JPS61202693A - Gene to code alpha-amino-epsilon-caprolactam racemase - Google Patents

Abstract

PURPOSE:To provide high racemase production ability, by forming a gene which has a specific amino acid sequence at N-end side and codes alpha-amino-epsilon- caprolactam racemase having a specific molecular weight. CONSTITUTION:DNA fragment of a mold capable of producing ACL is transformed with a plasmid bonded to a plasmid vector of Escherichia coli, to give a transformant of Escherichia coli lysine demanding strain. The transformant is selectively cultivated in a medium containing L-ACL hydrolase and D-ACL, and a transformant having ACL racemase activity is selected from the multiplied transformant. A plasmid is selected from the this transformant, to give recombinant DNA. The recombinant DNA has a restriction map shown by the figure.

Description

Detailed Description of the Invention [Industrial Field of Application] The present invention relates to α-amino-C-caprolactam (hereinafter referred to as ACL).
abbreviated as ) gene encoding racemase, D containing it
The present invention relates to an NA fragment and a recombinant DNA containing the DNA fragment.

[Conventional technology]

ACL racemase is a useful substance that is used in the production of monolysine from D-1 or DL-ACL as a raw material through its synergistic action with L-ACL hydrolase. (Reference 1) Conventionally, kcaligenes fae (4 = kcaligenes fae) which has the ability to produce ACL racemase
calis) has been reported (Reference 2).

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 improved.

Therefore, the present inventors attempted to clone the gene encoding the enzyme from a bacterium capable of producing the enzyme and use it to increase the productivity of the enzyme. As a general gene cloning method, a strain that has the ability to produce ACL racemase is mutated to create a strain lacking the enzyme, and this is used as a host, and self-cloning is performed using the recovery of the D-ACL assimilation ability of the host as an indicator. i using the method of ACL racemase antibody
Possible methods include the n5itu iI1 munoassay method (for example, Reference 3), or the colony hybridization method using as a probe an oligo DNA having a deduced nucleotide sequence based on the amino acid sequence of ACL racemase (for example, Reference 4).

However, no suitable plasmid or transformation method was found for transforming the ACL racemase-producing bacteria.
Furthermore, it has not been easy to prepare large quantities of purified enzymes for use in antibody preparation and amino acid sequencing.

[Problem that the invention seeks to solve]

The present inventors have devised an ingenuity in view of the above situation, and found that if the gene encoding ACL racemase on an intracellular plasmid is expressed in a host E. coli having an L-lysine auxotrophic mutation, L-ACL hydrolysis In the coexistence of the enzyme, the host proceeded with the D study, and as a result succeeded in acquiring a plasmid containing the gene encoding the enzyme.
Furthermore, they succeeded in identifying the N-terminal amino acid sequence of the enzyme and determining the DNA base sequence encoding it, thus completing the present invention.

[Means for solving problems]

That is, the present invention provides a DNA fragment containing a gene encoding ACL racemase and a recombinant DNA containing the DNA fragment.

The gene of the present invention has an amino acid sequence on the N-terminal side of Thr-Lys-8Ia-Leu-Tyr-Asp-A.
rg-Asp-Gly-Ala-Ala-11e-Gl
y-Asn-Leu-Gly-Lys-Leu-＾rg
-Phe-Phe-! l:ro-Leu-Ala-11
e-Ser-Gly-Gly-Arg-Gly-^1a
-Arg-Leu-11e-Glu-Glu-Asn-
Gly-Arg-Glu-Leu-11e-Asp-L
ea-Ser-Gly-Ala, with a molecular weight of 4.5
Encodes ACL racemase of ×104 daltons and approximately 1
, has 24 eb bases.

The recombinant DNA of the present invention can be produced as follows. That is, a transformant of an Escherichia coli lysine auxotrophic strain transformed with a plasmid in which a DNA fragment of a bacterium capable of producing ACL racemase was ligated to an E. coli plasmid vector was treated with L-ACL hydrolase and D-
The recombinant DNA of the present invention can be obtained by selectively culturing in a medium containing ACL, selecting a transformant having ACL racemase activity from the proliferated transformants, and extracting a plasmid from this transformant. obtain.

The present invention will be explained in detail below.

