JP3491695B2 - Method for producing N-acylneuraminic acid aldolase - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a microorganism belonging to a Gram-negative bacterium other than the genus Escherichia, into which a vector incorporating a DNA carrying genetic information for producing N-acylneuraminic acid aldolase (hereinafter abbreviated as NAL) has been introduced. And a novel method for producing NAL using the microorganism.

[0002]

2. Description of the Related Art N-acylneuraminic acid aldolase (NAL) is an enzyme number E.
It is an enzyme that is classified as C.4.1.3.3. And is called N-acylneuraminic acid: pyruvate lyase in the systematic name. This enzyme is an enzyme that decomposes sialic acid (N-acylneuraminic acid) into N-acylmannosamine and pyruvate,
It is an enzyme used for measuring changes in sialic acid content for tumor diagnosis. This enzyme is also used in animal tissues and gas gangrene
(Clostridium perfringens), Vibrio cholerae
It is known to exist in microorganisms such as e) and Escherichia coli. Furthermore, in the case of microorganisms, N
-It has been reported that the presence of acylneuraminic acid dramatically increases its productivity (Japanese Patent Publication No. 56-54153), but this N-acylneuraminic acid is very expensive and is industrially used. It was difficult to use on a scale. Therefore, in recent years, by applying gene recombination technology and culturing Escherichia bacteria into which a vector incorporating a DNA carrying NAL genetic information has been introduced, NAL can be obtained without adding expensive N-acylneuraminic acid to the medium. It is known that it can be produced (JP-A-61-1384,
JP-A-61-81786, JP-A-62-277, etc.). However, with these known methods, the productivity was not sufficient for industrially producing NAL. Furthermore, in order to culture Escherichia bacteria into which a vector incorporating a DNA carrying NAL genetic information derived from Escherichia bacteria has been introduced, it is basically necessary to remove enzymes such as catalase and ATPase originally produced by Escherichia bacteria. N from a wild strain that does not apply the gene recombination technology to
As with the purification of AL, much labor was required.

[0003]

The object of the present invention is to provide a large amount of NAL in a pure form at a low cost in a simpler method without using the above-mentioned enzyme which is a problem when NAL is used. To provide the means.

[0004]

[Means for Solving the Problems] In order to achieve the above-mentioned object, the present inventors have incorporated DNA carrying NAL genetic information into various vectors and used this recombinant plasmid to transform microorganisms belonging to various Gram-negative bacteria. Converted. as a result,
Some transformants showed significant N 2 addition without inducer.
I have found that it produces AL. Furthermore, the present inventors
When a microorganism belonging to a Gram-negative bacterium that grows at a lower temperature than an Escherichia bacterium is used as a NAL production host, NA
In the purification of L, it was found that most of the proteins derived from the host microorganism can be removed only by heating treatment, and it was possible to purify NAL by a simpler method.

That is, the present invention is a microorganism belonging to a Gram-negative bacterium other than Escherichia bacteria into which a recombinant plasmid in which a DNA carrying the genetic information for producing N-acylneuraminic acid aldolase derived from Escherichia bacteria is incorporated is introduced.

The present invention also relates to N derived from a bacterium belonging to the genus Escherichia.
-Acyl neuraminate aldolase producing microorganisms belonging to Gram-negative bacteria other than Escherichia genus introduced with a plasmid incorporating DNA carrying genetic information into a vector are cultured in a medium to produce N-acyl neuraminate aldolase in the culture, A method for producing N-acylneuraminic acid aldolase, which comprises collecting N-acylneuraminic acid aldolase from the culture.

D that carries the genetic information of NAL in the present invention
NA may be DNA derived from a bacterium belonging to the genus Escherichia, may be prepared from genomic DNA, or may be synthesized. Examples of the above DNA sequence include Agri.Bio.
DNA having the nucleotide sequence (Sequence ID No. 1) described in I. Chem., 50 (8), 2155 to 2158, 1986 can be mentioned. It should be noted that the DNA of the present invention can be produced by gene recombination technology without changing the basic characteristics of NAL encoded by the DNA at a specific site of the basic DNA.
Alternatively, it also includes those artificially mutated so as to improve its characteristics, for example, those caused by substitution, deletion, insertion and the like.

