JP4198261B2 - Novel esterolytic enzyme - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel esterolytic enzyme, a gene thereof, and a production method thereof.
[0002]
[Prior art]
An esterolytic enzyme is an enzyme widely distributed in various microorganisms and degrades an ester bond. Among them, lipase not only decomposes triglycerides into mono- and di-glycerides and fatty acids, but also catalyzes transesterification and esterification reactions in a non-aqueous solvent system. Since such reactions in non-aqueous solvent systems are very useful for organic synthesis, esterolytic enzymes (particularly lipases) have been used in various industrial production processes (Andree et al., J. Biol. Appl. Biochem. 2: 218-219 (1980) and Bloking et al., Trends Biotechnol. 9: 360-363 (1991)), have been used for the production of cocoa substitutes by transesterification of palm oil.
[0003]
On the other hand, research is being advanced to modify fats and oils or to add new added value by utilizing the substrate specificity of esterase (lipase). For example, lipids are absorbed in the human body in the form of 2-monoglycerides, so if a substituent having a certain function can be introduced at the 2-position of triglycerides, oils such as palm oil can be converted into functional foods. it can.
[0004]
However, in general, an esterolytic enzyme (lipase) is significantly affected by a functional group in the vicinity of an ester bond, and there is a problem that hydrolysis is difficult when the functional group is bulky.
[0005]
Therefore, a highly specific esterase (lipase) is required even for an ester having a bulky functional group in the vicinity.
[0006]
[Problems to be solved by the invention]
An object of the present invention is to provide an enzyme having a highly specific esterolytic (lipase) activity even for an ester having a bulky functional group in the vicinity, and the enzyme gene can be isolated and mass-produced. It aims to be.
[0007]
[Means for Solving the Problems]
This object is achieved by providing an enzyme having the following properties. That is, the present invention relates to an enzyme that hydrolyzes oleyl benzoate and p-nitrophenyl benzoate, and has an amino acid sequence set forth in SEQ ID NO: 1. An enzyme identical to the enzyme having this sequence is not found by homology search and is novel.
[0008]
In addition, the present invention provides an oleyl benzoate having an amino acid sequence represented by SEQ ID NO: 1 having one or more amino acid substitutions, deletions or insertions, and higher than an enzyme having the amino acid sequence of SEQ ID NO: 1. The present invention relates to an esterolytic enzyme having hydrolytic activity and p-nitrophenylbenzoate hydrolytic activity.
[0009]
Furthermore, the present invention relates to a gene encoding an esterolytic enzyme having the amino acid sequence set forth in SEQ ID NO: 1.
[0010]
In a preferred embodiment, the gene encoding the esterolytic enzyme has the DNA sequence set forth in SEQ ID NO: 1.
[0011]
In addition, the present invention provides an oleyl benzoate hydrolysis activity and p-nitrophenyl having a substitution, deletion, or insertion of one or more amino acids in the sequence of SEQ ID NO: 1 and higher than the enzyme of SEQ ID NO: 1. It also relates to a gene encoding an esterolytic enzyme having benzoate hydrolyzing activity.
[0012]
Furthermore, the present invention provides an oleyl benzoate and p-, comprising an esterolytic enzyme expression vector having a gene encoding the above esterolytic enzyme, a cell transformed with the vector, and a step of culturing the transformed cell. The present invention relates to a method for producing an esterolytic enzyme that hydrolyzes nitrophenylbenzoate.
[0013]
The enzyme having a specific amino acid sequence or a similar sequence of the present invention is used for synthesizing a compound having a bulky functional group near an ester bond, imparting a new function, or the like.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
The esterolytic enzyme of the present invention has the amino acid sequence set forth in SEQ ID NO: 1 and its similar sequence. Such an enzyme is described in detail in Examples, but first, oleyl benzoate, which is a bulky substrate, is used. (Hereinafter referred to as OB) as a sole carbon source, a microorganism that grows is selected, and then a microorganism capable of degrading p-nitrophenylbenzoate (hereinafter referred to as pNPB) is screened, and an enzyme produced by the obtained microorganism Is purified and the amino acid sequence is determined, or the gene encoding this enzyme is selected from the obtained microorganism and the amino acid sequence is determined. At present, when the gene recombination technique has become a general technique, the latter method is usually used.