As a DNA donor, the bacterium Achromobacter obae (Fiber Science Laboratory No. 77), which has the ability to produce ACL racemase, was used.
No. 6) is used.

Extraction and purification of DNA carrying genetic information encoding ACL racemase from bacterial cells can be carried out, for example, by the Saito method described in Reference 5.
This can be carried out according to a conventional method such as the Miura method.

In this case, the lysis process using a surfactant is carried out at 30-65°C.
If the time is 20 to 60 minutes, the yield of DNA can be further increased. Fragmentation of purified DNA was performed by Hindll, E.
A commercially available restriction enzyme such as C0RI can be used, and the enzyme reaction conditions can be based on, for example, the enzyme manufacturer's manual or the simple method described in Reference 6.

Vectors used in the present invention are not limited, but for example, pBR3
22 and pAcYc184, whose properties have been well investigated, are convenient. All are known plasmids, for example, available from ATCC, and each is AT
It has been deposited as CC37017 and ATCC37033. Replication and purification of the plasmid can be carried out by conventional methods such as those described in Reference 8.

The DNA fragments can be joined by a conventional method using T4 DNA ligase as described in Reference 7, for example. At this time, in order to suppress self-circularization of the vector, it is desirable to treat the restricted ends of the vector with alkaline phosphatase to remove phosphate residues.

The produced recombinant DNA is introduced into a host by a conventional transformation method. For example, the transformation method using Rubidium described in Reference 9 can be used.

As a host, a lysine-auxotrophic mutant strain of Escherichia coli that is unable to assimilate D-ACL as a nitrogen or carbon nutrient source is used.

For example, MM294 (ATCC3362
5) with common mutagens such as N-methyl-N'-nitro N-nitrosoguanidine (abbreviated as NT
G) to induce mutations, and can be obtained by using the usual penicillin screening method as described in Reference 10, for example.

The gene encoding ACL racemase, more precisely A
Transformants containing recombinant DNA containing the gene encoding the apoenzyme of CL racemase can be selected as follows.

In other words, it is a minimal medium that complements the host's nutritional requirements other than lysine, and it also contains 20 to 500 μg/tal of D-AC.
First, transformants that grow in a screening medium containing L, L-ACL, and a hydrolase are selected.

The amount of L-ACL hydrolase to be used is preferably 10 to 100 units/ml, as long as it does not allow the host to proliferate independently of its own auxotrophic markers by adding it to the medium. Used within the range of

The unit here refers to 0.4ml of 2mM containing the enzyme.
MnCLz 10%W/VL-ACL-HCI (
When the pH 8) was incubated at 40°C for 1 hour, 0.
Enzyme activity that produces 1 μmol of lysine is defined as 1 unit. The method described in Reference 11 can be used to prepare L-ACL hydrolase.

I can do it. Next, the ACL racemase activity of the transformants grown in this screening medium is examined, and transformants having the ability to produce ACL racemase are selected. As a method for measuring the activity, a photometer can be used, for example, as described in Reference 12. Confirmation that the activity is derived from a plasmid can be confirmed, for example, by re-transforming a host using a plasmid obtained by the ordinary plasmid small quantity extraction method described in Reference 13, and examining the activity of the resulting transformant. be.

The gene of the present invention and the plasmid pNN36 containing it can be produced by the following method. That is, A.C.
A lysine auxotrophic mutant strain of Escherichia coli was transformed using a plasmid in which a bacterial DNA fragment capable of producing L racemase was incorporated into the vector pBR322, and this transformant was injected with L-ACL hydrolase and D-ACL. A transformant having ACL racemase activity was obtained by selective culturing in a minimal medium that supplements nutritional requirements other than lysine, and pNN3, one of the plasmids extracted from the transformant, was digested with EcoRI. , the resulting DNA fragment was integrated into plasmid pACYC184, and A
A plasmid pNN32 having the ability to produce CL racemase was formed, and this composite plasmid was truncated using HindnI, and the old nd
I
Plasmid pNN36 is obtained by integrating into this site.

pNN32 is a transformant of Escherichia coli (Feikoken Bacteria No. 761
No. 2) has been deposited with the Institute of Fine Technology.

The gene encoding ACL racemase can be identified by DNA base sequence analysis and enzyme protein amino acid sequence analysis.

