WO2008004473A1

WO2008004473A1 - Novel protein complex, method for maturation of cobalt-type low-molecular-weight nitrile hydratase using the protein complex, matured cobalt-type low-molecular-weight nitrile hydratase, and method using the nitrile hydratase

Info

Publication number: WO2008004473A1
Application number: PCT/JP2007/062872
Authority: WO
Inventors: Michihiko Kobayashi; Yoshiteru Hashimoto; Kentaro Shiraki
Original assignee: University Of Tsukuba
Priority date: 2006-07-06
Filing date: 2007-06-27
Publication date: 2008-01-10
Also published as: JPWO2008004473A1; JP5120852B2

Abstract

[PROBLEMS] To provide a novel protein complex involved in the maturation of a cobalt-type low-molecular-weight nitrile hydratase. [MEANS FOR SOLVING PROBLEMS] A trimeric protein complex αe2 formed by an NhlA protein molecule having the amino acid sequence depicted in SEQ ID NO:1 (α-subunit of cobalt-type low-molecular-weight nitrile hydratase) and two NhlE protein molecules each having the amino acid sequence depicted in SEQ ID NO:2. The protein complex can be used for maturing a cobalt-type low-molecular-weight nitrile hydratase which has been purified in an immature state. The matured cobalt-type low-molecular-weight nitrile hydratase is highly reactive, and therefore can be used for the production of an amide compound on an industrial scale.

Description

Specification

Novel protein complex, cobalt type low molecular weight nitrile hydratase maturation method using the protein complex, matured cobalt type low molecular weight nitryl hydratase, and method using the nitrile hydratase

Technical field

[0001] The present invention relates to a novel protein complex and the like. More specifically, a novel protein complex involved in the maturation of a cobalt-type low molecular weight nitrile hydratase, a cobalt-type low molecular weight nitrile hydratase maturation method using the protein complex, a matured cobalt-type low molecular weight nitrile Background art relating to hydratase and methods using the nitrile hydratase

[0002] A nitrile hydratase is an enzyme having a nitrile hydration activity that hydrates a nitrile group and converts it into an amide group. For example, it is used as an enzyme in the conversion of acrylic nitrile to acrylamide. Currently, tens of thousands of tons of acrylamide are produced annually using this method. Nitrilehydratase is also used as an enzyme in the production of nicotinamide (NAD), which is a kind of vitamin.

[0003] The nitrile hydratase is classified into an iron type in which the active center metal causing the enzyme reaction is iron and a cobalt type in which the active center metal is cobalt. In general, the iron type has low stability, and the Kobanoleto type has high stability. For this reason, cobalt-type nitrile hydratase is widely used industrially.

[0004] Cobalt type nitrile hydratase is classified into high molecular weight (hereinafter referred to as "H type") and low molecular weight (hereinafter referred to as "L type"). In general, H type has high stability and high reactivity, so industrially, cobalt type high molecular weight (H type) nitrile hydratase is often used.

[0005] On the other hand, L-form is not widely used industrially at present because it is relatively difficult to purify a mature (activated) enzyme.

[0006] It should be noted that, in Patent Document 1, Syudonocardia 'thermofila JCM3095 (Pseudonosar A gene involved in the activation of nitrile hydratase from dia thermophila (JCM3095) has been disclosed. It is disclosed that this gene is a third open reading frame (ORF) different from the nitrile hydratase structural gene (β subunit).

[0007] In addition, as related documents, Patent Document 2 discloses a technique for activating nitrile hydratase using a metabolic inhibitor and the like, and Patent Document 3 includes cells containing nitrile hydratase, etc. A technique for improving the nitrile hydration activity of nitrile hydratase by contact with an oxidizing agent is disclosed in Non-Patent Document 1, which discloses a gene for ripening iron-type nitrile hydratase.

Patent Document 1: Japanese Patent Laid-Open No. 11-253168.

Patent Document 2: JP 2005-295815 A.

Patent Document 3: Japanese Patent Application Laid-Open No. 2004-350573.

Non-Patent Document 1: FEBS Letters 553 (2003) 391-396.