[0015]
OB and pNPB esterolytic enzymes can be obtained by screening from strains or soil samples stored in an available depository. For example, the cells are accumulated and cultured in a screening medium containing OB as a sole carbon source, and the grown strain is further cultured in the same medium to confirm the growth. After culture, OB is applied to an agar medium emulsified with β-cyclodextrin, and a strain having a clear zone forming ability is selected. Subsequently, the microorganism having the target enzyme can be selected by measuring the degradation activity of pNPB.
[0016]
The gene of the target enzyme can be selected, for example, by shotgun cloning. The selected microorganism is cultured, the genome is extracted by a conventional method, cleaved with an appropriate restriction enzyme, and an expression vector incorporated into an appropriate plasmid, for example, pBR322, pUC110, pUC18, pUC19, or the like is used in an appropriate host (for example, E. coli, yeast, Bacillus, etc.) (for example, transformation, electroporation, etc.), containing a selectable marker (eg, ampicillin, kanamycin, etc.) and an agar medium containing OB as the sole carbon source By screening for the ability to form a clear zone, a transformed strain having OB degrading activity can be obtained.
[0017]
The amino acid sequence is determined by confirming that the obtained transformant has the desired enzyme activity, extracting the plasmid, recovering the incorporated gene, and determining the base sequence. By culturing a vector (plasmid) having this gene, the target enzyme is produced in large quantities.
[0018]
Once the amino acid sequence or base sequence has been identified, the protein can be easily modified by substitution, deletion, or addition of one or more bases to the base sequence, and the activity is higher than that of the enzyme before modification. High enzymes can be obtained, which are also within the scope of the present invention.
[0019]
【Example】
EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, this invention is not limited to this Example.
[0020]
Examples of microorganisms that produce the target enzyme include microorganisms belonging to the genus Acinetobacter. For example, the Acinetobacter calcoaceticus subsp. Antiratus KM109 strain isolated by the present inventors (deposited at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology) The deposit number is FERM P-17224: hereinafter simply referred to as KM109 strain). Hereinafter, a method for screening a strain producing the target enzyme will be described first, and then isolation and identification of a gene from this strain will be described.
[0021]
(Screening of microorganisms producing enzymes that hydrolyze OB and pNPB)
A: Substrate synthesis The substrates OB and pNPB of the enzyme used in the present invention were synthesized by the following method.
[0022]
(Synthesis of OB)
43.5 g (Mw.268.48, 0.162 mol) of oleyl alcohol, 16.4 g (Mw. 101.19, 0.162 mol) of triethylamine, 0.59 g (Mw.122.17, 0.0048 mol) of 4-dimethylaminopyridine and 300 ml of hexane as a solvent under freezing Then, 25.0 g of benzoyl chloride (Mw. 140.57, 0.178 mol) was added dropwise. After 2 hours from the end of dropping, the temperature was gradually returned to room temperature and allowed to react overnight. After completion of the reaction, the product was purified by chromatography using 300 g of silica gel, eluting with 8 L of hexane. The purity of the product was confirmed by TLC (hexane: ethyl acetate = 9.1), UV absorption (San Gabriel, CA91778 USA, UVG-54), KMnO ₄ color development, and the product was ¹ H-NMR (HITACHI R -24B), IR (HORIBA FT-210-IR) confirmed the structure.
[0023]
(Synthesis of pNPB)
p-Nitrophenol 4.7g (Mw.139.11, 0.0338mol), pyridine 5.0ml (0.0618mol), 4-dimethylaminopyridine 0.21g (Mw.140.57, 0.00149mol) and hexane 150ml, benzene 50ml below freezing point While stirring well, 5.0 g (Mw. 140.57, 0.0356 mol) of benzoyl chloride was added dropwise. After 2 hours from the end of dropping, the temperature was gradually returned to room temperature and allowed to react overnight. After completion of the reaction, development on TLC (hexane: ethyl acetate = 20: 1), the product was confirmed by UV absorption, and the product was ¹ H-NMR (HITACHI R-24B), IR (HORIBA FT-210- IR) to confirm the structure.
