JP6531304B2 - Isoprene oligomer, polyisoprene, method for producing the same, rubber composition, and pneumatic tire - Google Patents

Description

The present invention relates to an isoprene oligomer, a polyisoprene, and a method for producing them, a rubber composition containing isoprene oligomer and / or polyisoprene, and a pneumatic tire using the rubber composition.

Heretofore, in rubber products, fillers of various materials and shapes are introduced into rubber compositions according to their applications, for the purpose of imparting new properties to the properties inherently possessed by rubber. By doing this, it is practiced to express desired characteristics. For example, in automobile tires, fillers such as silica and carbon black are introduced into the rubber phase which is an organic substance to improve the properties such as wear resistance, low heat buildup and wet grip performance. .

In mixing such a filler with a rubber phase in such a rubber composition, the rubber contained in the rubber phase for the purpose of enhancing the affinity between the two and further improving the low heat buildup and wet grip performance etc. The molecule is treated, for example, by reacting a compound having a nitrogen atom-containing group and a chlorosulphenyl group to introduce a functional group exhibiting affinity to the filler into the rubber molecule. It has been practiced to use a modified rubber (modified diene polymer) (for example, Patent Documents 1 and 2).

However, it is difficult to introduce the functional group at a predetermined position in the rubber molecule depending on the method of introducing the predetermined functional group to the rubber molecule, and as a result, the functional group is particularly functional at random positions of the main chain forming the rubber molecule It is known that groups are introduced. Thus, when a rubber molecule having a predetermined functional group introduced in the main chain is used, the bonding state of the rubber molecule and the filler becomes random, and not only the desired effect becomes difficult to obtain, but the functional group As a result of the deterioration of the properties as a rubber at the introduced portion, there is a problem that the properties as a whole of the rubber are impaired.

JP 2000-001573 A JP 2000-001575 A

An object of the present invention is to solve the above-mentioned problems, and to provide an isoprene oligomer, polyisoprene in which only the end portion of the molecule is substantially modified. Another object of the present invention is to provide a rubber composition containing the isoprene oligomer and / or the polyisoprene, and a pneumatic tire using the rubber composition for each component (for example, tread, sidewall) of a tire. .

（式（１）中、ｎは１〜１０の整数を表す。ｍは１〜３０の整数を表す。Ｙは、水酸基、ホルミル基、カルボキシ基、エステル基、カルボニル基又は下記式（２）で表される基を表す。）

This invention is an isoprene oligomer represented by following formula (1), Comprising: The atom in which at least one of the atom or atomic group contained in II part in following formula (1) contains a sulfur atom or a sulfur atom It relates to an isoprene oligomer substituted by a group.

(In the formula (1), n represents an integer of 1 to 10. m represents an integer of 1 to 30. Y represents a hydroxyl group, a formyl group, a carboxy group, an ester group, a carbonyl group or the following formula (2) Represents a group represented)

At least one of the atoms or atomic groups contained in the I-II moiety in the following formula (1-1) is substituted with a sulfur atom or an atomic group containing a sulfur atom, and the I-III moiety in the following formula (1-1) Preferably, the atoms or atomic groups contained in are not substituted.

It is preferable that the I-I part in the said Formula (1) is either of following formula (a)-(j).

（式（３）中、ｐは１〜１０の整数を表す。） The above isoprene oligomer is represented by the following formula (3), and is an allyl in which at least one atom or atomic group contained in the isoprene unit in the following formula (3) is substituted by a sulfur atom or an atomic group containing a sulfur atom It is preferable that it is obtained by being biosynthesized from the acid diphosphate and isopentenyl diphosphate.

(In the formula (3), p represents an integer of 1 to 10)

It is preferable to carry out the above biosynthesis using an enzyme having prenyltransferase activity.

（式（３）中、ｐは１〜１０の整数を表す。） It is preferable that the enzyme which has the said prenyltransferase activity is a protein in any one of the following [1]-[3].
[1] A protein consisting of an amino acid sequence represented by any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 [2] SEQ ID NOs: 2, 4, 6, 8, 10, 12, In the amino acid sequence represented by any one of SEQ ID NO: 14, which comprises a sequence containing 1 to 25 amino acid substitutions, deletions, insertions or additions, and is represented by the following formula (3), 3) catalyzing the reaction between isoprenyl diphosphate and allylic diphosphate in which at least one of the atoms or atom groups contained in the isoprene unit in 3) is substituted by a sulfur atom or an atom group containing a sulfur atom An active protein [3] consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 and It is represented by the following formula (3), Reaction of allylic diphosphate in which at least one atom or atom group contained in the isoprene unit in the formula (3) is substituted by a sulfur atom or an atom group containing a sulfur atom, and isopentenyl diphosphate With activity to catalyze

(In the formula (3), p represents an integer of 1 to 10)

（式（３）中、ｐは１〜１０の整数を表す。） The present invention is also an allyl represented by the following formula (3), wherein at least one atom or atomic group contained in an isoprene unit in the following formula (3) is substituted by a sulfur atom or an atomic group containing a sulfur atom The present invention relates to a process for producing the above-mentioned isoprene oligomer which is biosynthesized from dibasic diphosphate and isopentenyl diphosphate.

(In the formula (3), p represents an integer of 1 to 10)

It is preferable to carry out the above biosynthesis using an enzyme having prenyltransferase activity.

（式（４）中、ｎは１〜１０の整数を表す。ｍは１〜３０の整数を表す。ｑは３０〜４００００の整数を表す。Ｙは、水酸基、ホルミル基、カルボキシ基、エステル基、カルボニル基又は下記式（２）で表される基を表す。）

The present invention is also a polyisoprene represented by the following formula (4), wherein at least one of the atoms or atomic groups contained in the II-I moiety in the following formula (4) contains a sulfur atom or a sulfur atom It relates to polyisoprene substituted by atomic groups.

In the formula (4), n represents an integer of 1 to 10, m represents an integer of 1 to 30, and q represents an integer of 30 to 40000. Y represents a hydroxyl group, a formyl group, a carboxy group, an ester group , A carbonyl group or a group represented by the following formula (2))

At least one of the atoms or atomic groups contained in the II-II moiety in the following formula (4-1) is substituted with a sulfur atom or an atomic group containing a sulfur atom, and the II-III moiety in the following formula (4-1) Preferably, the atoms or atomic groups contained in are not substituted.

It is preferable that the II-I part in said Formula (4) is either of following formula (a)-(j).

The polyisoprene is preferably obtained by being biosynthesized from the isoprene oligomer and isopentenyl diphosphate.

The present invention also relates to a process for producing the above isoprene oligomer and the above polyisoprene biosynthesized from isopentenyl diphosphate.

The present invention also relates to a rubber composition comprising the above isoprene oligomer and / or the above polyisoprene.

The present invention also relates to a pneumatic tire produced using the above rubber composition.

The isoprene oligomer of the present invention is an isoprene oligomer represented by the above formula (1), wherein at least one of the atoms or atomic groups contained in the I-I portion in the above formula (1) is a sulfur atom or a sulfur atom Is an isoprene oligomer substituted by an atomic group containing The polyisoprene of the present invention is a polyisoprene represented by the above formula (4), wherein at least one of the atoms or atomic groups contained in the II-I portion in the above formula (4) is a sulfur atom or It is a polyisoprene substituted by an atomic group containing a sulfur atom. Therefore, the isoprene oligomer of the present invention and the polyisoprene of the present invention are substantially modified only by the terminal portion of the molecule (rubber molecule) with a sulfur atom or an atomic group containing a sulfur atom. It is excellent in affinity with the filler such as silica without being impaired in the characteristics originally possessed by Therefore, by blending the isoprene oligomer of the present invention and / or the polyisoprene of the present invention into a rubber composition, a rubber composition in which rubber molecules and a filler are combined at a higher level than before can be obtained. It is possible to provide a rubber composition excellent in the properties and wet grip performance (especially, abrasion resistance). In addition, by using the rubber composition for each member (for example, tread, sidewall) of a tire, for example, a pneumatic tire excellent in low heat buildup and wet grip performance (particularly, abrasion resistance) is provided. be able to.

In the process of artificially biosynthesizing a rubber molecule (polyisoprene), an enzyme such as prenyltransferase acts on a mixture of an initiating substrate such as farnesyl diphosphate (FPP) and a monomer such as isopentenyl diphosphate. As a result, isoprene oligomers are formed in which about 1 to 8 isoprene units are addition-polymerized with respect to the starting substrate. Thereafter, by mixing a latex component containing an enzyme which addition-polymerizes isopentenyl diphosphate with the isoprene oligomer, a polyisoprene in which a large number of isopentenyl diphosphates are linked to the oligomer is formed. It has been known.

Thus, addition polymerization by a natural enzyme is indispensable in each process of sequentially bonding a monomer to a starting substrate to make a rubber molecule.

For this reason, as a starting substrate or a monomer for biosynthesizing a rubber molecule (polyisoprene), it is necessary to use one which catalyzes a reaction by the enzyme to be used, and as a result, it is used as a raw material of rubber molecule (polyisoprene) The structures of starting substrates and monomers to be used were limited. In particular, with regard to the starting substrate, restriction due to the enzyme for generating oligomers has been limited to naturally occurring dimethylallyl diphosphate, geranyl diphosphate, farnesyl diphosphate, geranyl geranyl diphosphate and the like.

As a result, even in the case of artificially biosynthesized rubber molecules (polyisoprene), the degree of freedom in the structure is limited, and free molecular design for adding functionality not found in natural rubber has been difficult.

Therefore, for example, when it is desired to obtain a rubber molecule (polyisoprene) into which a functional group or the like is introduced, the rubber molecule (polyisoprene) which has been biosynthesized once as in the case of using a synthetic rubber as a raw material. For example, a treatment having a nitrogen atom-containing group and a compound having a chlorosulfenyl group is reacted to introduce a functional group having affinity to the filler into the rubber molecule.

On the other hand, according to the present invention, an isoprene oligomer or the like having a functionality added to the end by using farnesyl diphosphate or the like in which a part of the structure is modified as a starting substrate for producing isoprene oligomer or polyisoprene. It is based on finding that it is possible to produce polyisoprene.

In particular, the present invention is the case where the desired structure is introduced into the other part by maintaining the structure of part I of the following formula (I) with respect to the naturally occurring starting substrate farnesyl diphosphate etc. However, this is based on the finding that isoprene oligomers can be produced by using prenyltransferase, which is a naturally occurring oligomer-forming enzyme, or an enzyme in which a portion thereof is mutated. The reason for this is not necessarily clear, but it is considered that prenyltransferase causes adsorption to the structure of the I portion of the following formula (I) of the starting substrate and is relatively insensitive to the structures of other portions.

Based on this finding, it becomes possible to provide an isoprene oligomer or polyisoprene having a terminal end having desired properties, and an isoprene oligomer or isoprene oligomer having various functions added without impairing the properties of the isoprene oligomer or polyisoprene itself. It becomes possible to provide polyisoprene.

Furthermore, in addition to the above findings, the present inventors have incorporated sulfur sulphide or an isoprene oligomer in which modification with an atom group containing a sulfur atom is added at the end to the rubber composition or sulfur. A rubber composition is obtained in which the rubber molecules and the filler are composited in a higher dimension than when an isoprene oligomer or polyisoprene in which a modification other than the modification by an atom group containing atoms is added to the rubber composition is added to the rubber composition. It has been found that, for example, it is possible to provide a rubber composition which is excellent in low heat buildup, wear resistance, elongation at break, breaking strength, wet grip performance (especially wear resistance).

（式（１）中、ｎは１〜１０の整数を表す。ｍは１〜３０の整数を表す。Ｙは、水酸基、ホルミル基、カルボキシ基、エステル基、カルボニル基又は下記式（２）で表される基を表す。）

(Isoprene oligomer)
The isoprene oligomer of the present invention is an isoprene oligomer represented by the following formula (1), wherein at least one atom or atomic group contained in the I-I portion in the following formula (1) is a sulfur atom or a sulfur atom Is substituted by a group including Further, in the present specification, a group represented by the following formula (2) has three hydroxyl groups bonded to a phosphorus atom, but in an aqueous solution, part or all of these hydroxyl groups are dissociated (For example, it becomes a group represented by the following formula (5)). In the present specification, a group represented by the following formula (2) is a concept including a group in which part or all of such hydroxyl groups are dissociated.

(In the formula (1), n represents an integer of 1 to 10. m represents an integer of 1 to 30. Y represents a hydroxyl group, a formyl group, a carboxy group, an ester group, a carbonyl group or the following formula (2) Represents a group represented)

The isoprene oligomer of the present invention has a structure close to that of natural rubber and has high compatibility with rubber molecules. In addition, in the isoprene oligomer of the present invention, modification with a sulfur atom or a group containing a sulfur atom is added substantially only to the terminal part of the molecule. That is, the isoprene oligomer of the present invention has a hydroxyl group located at one end (end of polymerization termination), a formyl group, a carboxy group, an ester group, a carbonyl group or a group represented by the above formula (2). Since at least one of the atoms or atomic groups contained in the other end (the polymerization initiation end) is substituted by a sulfur atom or an atomic group containing a sulfur atom, silica is not impaired in the characteristics inherent to isoprene oligomers Strong interaction with fillers such as Thus, the isoprene oligomer of the present invention is highly compatible with the rubber, and further, has a strong interaction with the filler such as silica. A rubber composition in which a molecule and a filler are combined can be obtained, and for example, the low heat buildup, wet grip performance, and abrasion resistance (particularly, abrasion resistance) of the rubber composition can be improved.

In the isoprene oligomer of the present invention, polar groups and the like are present only at portions near the polymerization initiation end and the polymerization termination end. Therefore, compared with the case where the main chain portion has a polar group or the like or the polymerization termination terminal portion only has a polar group or the like, the dispersity of the filler such as silica is not impaired without impairing the characteristics originally possessed by the isoprene oligomer. For example, the effect of improving low heat buildup, wet grip performance, and abrasion resistance (particularly, abrasion resistance) is high.

In addition, the isoprene oligomer of the present invention exhibits excellent antibacterial activity. This is naturally occurring because at least one of the atoms or atomic groups contained in the I-I moiety in the above formula (1) constituting the isoprene oligomer is substituted by a sulfur atom or an atomic group containing a sulfur atom The structure is different from that of normal isoprene oligomers, and it has functions such as inhibition of enzyme or coenzyme possessed by bacteria, inhibition of nucleic acid synthesis, inhibition of cell membrane synthesis, inhibition of cell membrane synthesis, cell membrane disruption, cell membrane disruption, etc. It is guessed to be.

N of Formula (1) represents an integer of 1 to 10 (preferably 1 to 4, more preferably 2 to 3).
M of Formula (1) represents an integer of 1 to 30 (preferably 1 to 10, more preferably 1 to 8, more preferably 1 to 5).
Y in the formula (1) is represented by a hydroxyl group (-OH), a formyl group (-CHO), a carboxy group (-COOH), an ester group (-COOR), a carbonyl group (-COR) or the above formula (2) Represents a group.
R of an ester group (-COOR) and a carbonyl group (-COR) represents a C1-C30 (preferably C1-C17) alkyl group. As a C1-C30 alkyl group, a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group etc. are mentioned, for example.
As Y in the formula (1), a hydroxyl group and a carboxy group are preferable because they exhibit excellent antibacterial properties and strong interaction with a filler such as silica.