The vector in the present invention may be any vector as long as it can grow autonomously in a host microorganism, and a vector constructed for gene recombination is suitable. For example, pBR322, pUC19, Bluescript, pKT230, pTS1137, etc. can be used. pTS1137 was derived from the broad host vector R1b679A by the method of INOUYE et al. (J. Bacteriol., 166 (3), 739-745 (9
86). From R1b679A in Figure 3
The construction to pTS1137 is shown. A recombinant plasmid can be prepared by incorporating the above-mentioned DNA carrying the genetic information of NAL into these vectors. Examples of the recombinant plasmid include pNAL11, pNAL51, pNAL71 and the like.

[0009] The host microorganism is a microorganism belonging to Gram-negative bacteria other than Escherichia bacteria, the recombinant plasmid is stable and can autonomously grow, and the foreign D
Any microorganism can be used as long as it can express the trait of NA, and examples thereof include microorganisms belonging to the genus Pseudomonas and the genus Serratia. More preferably, microorganisms belonging to Gram-negative bacteria that grow at a lower temperature than Escherichia bacteria, for example, Pseudomonas putida
ida), Serratia liquef
aciens), Serratia plymuthic
a) and so on.

As a method for introducing the recombinant plasmid into the host microorganism, any method such as the competent cell method, the protoplast method, and the electroporation method may be used. The thus obtained transformant microorganism is cultured in a nutrient medium to produce a large amount of NA.
Stable production of L. Whether or not the target recombinant plasmid is introduced into the host microorganism is selected by selecting the target DNA.
Microorganisms capable of simultaneously expressing the drug resistance marker and NAL of the vector holding the vector may be searched. For example, a microorganism that grows in a selective medium based on the drug resistance marker and produces NAL may be selected.

Cultivation of a host microorganism which is a transformant may be carried out by selecting the culture conditions in consideration of the nutritional physiological properties of the host. Usually, in most cases, liquid culture is carried out, but industrially, aeration and agitation culture is carried out. It is advantageous to do so. As the nutrient source of the medium, those usually used for culturing microorganisms can be widely used. The carbon source may be any assimilable carbon compound, for example, glucose, sucrose, lactose,
Maltose, fructose, molasses, pyruvic acid, etc. are used. Any available nitrogen compound may be used as the nitrogen source, and for example, peptone, meat extract, yeast extract, casein hydrolyzate, soybean meal alkali extract, etc. are used. In addition, salts such as phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese and zinc, specific amino acids, specific vitamins and the like are used as necessary.

The culturing temperature can be appropriately changed within the range in which the bacterium grows and produces NAL, but host microorganisms that grow at a lower temperature than Escherichia bacteria, such as Pseudomonas putida, Serratia liquifaciens, Serratia primuchica, etc. When using, it is preferably about 20 to 30 ° C. Although the culturing time varies slightly depending on the conditions, it is sufficient to finish the culturing at an appropriate time in consideration of the time when the maximum yield of NAL is reached, and it is usually about 12 to 72 hours. The pH of the medium can be appropriately changed within the range in which the bacteria grow and produce NAL, but it is particularly preferably about pH 6.0 to 8.0.

[0013] A culture solution containing NAL-producing cells in the culture can be directly collected and used. However, in general, when NAL is present in the culture solution, it may be filtered, centrifuged, or the like. It is used after separating the NAL-containing solution and the microbial cells. When NAL is present in the microbial cells, the obtained culture is harvested by means such as filtration or centrifugation, and then the microbial cells are destroyed by an opportunistic method or an enzymatic method such as lysozyme, If necessary, a chelating agent such as EDTA and / or a surfactant is added to solubilize NAL, and the NAL is separated and collected as an aqueous solution.