[0024]
B. Measurement of pNPB hydrolysis activity Measurement of the hydrolysis activity of pNPB was performed as follows.
3 ml of a reaction solution containing 50 mM potassium phosphate (KPB, pH 6.5), 2.5 mM pNPB, 5% acetonitrile, and enzyme was preheated to 37 ° C., and pNPB (50 mM) dissolved in dry acetonitrile. ) The reaction was started by adding 150 μl. The reaction was stopped by adding 3 ml of ethanol, and the amount of p-nitrophenol produced was determined from the absorbance at _{400 nm} (ε _{400 nm} = 11,670). The amount of enzyme that produces 1 μmol of p-nitrophenol per minute was defined as 1 unit.
[0025]
C. Screening A soil / water sample obtained from Osaka Prefecture was placed in a 3 ml liquid medium containing 0.5% (NH ₄ ) ₂ SO ₄ , 0.1% KH ₂ PO ₄ , 0.05% MgSO ₄ , 0.5% NaCl, and 1.0% OB. pH7.2) was inoculated and cultured at 30 ° C. for 48 hours. If the medium becomes cloudy, 1% of the medium is planted twice on the same medium, 0.2% β-cyclodextrin (Nippon Food Processing Co., Ltd.) is added to the medium, and the mixture is stirred well to emulsify OB. And grown on a solid medium supplemented with 1.5% agar and cultured at 30 ° C. for 7 days. The growth state on the second day of culture and the ability to form a clear zone caused by decomposing OB on the seventh day were observed.
[0026]
A strain having a clear clear zone was selected and cultured in LB medium (50 ml) containing 0.5% OB at 30 ° C., 120 sp., 46 hours using a 500 ml Sakaguchi flask, and centrifuged (5,500 × g, 10 And 4 ° C.) to obtain bacterial cells and culture supernatant. The cells were suspended in KPB (50 mM, pH 6.5) containing 1 mM MgCl ₂ and then crushed with ultrasonic waves (Kaijo Electric Co., Ltd., 4280 type vibrator) for 30 seconds × 2 times. This was centrifuged again (12,000 × g, 20 minutes, 4 ° C.) to remove crushed cells, and a cell extract was obtained. Using pNPB as a substrate, the hydrolysis activity of this cell extract and culture supernatant was measured.
[0027]
In this screening, strains showing high activity for pNPB were selected, and further screened using pNPA, pNPB and OB as substrates. In addition, pNPA is p-nitrophenyl acetate, purchased from Tokyo Chemical Industry Co., Ltd., and the activity was measured by a method according to pNPB.
[0028]
The KM109 strain was isolated from this, and the enzyme produced by this strain was considered to be able to act on a bulky substrate with pNPB activity three times higher than pNPA activity. . Therefore, this strain was first identified, and then the esterase gene was isolated using this strain.
[0029]
(Identification of KM109 strain)
The mycological properties of the KM109 strain are shown below.
