JP3957873B2 - Pectate lyase gene - Google Patents

Description

【００３０】

【００３１】

【００３２】

【図面の簡単な説明】
【図１】ペクチン酸リアーゼ遺伝子クローニング用プライマー１及び２の塩基配列を示す図である。
【図２】プライマー１とプライマー２の間のＤＮＡ配列と推定アミノ酸配列及びプライマー３とプライマー４の位置を示す図である。
【図３】本発明ペクチン酸リアーゼ全領域を増幅するためのプライマー５とプライマー６を示す図である。実施例４で示したＰＣＲ増幅用プライマーで、生成する増幅断片（約１kbp）はＸｈｏＩで消化された後、ＳａｌＩとＳｍａＩで消化されたｐＨＳＰ６４に連結される。
【図４】ペクチン酸リアーゼ遺伝子（Ｐ１０３由来ｐａｌ）の分泌ベクター（ｐＨＳＰ６４）への導入と創製されたペクチン酸リアーゼ発現分泌ベクターｐＨＳＰ−１０３ＰＡＬを示す図である。
Ａｍｐ：アンピシリン耐性マーカー遺伝子
Ｔｅｔ：テトラサイクリン耐性マーカー遺伝子
【図５】Ｐ１０３組換えペクチン酸リアーゼ（実施例４）のpH−活性曲線を示す。５０mMグリシン−水酸化ナトリウム緩衝液で最大活性を示すpH約１０の値を１００％として、相対活性を表示した。
【図６】Ｐ１０３組換えペクチン酸リアーゼと他の酵素のアミノ酸配列の相同性を示す図である。
アミノ酸番号は、配列番号１の９４から２２８を基準として、他のアミノ酸配列と相同性を合わせた。＊印は共通のアミノ酸残基を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a gene encoding pectate lyase useful as a cleaning agent, food processing agent, fiber treatment agent and the like.
[0002]
[Prior art]
Pectate lyase (EC 4.2.2.2) is 1962, which has been first found in the culture solution of Bacillus polymyxa (Bacillus polymyxa) and Erwinia Karutobora (Erwinia cartovora), in need of calcium ions in the reaction (Starr & Moran, Science, 135, 920-921, 1962). Since then, many bacteria and fungi have been known to produce pectate lyase (Sakai, et al. Adv. Appl. Microbiol. 39, 213-294, 1993). Furthermore, as the technology of genetic recombination has developed, for example, Erwinia chrysanthemi EC16 (Tamaki, et al., J. Bacteriol., 170, 3468-3478, 1988), Xanthomonas campestrispv . Malvaccarum B414 (Liao et al., MPMI, 9, 14-21, 1996), Fusarium solani f. Sp. Pisi (Guo et al., J. Bacteriol., 177, 7070-7077, 1995, Arch. Biochem. Biophys., 332, 305-312, 1996, Gonzalez-candels & kolattukudy, J. Bacteriol., 174, 6343-6349, 1992), Aspergillus nidulans (Ho et al., Curr. Genet., 27, 142-149, 1995) etc. The base sequence is determined by cloning, and the deduced amino acid sequence is analyzed.
[0003]
There are relatively few reports on pectate lyase of Bacillus genus bacteria that have been well studied genetically and biochemically. Since Starr and Moran discovered pectate lyase in the culture of Bacillus polymyxa , Bacillus pumilus (Dave & Vaughn, J. Bacteriol., 198, 166-174, 1971), Bacillus subtilis (Chesson & Codner, J. Appl Bacteriol., 44, 347-364, 1978), an enzyme with an optimum pH of 8.5-9.3 produced by a thermophilic Bacillus bacterium (molecular weight unknown) (Karbassi & Luh, J. Food Sci. , 44, 1156-1161, 1979). Recently, Sakamoto et al. (Biosci. Biotech. Biochem., 58, 353-358, 1994) have purified pectate lyase having protopectinase activity from Bacillus subtilis IFO3134 (protopectinase-N). Those pectate lyase gene of Bacillus genus is determined Bacillus subtilis SO 113 (Nasser et al ., FEBS 335 319-326, 1993), Alkali-tolerant Bacillus sp. YA-14 (Kim et al., Biosci. Biotech. Biochem. 58, 947-949, 1994), and the nucleotide sequences of the SO 113 strain and the YA-14 strain show 99% homology and the amino acid sequences are completely identical. Met.