Disclosure of the invention

Problems to be solved by the invention

[0008] As described above, there are some reports on genes involved in nitrile hydratase maturation, but the mechanism of nitrile hydratase maturation has not been elucidated.

[0009] Therefore, in the present invention, the mechanism of maturation of cobalt-type low molecular weight nitryl hydratase is elucidated, and the industrial use of matured cobalt-type low molecular weight nitryl hydratase is made possible using the means. The main purpose.

Means for solving the problem

[0010] As a result of studying maturation of nitrile hydratase using cobalt-type low molecular weight nitryl hydratase derived from Rhodococcus rhodochrous Jl, the present inventors have found the following.

[0011] (1) Involved in the maturation of cobalt-type low molecular weight nitrile hydratase in the vicinity of the structural gene of cobalt-type low molecular weight nitryl hydratase (the gene encoding the subunit of the enzyme and the / 3 subunit) The presence of a gene. (2) The protein encoded by the gene cannot be purified by itself, but must be purified in the form of a complex with the _α subunit of cobalt-type low molecular weight nitrile hydratase.

(3) Protein complex strength consisting of the protein and the α subunit. Cobalt type low molecular weight nitrile hydratase must be involved in maturation.

Therefore, the present invention provides a protein complex of a protein having the amino acid sequence shown in SEQ ID NO: 1, two proteins each having the amino acid sequence shown in SEQ ID NO: 2, and a force-formed trimer. To do.

[0013] By using the protein complex, immature cobalt-type low molecular weight nitrile hydratase can be matured.

[0014] In addition, the use of the protein complex in the process of preparing a cobalt-type low molecular weight nitrile hydratase can produce a large amount of matured cobalt-type low molecular weight nitryl hydratase.

[0015] The matured cobalt-type low molecular weight nitrile hydratase can be used for the production of various amide compounds. For example, it can be used as an enzyme converting acrylonitrile to acrylamide. It can also be used as an enzyme that converts 3-cyanopyridine to nicotinamide.

[0016] Technical terms used in the present invention will be described below.

[0017] The cobalt-type low molecular weight nitrile hydratase is formed from an α subunit and a β subunit. The α subunit is a protein constituting the protein complex according to the present invention and having the amino acid sequence shown in SEQ ID NO: 1.

[0018] "Immature cobalt type low molecular weight nitryl hydratase" means a cobalt type low molecular weight nitryl hydratase having incomplete structural conversion and low enzyme activity.

[0019] The "matured cobalt type low molecular weight nitryl hydratase" means a cobalt type low molecular weight nitryl hydratase activated by structural transformation.

The invention's effect

[0020] By using the protein complex according to the present invention, immature cobalt type low molecular weight nitryl hydratase can be matured, and the enzymatic activity of cobalt type low molecular weight nitryl hydratase can be increased. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, preferred embodiments for carrying out the present invention will be described with reference to the drawings. The embodiments described below are examples of typical embodiments of the present invention, and the scope of the present invention is not construed to be narrow.

[0022] <About protein complex>

The novel protein complex according to the present invention will be described below with reference to FIG.

FIG. 1 is a diagram schematically showing a protein complex according to the present invention.

As shown in FIG. 1, the protein complex according to the present invention is a protein having the amino acid sequence IJ shown in SEQ ID NO: 1 (hereinafter referred to as “NhlA protein”, see “α” in the figure). It is a trimer formed from two proteins each having the amino acid sequence shown in SEQ ID NO: 2 (hereinafter referred to as “NhlE protein”, see “e” in the figure).

[0025] As described above, nitrile hydratase is formed from an α subunit and a β subunit. NhlA protein is a protein that constitutes the α subunit of cobalt-type low molecular weight nitrile hydratase (hereinafter referred to as “L-NHas ej”). NhlA protein is also composed of NhlE protein and trimer as described above. It has a function to mature immature L NHase.

[0026] It should be noted that the NhlA protein according to the present invention has the amino acid sequence shown in SEQ ID NO: 1 as long as it retains the above-mentioned function, but is not limited to a narrow range. That is, a protein having an amino acid sequence in which a part of the amino acid sequence shown in SEQ ID NO: 1 is substituted, deleted, inserted, etc. is also included in the NhlA protein according to the present invention.