DNA nucleotide sequencing can be performed, for example, by the usual method described in Reference 15, but it is more efficient to use commercially available M13 cloning kits and M13 sequencing kits, as well as commercially available nucleotide sequence input devices and automatic editing devices.

The N-terminal amino acid sequence of a protein can be easily determined using a commercially available protein sequencer using the Edman degradation method.

A transformant having the ability to produce ACL racemase is L-A
When used in combination with CL hydrolase, D-ACL or DL-ACL can be converted to L-lysine. The method can be performed, for example, according to Document 1.

L-ACL hydrolase is, for example, Cryptococcus
It can be produced from Candida fumicola (M1 Lab. Humicola No. 717) and Trichosporon cutaneum (M1 Lab. Humicola No. 714), and can be used as an enzyme in the synthesis reaction of L-lysine. In addition, it can also be used as a bacterial cell extract, treated bacterial cells, live bacterial cells, and culture (liquid) of these producing bacteria.

Furthermore, it is of course possible to use not only a transformant having the ability to produce ACL racemase, but also the ACL racemase produced by the transformant in the reaction.

EXAMPLES The present invention will be explained in more detail with reference to Examples below.

Example 1 (1) Extraction of DNA from ACL racemase producing bacteria
Mobaku name: Obae (Feikoken Bibori No. 776) as LB
Culture medium (tryptone 1%, yeast extract 0.5%, sodium chloride 1%, pH 7.5) in IA at 32°C.
．． 3ml containing 15mg lysozyme hydrochloride of late log phase bacterial cells cultured aerobically for 5 hours! (7) 0.15MN
a CI-0,1 MEDTA-50mMTri
Suspended in 5-HCI (pH 8), incubated at 37°C for 15 minutes, frozen and thawed, and then 25 ml
0゜1M Tris-HCI (pH9) 1%SD
S-0,1M NaCI was added and incubated at 60°C for 20 minutes to dissolve. Thereafter, the method of 5aito-λ M+ura (Reference 5) was applied to obtain 700μg of DN.
I got an A.

(2) Digestion and fractionation of DNA with restriction enzymes DNA obtained in (1) at 0.25 μg/μl under the reaction conditions described in Reference 6.
0.12 units/p It of restriction enzyme old ndl [
The digested product (125 μg of DNA) digested with I for 2 hours was centrifuged in a 12 ml 10-40% density gradient of 0.5
A fraction of each fraction was collected.

Centrifugation was carried out using a Hitachi Garlic 540T rotor at 20°C, 25.
00Orpm+ 24 hours. The length of each DNA was measured by electrophoresis, and 2 to 1 Ojb b fractions were ligated to a plasmid. Restriction enzymes manufactured by Takara Shuzo ■ were used, and the activity units were as defined in the attached manual.

(3) Preparation of vector DNA Plasmid vector pBR322 is derived from E. coli MM294.
Duplicated within. Replication and extraction and purification from bacterial cells were performed using LB containing 5 tag/l of tetracycline according to Reference 8.
Plasmid-containing bacterial cells cultured in a medium are treated with lysozyme/SD.
After lysis with S, the cells were extracted with phenol and purified by cesium achloride-ethidium chemical romide density gradient equilibrium centrifugation and biogel column chromatography. 520 μg of pBR322 was obtained per 11 mediums. In order to use it as a vector, it was completely degraded using the restriction enzyme ll1ndI[I under the reaction conditions described in Reference 7, and then treated with alkaline phosphatase according to Reference 7.

(4) Preparation of host E. coli MM294 in the logarithmic phase cultured in 5 ml of LB medium at 37°C for 3 hours was cultured according to the literature 1O.
After mutagenesis with 100 μg/ml NTG in M buffer, the lysine auxotrophic strain E, coli MM294Lys−, was isolated using the penicillin screening method. This strain was deficient in diaminopimelic acid decarboxylase activity.