[0004]
[Problems to be solved by the invention]
Although these conventional pectate lyases are available as some products, they are expensive and have problems such as the presence of various saccharide-degrading enzymes, and are not widely used in the industry.
[0005]
Therefore, the present invention provides a pectate lyase useful in a wide range of industries such as detergents, fiber treatments, etc., in order to enable single and mass production, a gene encoding it, a recombinant vector containing the gene, And it aims at providing the transformant containing this.
[0006]
[Means for Solving the Problems]
Therefore, the present inventor has searched for microorganisms that produce pectate lyase from nature, and has made various studies to establish its gene, its characteristics, and its mass production technology using genetic engineering techniques. With regard to a novel pectate lyase produced by Bacillus bacteria, the present inventors have succeeded in cloning the gene and establishing a production technique by gene recombination, thereby completing the present invention.
[0007]
That is, the present invention is one or several amino acids in the amino acid sequence or the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 by deletion, provides pectate lyase gene encoding substitution or addition of the amino acid sequence Is.
The present invention also provides a recombinant vector containing the pectate lyase gene and a transformant containing the recombinant vector.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
The gene of the present invention has an amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a sequence encoding an amino acid sequence in which one or several amino acids of the amino acid sequence are deleted, substituted or added. As long as pectate lyase activity is not lost, amino acid deletion, substitution or addition (hereinafter sometimes referred to as mutation) in the amino acid sequence is not particularly limited. In addition, 1 to several amino acids may be added to the N-terminus in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
[0009]
When the homology with other enzymes is examined between amino acid numbers 94 to 228 in SEQ ID NO: 1, as shown in FIG. 6, pectate lyase PelA derived from Aspergillus nidulans (Meng-Chen Ho, et al. Curr. Gcnet., 27. 142-149, 1995) has the highest homology, but only 43.9% homology. Therefore, an enzyme having homology higher than these when properly aligned between amino acid numbers 94 to 228 of SEQ ID NO: 1 is included in the enzyme encoded by the gene of the present invention. That is, the homology of amino acid sequences is preferably 45% or more, more preferably 70% or more, and particularly preferably 80% or more with respect to amino acid numbers 94 to 228 in SEQ ID NO: 1.
[0010]
Moreover, when the homology with other enzymes is examined between amino acid numbers 1 to 302 in SEQ ID NO: 1, pectate lyase derived from Glomerella cingulata (GenBank registration No. GCU32942) has the highest homology. There is only 39.9% homology between SEQ ID NOs: 14-287. Therefore, an enzyme having homology higher than those when appropriately aligned between amino acid numbers 14 to 287 of SEQ ID NO: 1 is included in the enzyme encoded by the gene of the present invention. That is, the homology of the amino acid sequence is preferably 40% or more, more preferably 70% or more, and particularly preferably 80% or more with respect to amino acid numbers 14 to 287 in SEQ ID NO: 1.
[0011]
The pectate lyase gene of the present invention may be any one as long as it encodes the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or the variant thereof, but the nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4 or the nucleotide sequence thereof Those having a base sequence in which one or several bases are deleted, substituted or added are preferred.
[0012]
The pectate lyase encoded by the gene of the present invention includes microorganisms belonging to the genus Bacillus isolated from soil (for example, Bacillus sp. KSM-P103 strain (FERM P-1588), Bacillus sp. KSM-P7 strain (FERM P-15985), and the like. Can be produced by culturing the mutant strain and collecting it from the obtained culture.
[0013]
The pectate lyase gene of the present invention can be cloned from, for example, Bacillus sp. KSM-P103 strain (FERM P-15588), Bacillus sp. KSM-P7 strain (FERM P-15985) or the like. Examples of the cloning means include known means such as a method of cloning a target gene by a shot gun method or a PCR method.
[0014]
In order to prepare a recombinant vector containing the pectate lyase gene, the pectate lyase gene may be incorporated into any vector suitable for expressing the gene in the target host. Examples of such vectors include pUC18, pBR322, and pUC19 when Escherichia coli is used as a host, and pUB110 and the like when Bacillus subtilis is used as a host.