[0027] As described above, the NhlE protein has a function of maturing immature L-NHase by forming a trimer with the NhlA protein.

[0028] It should be noted that the NhlE protein according to the present invention has the amino acid sequence shown in SEQ ID NO: 2 as long as it retains the above-described function, but is not limited to a narrow range. That is, a protein having an amino acid sequence in which a part of the amino acid sequence shown in SEQ ID NO: 2 is substituted, deleted, inserted, etc. is also included in the NhlE protein according to the present invention.

[0029] A gene encoding NhlE protein (hereinafter referred to as "nhlE gene") is a gene encoding nitrile hydratase (ie, the _α subunit and β subunit of this enzyme). The gene to encode. Hereinafter, they are referred to as “nhlA gene” and “nhlB gene”, respectively. ).

[0030] As those having these genes, for example, Achromobacter, Acinetobacter, Aeromonasj¾, Agrobactermm 禹, Bacillus j¾, itrobacterj¾, orynebactermmj¾, Enterobacter, Erwima, Klebsiella, Micrococcus, Nosediaj And microorganisms such as Pseudonocardia, Rhodococcus, Streptomyces, Thermophila, Rhizobium, and Xanthobacter. As a representative example, the nucleotide sequence in Rhodococcus rhodochrous Jl is shown in the sequence listing (SEQ ID NO: 3).

[0031] The plasmid according to the present invention, etc.>

Subsequently, examples of the plasmid according to the present invention will be described below with reference to FIG.

[0032] Fig. 2 is a schematic diagram showing a structural example of a plasmid according to the present invention.

[0033] As shown in Fig. 2, this plasmid has a sequence encoding the nhlA gene (of the nucleotide sequence shown in SEQ ID NO: 3, the 745th position is also the 1368th position lj, see "nhlA" in the figure). And a sequence encoding the nhlE gene (the 1370th to 1816th sequences of the nucleotide sequence shown in SEQ ID NO: 3; see “nhlE” in the figure).

[0034] For example, the protein complex according to the present invention can be obtained easily and in large quantities by transfecting this plasmid into a predetermined cultured cell, expressing it in a large amount in the cell, and then purifying it. In addition, as a preparation means of the protein complex based on this invention, a well-known method is widely applicable, The said preparation means is not limited narrowly only to the said means.

[0035] The plasmid shown in FIG. 2 can be prepared by a known method. For example, DNAs having the same sequence as the nhlA gene and nhlE gene are prepared, and those DNAs (including those linked in advance) and plasmid DNAs are restricted with restriction enzymes (see, for example, “Xba I” and “Sac I” in the figure). The plasmid according to the present invention can be prepared by treating with a ligating enzyme after the treatment with a.), That is, by incorporating the DNA into the plasmid DNA.

[0036] <About the maturation method of L_NHase>

A method for maturating L_NHase using the protein complex according to the present invention,

2

This will be described with reference to FIG. [0037] FIG. 3 shows a method for maturation of L NHase using the protein complex ae according to the present invention.

2

FIG. The tetramer α β and dimer ct shown in (I) of Fig. 3 have low enzyme activity.

twenty two

L NHase purified without being immature is shown. These tetramers are β, dimers

2 2 β is high in enzyme activity by using the protein complex according to the present invention.

2

It matures into a monomeric β (see (Π) in Fig. 2).

twenty two

[0038] In this way, the protein complex e according to the present invention is immaturely purified L

2

-NHase (tetramer β, dimer β / 3) can be matured after purification (translation)

twenty two

wear.

[0039] <L _ NHase maturation mechanism>

Here, the mechanism of maturation of L_NHase newly found by the present inventors will be described with reference to FIG.

[0040] FIG. 4 schematically shows a state in which plasmid 123 having three ○ RFs of nhlE gene 1, nhlA gene 2, and nhlB gene 3 was introduced into the host.

[0041] From the nhlA gene 2, and nhlB gene 3, tetramer 41 (α β) and dimer 42 _(alpha beta)

twenty two

L NHase is expressed. The inventors' research has shown that these enzyme activities are low and immature (see Example 3). On the other hand, the protein complex 5 e) according to the present invention is expressed from nhlE gene 1 and nhlA gene 2.