In the isoprene oligomer of the present invention, at least one of the atoms or atomic groups contained in the I-I moiety in the above formula (1) is substituted by a sulfur atom or an atomic group containing a sulfur atom. In the isoprene oligomer of the present invention, at least one of the atoms or atomic groups contained in the I-I moiety in the above formula (1) may be substituted by a sulfur atom or an atomic group containing a sulfur atom, In addition to the substitution, the atom or atomic group contained in the I-I portion in the above formula (1) may be further substituted by an atom other than a sulfur atom or another atomic group.

Examples of the atom or atomic group (atom or atomic group before substitution) contained in the I-I portion in the above formula (1) include, for example, a hydrogen atom, a methyl group, a methylene group, a carbon atom, a methine group, etc. It can be mentioned.

Examples of the atomic group containing a sulfur atom include a mercapto group, and a group having a cyclic structure containing a sulfur atom (eg, a group represented by the following formulas (s-1) to (s-5)) . Among them, a group having a cyclic structure containing a sulfur atom is preferable, a group having a saturated cyclic structure containing a sulfur atom is more preferable, and groups represented by the following formulas (s-1) to (s-5) Preferably, a group represented by the following formula (s-1) is particularly preferable. In addition, the cyclic structure containing a sulfur atom means the cyclic structure in which the sulfur atom forms a part of ring. In the cyclic structure, the number of sulfur atoms is preferably 1 to 3, and the number of carbon atoms is preferably 2 to 10, more preferably 2 to 5.

As atoms other than the said sulfur atom, a nitrogen atom, an oxygen atom, a silicon atom, a carbon atom etc. are mentioned, for example. Among them, oxygen atom is preferable. That is, in the isoprene oligomer of the present invention, the atom or atomic group contained in the I-I portion in the above formula (1) is substituted by a sulfur atom or an atomic group containing a sulfur atom and further substituted by an oxygen atom Is preferred.

As said other atomic group, For example, an ether group, an acetoxy group, an alkoxy group (preferably C1-C3 alkoxy group, more preferably a methoxy group), a hydroxyl group, an aryl group (preferably a phenyl group), an alkyl group (Preferably an alkyl group of 1 to 5 carbon atoms, more preferably an ethyl group, a tert-butyl group), an acetyl group, an N-alkyl-acetoamino group (the carbon number of alkyl is preferably 1 to 5), an azide group, etc. Can be mentioned. Among these, an alkoxy group, an ether group and an alkyl group are preferable. That is, in the isoprene oligomer of the present invention, the atom or atomic group contained in the I-I portion in the above formula (1) is substituted by a sulfur atom or an atomic group containing a sulfur atom, and further an alkoxy group or ether It is preferable to be substituted by at least one group (in particular, an ether group) selected from the group consisting of a group and an alkyl group.

（式（１−１）中のｎ、ｍ、Ｙは式（１）中のｎ、ｍ、Ｙと同一である。） In the isoprene oligomer of the present invention, at least one of the atoms or atomic groups contained in the I-I moiety in the above formula (1) is substituted by a sulfur atom or an atomic group containing a sulfur atom, the substitution may be Among repeating parts of the isoprene unit of the I-I moiety in the above formula (1), at least one atom or atomic group contained in the I-II moiety in the following formula (1-1) is a sulfur atom or a sulfur atom It is preferable that the atom or atomic group substituted by the containing atomic group and contained in the I-III part in following formula (1-1) is not substituted. This introduced the desired structure in the other part by maintaining the structure of the I part of the above formula (I) with respect to farnesyl diphosphate etc. which are the naturally occurring starting substrates of the present inventors. Even in this case, it is based on the finding that isoprene oligomers can be produced by using prenyltransferase, which is a naturally occurring oligomer-forming enzyme, or an enzyme mutated in part thereof.

(N, m and Y in the formula (1-1) are the same as n, m and Y in the formula (1))

As a specific example of the I-I part in the said Formula (1), the structure represented by following formula (a)-(j) is mentioned, for example. Among them, the structures represented by the following formulas (e) and (j) are preferable because they are highly effective in improving low heat buildup, wet grip performance, and abrasion resistance (particularly, abrasion resistance).

（式（３）中、ｐは１〜１０の整数を表す。） (Method for producing isoprene oligomer)
As a method for producing an isoprene oligomer of the present invention, for example, at least one of atoms or atomic groups represented by the following formula (3) and contained in an isoprene unit in the following formula (3) contains a sulfur atom or a sulfur atom Examples thereof include a method of biosynthesis from allylic diphosphate (hereinafter also referred to as an allylic diphosphate derivative) substituted by an atomic group and isopentenyl diphosphate. Furthermore, in the present specification, the allylic diphosphate derivative has three hydroxyl groups bonded to a phosphorus atom, but in an aqueous solution, part or all of these hydroxyl groups are dissociated (for example, as described above) It becomes like the group represented by Formula (5). In the present specification, the allylic diphosphate derivative is a concept including a compound in which part or all of such hydroxyl groups are dissociated.

(In the formula (3), p represents an integer of 1 to 10)

P of Formula (3) represents an integer of 1 to 10 (preferably 1 to 4, more preferably 2 to 3).

In the present specification, as the atom or atomic group contained in the isoprene unit in the formula (3) (atom or atomic group before substitution), an atom or atom contained in the I-I portion in the above formula (1) The same as the atomic group (atom or atomic group before substitution) can be mentioned.

In the present specification, the substitution of the atom or atomic group contained in the isoprene unit in the formula (3) is the same as the substitution described for the formula (1) above.

（式（３−１）中のｐは式（３）中のｐと同一である。） In addition, as described above, the substitution is preferably performed so as to maintain the structure of the I portion of the above formula (I). That is, at least one of the atoms or atomic groups contained in the IV moiety in the following formula (3-1) is substituted, and the atoms or atomic groups included in the V moiety in the following formula (3-1) are not substituted Is preferred. Thus, isoprene oligomers can be suitably produced by using prenyltransferase, which is a naturally occurring oligomer-forming enzyme, or an enzyme in which a portion of the enzyme is mutated.

(P in Formula (3-1) is the same as p in Formula (3))

なお、本明細書において、ＯＰＰは、リン原子に結合する３個の水酸基を有しているが、水溶液中では、これらの水酸基の一部又は全部が解離する（例えば、上記式（５）で表される基となる）。本明細書において、ＯＰＰとは、このような水酸基の一部又は全部が解離している基も含む概念である。 Specific examples of the allylic diphosphate derivative include, for example, compounds represented by the following formulas (A) to (J).

In the present specification, OPP has three hydroxyl groups bonded to a phosphorus atom, but in an aqueous solution, some or all of these hydroxyl groups dissociate (for example, in the above formula (5)) The basis to be represented). In the present specification, OPP is a concept including a group in which part or all of such hydroxyl groups are dissociated.

The allylic diphosphate derivatives represented by the above formulas (A) to (J) and the like are, for example, dimethylallyl diphosphate, geranyl diphosphate, farnesyl diphosphate, geranyl geranyl diphosphate, geraniol, farnesol, geranyl. The person skilled in the art can manufacture it from Geraniol et al. With reference to the method described in the examples.

As a method of biosynthesizing the isoprene oligomer of the present invention from the allylic diphosphate derivative and isopentenyl diphosphate, for example, a method of carrying out using an enzyme having prenyltransferase activity can be mentioned. Specifically, the allylic diphosphate derivative may be reacted with isopentenyl diphosphate in the presence of an enzyme having prenyltransferase activity.

In the present specification, an enzyme having prenyltransferase activity is a new enzyme in which a condensation reaction between an allylic substrate (allylic diphosphate) and isopentenyl diphosphate is catalyzed and the isoprene unit is increased by 1 unit. By synthesizing allylic diphosphate, it means an enzyme having an activity of catalyzing a reaction of sequentially linking isopentenyl diphosphate to an allylic substrate (allylic diphosphate). For example, enzymes that catalyze the following reaction may be mentioned.

The enzyme having the above prenyltransferase activity has already been confirmed in large numbers. Examples of enzymes having prenyltransferase activity include geranyl diphosphate synthetase, farnesyl diphosphate synthetase, geranyl geranyl diphosphate synthetase, Z-nonaprenyl diphosphate synthetase (Ishii, K. et al., ( 1986) Biochem, J., 233, 773.) Undecaprenyl diphosphate (UPP) synthetase (Takahashi, I. and Ogura, K. (1982) J. Biochem., 92, 1527 .; Keenman, M. V. and Allen, C. M. (1974) Arch. Biochem. Biophys., 161, 375.) and the like. Since the maximum number of isoprene units that can be produced by each enzyme (m in the above formula (1)) is determined, the enzyme used can be changed according to the target number of isoprene units (m in the above formula (1)) Just do it.

The enzymes having the above prenyl transferase activity are possessed by all species, for example, Micrococcus luteus B-P26 (Microccus luteus B-P26), Escherichia coli (Escherichia coli), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Arabidopsis・ Sarriana (Arabidopsis thaliana), Hevea brasiliensis (Hevea brasiliensis), Periploca sepium (Periploca sepium), Bacillus stearothermophilus (Bachilus Stearothermophilus), Sulfolobus acidocaldarius (Sulfolobus acidocalda ius, ATCC49426), and the like.

The isoprene oligomer of the present invention is obtained by reacting an allylic diphosphate derivative with isopentenyl diphosphate in the presence of an enzyme having prenyltransferase activity. The presence of an enzyme having prenyltransferase activity refers to a culture of the above organism, an organism isolated from the culture, a treated product of the organism, an enzyme purified from the culture or the organism, genetic engineering method A culture of an organism (transformant) transformed to express an enzyme having a prenyltransferase activity (this enzyme also includes a mutant enzyme described below), an organism separated from the culture, It means the situation in which the treated product of the organism, the culture, or the enzyme purified from the organism is present.
Here, the organism transformed to express an enzyme having prenyltransferase activity is a transformant produced by a conventionally known genetic engineering method, and the production method will be described later.

In order to obtain an organism of the above-mentioned organism, the organism may be cultured in an appropriate medium. The medium for this purpose is not particularly limited as long as the organism can grow, and may be a common medium containing a usual carbon source, a nitrogen source, an inorganic ion, and, if necessary, an organic nutrient source.

For example, as the carbon source, any of the above organisms can be used, and specifically, saccharides such as glucose, fructose, maltose and amylose, alcohols such as sorbitol, ethanol and glycerol, fumaric acid and citric acid Organic acids such as acetic acid and propionic acid, and salts thereof, carbohydrates such as paraffin, or mixtures thereof can be used.

Nitrogen sources include ammonium salts of inorganic salts such as ammonium sulfate and ammonium chloride, ammonium salts of organic acids such as ammonium fumarate and ammonium citrate, nitrates such as sodium nitrate and potassium nitrate, peptone, yeast extract, meat extract, corn steep Organic nitrogen compounds such as liquor or mixtures thereof can be used.

In addition, nutrients such as inorganic salts, trace metal salts, vitamins, and hormones, which are used in a common medium, can be appropriately mixed and used.

The culture conditions are also not particularly limited, and, for example, the culture may be carried out for about 12 to 480 hours while appropriately limiting pH and temperature in the range of pH 5 to 8 and temperature 20 to 60 ° C. under aerobic conditions. .

The culture of the above-mentioned organism means, for example, a culture solution obtained by culturing the above-mentioned organism under the above-mentioned culture conditions, a culture filtrate (culture supernatant) obtained by separating an organism (organism) from the culture by filtration etc. Can be mentioned. Further, examples of the organism separated from the culture include organisms (organisms) separated from the culture solution by filtration, centrifugation, and the like.

Examples of the treated products of the above-mentioned organisms include crushed organisms obtained by homogenizing organisms separated from the above culture, and crushed organisms obtained by sonication.

The above-mentioned culture product or an enzyme purified from the above-mentioned organism means, for example, known purification procedures such as salting out the above-mentioned culture product or the enzyme present in the above-mentioned organism, ion exchange chromatography, affinity chromatography, gel filtration chromatography and the like It is an enzyme obtained by performing The purity of the purified enzyme is not particularly limited.

By reacting an allylic diphosphate derivative with isopentenyl diphosphate in the presence of an enzyme having prenyltransferase activity, the isoprene oligomer of the present invention can be obtained. The reaction may be carried out by adding a culture of the above organism, a purified enzyme or the like to a solution containing a phosphoric acid derivative and isopentenyl diphosphate. The reaction temperature may be, for example, 20 to 60 ° C., the reaction time may be, for example, 1 to 16 hours, and the pH may be, for example, 5 to 8. Moreover, you may add magnesium chloride, surfactant, 2-mercaptoethanol etc. as needed.

In the isoprene oligomer of the present invention obtained by the above reaction, generally, Y in the above formula (1) is a group or a hydroxyl group represented by the above formula (2). The hydroxyl group is generated by hydrolysis of the group represented by the above formula (2).
An isoprene oligomer in which Y in the above formula (1) is a formyl group can be obtained, for example, by oxidizing an isoprene oligomer in which Y in the above formula (1) is a group represented by the above formula (2).
An isoprene oligomer in which Y in the above formula (1) is a carboxy group can be obtained, for example, by oxidizing an isoprene oligomer in which Y in the above formula (1) is a group represented by the above formula (2).
Further, for example, an isoprene oligomer in which Y in the above formula (1) is an ester group is obtained by oxidizing and esterifying an isoprene oligomer in which Y in the above formula (1) is a group represented by the above formula (2). can get.
Furthermore, for example, an isoprene oligomer in which Y in the above formula (1) is a carbonyl group is obtained by oxidizing and esterifying an isoprene oligomer in which Y in the above formula (1) is a group represented by the above formula (2). can get.

Since the isoprene oligomer of the present invention is obtained by biosynthesis except for organic synthesis of the allylic diphosphate derivative which is the starting substrate, it is possible to consider depletion of petroleum resources and environmental problems.

In the above formula (1) and the above formula (1-1), the I-I moiety corresponds to the structure derived from the allylic diphosphate derivative as the starting substrate, and the () _m moiety is a portion elongated by the enzyme reaction Correspond to the structure.

(Enzyme having prenyltransferase activity)
Next, an enzyme having prenyltransferase activity is described.
The base sequence and the amino acid sequence of farnesyl diphosphate synthetase from Bachilus stearothermophilus are shown in SEQ ID NOS: 1 and 2, respectively, of geranylgeranyl diphosphate synthetase from Sulfolobus acidocaldarius as an example of an enzyme having prenyltransferase activity possessed by the above organisms. The nucleotide sequence and the amino acid sequence are shown in SEQ ID NO: 3 and 4 in the sequence listing, respectively.