The NAL-containing solution thus obtained is concentrated under reduced pressure, membrane concentrated, salted out with ammonium sulfate, sodium sulfate or the like, or fractionated by a hydrophilic organic solvent such as methanol, ethanol or acetone. It should be made to precipitate by. Further, heating treatment and isoelectric point treatment are also effective refining means. In particular, when microorganisms belonging to Gram-negative bacteria that grow at a lower temperature than Escherichia, such as Pseudomonas putida, Serratia liquifaciens, Serratia promtica, etc. are used as host microorganisms, most of the host microorganism-derived substances are heated. The protein can be removed.

Further, by gel filtration using an adsorbent or a gel filtration agent, adsorption chromatography, ion exchange chromatography, and affinity chromatography,
A purified NAL can be obtained.

[0016]

EXAMPLES The present invention will be specifically described below with reference to examples. In the examples, NAL activity was measured as follows. That is, 50 mM phosphate buffer (pH 7.5), 20 mM N
Reacting the enzyme at 37 ° C. in acetylneuraminic acid, 0.2 mM NADH, 10 U / ml lactate dehydrogenase,
Lactate dehydrogenase was allowed to act on pyruvate generated by the enzymatic reaction, and the amount of decrease in NADH at 340 nm was recorded. ΔOD / min was calculated from the initial linear portion, and NAL activity was calculated from this. NAL1 unit is
The amount of enzyme that oxidizes 1 μmol of NADH per minute under these conditions was defined as the amount of enzyme.

Example 1 Production of NAL in Pseudomonas Bacteria (1) Preparation of Recombinant Plasmid Chromosomes from Escherichia coli JE1011 as described in Agric. Biol. Chem., 50 (8), 2155-2158 (1986). DN
A was extracted and the NAL gene was isolated. That is, Escherichia coli JE1011 (FERM P-13898) was added to LB medium (1% polypeptone, 0.5% yeast extract, 1% sodium chloride, pH 7.4) 1
00 ml was inoculated and cultured at 37 ° C. for 8 hours with shaking to obtain a culture solution. This culture solution was centrifuged at 12,000 rpm for 10 minutes to obtain about 1 g of wet cells, and then the recombinant cells were recombined from the cells.
ques (Rodriguez RL et al., Addison-Wesley Publin
About 1.5 mg of chromosomal DNA was prepared by the method described in shing Company, 1983). Next, 10 μg of this chromosomal DNA was cleaved by reacting 50 units of each of restriction enzymes HindIII (manufactured by Toyobo) and EcoRI (manufactured by Toyobo) at 37 ° C. for 16 hours. After cutting
The molecular weight was fractionated by agarose electrophoresis, and a DNA fragment of about 1.3 kb was purified from an agarose gel using a DNA purification kit, GENCLEAN II (Funakoshi). On the other hand, Bluescript KS (Stara
0.5 μg (made by tagene) was reacted with restriction enzyme Hind III and EcoRI10 unit at 37 ° C for 1 hour and completely cleaved, and then T4-DNA
Hind around 1.3 Kb purified from the above agarose gel by reacting with 1 unit of ligase (Toyobo) at 16 ℃ for 12 hours.
Ligated with the III-EcoRI fragment.

The ligated DNA is Escherichia coli (E
Transformation was performed using competent cells of scherichia coli) HB101. The obtained colony is ampicillin 50μ
Selection was performed on an LB agar medium containing g / ml to obtain a colony showing ampicillin resistance and expressing NAL activity. A 1.3 kb inserted DNA fragment was present in this plasmid, and this plasmid was designated as pNAL11. Figure 1
Described the construction of pNAL11.

Next, 0.5 μg of pNAL11 was added to the restriction enzyme H
inc II (Toyobo) and SmaI (Toyobo) 10 units and 3
After reacting at 7 ° C for 1 hour and completely cleaving, T4-DN
The reaction was carried out with 1 unit of A ligase at 16 ° C. for 12 hours, and the cut DNAs were ligated to obtain pNAL11R in which the 1.3 kb insert DNA was inserted in the reverse direction.