2

[0042] Next, protein complex 5 (ae) force tetramer 41 (αβ) and dimer 42 (α) L

2 2 2

Acts on NHase. As a result, L NHase of tetramer 6 (α β) was released with high enzyme activity.

twenty two

Appear.

[0043] In this manner, the protein complex 5 (ct e) according to the present invention is immature L NHase (4

2

Dimer 41 (hi β), dimer 42 (hi)), high enzyme activity ^^ 1½36 (tetramer 6 (hi β)

2 2 2

) Mature to).

2

[0044] <Maturation L-One NHase production method>

An example of the maturation L_NHase production method will be described with reference to FIG. In Fig. 5, STEP 1 shows in vivo (inside the host) and STEP 2 shows in vitro.

[0045] First, plasmid 12 having two ◯ RFs, nhlE gene 1 and nhlA gene 2, is introduced into a host, and the protein complex 5 (iii) according to the present invention is purified. NhlA gene 2 and nhl Introducing plasmid 23 with two 〇RFs of B gene 3 into the host, tetramer 41 (αβ), and

2 Purify L-NHase of 2 and dimer 42 (α β). As above, the tetramer 41 (a

L βase of 2β) and dimer 42 (ctβ) are immature with low enzymatic activity.

2

[0046] Then, the purified protein complex 5 (iii) and immature L-NHase (tetramer 41 (a

2 2 β) and dimer 42 (ひ j3)) are mixed in vitro. As a result, immature L_NHa

2

s _e (tetramer 41 (α β), dimer 42 (α force L— ^^ ½36 (tetramer 6 (

twenty two

Maturates to β)).

twenty two

[0047] Thus, using the protein complex 5 (iii) according to the present invention, matured L_NHase

2

Can be produced. The method for producing a matured L_NHase according to the present invention is not limited as long as protein complex 5 (iii) is used.

2

It includes all methods using protein complex 5 (iii) such as dripping.

2

[0048] Conventionally, in order to produce mature nitrile hydratase, it was necessary to introduce the gene together with the nitrile hydratase structural gene into the same host and purify it as mature nitrile hydratase.

[0049] However, by using the protein complex 5 (ae) according to the present invention, it is once immature.

2

Even purified L-NHase can be matured after purification. Therefore, using immature L NHase and separately purified protein complex 5 (ae),

2

Ripening L NHase can be produced.

[0050] The maturation L NHase production method according to the present invention is not limited to the form shown in FIG. 5, and includes a step of maturating immature L NHase using protein complex 5 (ae).

2

Is included.

[0051] <Industrial use of matured L_NHase>

Since the matured L_NHase obtained by the production method has high reactivity, it can be used for industrial production of all amide compounds.

[0052] For example, matured L_NHase according to the present invention is used in industrial production of acrylamide.

It can be used as a conversion enzyme from acrylonitrile to acrylamide. The acrylamide production method according to the present invention includes all methods including the step of using matured L_NHase as the converting enzyme. [0053] Further, the matured L NHase according to the present invention can be used as an enzyme for converting 3-cyanpyridine to nicotinamide in the industrial production of nicotinamide. The method for producing nicotinamide according to the present invention includes all methods including a step of using matured L NHase as the converting enzyme.

Example 1

[0054] In Example 1, the plasmids used in Examples 2 to 8 below were constructed.

[0055] Plasmid pL JK60 (J. Biol. Chem.,) Containing a cobalt-type low molecular weight nitrile hydratase (hereinafter referred to as "L NHase") gene derived from Rhodococcus rhodochrous Jl (see JP 05-219972). 271, 15796-15802 (1996) was used as a saddle type PCR, and the obtained PCR product was ligated to the plasmid PREIT19 (Japanese Patent Application No. 2004-380940) previously invented by the present inventor. Fig. 6 (m) schematically shows the gene site present in plasmid pLJK60.

[0056] Then, plasmids (I) to (V) below were constructed. The gene sites of these plasmids are schematically shown in FIGS. 6 (I) to (V).

