concentration to obtain the L-tagatose-6-phosphoric acid.

The synthesis method of the L-tagatose is characterized by comprising the following steps:

mixing D-galactose and reduced nicotinamide adenine dinucleotide disodium salt (NADH), adjusting the pH value of the solution to 6.5-8.5, adding D-galactose reductase (aldR) and galactitol dehydrogenase (GatDH), and reacting at 20-27 ℃ for 4-10 hours to generate L-tagatose;

the amino acid sequence of the D-galactose reductase is shown in SEQ ID No. 6.

6. The synthesis method according to claim 1 or 5, characterized in that: and after the reaction is finished, removing or recovering the enzyme, removing impurities from the reaction solution by anion exchange resin, and then desalting, concentrating and crystallizing to obtain the L-tagatose.

7. The method of synthesis according to claim 6, characterized in that: the enzyme is liquid enzyme or immobilized enzyme.

8. The method of synthesis according to claim 7, characterized in that:

if liquid enzyme is adopted, the pH value of a reaction system is adjusted to be strong acid after reaction, protein is precipitated, and the protein is removed by centrifugation;

if immobilized enzyme is adopted, the reaction is filtered and recycled.

9. The synthesis method according to claim 1 or 5, characterized in that: the reactant is added into tris (hydroxymethyl) aminomethane hydrochloric acid (Tris.HCl), hydroxyethyl piperazine ethanesulfoacid (Hepes) buffer solution or 3-morpholine propanesulfonic acid (MOPS) buffer solution for reaction.

10. The synthesis method according to claim 7 or 8, characterized in that: the immobilized enzyme is prepared by the following steps:

the desired enzymes were mixed or separately dissolved in 50mM potassium phosphate solution pH 8.0, followed by addition of 40mM phenoxyacetic acid and LX-1000EP epoxy resin to the buffer, stirring at room temperature for 5 hours, filtering off the immobilized enzyme, finally washing three times each with clear water and 25mM phosphate buffer pH 8.0, and then drying at low temperature for use.

Technical Field

The invention belongs to the field of biological enzyme synthesis, and particularly relates to a synthetic method of L-tagatose.

Background

L-tagatose (L-tagatose) is a rare sugar existing in nature, is a low-calorie functional sweetener, and is particularly an additive of various detergents, cosmetics and medicines. Meanwhile, L-tagatose is also a raw material for synthesizing a galactosidase inhibitor (L-deoxygalctojirimycin) and has a special effect on the chemotherapy of cancers. However, the high price limits the promotion (the price of L-tagatose is 20,000/g in Carbosynth, a sugar selling company known internationally).

There are several methods for preparing L-tagatose reported so far, but all have some defects, which make it impossible to produce and apply L-tagatose on a large scale at a low cost.

In a chemical preparation route, the synthesis of L-tagatose needs to undergo multi-step hydroxyl protection and deprotection, which not only leads to overlong preparation route and low overall yield, but also greatly troubles the purification of later-stage products due to chiral racemization problem in the preparation process.

The L-tagatose produced by fermentation has low product concentration and purity, so that the purification cost of the final product is too high, and the effective large-scale production cannot be realized.

Disclosure of Invention

The invention aims to provide a method for synthesizing L-tagatose, which realizes direct conversion from cheap galactitol or D-galactose to L-tagatose through multi-stage enzyme-linked catalysis, has short preparation route, simple reaction condition and high conversion rate, and therefore has remarkable advantages in production cost and environmental protection.

The purpose of the invention is realized by the following technical scheme:

the synthesis method of the L-tagatose comprises the following steps:

(1) mixing galactitol, adenosine disodium triphosphate ATP, nicotinamide adenine dinucleotide monosodium salt, magnesium salt and potassium salt, adjusting the pH value of a system to be 7.0-8.5 (the pH value range is maintained in the reaction process), adding galactitol dehydrogenase (GatDH) and ketohexokinase (TgK), reacting for 4-10 hours at room temperature, then adding barium oxalate, stirring for dissolving, adding absolute ethyl alcohol, mixing and precipitating L-tagatose-6-phosphoric acid, and centrifuging to collect white solid precipitate; re-dissolving white solid precipitate to remove Ba²⁺Ionizing and further purifying to obtain L-tagatose-6-phosphoric acid;

(2) taking L-tagatose-6-phosphoric acid, adding phosphohydrolase (APase), and reacting at 20-30 ℃ for 2-5 hours to generate L-tagatose;

preferably, the step (1) can also be simultaneously added with Lactate Dehydrogenase (LDH), acetate kinase (AcK),Sodium pyruvate, and a solution of acetic acid phosphate to convert NADH to NAD using Lactate Dehydrogenase (LDH)⁺Acetate kinase (AcK) regenerates ADP to ATP, thereby reducing the amount of coenzyme and ATP used;

wherein the enzyme activity ratio of galactitol dehydrogenase (GatDH), ketohexokinase (TgK), Lactate Dehydrogenase (LDH) and acetate kinase (AcK) is 2 (3-5) to (1-3) to (3-8), preferably 2:4:1.5: 5.

Wherein the molar ratio of the galactitol to the sodium pyruvate is (0.90-1.15) 1; other raw materials are either enzyme substrates or salts required for enzyme activity, and the required dosage is less;

re-dissolving the white solid precipitate in the step (1) to remove Ba²⁺Dissolving the white solid precipitate in Tris buffer solution with pH value of 1.0, and adding anhydrous sodium sulfate and Ba²⁺Carrying out ion reaction to generate white barium sulfate precipitate, and filtering to remove the white barium sulfate precipitate;

further purification as described in step (1) is to remove Ba²⁺Adjusting pH of the supernatant to neutral, loading onto anion resin exchange column, eluting with ammonia bicarbonate water solution (0-1N) with gradient concentration, and separating to obtain L-tagatose-6-phosphoric acid.

The synthesis method of the L-tagatose comprises the following steps:

mixing D-galactose and reduced nicotinamide adenine dinucleotide disodium salt (NADH), adjusting the pH value of the solution to 6.5-8.5, adding D-galactose reductase (aldR) and galactitol dehydrogenase (GatDH, EC 1.1.1.406), and reacting at 20-27 ℃ for 4-10 hours to generate L-tagatose;

the D-galactose reductase (aldR) used by the invention, the wild type (Unit ID: A0A2S9ZVX5, EC 1.1.1.21) of which is from Rhodotorula toruloides, and the wild type coenzyme is NADPH, can effectively utilize NADH after the site-specific modification of the invention, and the mutation sites are as follows: K247W, S248L and R253M, wherein the modified amino acid sequence is shown in SEQ ID No. 6.

The activity ratio of the D-galactose reductase to the galactitol dehydrogenase is 1 (2.5-4.0).

In the two routes, after the reaction (for route one, the reaction in the step (2)) is finished, removing or recovering the enzyme, removing impurities from the reaction solution by anion exchange resin, and then desalting, concentrating and crystallizing to obtain the L-tagatose;

the desalting can be carried out by prior art methods such as G25 size exclusion column desalting or osmotic membrane desalting.