The original substrate (starting substrate) of the enzyme possessed by the above-mentioned organism (enzyme having prenyltransferase activity) is allylic diphosphate. And, the above-mentioned allylic diphosphate derivative used as a starting substrate in the present invention originally functions as an inhibitor of the enzyme produced by the above-mentioned organism. Therefore, in the enzyme produced by the above organism, the enzyme activity for the above allylic diphosphate derivative is often low. Therefore, in the present invention, it is preferable to use a mutant-type enzyme in which the enzyme activity for the allylic diphosphate derivative is improved.

When a mutant enzyme is used, an organism (transformant) transformed to express the mutant enzyme may be produced by genetic engineering techniques.
The present inventors succeeded in improving the enzyme activity to the above allylic diphosphate derivative by producing mutant enzymes of the above farnesyl diphosphate synthetase from Bachilus stearothermophilus and geranylgeranyl diphosphate synthetase from Sulfolobus acidocaldarius. did.

The present inventors set an amino acid residue located five residues upstream of the first aspartate rich domain in an enzyme having prenyltransferase activity (in particular, farnesyl diphosphate synthetase, geranylgeranyl diphosphate synthetase). It has been found that substitution with other amino acid residues, particularly hydrophilic amino acid residues (preferably, glycine, serine, arginine, aspartic acid) can increase the enzyme activity for the above allylic diphosphate derivative. The allylic diphosphate derivative is more polar than allylic diphosphate because a sulfur atom or an atomic group containing a sulfur atom is introduced. Therefore, it is presumed that the activity is improved by reducing the hydrophobicity of the amino acid located in the vicinity of the highly polar part such as sulfur atom contained in the allylic diphosphate derivative. In the present specification, aspartate rich domain (also referred to as First Aspartate-Rich Motif: FARM) is common to trans-type prenyl diphosphate synthetases such as farnesyl diphosphate synthetase and geranylgeranyl diphosphate synthetase. Of the two aspartic acid-rich regions present, it refers to the upstream site.
Specific examples of FARM sequences include, for example, the amino acid sequence from position 86 to position 92 of the amino acid sequence described in SEQ ID NO: 2 (amino acid sequence of farnesyl diphosphate synthetase derived from Bachilus stearothermophilus), amino acid described in SEQ ID NO: 4 An amino acid sequence consisting of DDLPDS and DIMDD found in the amino acid sequence from position 82 to position 86 of the sequence (amino acid sequence of geranylgeranyl diphosphate synthetase from Sulfolobus acidocaldarius) can be mentioned.

Specifically, it is preferable to carry out any one of the following mutations (1) and (2).
(1) Tyrosine at position 81 of farnesyl diphosphate synthetase from Bachilus stearothermophilus is substituted with arginine, serine or aspartic acid (2) phenylalanine at position 77 of geranylgeranyl diphosphate synthetase from Sulfolobus acidocaldarius to glycine or serine Replace

Specifically, the following mutant enzymes were produced.
Mutant enzyme Y81R: substitution of arginine for tyrosine at position 81 of farnesyl diphosphate synthetase derived from Bachilus stearothermophilus (base sequence and amino acid sequence are shown in SEQ ID NOs: 5 and 6, respectively)
Mutant enzyme Y81S: substitution of serine at position 81 of farnesyl diphosphate synthetase from Bachilus stearothermophilus with serine (the base sequence and amino acid sequence are shown in SEQ ID NOS: 7, 8 in the sequence listing)
Mutant enzyme Y81D: substitution of aspartic acid with tyrosine at position 81 of farnesyl diphosphate synthetase derived from Bachilus stearothermophilus (base sequence and amino acid sequence are shown in SEQ ID NOs: 9 and 10, respectively)
Mutant enzyme F77G: Phenylalanine at position 77 of geranylgeranyl diphosphate synthetase from Sulfolobus acidocaldarius is substituted with glycine (the base sequence and amino acid sequence are shown in SEQ ID NOS: 11, 12 in the sequence listing, respectively)
Mutant enzyme F77S: Phenylalanine at position 77 of geranylgeranyl diphosphate synthetase from Sulfolobus acidocaldarius is substituted with serine (the base sequence and amino acid sequence are shown in SEQ ID NOs: 13 and 14, respectively).
Among the mutant enzymes, Y81D, which is a farnesyl diphosphate synthetase derived from Bachilus stearothermophilus, and F77G, which is a geranylgeranyl diphosphate synthetase derived from Sulfolobus acidocaldarius, are preferable.
Although the case of using farnesyl diphosphate synthetase derived from Bachilus stearothermophilus and geranylgeranyl diphosphate synthetase derived from Sulfolobus acidocaldarius as wild type enzymes was described here, other enzymes having prenyltransferase activity are used as wild type enzymes Even in such a case, mutant enzymes can be produced by the same method.

Specific examples of the enzyme having prenyltransferase activity include the following [1].
[1] A protein consisting of the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14

（式（３）中、ｐは１〜１０の整数を表す。） In addition, it is known that an enzyme may have an enzyme activity even when it contains one or more amino acid substitutions, deletions, insertions or additions in the original amino acid sequence. Therefore, specific examples of the enzyme having prenyltransferase activity include the following [2].
[2] 1 to 25 amino acid substitutions, deletions, insertions, or additions are included in the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 An allyl, which is composed of a sequence and represented by the following formula (3), wherein at least one atom or atomic group contained in an isoprene unit in the following formula (3) is substituted by a sulfur atom or an atomic group containing a sulfur atom Having activity of catalyzing the reaction between basic diphosphate (allylic diphosphate derivative) and isopentenyl diphosphate

(In the formula (3), p represents an integer of 1 to 10)

In order to maintain the activity of catalyzing the reaction between allylic diphosphate derivative and isopentenyl diphosphate, 1 to 25 amino acids, preferably 1 to 12 amino acids, more preferably 1 to 12 amino acids. It is preferable that it is an amino acid sequence containing substitution, deletion, insertion, or addition of 5 amino acids, more preferably 1 to 3 amino acids.

Also, it is known that a protein having an amino acid sequence with high sequence identity to the amino acid sequence of an enzyme having prenyltransferase activity may have similar activity. Therefore, specific examples of the enzyme having prenyltransferase activity include the following [3].
[3] It consists of an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 and which has allylic diphosphorus A protein having an activity of catalyzing the reaction of an acid derivative with isopentenyl diphosphate

In order to maintain the activity of catalyzing the reaction between the allylic diphosphate derivative and isopentenyl diphosphate, any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 The sequence identity with the amino acid sequence represented by is 90% or more, more preferably 95% or more, still more preferably 98% or more, particularly preferably 99% or more.

The sequence identity of the amino acid sequence and the base sequence can be determined according to the algorithm BLAST [Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)] or FASTA [Methods Enzymol. , 183, 63 (1990)] can be used.

As a method of confirming that the protein has the activity of catalyzing the reaction between the allylic diphosphate derivative and isopentenyl diphosphate, for example, a transformant expressing the protein by a conventionally known method is used. The transformant is used to produce the protein. Then, whether or not the reaction between allylic diphosphate derivative and isopentenyl diphosphate can be catalyzed using the protein is determined by HPLC (High Performance Liquid Chromatography), TLC (Thin-Layer Chromatography), etc. Or the method of confirming by quantifying a product and qualitatively is mentioned.

(DNA encoding an enzyme having prenyltransferase activity)
Moreover, following [1]-[3] is mentioned as a DNA which codes the enzyme which has prenyltransferase activity.
[1] DNA encoding the protein of the above [1] to [3]
[2] A DNA consisting of the nucleotide sequence represented by any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13
[3] It hybridizes under stringent conditions with a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence represented by any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13; And a DNA encoding a protein having an activity of catalyzing the reaction between allylic diphosphate derivative and isopentenyl diphosphate

The term "hybridize" as used herein is a step of hybridizing DNA to a DNA having a specific base sequence or a part of the DNA. Therefore, the DNA having the specific base sequence or the base sequence of a part of the DNA is useful as a probe for Northern or Southern blot analysis, or long enough to be used as an oligonucleotide primer for PCR (Polymerase Chain Reaction) analysis. The DNA may be The DNA used as a probe may be DNA of at least 100 bases or more, preferably 200 bases or more, more preferably 500 bases or more, but may be DNA of at least 10 bases or more, preferably 15 bases or more .

Methods of hybridization experiments of DNA are well known, for example, Molecular Cloning 2nd Edition, 3rd Edition (2001), Methods for General and Molecular Bacteriology, ASM Press (1994), Immunology methods manual, Academic press ( Conditions for hybridization can be determined and experiments can be performed according to many other standard textbooks as described in Molecular).

The above-mentioned stringent conditions include, for example, a filter on which DNA is immobilized and a probe DNA in 50% formamide, 5 × SSC (750 mM sodium chloride, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6) After overnight incubation at 42 ° C. in a solution containing 5 × Denhardt's solution, 10% dextran sulfate, and 20 μg / l denatured salmon sperm DNA, eg 0.2 × SSC solution at about 65 ° C. Conditions can be mentioned to wash the filter in, but lower stringency conditions can also be used. The change of stringency conditions is possible by adjusting the concentration of formamide (the lower the concentration of formamide, the lower the stringency), and the change of salt concentration and temperature conditions. As low stringent conditions, for example, 6 × SSCE (20 × SSCE, 3 mol / l sodium chloride, 0.2 mol / l sodium dihydrogen phosphate, 0.02 mol / l EDTA, pH 7.4), 0 After overnight incubation at 37 ° C. in a solution containing .5% SDS, 30% formamide, 100 μg / l denatured salmon sperm DNA, 50 ° C. 1 × SSC, 0.1% SDS solution The conditions used for washing can be mentioned. Further, as further lower stringency conditions, hybridization conditions may be performed using a solution of high salt concentration (for example, 5 × SSC) under the above-described low stringency conditions, followed by washing conditions.

The various conditions described above can also be set by adding or changing the blocking reagent used to reduce the background of the hybridization experiment. The addition of blocking reagents described above may involve changes in hybridization conditions in order to adapt the conditions.

As DNA that can be hybridized under the above-described stringent conditions, for example, SEQ ID NO: 1, 3, 5, 7, 9, 11 when calculated based on the above parameters using programs such as BLAST and FASTA. And at least 80%, preferably at least 90%, more preferably at least 95%, still more preferably at least 98%, particularly preferably at least 99%. The DNA which consists of a base sequence which has can be mentioned.

It is conventionally known that the DNA that hybridizes with the above-mentioned DNA under stringent conditions is a DNA encoding a protein having an activity of catalyzing a reaction between an allylic diphosphate derivative and isopentenyl diphosphate. The recombinant DNA expressing the DNA is prepared by the method described above, the recombinant DNA is introduced into a host cell, the resulting organism is cultured, and the protein is purified from the resulting culture. Then, using the protein, it can be confirmed whether or not the reaction between the allylic diphosphate derivative and isopentenyl diphosphate can be catalyzed by quantifying and characterizing the substrate or product by HPLC, TLC or the like. Methods are included.

The mutant enzyme and the DNA encoding the mutant enzyme are described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997). , Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985) or the like, using, for example, the base sequence represented by SEQ ID NO: 1 (base sequence of farnesyl diphosphate synthetase derived from Bachilus stearothermophilus), SEQ ID NO: 3 The site-specific mutation can be obtained by introducing a site-specific mutation into the base sequence represented by (base sequence of geranylgeranyl diphosphate synthetase derived from Sulfolobus acidocaldarius).

(Transformant)
Next, a method for producing an organism (transformant) transformed to express an enzyme having prenyltransferase activity is briefly described. Here, a method for producing a transformant transformed so as to express mainly the above mutant enzyme will be briefly described. Such a transformant can be produced by a conventionally known method if the above design concept is determined.

When introducing a mutation, first, a primer is designed so that the mutation can be introduced at a target site. Examples of the base sequence of the primer include the base sequences described in the Examples and the like. Next, using, for example, a DNA containing the base sequence represented by SEQ ID NO: 1 (base sequence of farnicyl diphosphate synthetase derived from Bachilus stearothermophilus) as a template DNA, a mutation was introduced by PCR using the above-mentioned primers. The linear DNA is amplified. Then, the obtained linear DNA is inserted downstream of the promoter of a suitable expression vector using a suitable restriction enzyme or the like to prepare a recombinant DNA. Then, a transformant can be obtained by introducing the recombinant DNA into a host cell compatible with the expression vector.

Also, for example, a DNA containing the base sequence represented by SEQ ID NO: 1 (base sequence of farnesyl diphosphate synthetase derived from Bachilus stearothermophilus) is inserted downstream of the promoter of a suitable expression vector using a suitable restriction enzyme etc. Then, using the expression vector as a template DNA, the DNA into which a mutation has been introduced by PCR or the like using the above-mentioned primers is circularized by a polymerase to prepare a recombinant DNA. Then, a transformant can be obtained by introducing the recombinant DNA into a host cell compatible with the expression vector.

In the case where no mutation is introduced, for example, a DNA containing the base sequence represented by SEQ ID NO: 1 (base sequence of farnesyl diphosphate synthetase derived from Bachilus stearothermophilus) downstream of the promoter of a suitable expression vector is suitably used. The recombinant DNA is prepared by insertion using a restriction enzyme or the like. Then, a transformant can be obtained by introducing the recombinant DNA into a host cell compatible with the expression vector.

In the above description, the case of using the DNA containing the known nucleotide sequence represented by SEQ ID NO: 1 (the nucleotide sequence of farnesyl diphosphate synthetase derived from Bachilus stearothermophilus) has been described, but other prenyltransferases derived from the above organisms A DNA encoding an enzyme having activity or an enzyme having prenyltransferase activity from organisms other than the above organisms may be used. In this case, a DNA encoding an enzyme having prenyltransferase activity is identified by screening using a known method, for example, using a part of the base sequence represented by SEQ ID NO: 1 as a probe. You can release it. A method of isolating a target DNA molecule using a DNA molecule as a probe is described in Molecular Cloning, 2nd edition, Cold Spring Harbor press (1989) and the like.
Furthermore, the enzyme having prenyltransferase activity derived from the above-mentioned organism is purified by the above-mentioned purification procedure, and the amino acid sequence of the purified enzyme is determined to identify and isolate the DNA encoding the enzyme. Good.

Any host cell can be used as long as it can express a target gene, such as a microorganism, yeast, animal cell, insect cell, plant cell, etc.

As an expression vector, one which can autonomously replicate in the above host cell or can be integrated into a chromosome and which contains a promoter at a position where it can transcribe the above recombinant DNA can be used.

When a prokaryote such as bacteria is used as a host cell, the above recombinant DNA is capable of autonomous replication in prokaryote, and at the same time, a DNA encoding an enzyme having a promoter, ribosome binding sequence, and prenyltransferase activity, transcription Preferably, it is a recombinant DNA constituted by a termination sequence. In addition, a gene that controls a promoter may be included.