(5) DNA ligation and transformation 0.5 μg of the DNA fragment obtained in (2) and 5 μg of the vector DNA obtained in (3) were combined with 0.5 units of T4 DNA based on the method of Reference 7. With ligase, add 100μJ of 6mM g Cjpz-6mMβ-
Mercaptoethanol-〇, 5mMATP-6mMTr
After overnight incubation at 4°C in 5-HCI (pH 7,6), 10 μl of this reaction solution was mixed with 200 μl of the bacterial solution prepared by making the host prepared in (4) competent by the method described in Reference 9. °C for 45 minutes, then 42 °C, 9
After incubation for 0 seconds and 1 minute at 4°C, L
Medium B was added to make 1 ml, and the mixture was cultured with shaking at 37° C. for 1 hour to perform transformation. The number of ampicillin-resistant and tetracycline-sensitive transformants in this transformant mix was 2.5 to 6.3 x 104 per μg of vector.

(6) Screening The cells in the transformant mix prepared in the previous section (5) were
Screening medium containing 0 μg/ml ampicillin (0.4% ammonium chloride, 0.1% ammonium chloride,
0.05%D-ACL, 0.3%KHz POs
, 0.7% N at HP Oa , 0
．． 05% NaCl, 1mM MgSO4, 0, 2mM
Mnclg, 0.1 mMcaclz, 50
μg/ml thiamin, 50. c+g/ml proline,
50 units/m It L-A CL hydrolase)
After washing twice by centrifugation using a microorganism, resuspension in 1 m/ml of the medium, and adding 50 μl of this bacterial suspension to 0.7% LMP agarose (Takara) preheated to 42°C.
Mix this with 3 ml of the above screening medium containing LO3) and 30 μg/ml ampicillin, and solidify this in a Thermo Petri dish with a diameter of 9 cm. The transformants were selectively cultured by static culture at 30°C. On the 3rd day of culture, an average of 0.2 colonies per Petri dish was observed, and the resulting colonies were transferred to the above-mentioned agarose/ampicillin-containing screening medium to reconfirm their proliferation ability, and then the plasmid was isolated according to the method of Reference 13. After extracting the plasmid and confirming that it was larger than the vector plasmid, the ACL racemase activity of the transformant was measured by the method described in the next section (7).

(7) Measurement of activity Transformants were incubated at -
In the evening, aerobic culture was carried out, and after washing with 50mM phosphate buffer (neutral), the same buffer was added to make 1mff1, and 1ml of 10% D-
After mixing with ACL (pH 8) and reacting at 40°C for 2 hours,
Incubate for 5 minutes in boiling water, 10, OOOX g
The AC
L racemase activity was examined. Table 1 shows the racemase activity of the transformant having the plasmid pNN3 among the E. coli transformants having the above activity.

Transformant E, coli MM294 containing pNN3
L31S-/I)NN3 has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Hui Industrial Science and Technology Research Institute No. 7611. A control experiment using Achromobacter obae (Hui Technological Research Institute No. 776) is also shown in Table 1.

Table 1. Racemase activity * Conversion rate - L / (D + L) (8) Preparation of pNN36 The obtained plasmid pNN3 was digested with the restriction enzyme EcoRI, and the resulting DNA fragment was ligated to the EcoR1 site of pACYC184 using T2CN A IJ gauze.
Perform a transformation reaction by mixing with the host competent cells prepared in the previous section (5), and add 0.7% LMP agarose and 5μ
Transformants that grow in a screening medium containing g/ml of tetracycline were obtained, plasmids were extracted from these transformants by the method described in Reference 13, and their sizes were compared by electrophoresis to find the smallest plasmid. pNN
It was named 32. pNN32 has the restriction site shown in FIG. 1(a), and the E. coli transformant containing this site H,co
li MM294Lys-/pNN32 has been deposited with the National Institute of Fine Technology under the title No. 7612.

was added to DNA polypolyase I (K) according to the method in Reference 14.
lenow fragment), and added linker pEcoRI (4640P) manufactured by Takara Shuzo.
After ligation, the EcoRI fragment digested with EC0RI was reacted with pAcYcl84, which had been digested with EcoRr and treated with alkaline phosphatase, and then subjected to a gauze reaction. Using this reaction product, the host described in the previous section (4) was injected using the method described in the previous section (5). Transformed with 0.7% LMP agarose and 5 μg/m
A transformant that grows in a screening medium containing 1 of tetracycline was obtained, and the 3.5*b foreign DNA in the complex plasmid contained in the transformant was found to be a complex (9) in which the 3.5*b foreign DNA was in the same direction as pNN32 with respect to pACYC184. Transformant E, coli MM29 with racemized pNN32
Transformants E and coli MM294Lys-/pNN36 carrying 4Lys-/pNN32 and pNN36 were
The cells were cultured in 00 ml of Sfreeling medium containing 5 μg/m 1 of tetracycline under the conditions described in (7) above, and the activity of the bacterial cells was examined. As shown in Table 1, a racemization reaction was confirmed.