The enzyme is liquid enzyme or immobilized enzyme;

if liquid enzyme is adopted, the pH value of a reaction system is adjusted to be strong acid after reaction, protein is precipitated, and the protein is removed by centrifugation;

if immobilized enzyme is adopted, the reaction is filtered and recycled.

Preferably, the reactant is added into tris (hydroxymethyl) aminomethane hydrochloride (Tris.HCl), hydroxyethyl piperazine ethanesulfonic acid (Hepes) buffer solution or 3-morpholine propanesulfonic acid (MOPS) buffer solution for reaction; phosphate buffer systems are not suitable for use herein and result in low reactivity of the enzyme.

The immobilized enzyme is prepared by the following steps:

mixing the required enzymes or dissolving the required enzymes in 50mM potassium phosphate solution with pH 8.0, then adding 40mM phenoxyacetic acid and LX-1000EP epoxy resin to the buffer solution, stirring for 5 hours at room temperature, filtering out the immobilized enzyme, finally washing with clear water and 25mM phosphate buffer solution with pH 8.0 for three times respectively, and drying at low temperature for later use;

the immobilized aldR and GatDH mixed enzyme has 70-85% of the activity of the corresponding liquid enzyme. The immobilized Apase activity is 85-94% of that of the corresponding liquid enzyme.

Compared with the prior art, the invention has the following advantages and effects:

1. the invention adopts cheap and large amount of D-galactose or galactitol in the market as raw material, and obtains expensive L-tagatose by continuous conversion of a plurality of enzymes. The preparation scheme has the advantages of various industrial applications, cheap used raw materials, short preparation route, simple reaction conditions and low waste discharge; the characteristics well meet the basic requirements of the current green industrial production.

2. One route of the invention takes the cheap galactitol (1200/kg, aladdin reagent net) as a raw material, converts the galactitol dehydrogenase (GatDH, EC 1.1.1.406) in the rhodobacter sphaeroides and the ketohexokinase (TgK, EC 2.7.1.209) of the propionibacterium into the L-tagatose-6-phosphate with high yield, and then dephosphorylates under the action of phosphohydrolase (APase, EC 3.1.3.1) to obtain the L-tagatose; coenzyme NAD + regeneration enzyme (LDH, EC 1.1.1.27) and ATP regeneration enzyme (AcK, EC 2.7.2.1) are introduced in the reaction, so that the dosage of the coenzyme can be obviously reduced.

3. In another preparation route of the invention, D-galactose is taken as a raw material, D-galactose reductase (aldR) and galactitol dehydrogenase (GatDH, EC 1.1.1.406) in rhodobacter sphaeroides are coupled to directly convert D-galactose into L-tagatose, and the reaction adopts D-galactose reductase (aldR, EC 1.1.1.21) and utilizes NADH as coenzyme to generate NAD⁺(the coenzyme for the aldR native enzyme is NADPH), while the dehydrogenase GatDH converts NAD⁺Reducing the coenzyme into NADH, thereby effectively realizing the recycling of the coenzyme in the two-step reaction, and only adding catalytic amount of the coenzyme; the scheme simplifies the process and effectively improves the conversion rate of two steps.

Drawings

FIG. 1 is an HPLC chromatogram of L-tagatose.

FIG. 2 is a schematic representation of L-tagatose¹H-NMR spectrum.

FIG. 3 shows the results of SDS-PAGE gel detection of the enzyme.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The information on the enzymes to which the present invention relates is as follows:

galactitol dehydrogenase (GatDH): derived from Rhodococcus sphaeroides (PDB:2WDZ, EC 1.1.1.406), the amino acid and DNA sequences of which are shown in SEQ ID No.1 and NO.7, respectively;

lactate Dehydrogenase (LDH): derived from Rhizobium radiobacter (Uniprot ID: A9CFS1, EC 1.1.1.27), and the amino acid and DNA sequences thereof are shown in SEQ ID No.2 and NO.8, respectively;

ketohexokinase (TgK): derived from Propionibacterium (Propionibacterium namnense) (Unit ID: F9NVE9, EC 2.7.1.209), the amino acid and DNA sequences of which are shown in SEQ ID No.3 and NO.9, respectively;

acetate kinase (AcK): is derived from Escherichia coli (E.coli) (Unit ID: P0A6A3, EC 2.7.2.1), and the amino acid and DNA sequences thereof are respectively shown in SEQ. ID. NO.4 and NO. 10;

alkaline phosphohydrolase (APase): is derived from Pseudomonas aeruginosa (Uniprot ID: P35482, EC 3.1.3.1), and has amino acid and DNA sequences shown in SEQ ID No.5 and NO.11, respectively.

Example 1: preparation of aqueous solution of acetic acid phosphoric acid

65ml of phosphoric acid (85%, 2.0mol) are dissolved in 500ml of ethyl acetate and then cooled to 0 ℃; to this solution 190ml of cooled acetic anhydride (2.0mol) was slowly added dropwise. The mixture was stirred at 0 ℃ for 8 hours and poured into a3 l reaction flask containing 500ml of water, 250 g of ice and 64 g of sodium bicarbonate. The mixture was continued to be stirred at low temperature until no bubbles were generated. The upper layer of ethyl acetate phase was separated and discarded, and the remaining aqueous phase was adjusted to pH 3 and extracted twice with 1.0L, 500mL of ethyl acetate to remove most of the residual acetic acid. Finally, the pH value of the aqueous solution containing the acetic acid phosphoric acid is adjusted to be neutral by sodium hydroxide for standby, and the enzyme activity test proves that 600ml of 1.1N acetic acid phosphoric acid aqueous solution is obtained.

Example 2: preparation of L-tagatose from galactitol

(1) Preparation of L-tagatose-6-phosphate from galactitol by liquid enzyme (GatDH, TgK)

To 1L of 50mM Tris-HCl solution (pH 8.0) was added 27.3 g of galactitol (150mM), 17.6 g of sodium pyruvate (160mM), 2.2 g of adenosine disodium triphosphate ATP (4mM), 1.38 g of nicotinamide adenine dinucleotide monosodium salt (2mM), 165ml of acetic acid phosphate solution (180mM), 2.8 g of magnesium chloride (30mM), 1.5 g of potassium chloride (20mM) in succession; after the pH value is adjusted to 8.0, adding galactitol for dehydrogenationThe reaction is initiated by the enzymes GatDH 2000U, ketohexokinase TgK 4000U, lactate dehydrogenase LDH 1500U and acetate kinase AcK 5000U; during the reaction, acid and alkali are added to maintain the pH value of the system between 7.0 and 8.5, the mixture is slowly stirred at room temperature for 8 hours, then 38.2 g of barium oxalate (150mmol) is added into the reaction solution, and the mixture is mixed with anhydrous ethanol with twice volume after being stirred and dissolved to precipitate L-tagatose-6-phosphoric acid. The white solid was collected by centrifugation and then slowly dissolved in Tris buffer pH 1.0, after which time 21.2 g of anhydrous sodium sulfate (150mmol) was added to remove Ba from the solution²⁺Ions, and the white precipitate is barium sulfate; then, barium sulfate was removed by filtration, the pH of the supernatant was adjusted to 7.0 with an aqueous NaOH solution, and the mixture was subjected to column chromatography (CRYSTALLINE CHEMICAL) using an anion-exchange resin D201 and eluted with a gradient (0-1N) aqueous ammonia hydrogen carbonate solution to obtain 39 g of a crude L-tagatose-6-phosphate (yield 98%).