Examples of expression vectors include pColdI (Takara Bio), pCDF-1b, pRSF-1b (all from Novagen), pMAL-c2x (New England Biolabs), pGEX-4T-1 (GE Healthcare Bio) Manufactured by Science), pTrcHis (manufactured by Invitrogen), pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-30 (manufactured by Qiagen), pET-3 to pET-52 (manufactured by Novagen), pKYP10 (JP-A-58-110600), pKYP200 [Agric. Biol. Chem. , 48, 669 (1984)], pLSA1 [Agric. Biol. Chem. , 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. Chem., USA, 82, 4306 (1985)], pBluescript II SK (+), pBluescript II KS (-) (manufactured by Stratagene), pTrS30 [prepared from Escherichia coli JM109 / pTrS30 (FERM BP-5407)], pTrS32 [p. E. coli JM109 / pTrS32 (FERM BP-5408), pPAC31 (WO 98/12343), pUC19 [Gene, 33, 103 (1985)], pSTV28 (Takara Bio Inc.), pUC118 (Takara Bio Inc.) And pPA1 (Japanese Patent Application Laid-Open No. 63-233798) and the like.

Any promoter may be used as long as it functions in host cells such as Escherichia coli. For example, trp promoter _(P trp), T7 promoter, lac promoter _(P lac), _{P L} promoter, _{P R promoter,} promoters from _{P SE} such promoters, E. coli or phage, such as, SPO1 promoter, SPO2 promoter, penP promoter Etc can be raised. In addition, a promoter in which two P _trp's are connected in series, a promoter such as tac promoter, lacT7 promoter, let I promoter, and the like, which are artificially designed and modified, can also be used.

Furthermore, the xylA promoter for expression in microorganisms belonging to the genus Bacillus [Appl. Microbiol. Biotechnol. , 35, 594-599 (1991)] and P54-6 promoter for expression in microorganisms belonging to the genus Corynebacterium [Appl. Microbiol. Biotechnol. 53, 674-679 (2000)] can also be used.
It is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosomal binding sequence, and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 bases).

Prokaryotes include Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, and Agrobacteria. (Agrobacterium), Alicyclobacillus, Anabena, Anacystis, Arthrobacter, Azotobacter, Azotobacter, Chromatium, Erwinia, Methylobac (Methyloba terium, Phormidium, Rhodobacter, Rhodopseudomonas, Rhodospirillum, Scenedesmus, Streptomyces, Synechococcus , Microorganisms belonging to the genus Zymomonas etc., such as Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli DH5α, Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485, E. coli JM 109 Escherichia coli HB101, Escherichia coli No. 49, E. coli W3110, E. coli NY 49, E. coli MP347, E. coli NM 522, E. coli BL21, Bacillus subtilis ATCC 33712, Bacillus megaterium, Brevibacterium ammoniagenes (Brevibacterium ammoniagenes), Brevibacterium immariophilum ATCC 14068, Brevibacterium saccharolyticum ATCC 14066, Brevibacterium flavum (Brevibacterium immariophilum) Brevibacterium flavum) ATCC 14067, Brevibacterium lactofermentum ATCC 13869, Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 14297, Corynebacterium acetoacidide film (Corynebacterium acetoichilum) Bacteria ammonia film (Microbacterium ammoniaphilum) ATCC 15354, Serratia ficaria (Serratia ficaria), Serratia fo Ticora (Serratia fonticola), Serratia liquefaciens (Serratia marcices), Serratia marcescens, Pseudomonas sp. D-0110, Agrobacterium radiobacter (Agrobacterium radiobacter), Agrobacterium lysozyme Jeans (Agrobacterium rhizogenes), Agrobacterium rubi (Agrobacterium rubi), Anabaena cylindrica (Anabaena cylindrica), Anabaena doriolum (Anabaena doliolum), Anabaena floss aqua (Anabaena) flos-aquae, Arthrobacter aurescens, Arthrobacter citreus, Arthrobacter globformis, Arthrobacter hydrocarboglutamicus , Arthrobacter mysolens, Arthrobacter nicotianae, Arthrobacter paraffineus, Arthrobacter protophormye (Arthro) actor protophormiae), Arthrobacter roseoparaffinus, Arthrobacter sulpheus (Arthrobacter sulfureus), Arthrobacter ureafaciens, Chromatium buderi (Chromatium buderi), (Chromatium tepidum), Chromatium vinosum (Chromatium vinosum), Chromatoum warmingi (Chromatium warmingii), Chromatium fluvaitatire (Chromatium fluviatile), Erwinia uredobara ( rwinia uredovora), erwinia carotovora, erwinia ananas, erwinia herbicola, erwinia punctata, erwinia terreus, methylobacteria (Methylobacterium rhodesianum), Methylobacterium exotorquens (Methylobacterium extorquens), Formidium sp. A) ATCC 29409, Rhodobacter capsulatus (Rhodobacterium capsulatus), R. b .. (Rhodospirillum rubrum), Rhodospirillum salexigens (Rhodospirillum salexigens), Rhodospirium salinarum (Rhodospirillum salinarum), Treptomyces ambofaciens (Streptomyces ambofaciens), Streptomyces aureofaciens (Streptomyces aureofaciens), Streptomyces aureus (Streptomyces aureus), Streptomyces fungididicus (Streptomyces fungidicus), Streptomyces fungidimicus Eggplant (Streptomyces griseochromogenes), Streptomyces griseus, Streptomyces lividans (Streptomyces lividans), Streptomyces oriboglyceus (Streptomyc) s olivogriseus), Streptomyces Rameusu (Streptomyces rameus), Streptomyces Tanashienshisu (Streptomyces tanashiensis), Streptomyces Binaseusu (Streptomyces vinaceus), Zymomonas mobilis (Zymomonas mobilis), or the like can be mentioned.

As a method for introducing recombinant DNA, any method can be used as long as it is a method for introducing DNA into the above-mentioned host cell. For example, a method using calcium ion [Proc. Natl. Acad. Sci. , USA, 69, 2110 (1972)], protoplast method (JP-A-63-284394), electroporation method [Nucleic Acids Res. , 16, 6127 (1988)], and the heat shock method etc. can be mentioned.

When a yeast strain is used as a host cell, for example, YEp13 (ATCC 37115), YEp24 (ATCC 37051), YCp50 (ATCC 37419), pHS19, pHS15, etc. can be used as an expression vector.

Any promoter may be used as long as it functions in the yeast strain. For example, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shock polypeptide promoter And promoters such as MFα1 promoter and CUP 1 promoter.

Host cells include, but are not limited to, Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Schwanniomyces, Pichia, or Candy. Yeast strains belonging to the genus Candida can be mentioned, and specifically, Saccharomyces cerevisiae, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichospolon pullulans Trichosporon pullulans), Shiwaniomaisesu-Arubiusu (Schwanniomyces alluvius), Pichia pastoris (Pichia pastoris), it is possible to increase the Candida utilis (Candida utilis) and the like.

As a method for introducing recombinant DNA, any method can be used as long as it is a method for introducing DNA into yeast, for example, electroporation [Methods Enzymol. , 194, 182 (1990)], spheroplast method [Proc. Natl. Acad. Sci. , USA, 81, 4889 (1984)], lithium acetate method [J. Bacteriol. , 153, 163 (1983)] and the like.

When animal cells are used as a host, examples of expression vectors include pcDNAI, pcDM8 (commercially available from Funakoshi), pAGE107 (Japanese Patent Laid-Open No. 3-22979), pAS3-3 (Japanese Patent Laid-open No. 2-227075), pCDM8 [Nature, 329, 840 (1987)], pcDNA I / Amp (manufactured by Invitrogen), pREP 4 (manufactured by Invitrogen), pAGE 103 [J. Biochem, 101, 1307 (1987)], pAGE210, pAMo, pAMoA and the like can be used.

Any promoter that can function in animal cells can be used, for example, a promoter of cytomegalovirus (CMV) IE (immediate early) gene, SV40 early promoter or metallothionein promoter, retrovirus Promoters, heat shock promoters, SR.alpha. Promoters and the like. In addition, the enhancer of IE gene of human CMV may be used together with the promoter.

Host cells include mouse myeloma cells, rat myeloma cells, mouse hybridoma cells, human cells Namalwa cells or Namalwa KJM-1 cells, human fetal kidney cells, human leukemia cells, African green monkey kidney cells And CHO cells which are Chinese hamster cells, HBT5637 (JP-A-63-299), and the like.
SP2 / 0, NSO, etc. as mouse / myeloma cells, YB2 / 0 etc. as rat / myeloma cells, HEK293 (ATCC CRL-1573) as human fetal kidney cells, BALL-1 etc. as human leukemia cells, Africa Examples of green monkey kidney cells include COS-1 and COS-7.

As a method for introducing recombinant DNA, any method can be used as long as it is a method for introducing DNA into animal cells. For example, electroporation [Cytotechnology, 3, 133 (1990)], calcium phosphate method 2-227075), Lipofection method [Proc. Natl. Acad. Sci. , USA, 84, 7413 (1987)] and Virology, 52, 456 (1973).

When insect cells are used as a host, for example, Baculovirus Expression Vectors, A Laboratory Manual, W. et al. H. Proteins are produced by methods described in Freeman and Company, New York (1992), Current Protocols in Molecular Biology, Molecular Biology, A Laboratory Manual, Bio / Technology, 6, 47 (1988), etc. can do.

That is, after co-introducing a recombinant gene transfer vector and a baculovirus into insect cells to obtain a recombinant virus in insect cell culture supernatant, further infecting insect cells with a recombinant virus to produce a protein it can.
Examples of gene transfer vectors used in the method include pVL1392, pVL1393 and pBlueBacIII (all manufactured by Invitrogen).

As the baculovirus, for example, Autographa californica nuclear polyhedrosis virus (Autographa californica nuclear polyhedrosis virus), which is a virus that infects night bugs, and the like can be used.
As insect cells, ovarian cells of Spodoptera frugiperda, ovarian cells of Trichoplusia ni, cultured cells derived from silkworm ovaries and the like can be used.

Sf9, Sf21 (Baculovirus Expression Vectors Laboratory Manual) etc. for Spodoptera frugiperda ovarian cells, High 5 for B. trichoplesia ni cells and BTI-TN-5B1-4 (manufactured by Invitrogen) As cultured cells derived from silkworm ovary, Bombyx mori N4 can be mentioned.
Examples of the method for co-introducing the above-mentioned recombinant gene transfer vector into the insect cell and the above-mentioned baculovirus for preparing a recombinant virus include the calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), the lipofection method [Proc. Natl. Acad. Sci. , USA, 84, 7413 (1987)] and the like.

When plant cells are used as host cells, examples of expression vectors include Ti plasmid, tobacco mosaic virus vector, and the like.
Any promoter may be used as long as it functions in plant cells, and examples include 35S promoter of cauliflower mosaic virus (CaMV), rice actin 1 promoter and the like.

Examples of host cells include tobacco, potato, tomato, carrot, soybean, rapeseed, alfalfa, rice, wheat, barley, and other plant cells.
As a method for introducing a recombinant vector, any method can be used as long as it is a method for introducing DNA into plant cells. For example, a method using Agrobacterium (JP-A-59-140885, JP-A-60) No.7-0080 (WO 94/00977), electroporation (Japanese Patent Application Laid-Open No. 60-251887), method using a particle gun (gene gun) (Japanese Patent Nos. 2606856 and 2517813), and the like.

The host may be any of microorganisms, yeast, animal cells, insect cells and the like, and plant cells and the like, but preferably a microorganism, more preferably a microorganism belonging to the genus Escherichia, further preferably a microorganism belonging to E. coli. be able to.
When expressed by yeast, animal cells, insect cells or plant cells, proteins to which sugars or sugar chains have been added can be obtained.

The resulting transformant is cultured in a culture medium, and an enzyme having prenyltransferase activity can be produced by accumulating and producing an enzyme having prenyltransferase activity in the culture. In addition, if necessary, the enzyme may be purified by the above-mentioned purification procedure.

The method of culturing the above-mentioned transformant in a culture medium can be carried out according to a conventional method used for culturing a host. For example, culture may be performed with the above-described medium composition and culture conditions.

（式（４）中、ｎは１〜１０の整数を表す。ｍは１〜３０の整数を表す。ｑは３０〜４００００の整数を表す。Ｙは、水酸基、ホルミル基、カルボキシ基、エステル基、カルボニル基又は下記式（２）で表される基を表す。）

(Polyisoprene)
Next, the polyisoprene of the present invention will be described. The polyisoprene of the present invention is a polyisoprene represented by the following formula (4), wherein at least one of the atoms or atomic groups contained in the II-I portion in the following formula (4) is a sulfur atom or a sulfur atom Is substituted by a group including The polyisoprene of the present invention is composed of a trans structural part and a cis structural part as represented by the following formula (4). In addition, a trans structure part means the repeating part (The part which united the II-I part and () _m part) of the isoprene unit of a trans structure. Moreover, a cis structure part means the repeating part (() _q part in following formula (4)) of the isoprene unit of a cis structure.
In the present specification, to modify the terminal portion of the molecule (rubber molecule) means to modify the I-I portion in the above formula (1) or II in the following formula (4) present at the end of the molecule (rubber molecule). The desired functional group is introduced into a predetermined part of the -I part, or the I-I part in the above formula (1) present at the end of the molecule (rubber molecule) or in the following formula (4) It means that a different structure is introduced into a predetermined part of the II-I part.

In the formula (4), n represents an integer of 1 to 10, m represents an integer of 1 to 30, and q represents an integer of 30 to 40000. Y represents a hydroxyl group, a formyl group, a carboxy group, an ester group , A carbonyl group or a group represented by the following formula (2))

The polyisoprene of the present invention has a structure close to that of natural rubber and has high compatibility with rubber molecules. In addition, the polyisoprene of the present invention is substantially modified only by the terminal part of the molecule with a sulfur atom or a group containing a sulfur atom. That is, the polyisoprene of the present invention has a hydroxyl group located at the end of the cis structure part, a formyl group, a carboxy group, an ester group, a carbonyl group or a group represented by the above formula (2), and 4) Since at least one of the atoms or atomic groups contained in the II-I moiety in 4) is substituted by a sulfur atom or an atomic group containing a sulfur atom, silica and the like can be obtained without impairing the inherent properties of polyisoprene. Strong interaction with the filler of Thus, the polyisoprene of the present invention is highly compatible with the rubber, and further, has a strong interaction with the filler such as silica. A rubber composition in which a molecule and a filler are combined can be obtained, and for example, the low heat buildup, wet grip performance, and abrasion resistance (particularly, abrasion resistance) of the rubber composition can be improved.

In the polyisoprene of the present invention, polar groups and the like are present only at the end portion of the cis structure portion or the portion near the end of the trans structure portion. Therefore, the dispersion of the filler such as silica is not impaired while the characteristics originally possessed by polyisoprene are inhibited as compared with the case where the main chain has a polar group etc. or the case where the terminal part of the cis structure only has a polar group etc. The property is high, and for example, the effect of improving low heat buildup, wet grip performance, and abrasion resistance (in particular, abrasion resistance) is high.