(I) nhlA gene (the 745th power of the nucleotide sequence shown in SEQ ID NO: 3 is also 1368th, see “nhlA” in FIG. 6; the same shall apply hereinafter), nhlB gene (the first of the nucleotide sequences shown in SEQ ID NO: 3) No. 681, No. 681, see “nhlB” in FIG. 6, the same shall apply hereinafter), and nhlE gene (1370, No. 1816 in the nucleotide sequence shown in SEQ ID NO: 3, see “nhlE” in FIG. 6, and so on) Z) Plasmid pREIT-nhlBAE with three 〇RFs.

(II) Plasmid pREIT_nhlBA with two ○ RFs of nhlA gene and nhlB gene.

(III) Plasmid pREIT-nhlAE having two ORFs of nhlA gene and nhlE gene.

(IV) Plasmid pREIT-nhlE having ORF of nhlE gene only.

(V) Plasmid pREIT-nhl AAE, which lacks the N-terminal 24 amino acids of the subunit of L_NHase and has the 25th Met force, starting with RF and two ORFs of the nhlE gene.

Example 2

[0057] In Example 2, the NhlE protein constituting the protein complex ct e (see Fig. 1) according to the present invention is used.

2

We investigated the involvement of proteins in L NHase maturation. Specifically, the specific activity of L-NHase expressed when the plasmid pREIT-nhlBA (II) constructed in Example 1 is introduced into the host, and the expression when the plasmid pREIT-nhlBAE (I) is introduced into the host. L_NHase The specific activities of were compared. The L NHase activity was measured as follows (the same applies hereinafter).

[0058] Cell-free extract diluted with water, 50 mM potassium phosphate buffer (pH 7.5), 20 mM 3_cyanopyridine In 0.5 mL, reacted at 20 ° C for 20 minutes, then 0.5 mL of acetonitrile The reaction was stopped by raising. Nicotinamide produced by the enzymatic reaction was analyzed by HPLC. Table 1 shows the HPLC analysis conditions. The amount of enzyme that produces l x mol of benzoic acid per minute was defined as limit.

[table 1]

Analysis conditions

[0059] First, using Rhodococcus fascians DSM43985 as a host, plasmid pREIT-nhlBA (II) was introduced to express L_NHase. Then, the large-scale expression of both subunits of / 3 is SDS-PAE (sodium dodecyl sulrate-polyacrylamide gel electropnoresis).

The results of DS-PAGE are shown in Fig. 71anel. However, the enzyme activity of L-NHase in the cell-free extract was very low, 0.1 U / mg, relative to the expression level of both α and 3 subunits. Table 2 shows the enzyme activities.

Similarly, the constructed plasmid pREIT-nhlBAE (I) was introduced using Rhodococcus fascians DSM43985 as a host to express L NHase. Then, a large amount of NhlE protein encoded by the nhlE gene was confirmed in addition to the α, 3 subunits. The result of SDS-PAGE is shown in Fig. 71ane3. In addition, although the expression levels of both α and β subunits in the cell-free extract were almost the same as in the case of the plasmid pREIT-nhlBA (II), the enzyme activity of L-NHase was 8.6 U. / mg markedly increased. The enzyme activity is shown in Table 2.

[0061] [Table 2] Plasmid expression L-NHase enzyme activity pREIT-nhlBA (II) 0.1 U / mg pREIT-nhlBAEil) 8.6 U / mg

[0062] From the above results, it was found that in order to express mature L_NHase with high enzymatic activity, not only the expression of both subunits / 3 but also the presence of NhlE protein is important.

Example 3

[0063] In Example 3, using Rhodococcus fascians DSM43985 as a host, L-NHase expressed when plasmid pREIT-n hlBAE (I) is introduced and expressed when plasmid pREIT-nhlBA (II) is introduced The difference in subunit structure and enzyme activity from L NHase was investigated.

[0064] From Rhodococcus fascians DSM43985 into which plasmid pREIT-nhlBAE (I) was introduced, tetramer (α β) and NHase could be purified. Purified tetramer (α β) and NHase

Fig. 8 (1) shows the retention capacity of 2 2 2 2 in gel filtration chromatography, and Fig. 91anel shows the results of SDS-PAGE. The specific activity of L-NHase was 320 U / mg. Specific activities are shown in Table 3.