(2) Hydrolyzing L-tagatose-6-phosphate with immobilized phosphohydrolase (APase) to obtain L-tagatose

26 g of L-tagatose-6-phosphate (100mM) and 77mg of magnesium chloride (2mM) were dissolved in 1L of 25mM Tris.HCl solution, pH 7.0, 2000U of immobilized phosphohydrolase APase was added thereto, and the mixture was slowly stirred at 25 ℃ for 4 hours, followed by direct filtration separation and recovery of the immobilized enzyme. The reaction solution was purified using a D201 anion resin exchange column (pure water as mobile phase), phosphoric acid impurities were adsorbed on the resin and L-tagatose was directly eluted, and finally desalted using a G25 size exclusion column and then suction-dried at low temperature to obtain 13.2G of white foam (yield 73%) as L-tagatose. The immobilized phosphohydrolase (APase) retained 95% of its initial activity after recovery.

HPLC analysis is carried out on the obtained product, and a Phenomenex (Philomena) Rezex RCM series monosaccharide analysis chromatographic column (8mm multiplied by 300mm,9 mu m) is adopted, and the use temperature is 70 ℃; the HPLC chromatogram of the product is shown in FIG. 1, which is obtained by using pure water as the mobile phase at a flow rate of 1ml/min and detecting with a refractive index detector (SHODEX RI-101).

Of the product¹The H-NMR spectrum is shown in FIG. 2, Varian 600 meganucleiMagnetic, D₂O is a solvent.

From the combination of FIGS. 1 and 2, it can be concluded that the product is L-tagatose.

Example 3: preparation of L-tagatose from D-galactose by liquid enzyme (aldR, GatDH)

Adding 18 g of D-galactose (100mM) and 0.71 g of reduced nicotinamide adenine dinucleotide disodium salt (NADH,1mM) into 1L of 50mM Tris-hydrochloric acid (Tris.HCl) solution with pH of 7.5, adjusting the pH value of the solution to 7.5, adding crude enzyme solution (1000U aldR and 3000U GatDH to start the reaction, maintaining the reaction solution at 25 ℃ and slowly stirring for 8 hours, adding acid and alkali to maintain the pH of the reaction system to be 6.5-8.5 during the reaction, adding HCl aqueous solution to adjust the pH to 1.0 after the reaction is finished, terminating the reaction, precipitating protein, filtering to remove, adjusting the reaction solution to pH 7.0, purifying by using D201 anion exchange resin, removing salt from the collected product solution by using a reverse osmosis membrane, concentrating and crystallizing (ethanol: water, 1.5:1, v: v) to obtain 13.5 g of L-tagatose (yield 75%).

HPLC spectrum of the product obtained and¹the H-NMR spectra are respectively as shown in FIG. 1 and FIG. 2, and the product is presumed to be L-tagatose.

Example 4: preparation of L-tagatose from D-galactose by immobilized Mixed enzyme (aldR, GatDH)

Similar to example 3, but mixed immobilized aldR, GatDH enzyme was used for catalysis instead of liquid enzyme. The immobilized enzyme can be recycled after the reaction.

Similarly, 18 g of D-galactose (100mM) and 0.71 g of reduced nicotinamide adenine dinucleotide disodium salt (NADH,1mM) were added to 1L of 50mM Tris-hydrochloric acid (Tris.HCl) solution having a pH of 8.0, and after the pH of the solution was adjusted to 8.0, 4000U of immobilized enzyme (aldR: GatDH activity ratio: 1:4) was added to start the reaction, and the reaction solution was slowly stirred at 25 ℃ for 10 hours while maintaining the system pH at 6.5 to 8.5 during the reaction. After the reaction is finished, filtering and recovering the immobilized enzyme (the immobilized enzyme is washed for three times by 50mM of a Tris buffer solution with the pH value of 8.0 and then is stored for standby at 4 ℃), adjusting the pH value of the filtrate to 7.0, purifying by using D201 anion exchange resin, desalting the collected product solution by using a reverse osmosis membrane, concentrating and crystallizing (ethanol: water, 1.5:1, v: v) to obtain 11 g of L-tagatose (the yield is 61%), wherein the immobilized mixed enzyme recovered by filtering has 80% of initial enzyme activity.

HPLC spectrum of the product obtained and¹the H-NMR spectra are respectively as shown in FIG. 1 and FIG. 2, and the product is presumed to be L-tagatose.

Example 5: fermentative production of enzymes

The enzyme used in the method is obtained by fermentation production, and the following basic operation flow for preparing the enzyme is as follows:

firstly, synthesizing a gene sequence (SEQ. ID. NO. 1-NO. 6) corresponding to the enzyme by a gene company (Anhui general organisms), then subcloning the gene sequence to pET28a plasmid through NdeI/XhoI enzyme cutting sites, transferring the plasmid into E.coli (BL21) (engine organisms) cells for plate culture, and finally selecting a single clone for liquid step-by-step amplification culture.

Firstly transferring a single colony on a plate into 5ml of LB culture solution (37 ℃) containing 50 mu M kanamycin for culture, inoculating the single colony into 250ml of LB culture solution containing the same antibiotics after the cells grow to a logarithmic phase, and finally transferring the single colony into a 5L culture fermentation tank for culture; when the OD600 of the cells was about 20, 0.5mM isopropyl-. beta. -D-thiogalactopyranoside (IPTG) was added to induce protein expression for 6 hours at 26 ℃ and then centrifuged (4000rpm,15min) to collect 30-55g of wet cells.

To verify the expression of the enzyme, a small amount of cells are taken and uniformly mixed with tris (hydroxymethyl) aminomethane hydrochloride (Tris.HCl) buffer (50mM, pH 8.0), then the cells are crushed by a freeze-thaw method, and after high-speed centrifugation, the supernatant is taken and SDS-PAGE protein gel (sodium dodecyl sulfate-polyacrylamide gel) is run to determine the soluble expression of the protein (figure 3);

and (3) uniformly mixing the residual cells with a buffer solution (10 g of wet cells are mixed with about 200ml of buffer solution on the market), then carrying out high-pressure cell disruption and high-speed centrifugation (16000rpm,45min) to remove cell walls, and finally directly carrying out subsequent use on the obtained enzyme-containing clear liquid (namely the crude enzyme liquid) (the activity of the crude enzyme liquid is 150-350U/ml, U is the enzyme amount required for converting 1 mu mol of substrate at room temperature for one minute) or further purifying and then immobilizing for use (solid enzyme reaction).

The LB medium is composed of: 1% tryptone, 0.5% yeast powder, 1% NaCl, 1% dipotassium hydrogen phosphate and 5% glycerol.