N and m in the formula (4) are the same as n and m in the formula (1).
Q of the said Formula (4) represents the integer of 30-40000 (preferably 800-30000, more preferably 1000-20000).
Y in the formula (4) is the same as Y in the formula (1). In addition, as Y, since the interaction with fillers, such as a silica, is strong, a hydroxyl group and a carboxy group are preferable.

Examples of the atom or atomic group contained in the II-I portion in the above formula (4) (atom or atomic group before being substituted) include the atom or atomic group contained in the I-I portion in the above formula (1) The same as (atom or atomic group prior to substitution) can be mentioned.

Moreover, substitution of the atom or atomic group contained in the II-I part in said formula (4) is the same as the substitution demonstrated about said formula (1).

（式（４−１）中のｎ、ｍ、ｑ、Ｙは式（４）中のｎ、ｍ、ｑ、Ｙと同一である） At least one of the atoms or atomic groups contained in the II-I moiety in the above formula (4) is substituted by a sulfur atom or an atomic group containing a sulfur atom, the substitution being as described for the isoprene oligomer Similarly, at least one of the atoms or atomic groups contained in the II-II moiety in the following formula (4-1) is substituted with a sulfur atom or an atomic group containing a sulfur atom, and II in the following formula (4-1) It is preferable that the atom or atomic group contained in -III part is not substituted.

(N, m, q and Y in the formula (4-1) are the same as n, m, q and Y in the formula (4))

As a specific example of the II-I part in said Formula (4), the structure represented by said Formula (a)-(j) is mentioned, for example. Among them, the structures represented by the above formulas (e) and (j) are preferable because they are highly effective in improving low heat buildup, wet grip performance, and abrasion resistance (particularly, abrasion resistance).

(Method of producing polyisoprene)
Examples of the method for producing the polyisoprene of the present invention include a method of biosynthesizing the isoprene oligomer of the present invention and isopentenyl diphosphate.

The polyisoprene of the present invention can be obtained by biosynthesis except for organic synthesis of the allylic diphosphate derivative which is the starting substrate, so it is possible to consider the depletion of petroleum resources and environmental problems.

Conventionally, in natural rubber latex, the condensation reaction between isoprene oligomer and isopentenyl diphosphate is catalyzed and isopentene diphosphate is Z type sequentially to isoprene oligomer (newly increased isoprene unit has cis structure) It is known that it contains an enzyme having an activity of catalyzing the following reaction that produces polyisoprene and a rubber elongation factor.

In the present invention, polyisoprene can be produced using this enzyme, rubber elongation factor and the like.
That is, as a method of biosynthesizing the polyisoprene of the present invention from the isoprene oligomer of the present invention and isopentenyl diphosphate, for example, a method of carrying out using an enzyme, a rubber elongation factor, etc. contained in natural rubber latex is mentioned Be Alternatively, an enzyme cloned from a natural rubber latex, a rubber elongation factor, or the like may be used.

That is, the isoprene oligomer of the present invention may be reacted with isopentenyl diphosphate in the presence of the enzyme and / or the rubber elongation factor. Specifically, for example, the reaction is carried out by adding natural rubber latex or an enzyme separated from natural rubber latex, a rubber elongation factor, etc. to a solution containing the isoprene oligomer of the present invention and isopentenyl diphosphate. It is good. The reaction temperature may be, for example, 20 to 40 ° C., the reaction time may be, for example, 1 to 72 hours, and the pH may be, for example, 6 to 8. Moreover, you may add magnesium chloride, surfactant, 2-mercaptoethanol etc. as needed.

In the polyisoprene of the present invention obtained by the above reaction, generally, Y in the above formula (4) is a group or a hydroxyl group represented by the above formula (2). The hydroxyl group is generated by hydrolysis of the group represented by the above formula (2).
In addition, polyisoprene in which Y in the above formula (4) is a formyl group can be obtained, for example, by oxidizing polyisoprene in which Y in the above formula (4) is a group represented by the above formula (2).
Moreover, polyisoprene in which Y in the above formula (4) is a carboxy group can be obtained, for example, by oxidizing polyisoprene in which Y in the above formula (4) is a group represented by the above formula (2).
In addition, polyisoprene in which Y in the above formula (4) is an ester group is obtained, for example, by oxidizing and esterifying polyisoprene in which Y in the above formula (4) is a group represented by the above formula (2). can get.
In addition, polyisoprene in which Y in the above formula (4) is a carbonyl group is obtained, for example, by oxidizing and esterifying polyisoprene in which Y in the above formula (4) is a group represented by the above formula (2). can get.

The origin of the natural rubber latex is not particularly limited. For example, Hevea brasiliensis, Ficus elastica, Ficus lyrata, Benjamin gum (Ficus benjamina), Indo borage (Ficus religiosa), Bengal borage (Ficus benghalensis), phylum (Lactarius chrysorrheus) and the like. Among them, Hevea brasiliensis is preferable because the molecular weight of the produced rubber is large and the amount of rubber contained in the latex is large.

The natural rubber latex is obtained, for example, by scratching the stem of Hevea brasiliensis in a groove shape with a knife or the like (tapping) and recovering the natural rubber latex flowing out of the cut milk duct.

Enzymes separated from natural rubber latex, rubber elongation factors, for example, are serum (Serum), liquid bottom phase (bottom fraction), rubber phase (rubber fraction), etc. which are separated by centrifuging natural rubber latex. It can be mentioned. The above-described enzymes, the above-mentioned rubber elongation factor, and the like are contained in the serum or liquid low phase or rubber phase.

In the above formula (4) and the above formula (4-1), the structure in which the II-I moiety is derived from the allylic diphosphate derivative which is the starting substrate, () _m moiety, () _q moiety extended by enzyme reaction Corresponds to the structure of the part. Moreover, the structure which united II-I part and () _m part respond | corresponds to the structure derived from the isoprene oligomer used when manufacturing polyisoprene.

(Rubber composition)
The rubber composition of the present invention comprises the isoprene oligomer of the present invention and / or the polyisoprene of the present invention. Therefore, the rubber composition of the present invention is excellent in low heat buildup, wet grip performance, and abrasion resistance (in particular, abrasion resistance). The polyisoprene of the present invention can be used as a rubber component.

The content of the polyisoprene of the present invention is preferably 20% by mass or more, more preferably 40% by mass or more, still more preferably 60% by mass or more, and may be 100% by mass in 100% by mass of the rubber component. .

Examples of rubber components that can be used other than the polyisoprene of the present invention include isoprene rubber (IR), natural rubber (NR), butadiene rubber (BR), styrene butadiene rubber (SBR), styrene isoprene butadiene rubber (SIBR), chloroprene Mention may be made of diene rubbers such as rubber (CR) and acrylonitrile butadiene rubber (NBR). The rubber component may be used alone or in combination of two or more. Among them, NR and BR are preferable.

When the isoprene oligomer of the present invention is blended in a rubber composition, it is preferable to use NR as the rubber component because the compatibility with the isoprene oligomer is high. By combining the isoprene oligomer of the present invention with NR, the effect of blending the isoprene oligomer of the present invention is more suitably obtained.

When the isoprene oligomer of the present invention is blended in a rubber composition, the content of NR in 100% by mass of the rubber component is preferably 20% by mass or more, more preferably 40% by mass or more, still more preferably 60% by mass or more It may be 100% by mass.

The content of the isoprene oligomer of the present invention is preferably 1 part by mass or more, more preferably 2 parts by mass or more, with respect to 100 parts by mass of the rubber component. If the amount is less than 1 part by mass, the effect obtained by blending the isoprene oligomer may not be sufficiently obtained. The content of the isoprene oligomer is preferably 20 parts by mass or less, more preferably 15 parts by mass or less. If it exceeds 20 parts by mass, the strength may be reduced, and the abrasion resistance may also be reduced.

As a filler which can be used by this invention, a silica, carbon black, clay, a calcium carbonate etc. are mentioned, for example.

In the present invention, it is preferred to use silica as the filler. By blending silica, the effects obtained by blending the isoprene oligomer of the present invention and / or the polyisoprene of the present invention are sufficiently obtained. The silica is not particularly limited, and examples thereof include dry method silica (anhydrous silicic acid), wet method silica (hydrous silicic acid) and the like, but wet method silica is preferable because it has many silanol groups.

In the present invention, it is also preferable to use carbon black as a filler. Also in this case, the effects obtained by blending the isoprene oligomer of the present invention and / or the polyisoprene of the present invention can be sufficiently obtained.

In the rubber composition of the present invention, in addition to the above-mentioned components, compounding agents generally used for producing a rubber composition, for example, a silane coupling agent, zinc oxide, stearic acid, various antioxidants, softening of oil and the like Agents, waxes, vulcanizing agents such as sulfur, vulcanization accelerators and the like can be appropriately blended.

As a method for producing the rubber composition of the present invention, a known method can be used. For example, the above-mentioned components are kneaded using a rubber kneading apparatus such as an open roll or a Banbury mixer and then vulcanized. It can be manufactured.

The rubber composition of the present invention can be suitably used for each component (for example, tread, sidewall, undertread, ply, breaker, carcass) of a tire.

(Pneumatic tire)
The pneumatic tire of the present invention can be produced by the usual method using the above rubber composition. That is, the rubber composition is extruded at the unvulcanized stage according to the shape of each member (for example, tread, sidewall) of the tire, and is molded by a usual method on a tire molding machine. It bonds together with a member and forms an unvulcanized tire. The unvulcanized tire can be heated and pressurized in a vulcanizer to produce a tire.

Although the present invention will be specifically described based on examples, the present invention is not limited to these.

(Preparation of starting substrate)
(Production Example 1)
(Synthesis of Compound Represented by Formula (A) Above)
The synthesis of the compound (29) represented by the above formula (A) is as shown in the following scheme. Chloroacetone (35) was reacted with ethyl mercaptan under base catalysis to obtain ketone (36). This ketone (36), together with diethoxycarbethoxymethyl phosphine oxide, was subjected to wittig reaction to obtain an E-form, Z-form unsaturated carboxylic acid ester. E form and Z form were separated and purified using a flash column. ¹ H-NMR was used for structural confirmation of E-form and Z-form. That is, in the chemical shift of the methyl group at the 3-position, in general, the methyl group in the cis position with respect to the ester carbonyl group is outfielder than the trans position. E form (37) is reduced with LiAlH ₄ to obtain alcohol (38). In addition, in this alcohol, the chemical shift of the methyl group at the 3-position causes the cis-position methyl group to be in a higher magnetic field than the trans position, contrary to the case of the ester. Furthermore, E form and Z form were able to be confirmed by this thing. This alcohol (38) is chlorinated with CCl _{4 in the} presence of Ph ₃ P by the technique of Poulter et al., Pyrophosphorylated with THP (tris (tetra-n-butyl) ammonium hydrogen diphosphate), The compound (29) represented by Formula (A) was obtained. Confirmation of intermediates and final products in each synthesis step was performed using TLC and instrumental analysis (IR, NMR).

(Production Example 2)
(Synthesis of Compound Represented by Formula (B) Above)
The synthesis of the compound (30) represented by the above formula (B) is as shown in the following scheme. Chloroacetone (35) was reacted with propyl mercaptan under a base catalyst to obtain ketone (40). The obtained ketone (40) was subjected to Wittig reaction with diethoxycarbethoxymethyl phosphine oxide to obtain an E-form, Z-form unsaturated carboxylic acid ester. E form and Z form were separated and purified by column. E form (41) was reduced with LiAlH ₄ to obtain alcohol (42). The alcohol (42) was chlorinated as usual and pyrophosphorylated using THP to obtain a compound (30) represented by the above formula (B). Confirmation of intermediates and final products in each synthesis step was performed using TLC and instrumental analysis (IR, NMR).

(Production Example 3)
(Synthesis of Compound Represented by Formula (C) Above)
The synthesis method of the compound (31) represented by the above formula (C) is as shown in the following scheme. Michael addition of ethyl mercaptan to methyl vinyl ketone (44) under base catalyst gave ketone (45). This ketone (45) was subjected to Wittig reaction together with diethoxycarbethoxymethylphosphine oxide to obtain an E-form, Z-form unsaturated carboxylic acid ester. E form and Z form were separated and purified by column. E form (46) was reduced with LiAlH ₄ to obtain alcohol (47). The alcohol (47) was chlorinated as usual and pyrophosphorylated using THP to obtain a compound (31) represented by the above formula (C). Confirmation of intermediates and final products in each synthesis step was performed using TLC and instrumental analysis (IR, NMR).

(Production Example 4)
(Synthesis of Compound Represented by Formula (D) Above)
The compound represented by the above formula (D) was prepared in the same manner as the other compounds.
Confirmation of intermediates and final products in each synthesis step was performed using TLC and instrumental analysis (IR, NMR).

(Production Example 5)
(Synthesis of Compound Represented by Formula (E) Above)
The synthesis method of the compound (34) represented by the above formula (E) is shown in the following scheme. 1,1-Dimethoxy-3-butanone (57) is reacted with 1,2-ethylene dithiol under acid catalysis to give ketone (58). The ketone (58) was subjected to Wittig reaction with diethoxycarbethoxymethyl phosphine oxide to obtain an E-form, Z-form unsaturated carboxylic acid ester. E form and Z form were separated and purified by the column. E form (59) was reduced with LiAlH ₄ to obtain alcohol (60). This alcohol (60) was chlorinated as usual and pyrophosphorylated using THP to obtain a compound (34) represented by the above formula (E). Confirmation of intermediates and final products in each synthesis step was performed using TLC and instrumental analysis (IR, NMR).

(Production Example 6)
(Synthesis of Compound Represented by Formula (F) Above)
The synthesis method of the compound (14) represented by the above formula (F) is shown in the following scheme. Geraniol (21) and dihydropyran were condensed under pyridinium p-toluenesulfonic acid (PPTS) catalyst to obtain an ether (22) in which the hydroxyl group of geraniol was protected by a tetrahydropyran ring. An oxidation reaction with t-butyl hydroperoxide (TBHP) was carried out using (22) as a catalyst for selenium dioxide, and a hydroxyl group was selectively introduced into carbon at the 8-position to obtain a trans alcohol (23). Chlorination of the hydroxyl group of (23) with N-chlorosuccinimide (NCS) in dichloromethane gave chloride (24). Sulfide synthesis was conducted using (24) with metallic sodium and ethanethiol to synthesize sulfide (69). Then, the tetrahydropyran ring was removed from (69) under PTS catalysis to give alcohol (70). From this point onward, the hydroxyl group was replaced by chlorine to obtain a chloride (71) by chlorination using the same synthesis method as described above. After this, chloride (71) was diphosphorylated using THP to obtain a compound (14) represented by the above formula (F). Confirmation of intermediates and final products in each synthesis step was performed using TLC and instrumental analysis (IR, NMR).