[0030]
A. Form (1) Cell shape Staphylococcus aureus (2) Cell size 0.9-1.6 × 1.5-2.5μm
(3) Presence or absence of mobility −
(4) Presence or absence of spores-
(5) Gram staining-
(6) Anti-acid staining −
[0031]
B. Growth condition in each medium
(1) Standard agar plate culture Light brown, glossy, smooth, perfect circle (2) Standard agar slope culture Light brown, glossy, smooth (3) Standard liquid culture The upper layer is slightly darker and the coating is thin on the liquid surface (4) Standard gelatin puncture culture No weak growth or liquefaction is observed along the puncture line (5) Litmus milk No change [0032]
C. Physiological properties (1) Reducing ability of nitrate
(2) Denitrification reaction −
(3) Methyl red test −
(4) Formation of acetylmethyl carbinol −
(5) Formation of indole −
(6) Production of hydrogen sulfide −
(7) Starch hydrolysis-
(8) Use of citric acid +
(9) Use of inorganic nitrogen source Nitrate: + Ammonium salt: +
(10) Production of pigment-
(11) Urease activity −
(12) Oxidase activity −
(13) Catalase activity +
(14) Range of growth
pH 4.0-, 4.5 +, 7.0 +,
9.5 +, 10.0-
Temperature 37 ℃ +, 41 ℃ + ^W , 45 ℃ −
(15) Attitude toward oxygen Aerobic (16) Production of dioxyacetone −
(17) Decomposition of hippuric acid +
(18) Degradation of amino acids Lysine-, arginine-, ornithine-
(19) Deamination of phenylalanine −
(20) Temperature resistance (85 ° C, 10 minutes)-
(21) Resistance of sodium chloride 2.0% +, 5.0%-
7.0% −, 10% −
(22) Growth of Sabouraud agar medium +
(23) Growth of 0.001% lysozyme medium +
(24) Degradation of tyrosine −
(25) Citric acid / ammonium agar −
Production in China (26) Degradability of casein −
(27) Degradability of gelatin −
(28) Growth in anaerobic media −
(29) Mackonki medium viability +
(30) Recitinase reaction-
(31) Alkali production ability in VP medium −
(32) Utilization of sugars and bioacidic L-arabinose +
D-xylose +
D-glucose +
D-Mannose +
D-fructose-
D-galactose +
Maltose −
Sucrose −
Lactose +
Trehalose −
D-Sorbit-
D-man knit-
Inosit −
Glycerin −
Starch −
Melibiose +
Salicin −
Ethanol +
(33) Growth on SS agar −
(34) Growth on KCN medium +
(35) ONPC −
(36) DNase −
[0033]
Based on the above mycological properties, the isolated strain was identified as Acinetobacter calcoaceticus subsp. Antiratus.
[0034]
(Isolation of enzyme gene of the present invention and determination of nucleotide sequence)
The obtained KM109 strain was cultured in LB medium, and genomic DNA was extracted according to the method of Rao et al. (Rao et al., Method Enzymol. 153, 166-198 (1987)). The genomic DNA is cleaved with the restriction enzyme EcoRI, ligated to the vector pUC19 (purchased from Takara Shuzo Co., Ltd.) cleaved with EcoRI, E. coli DH5α is transformed, and the color on the LB medium containing ampicillin, X-gal and IPTG White colonies (transformants) were selected by selection.
[0035]
First, the obtained white colonies were screened for tributyrin degrading activity. The white colonies selected above were transferred to tributyrin medium, cultured at 37 ° C for 1-2 days, and stored at 4 ° C for about one week. Among these, the one that formed the clear zone was transferred again to the tributyrin medium, and 56 colonies that surely formed the clear zone were obtained.
[0036]
Next, pNPB was used as a substrate, and transformants having pNPB degrading activity were screened. First, the 56 colonies were inoculated on LB + 1% Neugen agar medium, cultured at 37 ° C. overnight, and stored at 4 ° C. for one day. Next, 150 μl of 1 M KPB buffer (pH 7.5) and 150 μl of 50 mM pNPB (acetonitrile solution) were added to soft agar (0.7% agar, 3 ml), and the mixture was overlaid on the storage plate. After culturing at 37 ° C. for a day, the medium around the cells turned yellow and a strain that formed a clear zone was selected as a pNPB hydrolyzing active strain. Six colonies (KE1, KE3, KE10, KE31, KE32, KE33) were selected.
[0037]
As a result of extracting plasmids retained by these colonies and comparing them with restriction enzyme cleavage patterns, it was found that 4 colonies of KE1, KE10, KE32, and KE33 had the same fragment (1.2 kbp), The plasmid derived from the KE32 strain was named pKE32. The fragment (1.6 kbp) on the plasmid (pKE3) carried by KE3 almost overlaps with the inserted fragment on pKE32, but additionally had an extra EcoRI fragment of about 400 bp. Therefore, the pNPB activity of three types of KE3, KE31 and KE32 was confirmed.