Example 6: immobilization of enzymes

Ammonium sulfate solids were gradually added to the crude enzyme solutions of galactose reductase (aldR), galactitol dehydrogenase (GatDH) and phosphohydrolase (APase) collected above until enzyme precipitation (35% -60%, w/v ammonium sulfate/buffer). The enzyme solid was then collected by centrifugation (10000rpm,12min) and slowly dissolved in 25mM Tris buffer pH 8.0, and finally desalted on a G25 size exclusion column (purchased from Sigma) and separated on a DEAE Seplite FF anion exchange column to give the primary purified liquid enzymes aldR, GatDH, APase.

Primary purified liquid enzymes aldR and GatDH are mixed and fixed at one time by LX-1000EP epoxy resin (Xian blue Xiao Co.) according to a certain activity ratio; the purified enzyme APase was also immobilized singly using LX-1000 EP.

The basic method for fixing is as follows: mixing aldR with GatDH and dissolving in 1L 50mM potassium phosphate solution pH 8.0, adding 40mM phenoxyacetic acid and 300 g LX-1000EP epoxy resin to the buffer solution, stirring at room temperature for 4 hours, filtering out the immobilized enzyme, washing three times with clear water and 25mM phosphate buffer pH 8.0, and drying at low temperature. The immobilized aldR and GatDH mixed enzyme has 70-85% of the activity of the corresponding liquid enzyme.

The immobilization method of the phosphohydrolase (APase) is basically the same as the immobilization method of the mixed enzyme, and the enzyme activity after the immobilization of 2000U of the initially purified APase solution is 85-94% of that of the liquid enzyme.

The immobilization method of galactitol dehydrogenase (GatDH), ketohexokinase (TgK), Lactate Dehydrogenase (LDH), and acetate kinase (AcK) is basically the same as the above-described mixed enzyme immobilization.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> university of south China

Synthesis method of <120> L-tagatose

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 254

<212> PRT

<213> Rhodococcus sphaeroides (Rhodobacter sphaeroides)

<220>

<223> GatDH

<400> 1

Met Asp Tyr Arg Thr Val Phe Arg Leu Asp Gly Ala Cys Ala Ala Val

1 5 10 15

Thr Gly Ala Gly Ser Gly Ile Gly Leu Glu Ile Cys Arg Ala Phe Ala

20 25 30

Ala Ser Gly Ala Arg Leu Ile Leu Ile Asp Arg Glu Ala Ala Ala Leu

35 40 45

Asp Arg Ala Ala Gln Glu Leu Gly Ala Ala Val Ala Ala Arg Ile Val

50 55 60

Ala Asp Val Thr Asp Ala Glu Ala Met Thr Ala Ala Ala Ala Glu Ala

65 70 75 80

Glu Ala Val Ala Pro Val Ser Ile Leu Val Asn Ser Ala Gly Ile Ala

85 90 95

Arg Leu His Asp Ala Leu Glu Thr Asp Asp Ala Thr Trp Arg Gln Val

100 105 110

Met Ala Val Asn Val Asp Gly Met Phe Trp Ala Ser Arg Ala Phe Gly

115 120 125

Arg Ala Met Val Ala Arg Gly Ala Gly Ala Ile Val Asn Leu Gly Ser

130 135 140

Met Ser Gly Thr Ile Val Asn Arg Pro Gln Phe Ala Ser Ser Tyr Met

145 150 155 160

Ala Ser Lys Gly Ala Val His Gln Leu Thr Arg Ala Leu Ala Ala Glu

165 170 175

Trp Ala Gly Arg Gly Val Arg Val Asn Ala Leu Ala Pro Gly Tyr Val

180 185 190

Ala Thr Glu Met Thr Leu Lys Met Arg Glu Arg Pro Glu Leu Phe Glu

195 200 205

Thr Trp Leu Asp Met Thr Pro Met Gly Arg Cys Gly Glu Pro Ser Glu

210 215 220

Ile Ala Ala Ala Ala Leu Phe Leu Ala Ser Pro Ala Ala Ser Tyr Val

225 230 235 240

Thr Gly Ala Ile Leu Ala Val Asp Gly Gly Tyr Thr Val Trp

245 250

<210> 2

<211> 337

<212> PRT

<213> Rhizobium radiobacter (Agrobacterium fabrum)

<220>

<223> LDH

<400> 2

Met Arg Ile Val Val Tyr Ser Ala Lys Pro Tyr Asp Arg Gln Phe Leu

1 5 10 15

Asp Glu Ala Ala Arg Pro Gly Thr Asp Leu Gln Tyr Cys Glu Ala Arg

20 25 30

Leu Ser Pro Glu Thr Val Ala Leu Ala Asp Gly Ala Val Ala Ile Cys

35 40 45

Ala Phe Val Asn Asp Asp Leu Ser Arg Pro Val Leu Glu Lys Leu Ala

50 55 60

Gly Met Gly Val Arg Leu Val Ala Leu Arg Cys Ala Gly Phe Asn Gln

65 70 75 80

Val Asp Leu Ala Ala Ala Glu Lys Leu Gly Leu Thr Ile Ala Arg Val

85 90 95

Pro Ala Tyr Ser Pro Tyr Ala Val Ala Glu His Thr Met Ala Leu Ile

100 105 110

Leu Ser Leu Asn Arg Lys Ile His Arg Ala Tyr Asn Arg Val Arg Glu

115 120 125

Gly Asn Phe Ala Leu Asp Gly Leu Leu Gly Phe Asp Leu His Gly Lys

130 135 140

Thr Met Gly Ile Val Gly Thr Gly Lys Ile Gly Ala Ile Phe Ala Arg

145 150 155 160

Ile Ala Ala Gly Phe Gly Cys Arg Leu Ile Gly His Asp Leu His Pro

165 170 175

Asn Pro Asp Cys Glu Ala Leu Gly Met Thr Tyr Gly Thr Arg Glu Glu

180 185 190

Leu Phe Arg Thr Ser Asp Ile Val Ala Leu Met Cys Pro Leu Thr Arg

195 200 205

Glu Thr Arg His Leu Ile Arg Arg Glu Thr Leu Pro Leu Leu Lys Lys

210 215 220

Gly Val Met Leu Ile Asn Thr Ser Arg Gly Ala Ile Ile Asp Thr Pro

225 230 235 240

Ala Ala Ile Thr Gly Leu Lys Asp Gly Thr Ile Gly Ser Leu Gly Ile

245 250 255

Asp Val Tyr Glu Glu Glu Ala Asp Leu Phe Phe Glu Asp Leu Ser Asn

260 265 270

Asp Val Leu Arg Asp Asp Val Phe Ala Arg Leu Leu Thr Phe Pro Asn

275 280 285

Val Leu Val Thr Gly His Gln Gly Phe Phe Thr Gln Glu Ala Leu Lys

290 295 300

Asn Ile Ala Asp Thr Thr Ile Gly Asn Ile Glu Ser Phe Val Asp Thr

305 310 315 320

Gly Lys Ala Leu His Ala Val Ser Thr Glu Gln Leu Ala Gly Ala Val

325 330 335

Ser

<210> 3

<211> 576

<212> PRT

<213> Propionibacterium (Propionibacterium namnense)