(Production Example 7)
(Synthesis of Compound Represented by Formula (G) Above)
The synthesis method of the compound (16) represented by the above formula (G) is shown in the following scheme. Geraniol (21) and dihydropyran were condensed under pyridinium p-toluenesulfonic acid (PPTS) catalyst to obtain an ether (22) in which the hydroxyl group of geraniol was protected by a tetrahydropyran ring. An oxidation reaction with t-butyl hydroperoxide (TBHP) was carried out using (22) as a catalyst for selenium dioxide, and a hydroxyl group was selectively introduced into carbon at the 8-position to obtain a trans alcohol (23). Chlorination of the hydroxyl group of (23) with N-chlorosuccinimide (NCS) in dichloromethane gave chloride (24). Sulfide synthesis was conducted using (24) with metallic sodium and butanethiol to synthesize sulfide (75). Then, the tetrahydropyran ring was removed from (75) under PTS catalysis to give alcohol (76). From this point onward, the hydroxyl group was replaced with chlorine to obtain a chloride (77) by chlorination using the same synthesis method as described above. After this, chloride (77) was diphosphorylated using THP to obtain a compound (16) represented by the above formula (G). Confirmation of intermediates and final products in each synthesis step was performed using TLC and instrumental analysis (IR, NMR).

Production Example 8
(Synthesis of Compound Represented by Formula (H) Above)
The synthesis method of the compound (17) represented by the above formula (H) is shown in the following scheme. Geraniol (21) and dihydropyran were condensed under pyridinium p-toluenesulfonic acid (PPTS) catalyst to obtain an ether (22) in which the hydroxyl group of geraniol was protected by a tetrahydropyran ring. An oxidation reaction with t-butyl hydroperoxide (TBHP) was carried out using (22) as a catalyst for selenium dioxide, and a hydroxyl group was selectively introduced into carbon at the 8-position to obtain a trans alcohol (23). Chlorination of the hydroxyl group of (23) with N-chlorosuccinimide (NCS) in dichloromethane gave chloride (24). Sulfide synthesis was carried out using sodium metal and pentanethiol for (24) to synthesize sulfide (78). Then, the tetrahydropyran ring was removed from (78) under PTS catalysis to give alcohol (79). From this point onward, the hydroxyl group was replaced with chlorine by chlorination using the same synthesis method as described above to obtain a chloride (80). After this, chloride (80) was diphosphorylated using THP to obtain a compound (17) represented by the above formula (H). Confirmation of intermediates and final products in each synthesis step was performed using TLC and instrumental analysis (IR, NMR).

Production Example 9
(Synthesis of Compound Represented by Formula (I) Above)
The synthesis method of the compound (18) represented by the above formula (I) is shown in the following scheme. Geraniol (21) and dihydropyran were condensed under pyridinium p-toluenesulfonic acid (PPTS) catalyst to obtain an ether (22) in which the hydroxyl group of geraniol was protected by a tetrahydropyran ring. An oxidation reaction with t-butyl hydroperoxide (TBHP) was carried out using (22) as a catalyst for selenium dioxide, and a hydroxyl group was selectively introduced into carbon at the 8-position to obtain a trans alcohol (23). Chlorination of the hydroxyl group of (23) with N-chlorosuccinimide (NCS) in dichloromethane gave chloride (24). Sulfide synthesis was carried out using (24) with metallic sodium and hexanethiol to synthesize a sulfide (81). Then, the tetrahydropyran ring was removed from (81) under PTS catalysis to give alcohol (82). From this point onward, the hydroxyl group was replaced by chlorine to obtain a chloride (83) by chlorination using the same synthesis method as described above. After this, chloride (83) was diphosphorylated using THP to obtain a compound (18) represented by the above formula (I). Confirmation of intermediates and final products in each synthesis step was performed using TLC and instrumental analysis (IR, NMR).

Production Example 10
(Synthesis of Compound Represented by Formula (J) Above)
The compound represented by the above formula (J) was prepared in the same manner as the other compounds.
Confirmation of intermediates and final products in each synthesis step was performed using TLC and instrumental analysis (IR, NMR).

<Example of experiment using farnesyl diphosphate synthase from Bachilus stearothermophilus>
(Production Example 11)
(Preparation of mutagenizing enzyme)
A mutation was introduced into a farnesyl diphosphate synthetase derived from Bachilus stearothermophilus (the base sequence and the amino acid sequence are shown in SEQ ID NOS: 1 and 2 in the sequence listing, respectively) to prepare a mutation introducing enzyme.
The reagents used were Stratagene's QuickChange Site-Directed Mutagenesis Kit. The primers were designed to introduce a mutation at the target site. Primers for mutation introduction were purchased from Medical Biology Research Institute, Inc. (manufacturer: XX IDT). The designed primers are as shown below.
Primer sense primer for preparation of mutant type enzyme Y81R 5'-caaatcatcatggggatcaaagagcgcgtatggatcatttcaatcgcg-3 '(SEQ ID NO: 15)
Antisense primer 5'-cgcgattgaaatgatccatacgcgcttttatccatgatgatttg-3 '(SEQ ID NO: 16)
Primer sense primer for preparation of mutant type enzyme Y81S 5′-caaatcatcatggggatcaaagagctcgtatggatcatttcaatcgcg-3 ′ (SEQ ID NO: 17)
Antisense primer 5'-cgcgattgaaatgatccatacgagcttttgatcgatgatttg-3 '(SEQ ID NO: 18)
Primer sense primer for preparation of mutant type enzyme Y81D 5′-aatcatcatggatcaaagagtccgtatggatcatttcaatcgc-3 ′ (SEQ ID NO: 19)
Antisense primer 5'-gcgattgaaatgatccatacggactcttttatccatgatgatt-3 '(SEQ ID NO: 20)

The dsDNA template used was pEX (pEX / BS-FPS) into which a farnesyl diphosphate synthase derived from Bachilus stearothermophilus (hereinafter also referred to as a wild-type enzyme) was incorporated.
The pEX / BS-FPS was transferred from Prof. Furuyama Tanetoshi, Research Institute for Multidisciplinary Research in Tohoku University. 2 μl of 10x Pfu polymerase buffer, 2-20 ng of dsDNA template, 50 ng of sense primer, 50 ng of antisense primer, 0.4 μl of 2.5 mM each dNTP, 20 μl of ddH ₂ O, 0.4 ml of Pfu polymerase (2.5 U / μl) Mix and perform PCR reaction. The PCR reaction was carried out at 95 ° C. for 30 sec in one cycle and at 95 ° C. for 30 sec-55 ° C. for 1 min-68 ° C. for 8 min for 15 cycles. After PCR, 0.4 μl of Dpn I was added to the PCR reaction solution, and Dpn I treatment was performed at 37 ° C. for 1 hour. Heat shock method E. coli using 1-10 μl of Dpn I treatment solution. E. coli DH5α was transformed, and the transformant was coated on LB agar medium containing 50 μg / mL of ampicillin and cultured overnight at 37 ° C. to select transformants. The transformant was cultured overnight in LB medium containing 50 μg / ml of ampicillin, and a plasmid was prepared from the obtained culture medium by the alkaline SDS method. The plasmid was confirmed to be mutagenized using a sequencer.

Production Example 12
(Production of a protein having prenyltransferase activity)
Obtained E. coli. E. coli BL21 (DE3) / pEX / BS-FPS (wild type and mutant) were inoculated into a test tube containing 3 mL of LB medium containing 50 μg / mL of ampicillin, and shake cultured at 37 ° C. for 5 hours. Inoculate 1 mL of the obtained culture solution into a 500 mL Erlenmeyer flask containing 100 mL of LB medium containing 50 μg / mL of ampicillin, shake culture at 37 ° C. for 3 hours, and add IPTG to 0.1 mmol / L. And shake cultured at 30 ° C. for 18 hours. The culture solution was centrifuged to obtain wet cells.
The wet cells obtained above were disrupted by sonication, and then centrifuged to obtain a supernatant having a protein having prenyltransferase activity purified using a Butyl-Toyopearl column and a DEAE-Toyopearl column. . The purified protein was confirmed to be purified by SDS-PAGE.

(Example and Comparative Example)
(Preparation of isoprene oligomer)
10 mg of each purified protein, 50 mM Tris-HCl Buffer (pH 8.5), 50 mM ammonium chloride, 5 mM magnesium chloride, 50 mM 2-mercaptoethanol, 25 mM starting substrate (dimethylallyl diphosphate (DMAPP) , Geranyl diphosphate (GPP), each starting substrate prepared in Preparation Examples 1 to 10), 25 mM [1- ¹⁴ C] isopentenyl diphosphate, and the reaction solution is adjusted to 30 at a water bath of 55 ° C. I was allowed to react for a minute.
After completion of the reaction, hydrochloric acid and hexane were added, and the mixture was stirred and allowed to stand. Thereafter, the supernatant (hexane layer) was concentrated to dryness by evaporation. The structure was partially confirmed by NMR to obtain an isoprene oligomer.
The details (n and m in the formula (1)) of the obtained isoprene oligomer were as shown in Tables 1 and 2. In addition, Y was a hydroxyl group or a group represented by the above formula (2).
In addition, n and m in Formula (1) were computed based on the information of the used starting substrate, and the isoprene chain length by TLC. Moreover, Y identified the structure by NMR or IR.

(Example and Comparative Example)
(Comparison of activity of wild type enzyme and mutant type enzyme (relative activity by substrate))
The reaction was carried out using the starting substrate prepared in Preparation Examples 1 to 10, dimethylallyl diphosphate (DMAPP) and geranyl diphosphate (GPP) under the following conditions, and the activity of each mutant-type enzyme with respect to each starting substrate The activity against wild-type enzyme (farnesyl diphosphate synthetase derived from Bachilus stearothermophilus) geranyl diphosphate was expressed as an index of 100 (Table 3).
500 ng of purified protein, 50 mM phosphate buffer (pH 5.8), 50 mM ammonium chloride, 0.01% Triton X-100, 50 mM 2-mercaptoethanol, 25 mM starting substrate (prepared in Preparations 1 to 10) Prepare a reaction solution containing each starting substrate, dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP), 25 mM [1- ¹⁴ C] isopentenyl diphosphate, and in water bath at 55 ° C for 15 minutes. It was made to react. After the reaction, the activity of each enzyme was measured by quantifying the value of liquid scintillation and TLC.

(Example and Comparative Example)
(Preparation of polyisoprene)
10 μl of the latex component, 50 mM Tris-HCl buffer (pH 7.5), 2.5 mM magnesium chloride, 40 mM 2-mercaptoethanol, 40 mM potassium fluoride, 50 μM isoprene oligomer, 25 mM [1- ¹⁴ C] The reaction solution containing isopentenyl diphosphate was prepared, and was reacted in water bath at 30 ° C. for 3 days. After the reaction, the molecular weight was measured by GPC. And n and q in Formula (4) were computed based on the measured molecular weight and the information of the starting substrate used. The details of the obtained polyisoprene (n and q in the formula (4)) were as shown in Tables 4 and 5. In addition, Y was a hydroxyl group or a group represented by the above formula (2). In addition, identification of Y was performed in the same manner as in Example (preparation of isoprene oligomer).

As a latex component, a serum prepared by ultracentrifugation of a latex obtained from Hevea brasiliensis was used.

The starting isoprene oligomer (geranyl diphosphate, dimethylallyl diphosphate, Preparation Examples 1 to 5) was prepared using the mutant enzyme Y81R under the same conditions as in Example (Preparation of isoprene oligomer). The isoprene oligomers obtained using each of the starting substrates prepared in 10) were used.
In Tables 4 and 5 below, geranyl diphosphate, dimethylallyl diphosphate as the starting substrate, a compound represented by the above formula (A), a compound represented by the above formula (B), a compound represented by the above formula (A) The compound represented by C), the compound represented by the above formula (D), the compound represented by the above formula (E), the compound represented by the above formula (F), and the compound represented by the above formula (G) Using the compound, the compound represented by the above formula (H), the compound represented by the above formula (I), and the compound represented by the above formula (J), the same conditions as in Example (Preparation of isoprene oligomer) The isoprene oligomer obtained by using the mutant enzyme Y81R or the like in each of isoprene oligomer (GPP), isoprene oligomer (DMAPP), isoprene oligomer (A), isoprene oligomer (B), isoprene oligomer (C) Isoprene oligomer (D), isoprene oligomer (E), isoprene oligomer (F), isoprene oligomer (G), isoprene oligomer (H), isoprene oligomer (I), and isoprene oligomer (J).

Next, the isoprene oligomer of the present invention and polyisoprene were blended into a rubber composition, and in order to evaluate the performance, first, an isoprene oligomer and polyisoprene to be subjected to the evaluation were prepared. Basically, it was prepared according to the above-mentioned Example (preparation of isoprene oligomer), Example (preparation of polyisoprene), but in order to adjust the molecular weight of the resulting isoprene oligomer, polyisoprene, The reaction was carried out by adjusting the conditions described in (Preparation) and (Preparation of polyisoprene).

Production Example 13
(Preparation of isoprene oligomer for rubber composition evaluation)
First, an isoprene oligomer was prepared using the starting substrate prepared in Preparation Examples 1 to 10, dimethylallyl diphosphate (DMAPP), and geranyl diphosphate (GPP). In addition, the details (n, m, Y in Formula (1)) of the obtained isoprene oligomer were determined by the method similar to an Example (preparation of an isoprene oligomer).

(Preparation of isoprene oligomer (1-GPP))
An isoprene oligomer (1- GPP) was prepared using geranyl diphosphate as the starting substrate. The details (n, m in the formula (1)) of the obtained isoprene oligomer (1-GPP) were n = 3 and m = 2. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of isoprene oligomer (1-DMAPP))
An isoprene oligomer (1-DMAPP) was prepared using dimethylallyl diphosphate as the starting substrate. The details (n, m in Formula (1)) of the obtained isoprene oligomer (1-DMAPP) were n = 2 and m = 2. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of isoprene oligomer (1-A))
The isoprene oligomer (1-A) was prepared using the compound represented by the above-mentioned formula (A) as a starting substrate. The details (n and m in Formula (1)) of the obtained isoprene oligomer (1-A) were n = 2 and m = 2. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of isoprene oligomer (1-B))
The isoprene oligomer (1-B) was prepared using the compound represented by the above formula (B) as a starting substrate. The details (n and m in Formula (1)) of the obtained isoprene oligomer (1-B) were n = 2 and m = 2. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of isoprene oligomer (1-C))
The isoprene oligomer (1-C) was prepared using the compound represented by the above formula (C) as a starting substrate. The details (n, m in Formula (1)) of the obtained isoprene oligomer (1-C) were n = 2 and m = 2. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of isoprene oligomer (1-D))
Preparation of isoprene oligomer (1-D) was performed using the compound represented by the said Formula (D) as a starting substrate. The details (n and m in Formula (1)) of the obtained isoprene oligomer (1-D) were n = 2 and m = 2. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of isoprene oligomer (1-E))
The isoprene oligomer (1-E) was prepared using the compound represented by the above formula (E) as a starting substrate. The details (n, m in Formula (1)) of the obtained isoprene oligomer (1-E) were n = 2 and m = 2. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of isoprene oligomer (1-F))
Preparation of isoprene oligomer (1-F) was performed using the compound represented by said Formula (F) as a starting substrate. The details (n and m in Formula (1)) of the obtained isoprene oligomer (1-F) were n = 3 and m = 1. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of isoprene oligomer (1-G))
The isoprene oligomer (1-G) was prepared using the compound represented by the above formula (G) as a starting substrate. The details (n, m in Formula (1)) of the obtained isoprene oligomer (1-G) were n = 3 and m = 2. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of isoprene oligomer (1-H))
The isoprene oligomer (1-H) was prepared using the compound represented by the above formula (H) as a starting substrate. The details (n and m in Formula (1)) of the obtained isoprene oligomer (1-H) were n = 3 and m = 1. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of isoprene oligomer (1-I))
The isoprene oligomer (1-I) was prepared using the compound represented by the above formula (I) as a starting substrate. The details (n and m in the formula (1)) of the obtained isoprene oligomer (1-I) were n = 3 and m = 1. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of isoprene oligomer (1-J))
Preparation of isoprene oligomer (1-J) was performed using the compound represented by said Formula (J) as a starting substrate. The details (n and m in Formula (1)) of the obtained isoprene oligomer (1-J) were n = 3 and m = 1. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

Production Example 14
(Preparation of polyisoprene for evaluation of rubber composition)
Next, polyisoprene was prepared using the isoprene oligomer obtained in Production Example 13. In addition, the details (n, m, q, Y in Formula (4)) of the obtained polyisoprene were determined by the method similar to Example (preparation of polyisoprene).