Next, 0.5 μg of pNAL11R was added to the restriction enzyme B.
amHI (Toyobo) and Xho I (Toyobo) 10 units
After reacting at 37 ℃ for 1 hour and completely cutting,
pTS1137 cleaved with mHI and Xho I
Shown in. J. Bacteriol., 166 (3), 739-745 (1986)) 0.5 μg was reacted with 1 unit of T4-DNA ligase for 12 hours to ligate DNA. The ligated DNA was transformed with Escherichia coli HB101 competent cells. The obtained colonies were selected on LB agar medium containing 50 μg / ml of streptomycin to obtain streptomycin-resistant colonies expressing NAL activity. A 1.3 kb inserted DNA fragment was present in this plasmid, and this plasmid was designated as pNAL.
It was set to 51. The construction of pNAL51 is described in FIG.

(2) Introduction of pNAL51 into Pseudomonas putida As a host microorganism, Pseudomonas putida mt-2 (ATCC330
15) was used to introduce pNAL51 by electroporation using Gene Pulser (manufactured by BIO-RAD). The conditions for electroporation were according to the conditions of Pseudomonas putida in the Gene Pulsar User Manual. Transformants were selected on LB agar medium containing 200 μg / ml of streptomycin to obtain streptomycin-resistant transformants expressing NAL activity. This transformant, Pseudomonas putida (pNAL51), retained the recombinant plasmid pNAL51.

(3) Production of NAL by transformant 50 ml of LB medium was dispensed into a 500 ml Sakaguchi flask, and 121
After autoclave sterilization for 15 minutes at ℃, let it cool, then 200
The above transformant, Pseudomonas putida (pNAL51), was inoculated into a medium containing streptomycin at a concentration of μg / ml and shake-cultured at 30 ° C. for 18 hours. After culturing, the cells were disrupted by ultrasonic treatment, and the NAL activity per 1 ml of the medium was measured. As a control experiment, Pseudomonas putida and Escherichia coli HB101 (pNAL51) that do not retain pNAL51 were similarly cultured and the NAL activity was measured. The results obtained are shown in Table 1.

[0023]

[Table 1]

As is clear from Table 1, the transformant of the present invention, Pseudomonas putida (pNAL51), exhibited NAL productivity about 3 times higher than that using Escherichia bacteria as a host.

Example 2 Production of NAL by Serratia bacterium (1) Preparation of recombinant plasmid pNAL11 prepared in Example 1 was used. (2) Introduction of recombinant plasmid pNAL11 into Serratia spp. Bacteria As host microorganism, Serratia liquifaciens IFO
The recombinant plasmid pNAL11 was introduced by electroporation in the same manner as in Example 1 using 12979 and Serratia primica JCM 1244. The conditions for electroporation were the same as those for Pseudomonas putida. The transformants were selected on LB agar medium containing 100 μg / ml of ampicillin and showed resistance to ampicillin.
A transformant expressing NAL activity was obtained. These transformants, Serratia liquifaciens (pNAL11) and Serratia primtika (pNAL11), retained the recombinant plasmid pNAL11.

(3) Production of NAL by transformant 100 ml of LB medium was dispensed into a 500 ml Sakaguchi flask, and 12
Perform autoclave sterilization at 1 ° C for 15 minutes, allow to cool, then
The above transformants and Serratia liquifaciens were added to a medium containing ampicillin at 0 μg / ml.
(pNAL11), Serratia primica (pNAL11) inoculated, 30
The cells were shake-cultured at 24 ° C for 24 hours. After culturing, by ultrasonic disruption,
The cells were disrupted and the NAL activity per 1 ml of medium was measured.
As a control experiment, NAL activity was measured by similarly culturing Serratia liquifaciens, Serratia primtika and Escherichia coli HB101 (pNAL11) which did not retain pNAL11. The obtained results are shown in Table 2.

[0027]

[Table 2]

As is clear from Table 2, the transformant of the present invention, Serratia liquifaciens (pNAL11), is about 7.5 times as high as that using Escherichia bacteria as a host, and Serratia primtica (pNAL11) is about 3.2 times. It showed twice as much NAL productivity.