<220>

<223> TgK

<400> 3

Met Thr Arg Leu Val Asn Asn Pro Asp Asp Phe Pro Ser Gln Ala Val

1 5 10 15

Ala Gly Leu Val Ser Ala Phe Pro Asn Tyr Val Arg Pro Val Phe Gly

20 25 30

Gly Val Val Arg Ala Ala Arg Thr Asp Arg Lys Val Ala Leu Val Val

35 40 45

Gly Gly Gly Ser Gly His Tyr Pro Ala Phe Ala Gly Trp Val Gly Pro

50 55 60

Gly Phe Ala Asp Gly Ala Val Cys Gly Asn Ile Phe Ser Ser Pro Ser

65 70 75 80

Ala Ser Gln Ala Tyr Ala Val Cys Lys Ala Ala Asp Arg Gly Ala Gly

85 90 95

Val Leu Ile Gly Phe Gly Asn Tyr Ala Gly Asp Val Leu His Phe Gly

100 105 110

Gln Ala Ala Glu Arg Leu Arg Ser Glu Gly Ile Asp Ala Arg Cys Leu

115 120 125

Leu Val Thr Asp Asp Ile Ala Ser Gly Gln Glu His Leu Lys Arg Arg

130 135 140

Gly Ile Ala Gly Asp Leu Pro Val Phe Lys Val Thr Ala Ala Ala Cys

145 150 155 160

Glu Glu Gly Arg Arg Ile Asp Glu Val Val Ala Val Phe Asp Arg Val

165 170 175

Asn Asp Arg Thr Arg Ser Leu Gly Val Ala Phe Ala Gly Cys Thr Leu

180 185 190

Pro Gly Cys Asp Glu Pro Leu Phe Arg Val Glu Ala Thr Gln Met Gly

195 200 205

Val Gly Met Gly Ile His Gly Glu Pro Gly Ile His Asp Glu Ala Leu

210 215 220

Gly Thr Ala Asp Glu Val Ala Ala Met Leu Val Asp Arg Leu Leu Ala

225 230 235 240

Asp Arg Pro Asn Asn Ser Gly Thr Arg Val Val Pro Ile Val Asn Gly

245 250 255

Leu Gly Ser Thr Lys Tyr Glu Glu Leu Phe Val Leu Trp Asn Ser Val

260 265 270

Ser Arg Arg Leu Glu Asp Ala Gly Leu Thr Ile Val Asp Pro Gln Val

275 280 285

Gly Glu Phe Val Thr Ser Leu Asp Met Ala Gly Val Ser Leu Thr Leu

290 295 300

Val Trp Leu Asp Asp Val Ile Glu Pro Leu Trp Leu Ala Ala Cys Asp

305 310 315 320

Thr Pro Ala Phe Arg Arg Gly Thr Val Ala Gln Val Asp Phe Asp Thr

325 330 335

Thr Pro Leu Pro Gln Glu Val Glu Gln Ile Ser Ile Thr Lys Pro Gly

340 345 350

Ser His Val Ser Gln Arg Leu Ala Gly Val Ile Val Gln Ala Leu Glu

355 360 365

Ala Val Ala Thr Arg Leu Ser Glu Arg Ser Gly Glu Leu Gly Arg Leu

370 375 380

Asp Ser Val Ala Gly Asp Gly Asp His Gly Ile Gly Met Thr Arg Gly

385 390 395 400

Ser Gln Ala Ala Leu Ala Glu Ala Arg Arg Val Arg Gly Asp Gly Ala

405 410 415

Gly Ala Ala Thr Thr Leu Ser Ala Ala Gly Leu Ala Trp Ser Glu His

420 425 430

Ala Gly Gly Thr Ser Gly Ala Leu Trp Gly Ala Val Leu Thr Gly Phe

435 440 445

Gly Ala Val Leu Gly Asp Glu Asp Arg Ala Asp Lys Asp Ala Ile Arg

450 455 460

Gln Ala Ala Arg Ala Ala Leu Asp Ala Val Thr Arg Leu Gly Gly Ala

465 470 475 480

Lys Ala Gly Asp Lys Thr Met Val Asp Ala Met Ile Pro Phe Val Thr

485 490 495

Thr Leu Glu Ser Ser Ala Asp Ala Leu Pro Gln Ala Trp Glu Ser Ala

500 505 510

Cys Arg Ala Ala Asp Ala Ala Ala Gln Ala Thr Ser Glu Met Thr Ala

515 520 525

Lys Ile Gly Arg Ala Arg Pro Leu Gly Glu Lys Ser Leu Gly Thr Pro

530 535 540

Asp Pro Gly Ala Met Ser Phe Cys Glu Val Val Val Ala Val Gly Ser

545 550 555 560

Val Leu Ser Thr Ser Ala Ser Ser Asn Gly Gly Leu Arg Ala Gln Arg

565 570 575

<210> 4

<211> 400

<212> PRT

<213> Escherichia coli (E. coli)