[0065] On the other hand, Rhodococcus fascians DSM43985 and others introduced the plasmid pREIT-nhlBA (II) have two types of tetramer (Hβ) _ ^ «½36 and dimer (HJ3) L_NHase. L_NHa

twenty two

se could be purified. Gel filtration chromatograph of purified tetramer (β) _1 «½36

twenty two

Figure 8 (2) shows the retention capacity in Fig. 1, and Fig. 91ane2 shows the results of SDS-PAGE. In addition, the retention capacity of the dimer (ひ / 3) and the gel filtration chromatography of the gel 36 is shown in FIG. 8 (3), and the result of SDS-PAGE is shown in FIG. 91ane3.

[0066] When these specific activities were examined, the specific activity of L_NHase of the tetramer (β) was 83U /

twenty two

The specific activity of L-NHase in dimer (α) was as low as 4U / mg. The specific activity is shown in Table 3.

[0067] [Table 3] Plasmid expression L 1 NHase enzyme expression L—NHase enzyme activity pREIT-nhlBAE (I) Tetramer (a ₂ jS ₂ ) 320 U / mg

Tetramer (Hi ₂ jS ₂ ) 83U / mg

pRE 【T-nWBA (II)

Dimer () 4U / mg

[0068] Based on the above results and the results of Example 2, it was found that NhlE protein is essential for mature complete L NHase.

Example 4

[0069] In Example 4, purification of NhlE protein was attempted.

[0070] From Rhodococcus fascians DSM43985 into which plasmid pREIT-nhlBAE (I) was introduced, purification of NhlE protein was attempted using the band on SD S-PAGE as an index. As a result, the NhlE protein was not confirmed to be purified alone, and could be purified in the form of a complex with other proteins. Figure 8 (4) shows the retention capacity of the purified complex in liquid chromatography.

Example 5

In Example 5, the subunit structure of the complex purified in Example 4 was examined.

[0072] In Example 4, the mobility of other proteins complexed with the NhlE protein coincided with those of the subunits on SDS-PAGE. The result of SDS-PAGE is shown in Fig. 91ane4.

[0073] Thus, when the N-terminal amino acid sequences of the NhlE protein and the protein that formed a complex were determined, they completely matched the subunit. The molecular weight of this complex was found to be 55.3 kDa by gel filtration. Here, the molecular weights of the subunit and NhlE protein are 22.8 kDa and 16.9 kDa, respectively.

[0074] From the above results, the subunit structure of this complex is considered to be e. This compound

2

The coalescence did not have L_NHase activity.

Example 6

[0075] In Example 6, expression with NhlE protein alone was attempted.

[0076] nhlE gene constructed in Example 1 using Rhodococcus fascians DSM43985 as a host Plasmid pREIT-nhlE (IV) with only ORF was introduced and expression of NhlE protein was tried, but no expression was confirmed.

[0077] On the other hand, when the plasmid pREIT-nhlAE (III) having two ORFs of the nhlA gene and the nhlE gene constructed in Example 1 was introduced using Rhodococcus fascians DSM43985 as the host, expression of the protein complex e Was confirmed.

2

[0078] Furthermore, with Rhodococcus fascians DSM43985 as the host, the N-terminal 24 amino acids of the L_N Hase subunit constructed in Example 1 were deleted, and the ORF of the nhlE gene and the ○ RF starting from the 25th Met Force introduced with plasmid pREIT-nhl A AE (V) Expression with Nh IE protein alone was not confirmed.

[0079] From the above results, it was found that the subunit is required for the expression of the NhlE protein, or the subunit is required for the stabilization of the expressed NhlE protein. Example 7

[0080] In Example 7, the effect of the protein complex a e in vitro was examined.

2

[0081] Rhodococcus fascians DSM43985 introduced with plasmid pREIT-nhlBA (II) Low specific activity, tetramer (β) enzyme (specific activity 83U / mg), and dimer with very low specific activity (

twenty two

a / 3) Enzyme (specific activity 4U / mg) was purified. A protein complex was mixed with each of the obtained enzymes, and the mixed solution was fractionated using a gel filtration column.

2

[0082] First, a mixed solution of a tetrameric enzyme having a low specific activity and a protein complex is separated.