[0015]
In order to transform a host using the recombinant vector thus obtained, a conventional method such as a protoplast method, a competent cell method or the like is performed. Although it does not restrict | limit especially as a host, A microorganism is preferable, Gram positive bacteria, such as Bacillus genus and Streptomyces genus; Gram negative bacteria, such as Escherichia coli ; Fungi, such as Saccharomyces genus yeast, Aspergillus genus mold, etc. It is done.
[0016]
The obtained transformant was inoculated into a medium containing an assimilable carbon source, nitrogen source and other essential nutrients, cultured according to a conventional method, and in accordance with a general enzyme collection and purification method from the obtained culture broth The pectate lyase can be obtained by the method.
[0017]
【Example】
EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.
[0018]
Example 1: Culture of Bacillus sp. KSM-P103 strain and Bacillus sp. KSM-P7 strain Bacillus sp. KSM-P103 strain and Bacillus sp. KSM-P7 strain were cultured at 30 ° C. by adding 50 ml of liquid medium to a Sakaguchi flask. Shaking culture was performed for 1 day. The medium composition is 3% (w / v) polypeptone S, 0.5% yeast extract, 1% fish meat extract, 0.15% dipotassium phosphate, 0.001% calcium chloride, 0.5% pectin, 0. 5% sodium carbonate (separately sterilized) was used.
[0019]
Example 2: Purification of Bacillus sp. KSM-P103 strain and Bacillus sp. KSM-P7 strain-derived pectate lyase The culture solution of Bacillus sp. KSM-P103 strain and Bacillus sp. KSM-P7 strain was centrifuged (10,000 × g, 15 minutes, 4 ° C.) and ammonium sulfate was gradually added to the supernatant (900 ml) so as to be 65% saturated. After being left overnight at 5 ° C., the precipitate was collected by centrifugation (10,000 × g, 15 minutes, 4 ° C.). This was dissolved in a small amount of a basic buffer [10 mM Tris-HCl buffer (pH 7.5) containing 1 mM calcium chloride] and dialyzed against the buffer overnight. The obtained dialysis internal solution (105 ml) was applied to a DEAE-Bio-GelA (Bio-Rad) column (2.5 × 16 cm) which had been equilibrated with a basic buffer in advance. Activity was observed in the fractions washed and eluted with the basic buffer and collected. Next, this non-adsorbed fraction was applied to a CM-Bio-GelA (Bio-Rad) column (2.5 × 22 cm) that had been equilibrated in advance with a basic buffer. After washing and elution with about 300 ml of basic buffer solution, the adsorbed protein was eluted by a concentration gradient elution method using a basic buffer solution (400 ml each) containing 0 to 0.1 M potassium chloride. The pectate lyase derived from the KSM-P103 strain and the KSM-P7 strain was eluted as a single peak at a potassium chloride concentration of about 70 to 80 mM, and was detected as a uniform protein in SDS-PAGE and PAGE by the Taber method. These fractions were collected and concentrated by ultrafiltration (YM3 membrane, manufactured by Amicon) and stored frozen at −20 ° C. as a 20% glycerol solution. In the case of the purified pectate lyase enzyme derived from the KSM-P103 strain obtained by the above operation, the activity yield was about 42%, and the purification factor was about 500 times. On the other hand, in the case of the purified pectate lyase enzyme derived from the KSM-P7 strain, the activity yield was about 25% and the purification ratio was about 180 times.
Each purified enzyme was blotted on a ProSorb filter (manufactured by PerkinElmer), and the amino terminal sequence was determined up to the 18th amino acid using a protein sequencer (476A type, manufactured by Applied Biosystems). As a result, KSM-P103 strain and KSM -The enzyme derived from the P7 strain was ANFNQQGFSTLNGGTTGG.
[0020]
Example 3: Cloning of pectate lyase gene (1) Preparation of chromosomal DNA of Bacillus sp. KSM-P103 strain and Bacillus sp. KSM-P7 strain Bacillus sp. KSM-P103 strain and Bacillus sp. KSM-P103 strain were shake-cultured in a liquid medium. The culture solution was centrifuged, and the cells were collected. Chromosomal DNA was prepared from the obtained cells by the method of Saito and Miura (Biochim. Biophys. Acta, 72, 619-629, 1963).