<220>

<223> AcK

<400> 4

Met Ser Ser Lys Leu Val Leu Val Leu Asn Cys Gly Ser Ser Ser Leu

1 5 10 15

Lys Phe Ala Ile Ile Asp Ala Val Asn Gly Glu Glu Tyr Leu Ser Gly

20 25 30

Leu Ala Glu Cys Phe His Leu Pro Glu Ala Arg Ile Lys Trp Lys Met

35 40 45

Asp Gly Asn Lys Gln Glu Ala Ala Leu Gly Ala Gly Ala Ala His Ser

50 55 60

Glu Ala Leu Asn Phe Ile Val Asn Thr Ile Leu Ala Gln Lys Pro Glu

65 70 75 80

Leu Ser Ala Gln Leu Thr Ala Ile Gly His Arg Ile Val His Gly Gly

85 90 95

Glu Lys Tyr Thr Ser Ser Val Val Ile Asp Glu Ser Val Ile Gln Gly

100 105 110

Ile Lys Asp Ala Ala Ser Phe Ala Pro Leu His Asn Pro Ala His Leu

115 120 125

Ile Gly Ile Glu Glu Ala Leu Lys Ser Phe Pro Gln Leu Lys Asp Lys

130 135 140

Asn Val Ala Val Phe Asp Thr Ala Phe His Gln Thr Met Pro Glu Glu

145 150 155 160

Ser Tyr Leu Tyr Ala Leu Pro Tyr Asn Leu Tyr Lys Glu His Gly Ile

165 170 175

Arg Arg Tyr Gly Ala His Gly Thr Ser His Phe Tyr Val Thr Gln Glu

180 185 190

Ala Ala Lys Met Leu Asn Lys Pro Val Glu Glu Leu Asn Ile Ile Thr

195 200 205

Cys His Leu Gly Asn Gly Gly Ser Val Ser Ala Ile Arg Asn Gly Lys

210 215 220

Cys Val Asp Thr Ser Met Gly Leu Thr Pro Leu Glu Gly Leu Val Met

225 230 235 240

Gly Thr Arg Ser Gly Asp Ile Asp Pro Ala Ile Ile Phe His Leu His

245 250 255

Asp Thr Leu Gly Met Ser Val Asp Ala Ile Asn Lys Leu Leu Thr Lys

260 265 270

Glu Ser Gly Leu Leu Gly Leu Thr Glu Val Thr Ser Asp Cys Arg Tyr

275 280 285

Val Glu Asp Asn Tyr Ala Thr Lys Glu Asp Ala Lys Arg Ala Met Asp

290 295 300

Val Tyr Cys His Arg Leu Ala Lys Tyr Ile Gly Ala Tyr Thr Ala Leu

305 310 315 320

Met Asp Gly Arg Leu Asp Ala Val Val Phe Thr Gly Gly Ile Gly Glu

325 330 335

Asn Ala Ala Met Val Arg Glu Leu Ser Leu Gly Lys Leu Gly Val Leu

340 345 350

Gly Phe Glu Val Asp His Glu Arg Asn Leu Ala Ala Arg Phe Gly Lys

355 360 365

Ser Gly Phe Ile Asn Lys Glu Gly Thr Arg Pro Ala Val Val Ile Pro

370 375 380

Thr Asn Glu Glu Leu Val Ile Ala Gln Asp Ala Ser Arg Leu Thr Ala

385 390 395 400

<210> 5

<211> 368

<212> PRT

<213> Pseudomonas aeruginosa (Pseudomonas aeruginosa)

<220>

<223> APase

<400> 5

Met Phe Lys Arg Ser Leu Ile Ala Ala Ser Leu Ser Val Ala Ala Leu

1 5 10 15

Val Ser Ala Gln Ala Met Ala Val Thr Gly Gly Gly Ala Ser Leu Pro

20 25 30

Ala Glu Leu Tyr Lys Gly Ser Ala Asp Ser Ile Leu Pro Ala Asn Phe

35 40 45

Ser Tyr Ala Val Thr Gly Ser Gly Thr Gly Lys Asn Ala Phe Leu Thr

50 55 60

Asn Asn Ser Ser Leu Phe Gly Thr Thr Gly Thr Val His Tyr Ala Gly

65 70 75 80

Ser Asp Ser Val Leu Ser Gly Ser Glu Leu Thr Thr Tyr Asn Ser Asn

85 90 95

Tyr Asn Gly Thr Tyr Gly Pro Leu Ile Gln Ile Pro Ser Val Ala Thr

100 105 110

Ser Val Thr Val Pro Tyr Arg Lys Asp Gly Asn Thr Thr Leu Asn Leu

115 120 125

Thr Ser Ala Gln Leu Cys Asp Ala Phe Ser Gly Ala Lys Thr Thr Trp

130 135 140

Gly Gln Leu Leu Gly Thr Thr Asp Ser Thr Pro Ile Arg Ile Val Tyr

145 150 155 160

Arg Thr Gly Ser Ser Gly Thr Thr Glu Leu Phe Thr Arg His Leu Asn

165 170 175

Ser Ile Cys Pro Thr Arg Phe Ala Thr Asn Ser Thr Phe Thr Asn Ala

180 185 190

Arg Leu Pro Ala Gly Gly Thr Leu Pro Ser Asn Trp Val Gly Val Ala

195 200 205

Ala Thr Ser Thr Val Val Ser Thr Val Lys Ala Thr Asn Gly Ser Leu

210 215 220

Gly Tyr Val Ser Pro Asp Ala Val Asn Ile Asn Ser Asn Ala Glu Val

225 230 235 240

Ser Arg Val Asn Gly Asn Leu Pro Thr Gln Ala Asn Val Ser Thr Ala

245 250 255

Leu Gly Ser Val Ala Pro Pro Ala Asn Ala Ala Asp Arg Ala Asp Pro

260 265 270

Ser Lys Trp Val Pro Val Phe Thr Asn Pro Ser Ala Gly Tyr Ser Ile

275 280 285

Val Gly Tyr Thr Asn Phe Val Phe Gly Gln Cys Tyr Lys Asp Ala Ser

290 295 300

Val Ser Thr Asp Val Arg Ala Phe Ile Asn Lys His Tyr Gly Gly Thr

305 310 315 320

Thr Thr Asn Ala Ala Val Ala Ala His Gly Phe Ile Pro Leu Thr Pro

325 330 335

Ala Trp Lys Ser Ala Ile Val Ser Ala Phe Tyr Thr Gly Thr Ser Glu

340 345 350

Asn Leu Ala Ile Gly Asn Thr Asn Val Cys Asn Thr Lys Gly Arg Pro

355 360 365

<210> 6

<211> 290

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> aldR

<400> 6

Met Gly Ala Glu His Ala Val Ala Ser Pro Phe Thr Leu Ala Ser Ser

1 5 10 15

Val Lys Leu Arg Asn Gly Ala Gln Met Pro Arg Leu Gly Phe Gly Val

20 25 30

Phe Gln Ser Thr Asn Ala Lys Ala Ser Thr Ala His Ala Leu Thr Met

35 40 45

Gly Tyr Arg His Ile Asp Ser Ala Arg Tyr Tyr His Asn Glu Glu Glu

50 55 60

Val Cys Ala Ala Val Gln Lys Phe Ser Gly Gly Asn Leu Pro Asn Glu

65 70 75 80

Gly Thr Gly Lys Val Trp Leu Thr Thr Lys Val Met Gly Gln Glu His

85 90 95

Gly Thr Asp Gln Thr Asn Lys Ala Val Asp Glu Ser Val Ala Ile Ala

100 105 110

Lys Lys Tyr Gly Leu Thr Trp Asp Leu Phe Leu Leu His Asp Pro Thr

115 120 125

Ala Gly Lys Gln Lys Arg Leu Glu Ala Trp Lys Val Leu Ile Glu Lys

130 135 140

Arg Asp Gln Gly Leu Ile Lys Ser Ile Gly Val Ser Asn Phe Gly Val

145 150 155 160

Lys His Leu Glu Gln Ile Lys Glu Ala Gly Leu Glu Thr Pro Glu Val

165 170 175

Asn Gln Ile Glu Leu His Pro Phe Leu Gln Gln Arg Asp Ile Val Glu

180 185 190

Tyr Cys Glu Lys Glu Gly Ile Val Val Glu Ala Tyr Cys Pro Ile Leu

195 200 205

Arg Gly Lys Arg Phe Asp Asp Pro Thr Leu Val Glu Leu Ser Lys Lys

210 215 220

His Ser Val Thr Val Pro Gln Ile Leu Ile Arg Trp Ser Leu Gln Lys

225 230 235 240

Gly Phe Val Pro Leu Pro Trp Leu Asp Thr Pro Gly Met Ile Gln Ala

245 250 255

Asn Ala Asp Leu Trp Asp Phe Glu Leu Asp Glu Gly Asp Met Gln Gln

260 265 270

Met Glu Lys Leu Asp Glu Gly Tyr Ala Val Ser Trp Asn Pro Val Asn

275 280 285

Val Glu

290

<210> 7

<211> 765

<212> DNA

<213> Rhodococcus sphaeroides (Rhodobacter sphaeroides)