Purification and N-terminal amino acid sequencing of αI ACL racemase 0.5% DL-ACL, 0.2% glucose.

0.2%KHz P 04. 0.04%M
g S Oa 'mu7 Hz o, 0.01% ammonium sulfate, 0.003% Mn
el.

-4H20, 0,005% FeSO4, 7H20-containing medium (neutral) 4 (L) was cultured at 30°C for 20 hours and collected by centrifugation. The bacterial cells were added to 18 μl of 2.51 m1DN.
asel-〇. 1 11μlm1RNas eA-0
, 25 ML sucrose-2X 10-'M pyridoxal phosphate-〇, O 1% β-mercaptoethanol-suspended in 10 mM phosphate buffer (pH 6,8), and suspended in a Manton-Gaulin laboratory.
After disrupting the bacterial cells with a microorganism (ory hos+-ogenizer), DEAE-Toyopearl,
Heat treatment, Sephadex G200, Sephadex G7
5, Octyl-Sepharose CL-4B.

Purification was performed using thiopropyl-Sepharose 6B to obtain 8 mg of electrophoretically single purified enzyme.

BioRad Molecular Weight Marker Kit 5DS-PAGE Standard (LOW) (161
-0304) as a marker, 5DS-polyacrylamide electrophoresis was performed with 5% concentrating gel and 10% separating gel according to the method described in Reference 17, and the results shown in Figure 2 were obtained. Based on this, a calibration curve ( From Figure 3), the molecular weight of ACL racemase was calculated to be 4.5 x 104. However, if the molecular weight of the molecular weight marker ovalbumin used is not 45,000 as described in the instruction manual, but 43,000 as described in Reference 21.22, the molecular weight of ACL racemase is 4.3 × 104. Calculated.

Further, the purified enzyme was hydrolyzed in 6NHC1 containing 2% thioglycolic acid under vacuum sealing at 110°C for 22 or 48 hours, and the amino acid composition was analyzed using an automatic amino acid analyzer Hitachi 835. The results are shown in Table 2.

Thr, Set, and Trp amounts were calculated by θ time extrapolation.

In addition, purified enzyme was added to 2 mg/m1.
．． Dissolved in 1% SDS solution, 60 μl of which was added to Applied Biosystems (Applied Biosystems).
systems Inc.) protein sequencer (p
rotein 5equencer) 470 A was used in the instruction manual calligraphy to perform Edman disassembly, and PT was sequentially practiced.
I decided to use H-amino acids from Shimadzu ■High-Speed Sea Urchin. N-terminal T
hr was determined from the amino acid sequence and the base sequence in the next section (11).

Total ioo, o.

(11) Partial determination of the primary structure of the gene After extracting the EcoRI digest of PNN36 with phenol,
% agarose gel electrophoresis and separated 3,5
The Kb DNA fragment was isolated from the gel by electrolysis and then purified by phenol-chloroform extraction. The DNA fragment thus obtained was circularized, ultrasonic cut, end-repaired, and fractionated according to the literature 19.20, using the M13 cloning kit N manufactured by Amersham.
4501 to create a recombinant phage library, and the primary structure of the 134 DNA fragment was analyzed by the dideoxy method using Takara Shuzo's M13 Sequencing Kit 6010A according to the attached instructions. The base sequence of 3.5Ke DNA was determined using the DNA binding editing program GRASE manufactured by Mitsui Knowledge Development.

Within this sequence, a nucleotide sequence encoding the amino acid sequence obtained in the above αl was found, and is shown in FIG.

Also, the translation start code must be ATG, and A
9b upstream of TG is AGGA, which is identical to the SD sequence of E. coli.
It was found that there is.

(12) Determination of restriction sites Among the restriction enzyme recognition sites, 5phl,
oI, M 1 u 1, Pstl, BglII, Nco
It was experimentally confirmed that the I and xmn■ sites can be cleaved with restriction enzymes. The results are shown in FIG.