(Preparation of polyisoprene (1-GPP))
Polyisoprene (1-GPP) was prepared using the isoprene oligomer (1-GPP) prepared in Preparation Example 13. The detail (n, m, q in Formula (4)) of the obtained polyisoprene (1- GPP) was n = 3, m = 2, q = 3000-15000. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of polyisoprene (1-DMAPP))
Preparation of polyisoprene (1-DMAPP) was performed using the isoprene oligomer (1-DMAPP) prepared in Preparation Example 13. The details (n, m, q in Formula (4)) of the obtained polyisoprene (1-DMAPP) were n = 2, m = 2 and q = 3 000-1500. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of polyisoprene (1-A))
Polyisoprene (1-A) was prepared using the isoprene oligomer (1-A) prepared in Preparation Example 13. The details (n, m, q in the formula (4)) of the obtained polyisoprene (1-A) were n = 2, m = 2 and q = 1000 to 5000. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of polyisoprene (1-B))
Polyisoprene (1-B) was prepared using the isoprene oligomer (1-B) prepared in Preparation Example 13. The details (n, m, q in Formula (4)) of the obtained polyisoprene (1-B) were n = 2, m = 2 and q = 1000-5000. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of polyisoprene (1-C))
Polyisoprene (1-C) was prepared using the isoprene oligomer (1-C) prepared in Preparation Example 13. The details (n, m, q in the formula (4)) of the obtained polyisoprene (1-C) were n = 2, m = 2, q = 1000 to 8000. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of polyisoprene (1-D))
Polyisoprene (1-D) was prepared using the isoprene oligomer (1-D) prepared in Preparation Example 13. The details (n, m, q in the formula (4)) of the obtained polyisoprene (1-D) were n = 2, m = 2 and q = 1000 to 12000. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of polyisoprene (1-E))
Preparation of polyisoprene (1-E) was performed using the isoprene oligomer (1-E) prepared in Preparation Example 13. The details (n, m, q in the formula (4)) of the obtained polyisoprene (1-E) were n = 2, m = 2 and q = 1000 to 12000. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of polyisoprene (1-F))
Polyisoprene (1-F) was prepared using the isoprene oligomer (1-F) prepared in Preparation Example 13. The details (n, m, q in Formula (4)) of the obtained polyisoprene (1-F) were n = 3, m = 1, q = 4000-10000. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of polyisoprene (1-G))
Polyisoprene (1-G) was prepared using the isoprene oligomer (1-G) prepared in Preparation Example 13. The details (n, m, q in the formula (4)) of the obtained polyisoprene (1-G) were n = 3, m = 2 and q = 1500 to 7,000. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of polyisoprene (1-H))
Polyisoprene (1-H) was prepared using the isoprene oligomer (1-H) prepared in Preparation Example 13. The details (n, m, q in Formula (4)) of the obtained polyisoprene (1-H) were n = 3, m = 1, and q = 2000-7000. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of polyisoprene (1-I))
Polyisoprene (1-I) was prepared using the isoprene oligomer (1-I) prepared in Production Example 13. The details (n, m, q in Formula (4)) of the obtained polyisoprene (1-I) were n = 3, m = 1, q = 1500-5000. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of polyisoprene (1-J))
Polyisoprene (1-J) was prepared using the isoprene oligomer (1-J) prepared in Preparation Example 13. The details (n, m, q in the formula (4)) of the obtained polyisoprene (1-J) were n = 3, m = 1, q = 1500 to 5000. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

<Example of experiment using geranylgeranyl diphosphate synthetase from Sulfolobus acidocaldarius>
Production Example 15
(Preparation of mutagenizing enzyme)
A mutation was introduced into Sulfolobus acidocaldarius-derived geranylgeranyl diphosphate synthetase (the base sequence and the amino acid sequence are shown in SEQ ID NOS: 3 and 4 in the sequence listing, respectively) to prepare a mutagenesis enzyme.
The reagents used were Stratagene's QuickChange Site-Directed Mutagenesis Kit. The primers were designed to introduce a mutation at the target site. Primers for mutation introduction were purchased from Medical Biology Research Institute, Inc. (manufacturer: XX IDT). The designed primers are as shown below.
Primer sense primer for preparation of mutant type enzyme F77G 5′-ataatatcatcatgccacaagcgtaccagtatgaagaacttcaatagctgca-3 ′ (SEQ ID NO: 21)
Antisense primer 5'-tgcagctattgaagttcttcatactggtacgcttgtgcatgatgatattat-3 '(SEQ ID NO: 22)
Primer sense primer for preparation of mutant type enzyme F77S 5′-ataatatcatcatgcacaagcgtactagtatgaagaacttaatagctgca-3 ′ (SEQ ID NO: 23)
Antisense primer 5'-tgcagctattgaagttcttcatactagtctgcttgtgcatgatgatattat-3 '(SEQ ID NO: 24)

The dsDNA template used pUC119 (pUC119 / SA-GGPS) into which the geranylgeranyl diphosphate synthetase (hereinafter, also referred to as a wild-type enzyme) derived from Sulfolobus acidocaldarius was incorporated.
PUC119 / SA-GGPS was transferred from Prof. Tokuzo Nishino, Faculty of Engineering, Tohoku University. 2 μl of 10x Pfu polymerase buffer, 2-20 ng of dsDNA template, 50 ng of sense primer, 50 ng of antisense primer, 0.4 μl of 2.5 mM each dNTP, 20 μl of ddH ₂ O, 0.4 ml of Pfu polymerase (2.5 U / μl) Mix and perform PCR reaction. The PCR reaction was carried out at 95 ° C. for 30 sec in one cycle and at 95 ° C. for 30 sec-55 ° C. for 1 min-68 ° C. for 8 min for 15 cycles. After PCR, 0.4 μl of Dpn I was added to the PCR reaction solution, and Dpn I treatment was performed at 37 ° C. for 1 hour. Heat shock method E. coli using 1-10 μl of Dpn I treatment solution. E. coli DH5α was transformed, and the transformant was coated on LB agar medium containing 50 μg / mL of ampicillin and cultured overnight at 37 ° C. to select transformants. The transformant was cultured overnight in LB medium containing 50 μg / ml of ampicillin, and a plasmid was prepared from the obtained culture medium by the alkaline SDS method. The plasmid was confirmed to be mutagenized using a sequencer.

Production Example 16
(Production of a protein having prenyltransferase activity)
Obtained E. coli. E. coli BL21 (DE3) / pUC119 / SA-GGPS (wild type and mutant) was inoculated into a test tube containing 3 mL of LB medium containing 50 μg / mL of ampicillin, and shake cultured at 37 ° C. for 5 hours. Inoculate 1 mL of the obtained culture solution into a 500 mL Erlenmeyer flask containing 100 mL of LB medium containing 50 μg / mL of ampicillin, shake culture at 37 ° C. for 3 hours, and add IPTG to 0.1 mmol / L. And shake cultured at 30 ° C. for 18 hours. The culture solution was centrifuged to obtain wet cells.
The wet cells obtained above were disrupted by sonication, and then centrifuged to obtain a supernatant having a protein having prenyltransferase activity purified using a Butyl-Toyopearl column and a DEAE-Toyopearl column. . The purified protein was confirmed to be purified by SDS-PAGE.

(Example and Comparative Example)
(Preparation of isoprene oligomer)
10 mg of each purified protein, 50 mM phosphate buffer (pH 5.8), 5 mM magnesium chloride, 50 mM ammonium chloride, 0.01% Triton X-100, 50 mM 2-mercaptoethanol, 25 mM starting substrate (dimethyl Preparing a reaction solution containing allyl diphosphate (DMAPP), geranyl diphosphate (GPP), each starting substrate prepared in Preparation Examples 1 to 10, 25 mM [1- ¹⁴ C] isopentenyl diphosphate; The reaction was performed for 30 minutes in a water bath at 55 ° C.
After completion of the reaction, hydrochloric acid and hexane were added, and the mixture was stirred and allowed to stand. Thereafter, the supernatant (hexane layer) was concentrated to dryness by evaporation. The structure was partially confirmed by NMR to obtain an isoprene oligomer.
The details of the obtained isoprene oligomer (n, m in the formula (1)) were as shown in Tables 6 and 7. In addition, Y was a hydroxyl group or a group represented by the above formula (2).
In addition, n and m in Formula (1) were calculated based on the information of the used starting substrate and the isoprene chain length by TLC. Moreover, Y identified the structure by NMR or IR.

(Example and Comparative Example)
(Comparison of activity of wild type enzyme and mutant type enzyme (relative activity by substrate))
The reaction was carried out using the starting substrate prepared in Preparation Examples 1 to 10, dimethylallyl diphosphate (DMAPP) and geranyl diphosphate (GPP) under the following conditions, and the activity of each mutant-type enzyme with respect to each starting substrate The activity of the wild-type enzyme (geranylgeranyl diphosphate synthetase from Sulfolobus acidocaldarius) was displayed as an index of 100 (Table 8).
500 ng of purified protein, 50 mM phosphate buffer (pH 5.8), 50 mM ammonium chloride, 0.01% Triton X-100, 50 mM 2-mercaptoethanol, 25 mM starting substrate (prepared in Preparations 1 to 10) Prepare a reaction solution containing each starting substrate, dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP), 1 mM [1- ¹⁴ C] isopentenyl diphosphate, for 30 minutes in a waterbath at 55 ° C. It was made to react. After the reaction, the activity of each enzyme was measured by quantifying the value of liquid scintillation and TLC.

(Example and Comparative Example)
(Preparation of polyisoprene)
10 μl of the latex component, 50 mM Tris-HCl buffer (pH 7.5), 2.5 mM magnesium chloride, 40 mM 2-mercaptoethanol, 40 mM potassium fluoride, 50 μM isoprene oligomer, 25 mM [1- ¹⁴ C] The reaction solution containing isopentenyl diphosphate was prepared, and was reacted in water bath at 30 ° C. for 3 days. After the reaction, the molecular weight was measured by GPC. And n and q in Formula (4) were computed based on the measured molecular weight and the information of the starting substrate used. The details of the obtained polyisoprene (n and q in the formula (4)) were as shown in Tables 9 and 10. In addition, Y was a hydroxyl group or a group represented by the above formula (2). In addition, identification of Y was performed in the same manner as in Example (preparation of isoprene oligomer).

As a latex component, a serum prepared by ultracentrifugation of a latex obtained from Hevea brasiliensis was used.

The starting isoprene oligomer (geranyl diphosphate, dimethylallyl diphosphate, Preparation Examples 1 to 5) was prepared using the mutant enzyme F77G or the like under the same conditions as in Example (Preparation of isoprene oligomer). The isoprene oligomers obtained using each of the starting substrates prepared in 10) were used.
In Tables 9 and 10 below, geranyl diphosphate, dimethylallyl diphosphate as the starting substrate, a compound represented by the above formula (A), a compound represented by the above formula (B), a compound represented by the above formula (A) The compound represented by C), the compound represented by the above formula (D), the compound represented by the above formula (E), the compound represented by the above formula (F), and the compound represented by the above formula (G) Using the compound, the compound represented by the above formula (H), the compound represented by the above formula (I), and the compound represented by the above formula (J), the same conditions as in Example (Preparation of isoprene oligomer) The isoprene oligomer obtained using the mutant enzyme F77G or the like in each of isoprene oligomer (GPP), isoprene oligomer (DMAPP), isoprene oligomer (A), isoprene oligomer (B), and isoprene oligomer (C), respectively. Isoprene oligomer (D) isoprene oligomer (E), isoprene oligomer (F), isoprene oligomer (G), isoprene oligomer (H), isoprene oligomer (I), and isoprene oligomer (J).

Next, the isoprene oligomer of the present invention and polyisoprene were blended into a rubber composition, and in order to evaluate the performance, first, an isoprene oligomer and polyisoprene to be subjected to the evaluation were prepared. Basically, it was prepared according to the above-mentioned Example (preparation of isoprene oligomer), Example (preparation of polyisoprene), but in order to adjust the molecular weight of the resulting isoprene oligomer, polyisoprene, The reaction was carried out by adjusting the conditions described in (Preparation) and (Preparation of polyisoprene).

Production Example 17
(Preparation of isoprene oligomer for rubber composition evaluation)
First, an isoprene oligomer was prepared using the starting substrate prepared in Preparation Examples 1 to 10, dimethylallyl diphosphate (DMAPP), and geranyl diphosphate (GPP). In addition, the details (n, m, Y in Formula (1)) of the obtained isoprene oligomer were determined by the method similar to an Example (preparation of an isoprene oligomer).