Example 3 Production of NAL by Serratia bacterium (1) Preparation of recombinant plasmid pNAL11 prepared in Example 1 (1 μg) was treated with a restriction enzyme,
HindIII and ScaI (Toyobo) 10 units each at 37 ℃, 1
After reacting for a period of time and completely cleaving, molecular weight fractionation was performed by agarose gel electrophoresis, DNA purification kit, GENECLEA
About 1.1 kb of HindIII-ScaI from N-II agarose gel
The fragment was purified. On the other hand, 0.5 μg of Bluescript KS was completely cleaved with 10 units each of HindIII and SmaI restriction enzymes, and then reacted with 1 unit of T4-DNA ligase at 16 ° C for 12 hours. -ScaI
Ligated with the fragment. The ligated DNA was transformed with Escherichia coli HB101 competent cells. The resulting colonies were LB containing 50 μg / ml ampicillin.
By selecting on an agar medium, a colony showing ampicillin resistance and expressing NAL activity was obtained. A 1.1 kb insert fragment DNA was present in this plasmid, and this plasmid was designated as pNAL71. Figure 4 shows pNAL71
Described the construction of.

(2) Introduction of recombinant plasmid pNAL71 into Serratia bacteria As a host microorganism, Serratia liquefiaciens IFO
Using 12979 and Serratia primica JCM 1244, the recombinant plasmid pNAL71 was introduced by the electroporation method as in Example 1. The conditions for electroporation were the same as those for Pseudomonas putida. The transformants were selected on LB agar medium containing 100 μg / ml of ampicillin and showed resistance to ampicillin.
A transformant expressing NAL activity was obtained. These transformants, Serratia liquifaciens (pNAL71) and Serratia primtika (pNAL71), retained the recombinant plasmid pNAL11.

(3) Production of NAL by transformant 100 ml of LB medium was dispensed into a 500 ml Sakaguchi flask, and 12
Perform autoclave sterilization at 1 ° C for 15 minutes, allow to cool, then
The above transformants and Serratia liquifaciens were added to a medium containing ampicillin at 0 μg / ml.
(pNAL71), Serratia primica (pNAL71) inoculated, 30
The cells were shake-cultured at 24 ° C for 24 hours. After culturing, by ultrasonic disruption,
The cells were disrupted and the NAL activity per 1 ml of medium was measured.
As a control experiment, NAL activity was measured by similarly culturing Serratia liquifaciens, Serratia primtika and Escherichia coli HB101 (pNAL71) that did not retain pNAL71. The results obtained are shown in Table 3.

[0032]

[Table 3]

As is clear from Table 3, the transformant of the present invention, Serratia liquifaciens (pNAL71), is about 7.8 times higher than that using Escherichia bacterium as a host, and Serratia primtika (pNAL71) is about 3.3 times. It showed twice as much NAL productivity.

Example 4 Production of NAL by Serratia spp. LB medium 61 was dispensed into 101 jar-famentor,
Autoclave for 15 minutes at 1 ° C, let stand to cool, and then use 100 mg / ml ampicillin (manufactured by Nacalai Tex) separately sterile filtered.
6 ml was added. Add the same composition as above to this medium beforehand.
Serratia liquefaciens cultured at 30 ℃ for 24 hours with shaking
60 ml of the culture solution of (pNAL71) was inoculated, and the culture was carried out at 30 ° C. for 24 hours with aeration and stirring. The NAL activity after the culture was 5.8 U / ml.

6 l of the culture broth was collected by centrifugation, suspended in 200 ml of 50 mM phosphate buffer (pH 7.5), crushed with a Dynomill crusher by a conventional method, and then centrifuged at 15,000 rpm for 20 minutes. A NAL crude enzyme solution was obtained. The crude enzyme solution thus obtained was subjected to nucleic acid removal treatment with protamine and then subjected to salting out fractionation with ammonium sulfate. Salting out the precipitate
It was dissolved in 50 ml of 50 mM phosphate buffer (pH 7.5) and heated at 55 ° C for 16 hours. Sephadex G for heating solution
After desalting with -25, the product was subjected to DEAE Sepharose CL-6B chromatography and a NAL active fraction was obtained.

The NAL fraction after DEAE Sepharose chromatography showed a single band on SDS-PAGE electrophoresis and its specific activity was 58.4 U / mg. The activity yield is
It was 55.8%. Table 4 shows a summary of the above purification.