<220>

<223> GatDH

<400> 7

atggattatc gcaccgtgtt tcgcctggat ggcgcgtgcg cggcggtgac cggcgcgggc 60

agcggcattg gcctggaaat ttgccgcgcg tttgcggcga gcggcgcgcg cctgattctg 120

attgatcgcg aagcggcggc gctggatcgc gcggcgcagg aactgggcgc ggcggtggcg 180

gcgcgcattg tggcggatgt gaccgatgcg gaagcgatga ccgcggcggc ggcggaagcg 240

gaagcggtgg cgccggtgag cattctggtg aacagcgcgg gcattgcgcg cctgcatgat 300

gcgctggaaa ccgatgatgc gacctggcgc caggtgatgg cggtgaacgt ggatggcatg 360

ttttgggcga gccgcgcgtt tggccgcgcg atggtggcgc gcggcgcggg cgcgattgtg 420

aacctgggca gcatgagcgg caccattgtg aaccgcccgc agtttgcgag cagctatatg 480

gcgagcaaag gcgcggtgca tcagctgacc cgcgcgctgg cggcggaatg ggcgggccgc 540

ggcgtgcgcg tgaacgcgct ggcgccgggc tatgtggcga ccgaaatgac cctgaaaatg 600

cgcgaacgcc cggaactgtt tgaaacctgg ctggatatga ccccgatggg ccgctgcggc 660

gaaccgagcg aaattgcggc ggcggcgctg tttctggcga gcccggcggc gagctatgtg 720

accggcgcga ttctggcggt ggatggcggc tataccgtgt ggtga 765

<210> 8

<211> 1014

<212> DNA

<213> Rhizobium radiobacter (Agrobacterium fabrum)

<220>

<223> LDH

<400> 8

atgcgcattg tggtgtatag cgcgaaaccg tatgatcgcc agtttctgga tgaagcggcg 60

cgcccgggca ccgatctgca gtattgcgaa gcgcgcctga gcccggaaac cgtggcgctg 120

gcggatggcg cggtggcgat ttgcgcgttt gtgaacgatg atctgagccg cccggtgctg 180

gaaaaactgg cgggcatggg cgtgcgcctg gtggcgctgc gctgcgcggg ctttaaccag 240

gtggatctgg cggcggcgga aaaactgggc ctgaccattg cgcgcgtgcc ggcgtatagc 300

ccgtatgcgg tggcggaaca taccatggcg ctgattctga gcctgaaccg caaaattcat 360

cgcgcgtata accgcgtgcg cgaaggcaac tttgcgctgg atggcctgct gggctttgat 420

ctgcatggca aaaccatggg cattgtgggc accggcaaaa ttggcgcgat ttttgcgcgc 480

attgcggcgg gctttggctg ccgcctgatt ggccatgatc tgcatccgaa cccggattgc 540

gaagcgctgg gcatgaccta tggcacccgc gaagaactgt ttcgcaccag cgatattgtg 600

gcgctgatgt gcccgctgac ccgcgaaacc cgccatctga ttcgccgcga aaccctgccg 660

ctgctgaaaa aaggcgtgat gctgattaac accagccgcg gcgcgattat tgataccccg 720

gcggcgatta ccggcctgaa agatggcacc attggcagcc tgggcattga tgtgtatgaa 780

gaagaagcgg atctgttttt tgaagatctg agcaacgatg tgctgcgcga tgatgtgttt 840

gcgcgcctgc tgacctttcc gaacgtgctg gtgaccggcc atcagggctt ttttacccag 900

gaagcgctga aaaacattgc ggataccacc attggcaaca ttgaaagctt tgtggatacc 960

ggcaaagcgc tgcatgcggt gagcaccgaa cagctggcgg gcgcggtgag ctaa 1014

<210> 9

<211> 1731

<212> DNA

<213> Propionibacterium (Propionibacterium namnense)

<220>

<223> TgK

<400> 9

atgacccgcc tggtgaacaa cccggatgat tttccgagcc aggcggtggc gggcctggtg 60

agcgcgtttc cgaactatgt gcgcccggtg tttggcggcg tggtgcgcgc ggcgcgcacc 120

gatcgcaaag tggcgctggt ggtgggcggc ggcagcggcc attatccggc gtttgcgggc 180

tgggtgggcc cgggctttgc ggatggcgcg gtgtgcggca acatttttag cagcccgagc 240

gcgagccagg cgtatgcggt gtgcaaagcg gcggatcgcg gcgcgggcgt gctgattggc 300

tttggcaact atgcgggcga tgtgctgcat tttggccagg cggcggaacg cctgcgcagc 360

gaaggcattg atgcgcgctg cctgctggtg accgatgata ttgcgagcgg ccaggaacat 420

ctgaaacgcc gcggcattgc gggcgatctg ccggtgttta aagtgaccgc ggcggcgtgc 480

gaagaaggcc gccgcattga tgaagtggtg gcggtgtttg atcgcgtgaa cgatcgcacc 540

cgcagcctgg gcgtggcgtt tgcgggctgc accctgccgg gctgcgatga accgctgttt 600

cgcgtggaag cgacccagat gggcgtgggc atgggcattc atggcgaacc gggcattcat 660

gatgaagcgc tgggcaccgc ggatgaagtg gcggcgatgc tggtggatcg cctgctggcg 720

gatcgcccga acaacagcgg cacccgcgtg gtgccgattg tgaacggcct gggcagcacc 780

aaatatgaag aactgtttgt gctgtggaac agcgtgagcc gccgcctgga agatgcgggc 840

ctgaccattg tggatccgca ggtgggcgaa tttgtgacca gcctggatat ggcgggcgtg 900

agcctgaccc tggtgtggct ggatgatgtg attgaaccgc tgtggctggc ggcgtgcgat 960

accccggcgt ttcgccgcgg caccgtggcg caggtggatt ttgataccac cccgctgccg 1020

caggaagtgg aacagattag cattaccaaa ccgggcagcc atgtgagcca gcgcctggcg 1080

ggcgtgattg tgcaggcgct ggaagcggtg gcgacccgcc tgagcgaacg cagcggcgaa 1140

ctgggccgcc tggatagcgt ggcgggcgat ggcgatcatg gcattggcat gacccgcggc 1200

agccaggcgg cgctggcgga agcgcgccgc gtgcgcggcg atggcgcggg cgcggcgacc 1260

accctgagcg cggcgggcct ggcgtggagc gaacatgcgg gcggcaccag cggcgcgctg 1320

tggggcgcgg tgctgaccgg ctttggcgcg gtgctgggcg atgaagatcg cgcggataaa 1380

gatgcgattc gccaggcggc gcgcgcggcg ctggatgcgg tgacccgcct gggcggcgcg 1440

aaagcgggcg ataaaaccat ggtggatgcg atgattccgt ttgtgaccac cctggaaagc 1500

agcgcggatg cgctgccgca ggcgtgggaa agcgcgtgcc gcgcggcgga tgcggcggcg 1560

caggcgacca gcgaaatgac cgcgaaaatt ggccgcgcgc gcccgctggg cgaaaaaagc 1620

ctgggcaccc cggatccggg cgcgatgagc ttttgcgaag tggtggtggc ggtgggcagc 1680

gtgctgagca ccagcgcgag cagcaacggc ggcctgcgcg cgcagcgctg a 1731

<210> 10

<211> 1203

<212> DNA

<213> Escherichia coli (E. coli)