(Preparation of isoprene oligomer (2-GPP))
An isoprene oligomer (2-GPP) was prepared using geranyl diphosphate as the starting substrate. The details (n, m in the formula (1)) of the obtained isoprene oligomer (2-GPP) were n = 3 and m = 1. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of isoprene oligomer (2-DMAPP))
An isoprene oligomer (2-DMAPP) was prepared using dimethylallyl diphosphate as the starting substrate. The details (n, m in Formula (1)) of the obtained isoprene oligomer (2-DMAPP) were n = 2 and m = 2. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of isoprene oligomer (2-A))
The isoprene oligomer (2-A) was prepared using the compound represented by the above formula (A) as a starting substrate. The details (n, m in Formula (1)) of the obtained isoprene oligomer (2-A) were n = 2 and m = 1. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of isoprene oligomer (2-B))
Preparation of isoprene oligomer (2-B) was performed using the compound represented by said Formula (B) as a starting substrate. The details (n and m in Formula (1)) of the obtained isoprene oligomer (2-B) were n = 2 and m = 1. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of isoprene oligomer (2-C))
The isoprene oligomer (2-C) was prepared using the compound represented by the above formula (C) as a starting substrate. The details (n and m in the formula (1)) of the obtained isoprene oligomer (2-C) were n = 2 and m = 1. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of isoprene oligomer (2-D))
The isoprene oligomer (2-D) was prepared using the compound represented by the above formula (D) as a starting substrate. The details (n and m in Formula (1)) of the obtained isoprene oligomer (2-D) were n = 2 and m = 1. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of isoprene oligomer (2-E))
The isoprene oligomer (2-E) was prepared using the compound represented by the above formula (E) as a starting substrate. The details (n and m in the formula (1)) of the obtained isoprene oligomer (2-E) were n = 2 and m = 1. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of isoprene oligomer (2-F))
The isoprene oligomer (2-F) was prepared using the compound represented by the above formula (F) as a starting substrate. The details (n and m in the formula (1)) of the obtained isoprene oligomer (2-F) were n = 3 and m = 1. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of isoprene oligomer (2-G))
The isoprene oligomer (2-G) was prepared using the compound represented by the above formula (G) as a starting substrate. The details (n, m in Formula (1)) of the obtained isoprene oligomer (2-G) were n = 3 and m = 1. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of isoprene oligomer (2-H))
The isoprene oligomer (2-H) was prepared using the compound represented by the above formula (H) as a starting substrate. The details (n and m in Formula (1)) of the obtained isoprene oligomer (2-H) were n = 3 and m = 1. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of isoprene oligomer (2-I))
The isoprene oligomer (2-I) was prepared using the compound represented by the above formula (I) as a starting substrate. The details (n and m in the formula (1)) of the obtained isoprene oligomer (2-I) were n = 3 and m = 1. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of isoprene oligomer (2-J))
Preparation of isoprene oligomer (2-J) was performed using the compound represented by said Formula (J) as a starting substrate. The details (n, m in Formula (1)) of the obtained isoprene oligomer (2-J) were n = 3 and m = 1. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

Production Example 18
(Preparation of polyisoprene for evaluation of rubber composition)
Next, polyisoprene was prepared using the isoprene oligomer obtained in Production Example 17. In addition, the details (n, m, q, Y in Formula (4)) of the obtained polyisoprene were determined by the method similar to Example (preparation of polyisoprene).

(Preparation of polyisoprene (2-GPP))
Preparation of polyisoprene (2-GPP) was carried out using the isoprene oligomer (2-GPP) prepared in Preparation Example 17. The details (n, m, q in the formula (4)) of the obtained polyisoprene (2-GPP) were n = 3, m = 1, q = 1000 to 12000. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of polyisoprene (2-DMAPP))
Polyisoprene (2-DMAPP) was prepared using the isoprene oligomer (2-DMAPP) prepared in Preparation Example 17. The details (n, m, q in Formula (4)) of the obtained polyisoprene (2-DMAPP) were n = 2, m = 2 and q = 3 000-1500. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of polyisoprene (2-A))
Preparation of polyisoprene (2-A) was performed using the isoprene oligomer (2-A) prepared in Production Example 17. The details (n, m, q in the formula (4)) of the obtained polyisoprene (2-A) were n = 2, m = 1, q = 1000 to 18,000. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of polyisoprene (2-B))
Polyisoprene (2-B) was prepared using the isoprene oligomer (2-B) prepared in Production Example 17. The details (n, m, q in Formula (4)) of the obtained polyisoprene (2-B) were n = 2, m = 1, q = 3000 to 10000. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of polyisoprene (2-C))
Polyisoprene (2-C) was prepared using the isoprene oligomer (2-C) prepared in Production Example 17. The details (n, m, q in Formula (4)) of the obtained polyisoprene (2-C) were n = 2, m = 1, q = 3000 to 10000. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of polyisoprene (2-D))
Polyisoprene (2-D) was prepared using the isoprene oligomer (2-D) prepared in Production Example 17. The details (n, m, q in Formula (4)) of the obtained polyisoprene (2-D) were n = 2, m = 1, q = 3000-17000. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of polyisoprene (2-E))
Polyisoprene (2-E) was prepared using the isoprene oligomer (2-E) prepared in Production Example 17. The details (n, m, q in the formula (4)) of the obtained polyisoprene (2-E) were n = 2, m = 1, q = 1000 to 8000. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of polyisoprene (2-F))
Polyisoprene (2-F) was prepared using the isoprene oligomer (2-F) prepared in Production Example 17. The details (n, m, q in Formula (4)) of the obtained polyisoprene (2-F) were n = 3, m = 1, and q = 800-8000. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of polyisoprene (2-G))
Polyisoprene (2-G) was prepared using the isoprene oligomer (2-G) prepared in Production Example 17. The details (n, m, q in Formula (4)) of the obtained polyisoprene (2-G) were n = 3, m = 1, and q = 3000-12000. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of polyisoprene (2-H))
Polyisoprene (2-H) was prepared using the isoprene oligomer (2-H) prepared in Production Example 17. The details (n, m, q in Formula (4)) of the obtained polyisoprene (2-H) were n = 3, m = 1, and q = 3000-17000. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of polyisoprene (2-I))
Preparation of polyisoprene (2-I) was performed using the isoprene oligomer (2-I) prepared in Production Example 17. The details (n, m, q in the formula (4)) of the obtained polyisoprene (2-I) were n = 3, m = 1 and q = 1500 to 10000. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

(Preparation of polyisoprene (2-J))
Polyisoprene (2-J) was prepared using the isoprene oligomer (2-J) prepared in Production Example 17. The details (n, m, q in Formula (4)) of the obtained polyisoprene (2-J) were n = 3, m = 1, q = 4500 to 18000. In addition, Y was a hydroxyl group or a group represented by the above formula (2).

<Evaluation of rubber composition>
Next, the isoprene oligomer of the present invention and polyisoprene were blended into a rubber composition, and performance evaluation was performed.

Hereinafter, various medicines used by an example , a reference example, and comparative examples 1-4 are summarized and explained.
NR: TSR20
BR: BR01 manufactured by JSR Corporation
Carbon black: Diamond black (N220) manufactured by Mitsubishi Chemical Corporation
Isoprene oligomer: isoprene oligomer polyisoprene obtained in Production Examples 13 and 17: polyisoprene zinc obtained in Production Examples 14 and 18: Zinc oxide No. 1 stearic acid manufactured by Mitsui Metal Mining Co., Ltd. Stearic acid: NOF Corporation ) Stearic acid anti-aging agent: Noclac 6C (N- (1,3-dimethylbutyl) -N'-phenyl-p-phenylenediamine) manufactured by Ouchi Shinko Chemical Co., Ltd.
Wax: Sunnock wax sulfur manufactured by Ouchi Emerging Chemical Industry Co., Ltd. Powder sulfur vulcanization accelerator NS manufactured by Tsurumi Chemical Co., Ltd. NS: Noxella NS manufactured by Ouchi Emerging Chemical Industry Co., Ltd. (N-tert- Butyl-2-benzothiazylsulfenamide)
Silica: Nip seal AQ (wet silica) manufactured by Nippon Silica Co., Ltd.
Silane coupling agent: Si266 (bis (3-triethoxysilylpropyl) disulfide) manufactured by Degussa
Vulcanization accelerator DPG: Noxceler D (N, N-diphenylguanidine) manufactured by Ouchi Shinko Chemical Co., Ltd.

Examples , Reference Examples and Comparative Examples 1 to 4
According to the compounding prescription shown to Tables 11-14, materials other than sulfur and a vulcanization accelerator were knead | mixed using a 1.7 L Banbury mixer, and the kneaded material was obtained. Next, sulfur and a vulcanization accelerator were added to the obtained kneaded product, and the mixture was kneaded using an open roll to obtain an unvulcanized rubber composition. The obtained unvulcanized rubber composition was vulcanized at 150 ° C. for 30 minutes at a pressure of 80 kgf / cm ² using a steam vulcanization press to obtain a vulcanized rubber composition.

The following evaluation was performed about the obtained vulcanized rubber composition. The results are shown in Tables 11-14. The reference combination in Tables 11 and 12 was Comparative Example 2, and the reference combination in Tables 13 and 14 was Comparative Example 4.

(Viscoelastic test)
The tan δ was measured at 70 ° C. and a strain of 2% (initial elongation) at 70 ° C. using a viscometer manufactured by Iwamoto Seisakusho Co., Ltd., and the tan δ of the standard composition was expressed as an index of 100. The larger the index, the greater the heat generation. The heat resistance (low heat buildup) was considered to be improved when the index was 100 or less. That is, the smaller the index, the better the low heat buildup.

(Rambone wear test)
A wear test was carried out for 5 minutes under the conditions of a load of 3 kg, a slip ratio of 40% and a sand amount of 15 g / min using a Lambourn wear tester manufactured by Iwamoto Manufacturing Co., Ltd. The shape of the sample was 5 mm in thickness and 50 mm in diameter, and the grindstone used was a GC type abrasive of grain size # 80. The test results were indexed as 100 (standard) for the standard formulation. The larger the index, the better the abrasion resistance, and when the index exceeded 100, the abrasion resistance was considered to be improved.

(Tensile test)
A tensile test is carried out using a No. 3 dumbbell-shaped test piece consisting of the above-mentioned vulcanized rubber sheet according to JIS K6251 "Vulcanized rubber and thermoplastic rubber-Determination of tensile properties", and breaking strength (TB) (MPa) ) And elongation at break (EB) (%) were measured. The test results were indexed as 100 (standard) for the standard formulation. The larger the breaking strength and the breaking elongation, the better the breaking strength and the breaking elongation.

From Tables 11 and 12, an example using an isoprene oligomer in which at least one of the atoms or atomic groups contained in the I-I moiety in the above formula (1) is substituted by a sulfur atom or an atomic group containing a sulfur atom In the reference example, it was excellent in low heat build-up, wear resistance, and elongation at break (particularly, wear resistance).

From Tables 13 and 14, an example using polyisoprene in which at least one of the atoms or atomic groups contained in the II-I moiety in the above formula (4) is substituted by a sulfur atom or an atomic group containing a sulfur atom In the reference example, it was excellent in low heat buildup, wear resistance, and breaking strength (particularly, wear resistance).

(Sequence table free text)
SEQ ID NO: 1: Nucleotide sequence of farnesyl diphosphate synthetase from Bachilus stearothermophilus (wild-type enzyme) SEQ ID NO: 2 amino acid sequence of farnesyl diphosphate synthetase from Bachilus stearothermophilus (wild-type enzyme) SEQ ID NO: 3: Geranylgeranill from Sulfolobus acidocaldarius Nucleotide sequence of diphosphate synthetase (wild-type enzyme) SEQ ID NO: 4: Amino acid sequence of geranylgeranyl diphosphate synthetase (wild-type enzyme) from Sulfolobus acidocaldarius SEQ ID NO: 5: Nucleotide sequence of mutant enzyme Y81 R SEQ ID NO: 6: Mutation No. 7: Base sequence of mutant enzyme Y81S SEQ ID NO: 8: Amino acid sequence of mutant enzyme Y81S 9: Nucleotide sequence of mutant enzyme Y81D SEQ ID NO: 10: Amino acid sequence of mutant enzyme Y81D SEQ ID NO: 11: Nucleotide sequence of mutant enzyme F77G SEQ ID NO: 12: Amino acid sequence of mutant enzyme F77G SEQ ID NO: 13: Mutant enzyme F77S Nucleotide sequence of SEQ ID NO: 14: Amino acid sequence of mutant enzyme F77S SEQ ID NO: 15: Sense primer for preparation of mutant enzyme Y81R SEQ ID NO: 16: Antisense primer for preparation of mutant enzyme Y81 R SEQ ID NO: 17: Sense for preparation of mutant enzyme Y81 S Primer SEQ ID NO: 18: Antisense primer for preparation of mutant enzyme Y81S SEQ ID NO: 19: Sense primer for preparation of mutant enzyme Y81D SEQ ID NO: 20: Antisense primer for preparation of mutant enzyme Y81D SEQ ID NO: 21: Sense for preparation of mutant enzyme F77G Primer SEQ ID NO: 22: Modified Type enzyme F77G fabrication antisense primer SEQ ID NO 23: mutated enzyme F77S produced sense primer SEQ ID NO 24: mutated enzyme F77S fabrication antisense primer

Claims (8)

It is an isoprene oligomer represented by following formula (1), Comprising: The isoprene oligomer whose I-I part in following formula (1) is a following formula (e) or (j) .

(In the formula (1), n represents an integer of 1 to 10. m represents an integer of 1 to 30. Y represents a hydroxyl group, a formyl group, a carboxy group, an ester group, a carbonyl group or the following formula (2) Represents a group represented)

Represented by the following formula (3), and allylic diphosphate is a following formula (3) isoprene units the formula in (e) or (j), according to claim 1 Symbol for biosynthesis from isopentenyl diphosphate Method for producing isoprene oligomer of

(In the formula (3), p represents an integer of 1 to 10) The method for producing isoprene oligomer according to claim 2, wherein the biosynthesis is performed using an enzyme having prenyltransferase activity. The method for producing an isoprene oligomer according to claim 3, wherein the enzyme having prenyl transferase activity is a protein according to any one of the following [1] to [3].
[1] A protein consisting of an amino acid sequence represented by any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 [2] SEQ ID NOs: 2, 4, 6, 8, 10, 12, In the amino acid sequence represented by any one of SEQ ID NO: 14, which comprises a sequence containing 1 to 25 amino acid substitutions, deletions, insertions or additions, and is represented by the following formula (3), A protein having an activity of catalyzing a reaction between allylic diphosphate in which the isoprene unit in 3) is the formula (e) or (j) and isopentenyl diphosphate [3] SEQ ID NOs: 2, 4, 6 And an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence represented by any one of SEQ ID NOs: 8, 10, 12, 14 and represented by the following formula (3) allyl isoprene units in is the formula (e) or (j) Protein having a diphosphate, and the activity for catalyzing a reaction between isopentenyl diphosphate

(In the formula (3), p represents an integer of 1 to 10) It is a polyisoprene represented by following formula (4), Comprising: The polyisoprene whose II-I part in following formula (4) is a following formula (e) or (j) .

In the formula (4), n represents an integer of 1 to 10, m represents an integer of 1 to 30, and q represents an integer of 30 to 40000. Y represents a hydroxyl group, a formyl group, a carboxy group, an ester group , A carbonyl group or a group represented by the following formula (2))

1 SL and placing isoprene oligomer claims, method for producing a polyisoprene according to claim 5 wherein biosynthesized from isopentenyl diphosphate. Claim 1 Symbol placement isoprene oligomers and / or claim 5 rubber composition comprising a polyisoprene according. A pneumatic tire produced using the rubber composition according to claim 7 .