The PSt1 site shown in FIG. 3 corresponds to the right side of the two PStI sites in FIG. 4.

Reference 1, T, Fukumura; Agr, Biol
,Chem,.

41,. 1327-1330 (1977) 2, T.
Fukumura; Agr, Biol, Chem,
.

68, P428-436 (1979) 46 Ibid., 68
．． P379-389 (1979) 5, Hinata Saito: Protein nucleic acid enzyme.

11, P446 to 450 (1966) 6, R, W, D
avis et al.; QdvancedBacteria
l Genetics, A Manual forG
enetic Engineering+ p 227
~230Cold Spring Harbor La
boratory, New York.

7, Ibid., p138-139 8, T+Maniatis et al. 49; Molec
ularCloning, ^Laborator
y Manual.

p86-96 (1982) Cold Spring Harbor Labor
atory, New York.

9; Katsuya Shigesada: Cell Engineering 2, pp.616-626 (1983) 10. “Experimental Methods of Microbial Genetics” edited by Tatsuo Ishikawa, p.86-8B
(1982) Kyoritsu Shuppan If, T, Fukumura et al.; F PB
S Letters.

89, P298-300 (197B) 12, S,
A, Ahmed et al.; Agr. Biol, Chem.

47.1887-1893 (1983) 13, H,
C, Birnboim & J, Doly; Nuc
le'rcAcidSRes, 7.1513-1523
(1979) 14, T., Maniatis et al. 5;
Mo1ecular C1oniB, and hLaboratory Manua
lp 392-397 (19B2) 15.1. [
, edited by Grossman et al.; Methods in E
nzyn+ol.

y1 65.499-560 (1980); y1 65,560-580 (1980) 16, S, A,
Ahmed et al; F E B S Letter
s150゜Pa p370-374 (1982) 17, edited by Takeshi Horio et al.; Basic experimental methods for proteins and enzymes p3
19-324 (1982) Nankodo.

1B, C, HJ, former rS et al.; Methods
in Enzymology.

91, P486-493 (1983) 19, Sakae Soeda et al., Chemistry and Biology.

22.1541-547 (1984) 20, M13
Cloning and Sequencing 1
land book (p 1/129/83/12)A
sersham international plc
.

21. K., Weber and M., 0sborn.
; J. Biol. Chem.

244, P4406-4412 (1969) 22,
Fraser, T. and Bruce, J.
; Proc, Natl, Acad.

Sci, USA.

75, F5936-5940 (1978)

[Brief explanation of the drawing]

Figure 1(a) shows the restriction map of plasmid pNN32.
Figure (b) shows the restriction map of plasmid pNN36. FIG. 2 shows the results of electrophoresis of α-amino-ε-caprolactam racemase, and FIG. 3 shows a calibration curve prepared based on FIG. 2. FIG. 4 shows the N-terminal amino acid sequence of α-amino-ε-caprolactam racemase, the DNA encoding it, and the nucleotide sequence in its vicinity. FIG. 5 shows a restriction map of the DNA fragment of the invention.

Claims (7)

[Claims] (1) The amino acid sequence on the N-terminal side is [There is an amino acid sequence], and the molecular weight is 4.5 x 10^4 Daltons.
A gene encoding amino-ε-caprolactam racemase. (2) A 3.5 Kb DNA fragment containing the gene encoding α-amino-ε-caprolactam racemase. (3) The restriction map of DNA fragments is ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ The DNA fragment according to claim (2). (4) The molecular weight of α-amino-ε-caprolactam racemase is 4.5×10^4 Daltons, and the amino acid sequence on the N-terminal side is [there is an amino acid sequence]. ) DNA fragment described in section 2. (5) The base sequence at the 5' end of the gene encoding α-amino-ε-caprolactam racemase is [There is a base acid sequence], and the base of the gene is approximately 1.2 Kb. A DNA fragment according to scope item (2). (6) The restriction map is ▲ Contains mathematical formulas, chemical formulas, tables, etc. ▼ and the recombinant DNA fragment obtained by integrating a 3.5 Kb DNA fragment containing the gene encoding α-amino-ε-caprolactam racemase. DNA. (7) The recombinant DNA according to claim (6), wherein the recombinant DNA is plasmid pNN36.