[0037]

[Table 4]

Comparative Example 1 For comparison, Escherichia coli HB101 (pNAL7
Using 1), the same purification was carried out on the same scale as in Example 4. The results are shown in Table 5.

[0039]

[Table 5]

As is clear from Tables 4 and 5, according to the present invention, even with the same culture scale, about 9-fold NAL can be obtained more easily and with higher purity.

[0041]

INDUSTRIAL APPLICABILITY In the present invention, a large amount of N can be obtained by using a microorganism belonging to Gram-negative bacteria other than Escherichia, Pseudomonas bacteria or Serratia bacteria as a host.
It has become possible to produce AL. Furthermore, by using as a host a microorganism belonging to Gram-negative bacteria that grows at a lower temperature than the Escherichia bacterium, by producing NAL,
It has become possible to produce NAL easily and with high purity.

[0042]

[Sequence list]

SEQ ID NO: 1 Sequence length: 1243 Sequence type: nucleic acid (DNA) Number of chains: double-stranded Topology: linear Sequence type: genomic DNA origin Organism name: Escherichia coli Share name: JE1011 Array AAGCTTTCTG TATGGGGTGT TGCTTAATTG ATCTGGTATA ACAGGTATAA AGGTATATCG 60 TTTATCAGAC AAGCATCACT TCAGAGGTAT TT ATG GCA ACG AAT TTA CGT GGC 113                                     Met Ala Thr Asn Leu Arg Gly                                       1 5 GTA ATG GCT GCA CTC CTG ACT CCT TTT GAC CAA CAA CAA GCA CTG GAT 161 Val Met Ala Ala Leu Leu Thr Pro Phe Asp Gln Gln Gln Ala Leu Asp          10 15 20 AAA GCG AGT CTG CGT CGC CTG GTT CAG TTC AAT ATT CAG CAG GGC ATC 209 Lys Ala Ser Leu Arg Arg Leu Val Gln Phe Asn Ile Gln Gln Gly Ile      25 30 35 GAC GGT TTA TAC GTG GGT GGT TCG ACC GGC GAG GCC TTT GTA CAA AGC 257 Asp Gly Leu Tyr Val Gly Gly Ser Thr Gly Glu Ala Phe Val Gln Ser  40 45 50 55 CTT TCC GAG CGT GAA CAG GTA CTG GAA ATC GTC GCC GAA GAG GCG AAA 305 Leu Ser Glu Arg Glu Gln Val Leu Glu Ile Val Ala Glu Glu Ala Lys                  60 65 70 GGT AAG ATT AAA CTC ATC GCC CAC GTC GGT TGC GTC AGC ACC GCC GAA 353 Gly Lys Ile Lys Leu Ile Ala His Val Gly Cys Val Ser Thr Ala Glu              75 80 85 AGC CAA CAA CTT GCG GCA TCG GCT AAA CGT TAT GGC TTC GAT GCC GTC 401 Ser Gln Gln Leu Ala Ala Ser Ala Lys Arg Tyr Gly Phe Asp Ala Val          90 95 100 TCC GCC GTC ACG CCG TTC TAC TAT CCT TTC AGC TTT GAA GAA CAC TGC 449 Ser Ala Val Thr Pro Phe Tyr Tyr Pro Phe Ser Phe Glu Glu His Cys     105 110 115 GAT CAC TAT CGG GCA ATT ATT GAT TCG GCG GAT GGT TTG CCG ATG GTG 497 Asp His Tyr Arg Ala Ile Ile Asp Ser Ala Asp Gly Leu Pro Met Val 120 125 130 135 GTG TAC AAC ATT CCA GCC CTG AGT GGG GTA AAA CTG ACC CTG GAT CAG 545 Val Tyr Asn Ile Pro Ala Leu Ser Gly Val Lys Leu Thr Leu Asp Gln                 140 145 150 ATC AAC ACA CTT GTT ACA TTG CCT GGC GTA GGT GCG CTG AAA CAG ACC 593 Ile Asn Thr Leu Val Thr Leu Pro Gly Val Gly Ala Leu Lys Gln Thr             155 160 165 TCT GGC GAT CTC TAT CAG ATG GAG CAG ATC CGT CGT GAA CAT CCT GAT 641 Ser Gly Asp Leu Tyr Gln Met Glu Gln Ile Arg Arg Glu His Pro Asp         170 175 180 CTT GTG CTC TAT AAC GGT TAC GAC GAA ATC TTC GCC TCT GGT CTG CTG 689 Leu Val Leu Tyr Asn Gly Tyr Asp Glu Ile Phe Ala Ser Gly Leu Leu     185 190 195 GCG GGC GCT GAT GGT GGT ATC GGC AGT ACC TAC AAC ATC ATG GGC TGG 737 Ala Gly Ala Asp Gly Gly Ile Gly Ser Thr Tyr Asn Ile Met Gly Trp 200 205 210 215 CGC TAT CAG GGG ATC GTT AAG GCG CTG AAA GAA GGC GAT ATC CAG ACC 785 Arg Tyr Gln Gly Ile Val Lys Ala Leu Lys Glu Gly Asp Ile Gln Thr                 220 225 230 GCG CAG AAA CTG CAA ACT GAA TGC AAT AAA GTC ATT GAT TTA CTG ATC 833 Ala Gln Lys Leu Gln Thr Glu Cys Asn Lys Val Ile Asp Leu Leu Ile             235 240 245 AAA ACG GGC GTA TTC CGC GGC CTG AAA ACT GTC CTC CAT TAT ATG GAT 881 Lys Thr Gly Val Phe Arg Gly Leu Lys Thr Val Leu His Tyr Met Asp         250 255 260 GTC GTT TCT GTG CCG CTG TGC CGC AAA CCG TTT GGA CCG GTA GAT GAA 929 Val Val Ser Val Pro Leu Cys Arg Lys Pro Phe Gly Pro Val Asp Glu     265 270 275 AAA TAT CTG CCA GAA CTG AAG GCG CTG GCC CAG CAG TTG ATG CAA GAG 977 Lys Tyr Leu Pro Glu Leu Lys Ala Leu Ala Gln Gln Leu Met Gln Glu 280 285 290 295 CGC GGG TGAGTTGTTT CCCCTCGCTC GCCCCTACCG GTGAGGGGAA ATAAACGCAT 1033 Arg Gly     297 CTGTACCCTA CAATTTTCAT ACCAAAGCGT GTGGGCATCG CCCACCGCGG GAGACTCACA 1093 ATGAGTACTA CAACCCAGAA TATCCCGTGG TATCGCCATC TCAACCGTGC ACAATGGCGC 1153