2 2 2

I drew it. As a result, the size of the tetramer (ひ β) enzyme remained unchanged.

twenty two

Specific activity increased to 328.4 U / mg. Tetramer (3/3) enzyme and tamper when mixed (0 hours)

twenty two

Fig. 10A shows the retention capacity of gel composite chromatography in gel filtration chromatography.

2

Gel filtration chromatography of tetramer (β) enzyme and protein complex after 12 hours

2 2 2

The retention capacity at 1 is shown in FIG. 10B. Each specific activity is shown in Table 4. This is the same level as the specific activity of the tetramer (αβ) enzyme purified from Rhodococcus fascians DSM43985 introduced with plasmid pREIT-BAE (I).

twenty two

[0083] On the other hand, very low dimer specific activity _(alpha) mixed solvent of the enzyme and protein complex _{alpha e}

2

The liquid was fractionated. As a result, a new tetramer (α) enzyme was produced. During mixing (0 hours) Retention of dimer (α) enzyme and protein complex ae in gel filtration chromatography

2

Figure 10C shows the retention capacity of tetramer (α β) enzyme and protein complex 12 hours after mixing.

twenty two

The retention capacity in gel filtration chromatography is shown in FIG. 10D. Newly generated 4

2

The specific activity of the monomeric (β) enzyme was 326.2 U / mg. Specific activity is shown in Table 4. This too

twenty two

This is the same level as the specific activity of the tetramer (β) enzyme purified from Rhodococcus fascians DSM43985 introduced with plasmid pREIT-BAE (I).

twenty two

[Table 4]

[0085] From the results described above, the protein complex according to the present invention has an enzyme activity in vitro.

2

It was found that L-NHase expressed in an immature state with low sex was changed to matured L_NHase with high enzymatic activity after translation.

Example 8

[0086] In Example 8, the cobalt ion content of the following enzyme purified using Rhodococcus fascians DSM43985 as a host was measured.

(a) A tetramer (symbol β) enzyme purified by introducing pREIT-nhlBAE (I).

twenty two

(b) Tetramer (β) enzyme purified by introducing pREIT-nhlBA (II).

twenty two

(c) Dimer (H / 3) enzyme purified by introducing pREIT-nhlBA (II).

(d) 4 amounts after treatment of the tetrameric enzyme in (b) with a protein complex in vitro

2

Body ( _α β) enzyme.

twenty two

(e) Dimeric enzyme of (c) produced in vitro after treatment with protein complex e

2

Tetramer (symbol β) enzyme.

twenty two

[0087] The cobalt ion content of each of the enzymes (a) to (e) and the protein complex e

2

It was measured. The results are shown in Table 5.

[0088] [Table 5] Expression l_- Plasmid Co ion content after NHase ae ₂ treatment

Enzyme Co ion content

(a) Tetramer ^ .06mo \ / β

pl¾EIT-nhlBAEE (I)

(a _2J S ₂ ) (2.12mol / Qf _{2 /} S ₂ )

(b) Tetramer 0.0360101 / 0 () 5 (ά) 1.16ιηοΙ / ο; β (a _2i 8 ₂ ) (0.072mol / o; 2 / S2) (2.32mol / a _2i 8 ₂ ) pREIT-nWBA ( II)

(c) Dimer (e) 1.18mol / Qf ^

0.030mol / h β

ί β) (2.36mol / or ₂ ^ ₂ )

Treat with (b) 0.92mol / ae ₂ 0.40mol / ae ₂ pREIT-nhlAE (HI)

Process with ( _c )

_{_{0.92mol / ae 2 0.42mol / ae 2}} ae

[0089] (a) Tetramer purified by introducing pREIT-BAE (I))

twenty two

It contains 1.06 mol (2.12 mol / β) of cobalt ions, while (b) pREIT-

twenty two

A tetramer (α β) enzyme purified by introducing ΒΑ (ΒΑ) and (c) pREIT-BA (II) was introduced.

twenty two

The purified dimer (a i3) enzyme contained only 0.036 mol (0.072 mol / min β) and 0.030 mol cobalt ions per a i3 subunit, respectively. (B) with the enzyme

twenty two

The protein complex to be processed e and the protein complex to be processed with the enzyme of (c)

Both 2 2 cobalt ion contents were 0.92 mol per e subunit.