[0038]
(Confirmation of pNPB hydrolysis activity)
For KE3, KE31, KE32 and pUC19 as a control, the pNPB degradation activity was confirmed by absorbance measurement. First, the transformed strain was inoculated into 3 ml of LB liquid medium containing 50 μg / ml of ampicillin and cultured at 120 ° C. for 16 hours at 37 ° C. 3 ml of this culture solution was inoculated into 250 ml of LB liquid medium containing 50 μg / ml of ampicillin and OB 5%, and cultured at 37 ° C. for 47 hours at 170 sp.pm. The cells were collected by centrifugation, suspended in 10 ml of 50 mM KPB buffer (pH 6.5) per gram of bacterial cells, and the enzyme solution was prepared by ultrasonic disruption (30 seconds × 4, 4 ° C.).
[0039]
The pNPB degradation reaction was carried out at 37 ° C. for 10 minutes by adding 25 μl of 50 mM pNPB to a test tube containing 10 μl of enzyme solution and 465 μl of KPB buffer (pH 6.5). The reaction was stopped by adding 500 μl of ethanol, and the activity was calculated from the results of absorbance measurement at 400 nm. In KE3 7.68 × 10 ^- it was ¹ units / ml, the ^{KE31 2.45 × 10 -2 units / ml} , the KE32 1.24units / ml, 1.39 × 10 -2 units / ml in pUC19.
[0040]
The OB hydrolytic ability of KE3 was measured by liquid chromatography. The hydrolysis reaction was performed by adding 100 μl of OB to 5 ml of the enzyme solution, and in a 50 ml vial at 37 ° C. and 170 sp.pm for 18 hours. The reaction was stopped by adding 2 ml of ethyl acetate to 1 ml of the reaction solution. The ethyl acetate layer was extracted, evaporated, dissolved in 200 μl of acetonitrile, 10 μl of which was subjected to HPLC. A calibration curve was prepared with oleyl alcohol, and the activity was measured by the amount of oleyl alcohol produced. The column was measured using a CAPCELPAK C ₁₈ SG120 (Shiseido) and 95% acetonitrile (0.5% TFA) as a solvent at a wavelength of 210 nm. The retention time of oleyl alcohol was 12 minutes. When the enzyme activity was calculated from the HPLC results, it was 8.70 nmoles / h (per 1 ml of reaction solution for 18 hours) for KE3.
[0041]
Comparison of the activities of clones KE3, KE31, and KE32 revealed that KE32 had the greatest pNPB-degrading activity, but it was estimated that the fragment on pKE3 covers a wider region, so both bases of pKE3 and pKE32 The sequence was determined. The sequence of the structural gene and the deduced amino acid sequence are as described in SEQ ID NO: 1.
[0042]
As a result of homology search, a sequence identical to this amino acid sequence was not found, and it was confirmed that the enzyme having this sequence is novel.
[0043]
【The invention's effect】
The present invention provides an enzyme useful for hydrolyzing a bulky substrate, its structural gene. As a result, a large amount of an enzyme that is considered useful for the production of an optically active substance necessary for an intermediate raw material for pharmaceuticals can be supplied.
[0044]
[Sequence Listing]

Claims (6)

An enzyme that hydrolyzes oleyl benzoate and p-nitrophenyl benzoate, comprising an esterolytic enzyme consisting of the amino acid sequence set forth in SEQ ID NO: 1, or one or more amino acids in the amino sequence set forth in SEQ ID NO: 1. An esterolytic enzyme having a substitution, deletion, or insertion and having oleyl benzoate hydrolyzing activity and p-nitrophenyl benzoate hydrolyzing activity . A gene encoding the esterolytic enzyme according to claim 1 . Gene encoding the esterolytic enzyme comprising DNA sequence set forth in SEQ ID NO: 1, gene of claim 2. An expression vector for an esterolytic enzyme comprising the gene according to claim 2 or 3 . A transformed cell comprising the expression vector according to claim 4 . A method for producing an esterolytic enzyme comprising hydrolyzing oleyl benzoate and p-nitrophenyl benzoate, comprising a step of culturing the transformed cell according to claim 5 .