JPS63129984A - Alpha-amino-epsilon-caprolactam racemase - Google Patents

Abstract

PURPOSE:To improve the productivity of a microbial strain capable of producing alpha-amino-epsilon-caprolactam (ACL), by producing an ACL racemase having a specific amino acid sequence and a DNA sequence coding said racemase. CONSTITUTION:A gene DNA coding alpha-amino-epsilon-caprolactam (ACL) racemase is produced from a chromosome DNA of Achromobacter oobae (FERM P-776) or from a composite plasmid DNN36. A recombinant E.coli transformed with a composite plasmid DNN40 having structural gene of Achromobacter oobae or enzyme can be used as a bacterial strain capable of producing ACL racemase. The objective ACL racemase can be easily extracted from the recombinant E.coli and purified.

Description

Detailed Description of the Invention [Industrial Field of Application] The present invention relates to α-amino-ε-caprolactam (hereinafter referred to as ACL).
)・Relates to racemase and the DNA sequence encoding it.

[Prior art] ACL racemase is a useful substance used in the production of L-lysine using D or DL-ACL as a compound due to its synergistic action with L-ACL hydrolase (Ding, Fuk
umura,Agr,Biol,chcm,,41,
p1327-1330 (1977)).

Conventionally, Achromobacter obae (Achromobacter obae) has been used as a microorganism that improves ACL racemase production ability.
Several strains have been reported, including terobae).

Fukumura, Agr, Biol, Chem,,
41, ri 1321-1325 (1977)).

If the gene encoding ACL racemase is extracted from these known microorganisms and genetic engineering techniques are applied, it is expected that the productivity of the enzyme will be dramatically improved.

[Problems to be solved by the invention] In view of the above situation, the inventors have devised Ω1 and solved A.
The inventors succeeded in determining the entire amino acid sequence of the CL racemase protein and the base sequence of the gene DNA encoding it, thereby completing the present invention.

[Means for solving the problems] That is, the present invention provides AC
We provide L racemase and genetic DNA encoding it.

The amino acid sequence of the enzyme of the present invention is as shown in FIG. 1, and an example of the base sequence of the gene DNA encoding it is as shown in FIG.

The present invention will be explained in detail below.

Genetic DNA encoding ACL racemase can be obtained from the chromosomal DNA of Achromobacter obae (Feikokukenboku No. 776) or from the composite plasmid pNN36 described in JP-A-61-202693. It is more convenient to use pNN36.

〇L racemase producing bacteria include the bacterium Acro [Bacter obae (Feikoken Bacterial No. 776)] or the silk-modified canine enteric bacterium transformed with complex plasmid DNN40 having the structural gene of the enzyme (patent application). 1986-80469>
can be used, but it is more convenient to use recombinant E. coli. ACL racemase can be easily extracted and purified from this recombinant E. coli.

The method for purifying the ACL racemase used in the present invention is not particularly limited, but may be
9>Centrifuge the supernatant of the French press crushed liquid at 65°C11.
After heat denaturation for 0 minutes, centrifugation, the centrifuged supernatant was applied to a column using anion exchange resin uDEAF TOYOPEARL (manufactured by Toyo Soda Co., Ltd.), and the active fraction obtained by chromatography was transferred to a high-performance liquid A simple method of purification is through chromatography (hereinafter abbreviated as HPLC).

When preparing a purified enzyme with a higher ω content, the active fraction after chromatography using “DEAE Toyopearl” is subjected to Gelshi filtration column chromatography using “Sephacryl S-200” (manufactured by Pharmacia), or It can be purified by crystallization using ammonium sulfate.

For example, the amino acid sequence of a protein is given by Susumu Amizawa, Biochemistry, 57.
．． 472-480 (1985). - For example, a purified enzyme is digested with lysyl endopeptidase, the digested product is fractionated by HPLC, and the fractionated protein fragments are run on a commercially available protein sequencer using the Edman degradation method to determine the amino acid sequence of the protein fragment. be able to. The amino acid sequences obtained in this way are compared with the base sequence of the gene DNA and linked, and furthermore, both front sequences can be mutually confirmed. Even if the amino acid sequence is unknown, the amino acid sequence can be estimated from the DNA base sequence.

For example, the analysis of the WW sequence of DNA is performed using 5an(J(!r'
, F.

et at, Proc, Hat, ^cad, S
ci, U, S, 74.5463-67 (1977
), but it is more efficient to use commercially available M13 cloning kits and M13 sequencing kits, as well as commercially available base sequence input devices and automatic editing devices.

The activity can be measured using an optical rotation needle according to the method disclosed in JP-A-60-244289.

[Example] EXAMPLES The present invention will be described in more detail below with reference to Examples.