2

[0090] However, the amount of 4b after treating the enzyme of (b) and (c) with protein complex e, respectively.

2

The cobalt ion content of the body (H / 3) enzyme (d) and (e) is

twenty two

It increased to 1.16 mol (2.32 mol / β) and 1.18 mol (2.36 mol / β) per tablet.

2 2 2 2

[0091] On the other hand, the cobalt complex of the protein complex e after treatment with the enzymes (b) and (c)

2

The contents of N were reduced to 0.40 mol and 0.42 mol per α β subunit, respectively.

2

[0092] From the above results, the protein complex e has a low cobalt ion content and a specific activity.

2

Low enzyme L NHase was found to increase enzyme activity by passing its own cobalt ions.

Industrial applicability

[0093] By immature immature cobalt-type low molecular weight nitrile hydratase, which has heretofore been difficult to use industrially, to mature state of high enzyme activity, It can be used for production. In addition, the method for maturation of cobalt type low molecular weight nitrile hydratase using the protein complex according to the present invention can be carried out regardless of in vivo or in vitro, so that it can be used according to various situations. In addition, it can be used as a process for industrial production of cobalt-type low molecular weight nitrile hydratase, and for industrial production of all amino compounds.

Brief Description of Drawings

[0094] Fig. 1 is a diagram schematically showing a protein complex ct e according to the present invention.

2

FIG. 2 is a diagram schematically showing a plasmid 12 according to the present invention.

FIG. 3 is a diagram schematically showing an L NHase maturation method according to the present invention.

FIG. 4 is a diagram schematically showing the mechanism of L NHase maturation.

FIG. 5 is a diagram schematically showing an example of a method for producing mature L NHase according to the present invention.

FIG. 6 is a diagram schematically showing the gene site of the plasmid used and prepared in Example 1.

FIG. 7 is a drawing-substituting photograph showing the results of SDS-PAGE for the sample purified in Example 2.

FIG. 8 is a graph showing the retention capacity in the gel filtration chromatograph for the samples purified in Example 3 and Example 4.

FIG. 9 is a drawing-substituting photograph showing the results of SDS-PAGE for the samples purified in Example 3 and Example 4.

FIG. 10 is a graph showing the retention capacity in gel filtration chromatography of the sample purified in Example 7.

Explanation of symbols

[0095] e NhlE protein

a a subunit

1 nhlE to child

2 nhlA gene

3 nhlB gene

12 Plasmid with two ORFs of nhlE gene and nhlA gene 3 Plasmid tetramer immature L—NHase with three ORFs of nhlE, nhlA, and nhlB genes

Dimer immature L— NHase

Protein complex e

2

Mature L_NHase

Plasmid with two ORFs of nhlA gene and nhlB gene

Claims

The scope of the claims

[1] a protein having an amino acid sequence 'J shown in SEQ ID NO: 1,

A protein complex of two proteins each having the amino acid sequence shown in SEQ ID NO: 2 and a force-formed trimer.

[2] A cobalt type low molecular weight nitrile hydratase maturation method comprising at least a step of maturing immature cobalt type low molecular weight nitrile hydratase using the protein complex according to claim 1.

[3] A method for producing a matured cobalt-type low molecular weight nitrile hydratase comprising at least a step of maturing an immature cobalt-type low molecular weight nitrile hydratase using the protein complex according to claim 1.

[4] A matured cobalt-type low molecular weight nitrinohydratase produced by the method according to claim 3.

[5] A method for producing acrylamide comprising at least a step of using the matured cobalt-type low molecular weight nitrile hydratase according to claim 4 as an enzyme converting acrylonitrile to acrylamide.

[6] The matured cobalt-type low molecular weight nitrile hydratase according to claim 4,

3_ A method for producing nicotinamide comprising at least a step of using it as a converting enzyme from cyanopyridine to nicotinamide.

[7] From the 1370th to 1816th sequence among the base sequence shown in SEQ ID NO: 3,

A plasmid comprising at least the 745th to 1368th sequences of the base sequence shown in SEQ ID NO: 3, and