<220>

<223> AcK

<400> 10

atgagcagca aactggtgct ggtgctgaac tgcggcagca gcagcctgaa atttgcgatt 60

attgatgcgg tgaacggcga agaatatctg agcggcctgg cggaatgctt tcatctgccg 120

gaagcgcgca ttaaatggaa aatggatggc aacaaacagg aagcggcgct gggcgcgggc 180

gcggcgcata gcgaagcgct gaactttatt gtgaacacca ttctggcgca gaaaccggaa 240

ctgagcgcgc agctgaccgc gattggccat cgcattgtgc atggcggcga aaaatatacc 300

agcagcgtgg tgattgatga aagcgtgatt cagggcatta aagatgcggc gagctttgcg 360

ccgctgcata acccggcgca tctgattggc attgaagaag cgctgaaaag ctttccgcag 420

ctgaaagata aaaacgtggc ggtgtttgat accgcgtttc atcagaccat gccggaagaa 480

agctatctgt atgcgctgcc gtataacctg tataaagaac atggcattcg ccgctatggc 540

gcgcatggca ccagccattt ttatgtgacc caggaagcgg cgaaaatgct gaacaaaccg 600

gtggaagaac tgaacattat tacctgccat ctgggcaacg gcggcagcgt gagcgcgatt 660

cgcaacggca aatgcgtgga taccagcatg ggcctgaccc cgctggaagg cctggtgatg 720

ggcacccgca gcggcgatat tgatccggcg attatttttc atctgcatga taccctgggc 780

atgagcgtgg atgcgattaa caaactgctg accaaagaaa gcggcctgct gggcctgacc 840

gaagtgacca gcgattgccg ctatgtggaa gataactatg cgaccaaaga agatgcgaaa 900

cgcgcgatgg atgtgtattg ccatcgcctg gcgaaatata ttggcgcgta taccgcgctg 960

atggatggcc gcctggatgc ggtggtgttt accggcggca ttggcgaaaa cgcggcgatg 1020

gtgcgcgaac tgagcctggg caaactgggc gtgctgggct ttgaagtgga tcatgaacgc 1080

aacctggcgg cgcgctttgg caaaagcggc tttattaaca aagaaggcac ccgcccggcg 1140

gtggtgattc cgaccaacga agaactggtg attgcgcagg atgcgagccg cctgaccgcg 1200

taa 1203

<210> 11

<211> 1107

<212> DNA

<213> Pseudomonas aeruginosa (Pseudomonas aeruginosa)

<220>

<223> APase

<400> 11

atgtttaaac gcagcctgat tgcggcgagc ctgagcgtgg cggcgctggt gagcgcgcag 60

gcgatggcgg tgaccggcgg cggcgcgagc ctgccggcgg aactgtataa aggcagcgcg 120

gatagcattc tgccggcgaa ctttagctat gcggtgaccg gcagcggcac cggcaaaaac 180

gcgtttctga ccaacaacag cagcctgttt ggcaccaccg gcaccgtgca ttatgcgggc 240

agcgatagcg tgctgagcgg cagcgaactg accacctata acagcaacta taacggcacc 300

tatggcccgc tgattcagat tccgagcgtg gcgaccagcg tgaccgtgcc gtatcgcaaa 360

gatggcaaca ccaccctgaa cctgaccagc gcgcagctgt gcgatgcgtt tagcggcgcg 420

aaaaccacct ggggccagct gctgggcacc accgatagca ccccgattcg cattgtgtat 480

cgcaccggca gcagcggcac caccgaactg tttacccgcc atctgaacag catttgcccg 540

acccgctttg cgaccaacag cacctttacc aacgcgcgcc tgccggcggg cggcaccctg 600

ccgagcaact gggtgggcgt ggcggcgacc agcaccgtgg tgagcaccgt gaaagcgacc 660

aacggcagcc tgggctatgt gagcccggat gcggtgaaca ttaacagcaa cgcggaagtg 720

agccgcgtga acggcaacct gccgacccag gcgaacgtga gcaccgcgct gggcagcgtg 780

gcgccgccgg cgaacgcggc ggatcgcgcg gatccgagca aatgggtgcc ggtgtttacc 840

aacccgagcg cgggctatag cattgtgggc tataccaact ttgtgtttgg ccagtgctat 900

aaagatgcga gcgtgagcac cgatgtgcgc gcgtttatta acaaacatta tggcggcacc 960

accaccaacg cggcggtggc ggcgcatggc tttattccgc tgaccccggc gtggaaaagc 1020

gcgattgtga gcgcgtttta taccggcacc agcgaaaacc tggcgattgg caacaccaac 1080

gtgtgcaaca ccaaaggccg cccgtag 1107

<210> 12

<211> 873

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> aldR

<400> 12

atgggcgcgg aacatgcggt ggcgagcccg tttaccctgg cgagcagcgt gaaactgcgc 60

aacggcgcgc agatgccgcg cctgggcttt ggcgtgtttc agagcaccaa cgcgaaagcg 120

agcaccgcgc atgcgctgac catgggctat cgccatattg atagcgcgcg ctattatcat 180

aacgaagaag aagtgtgcgc ggcggtgcag aaatttagcg gcggcaacct gccgaacgaa 240

ggcaccggca aagtgtggct gaccaccaaa gtgatgggcc aggaacatgg caccgatcag 300

accaacaaag cggtggatga aagcgtggcg attgcgaaaa aatatggcct gacctgggat 360

ctgtttctgc tgcatgatcc gaccgcgggc aaacagaaac gcctggaagc gtggaaagtg 420

ctgattgaaa aacgcgatca gggcctgatt aaaagcattg gcgtgagcaa ctttggcgtg 480

aaacatctgg aacagattaa agaagcgggc ctggaaaccc cggaagtgaa ccagattgaa 540

ctgcatccgt ttctgcagca gcgcgatatt gtggaatatt gcgaaaaaga aggcattgtg 600

gtggaagcgt attgcccgat tctgcgcggc aaacgctttg atgatccgac cctggtggaa 660

ctgagcaaaa aacatagcgt gaccgtgccg cagattctga ttcgctggag cctgcagaaa 720

ggctttgtgc cgctgccgtg gctggatacc ccgggcatga ttcaggcgaa cgcggatctg 780

tgggattttg aactggatga aggcgatatg cagcagatgg aaaaactgga tgaaggctat 840

gcggtgagct ggaacccggt gaacgtggaa tga 873