Example 1 (1) Purification of ACL racemase: A DNA fragment having the structural gene of ACL racemase was
Commercially available plasmid pKK2 with tac promoter
ACL racemase high-producing E. coli JM105 (
pNN40) (Japanese Patent Application No. 61-80469) was added to 2YT medium (1.6% tryptone, 1% yeast extract, 0.5%
Cultures were grown overnight in Sakaro flasks at 37°C in sodium chloride, pH 7.5. This was diluted 300 times with 2YT medium, cultured aerobically, and when the bacterial concentration reached 5 x 108/Inl, IPTG (1sopropylβ-D-t
hio-OatactO3ide) was added to the medium to a concentration of 1 mM, and after further culturing for 14 hours, the cells were collected by centrifugation. 80g of collected bacteria is 10μg/mf! DNAs of
After suspending e in 2 g of 0.25 M sucrose-2X10-4 M pyridoxal phosphate-1.5% mercaptoethanol-M potassium phosphate buffer (pH 7.2), press in a continuous French press (Lanton Gaulin). ,A
The bacterial cells were disrupted using PV Gaulin (manufactured by Gaulin). The centrifuged supernatant of this disrupted solution was used to obtain a protein concentration of 2 mg/d! using the same solution in which the bacteria were suspended. After diluting to 65°C
Heated for 10 minutes. A commercially available protein assay kit (manufactured by Bio-Rad) was used to quantify the protein concentration.

9. of heat-treated liquid. OOOXg, on centrifugation for 15 minutes? ff
10x ff1 (7) 0.25M 1m -2X 1
0-”M pyridoxal phosphate-0.01% mercaptoethanol-10mM potassium phosphate buffer (pH 7,
After dialysis against 2> at 4°C for 2 days, it was charged into 11 DEAE Toyopearl 650M which had been equilibrated with a solution containing the same components as the external dialysis solution.

After washing the column with 1g of 0.05M KCl
It was eluted with MKCI and fractionated using a fraction collector. For both components having ACL racemase activity, Takeshi Horio et al. B [Basic Experimental Methods for Proteins and Enzymes J I) 275
Purity was examined by 5DS-polyacrylamide gel electrophoresis according to the method described in J.-279 (1981). Molecular weight 4
In addition to the band of ACL racemase with a size equivalent to 5,000,000, the enzyme was purified by applying reversed phase 1-I PLC to purify the enzyme.

That is, protein reservation 80/! ! 7 was dissolved in 0.1% TFA (trifluoroacetic acid) and applied to a reverse phase column "Inerts".
The enzyme protein having a MW of 45,000 yen was fractionated by elution using a 45-60% acetonitrile concentration gradient.

(2) Fragmentation of enzyme protein: The enzyme protein purified and separated in the previous section was mixed with 3mM dithiothreitol-6M urea-0.5MT to give a concentration of 4μ3/μg.
Dissolved in ris-HCI (pH 8.4>, 45°C, 2
After incubating for 4 hours under a nitrogen blanket, 1/4 volume of 40 mM iodoacetamide solution was added and incubated in the dark at room temperature for 20 minutes. To this was added 2.5 times the amount of 4M urea-0,2M2-amino-2-methyl-1,3-
Propanediol °(p)-19,5> and 0.04△U
Add lysyl endopeptidase (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.),
Incubated overnight at 37°C. Dissolve the reaction solution equivalent to 80μ3 of protein in 0.1% TFA and apply it to the reverse phase column.
TSK-ODS-80TM column'' (4,6X150m
(manufactured by Toyo Soda Co., Ltd.) and eluted by applying a concentration gradient of 20 to 60% acetonitrile. The elution pattern is shown in FIG.

(3) Determination of the amino acid sequence of protein fragments: Both fractions containing the protein fragments were separated from each other by Applied Biosystems.
P T
H amino acids from Shimadzu ■High-speed liquid chromatography LC-4A
” using C, 11, W, 1lir et at
,.

Methods in Enzymology, 91
．． 486-493 (1983). The 47 amino acids on the N-terminal side carry purified enzyme at 2 m9/ri.
Dissolve dl in 0.1% SDS solution and add 6
0 μρ was applied to the sequencer.

(4) Analysis of the primary structure of genetic DNA: Composite plasmid pNN32 containing genetic DNA
plasmid p containing the ACL racemase structural gene based on the method of JP-A No. 61-202693.
NN36 was constructed, and a 3.5 Kb ECORI-DNA fragment containing the gene was prepared from it. This fragment of DNA
The base sequence was analyzed using the so-called shotgun sea fencing method described in JP-A-61-202693.

(5) Determination of amino acid sequence of protein and base sequence of gene DNA: In the DNA sequencing described in item (4) of the previous section, G-C
In parts of the DNA base sequence where there are many nucleotides, the autoradiograph ladder is difficult to read, making it difficult to determine the base sequence using only the shotgun method.

Then, the deciphered female part was analyzed with reference to the amino acid sequence of the protein fragment determined by the sea fencing method described in item (3) of the previous section, and the nucleotide sequence was determined. The N-terminal and C-terminal base sequences were also determined from the amino acid sequence of the protein fragment.

Figure 2 shows the entire determined gene DNA sequence and the positions of the corresponding protein fragments. The number inside the circle in the diagram is number 3.
The peaks in the figure are shown.

[Brief explanation of the drawing]

FIG. 1 shows the entire amino acid sequence of α-amino-ε-caprolactam racemase, and FIG. 2 shows the entire base sequence of the gene DNA encoding said racemase. Figure 3 shows the elution pattern of the racemase.

Claims (3)

[Claims] (1) α-Amino-ε-caprolactam racemase having the amino acid sequence shown in FIG. 1 and its equivalent. (2) DN encoding α-amino-ε-caprolactam racemase having the amino acid sequence shown in Figure 1
A array. (3) The DNA sequence according to claim (2), which is the base sequence shown in FIG.