[Brief description of drawings]

FIG. 1 shows a construction diagram of plasmid pNAL11.

FIG. 2 shows a construction diagram of plasmid pNAL51.

FIG. 3 shows a construction diagram of plasmid pTS1137.

FIG. 4 shows a construction diagram of plasmid pNAL71. >

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI (C12N 1/21 C12N 15/00 ZNAA C12R 1: 425) (C12N 9/88 C12R 1:38) (C12N 9/88 C12R 1 : 425) (C12N 15/09 ZNA C12R 1:19) (56) Reference Nulic Acids Research arch, 1985, Vol. 13, No. 24, p. 8843-8852 Agric. Biol. Chem. , 1986, Vol. 50, No. 8, pp. 2155-2158 (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 15/09-15/90 C12N 1/21 C12N 9/88

Claims (2)

(57) [Claims] 1. A Shudomo introduced a recombinant plasmid incorporating the DNA of the vector carrying the N- acyl neuraminic acid aldolase-producing genetic information derived from bacteria belonging to the genus Escherichia
A microorganism belonging to the genus Solanum or the bacterium Serratia . 2. A Pseudomona in which a recombinant plasmid in which a DNA carrying genetic information for producing N-acylneuraminic acid aldolase derived from a bacterium belonging to the genus Escherichia is incorporated is introduced.
A microorganism belonging to the genus Solanum or a bacterium of the genus Serratia is cultured in a medium to produce N-acylneuraminic acid aldolase in the culture, and N-acylneuraminic acid aldolase is collected from the culture. Method for producing acid aldolase.