[0021]
(2) Preparation of Primer Primer 1 and Primer 2 were synthesized from the amino terminal sequence of the purified enzyme obtained in Example 2 and the amino acid sequence of the pectate lyase conserved region (FIG. 1).
(3) Cloning Using these primers 1 and 2, PCR was performed using the chromosomal DNA (0.5 μg) of Bacillus sp. KSM-P103 strain as a template. The obtained amplified fragment was purified with a PCR fragment purification kit (manufactured by Boehringer Mannheim), then introduced into the Sma I site of the plasmid vector pUC19 and cloned. When the base sequences of the four clones obtained were determined, a part of the target pectate lyase gene sequence was detected, and the amino acid sequence could also be estimated (see FIG. 2).
Next, inverse PCR was performed to amplify the upstream and downstream regions of the above-mentioned PCR amplified fragment. The base sequences found in FIG. 2, primer 3 and primer 4 were used. The Bacillus sp KSM-P103 strain chromosome of (1 [mu] g), previously digested with Hin dIII, phenol / chloroform extracted and, as a template by intramolecular coupling using T4DNA ligase (circularization DNA binding). PCR was performed using the Long Template System PCR kit (TaKaRa). As a result, an amplified fragment of about 4.5 kbp was detected. When this DNA fragment and primers 1-2 were sequenced by direct sequencing, the amino acid sequence and base sequence of pectate lyase consisting of 302 amino acids from the N-terminal amino acid sequence to the amino acid before the stop codon (TAA) (SEQ ID NOs: 1 and 3) were determined. The molecular weight of pectate lyase (secreted mature enzyme) produced by Bacillus sp. KSM-P103 strain is estimated to be 33,212 Da (about 33 kDa) from this sequence. Further, the amino acid sequence and base sequence of pectate lyase consisting of 302 amino acids from the N-terminal amino acid sequence of pectate lyase produced by Bacillus sp. KSM-P7 strain to the amino acid before the stop codon (TAA) by the same method ( SEQ ID NOs: 2 and 4) were determined. The molecular weight of pectate lyase (secreted mature enzyme) produced by Bacillus sp. KSM-P7 strain is estimated to be 33,355 Da (about 33 kDa) from this sequence.
[0022]
Example 4: Construction of recombinant plasmid and production of pectate lyase by Bacillus subtilis First, the N-terminal amino acid Ala of pectate lyase produced by Bacillus sp. KSM-P103 and the signal sequence derived from the host vector used were An upstream primer (primer 5, see FIG. 3) was designed to be ligated. The primer on the downstream side (primer 6, see FIG. 3) was designed as a 26 bp sequence located 73 bp downstream from the stop codon (TAA) of the pectate lyase gene. The Sal I site present on the 3 'side of the signal sequence coding part of the used vector pHSP64 (Sumitomo et al., Biosci. Biotech. Biochem., 56, 872-877, 1992) and the Sma I site located 11 bp downstream thereof. A PCR-amplified DNA fragment was inserted between them.
[0023]
That is, PCR was performed using primers 5 and 6 and the chromosomal DNA of the Bacillus sp. KSM-P103 strain as a template, and an approximately 1 kb pDNA fragment was amplified. This was digested with Xho I and ligated with ligase and pHSP64 cleaved with Sal I and Sma I (see FIG. 4).
This recombinant plasmid was named pHSP-103PAL.
[0024]
Next, Bacillus subtilis ISW1214 was transformed with pHSP-103PAL and cultured in a liquid medium at 30 ° C. for 3 days to produce a significant amount of pectate lyase outside the cells. Further, a large amount of pectate lyase was produced outside the cells by the same method as described above using the pectate lyase structural gene derived from Bacillus sp. KSM-P7.
[0025]
Reference Example 1: Characteristics of recombinant pectate lyase The two crude pectate lyase enzyme solutions obtained in Example 4 were completely purified according to Example 2.
[Standard enzyme activity assay]
In a test tube, 0.2 ml of 0.5 M glycine-sodium hydroxide buffer (pH 10.5), 0.1 ml of 4 mM calcium chloride solution, 0.2 ml of 1% (w / v) polygalacturonic acid aqueous solution (ICN) Manufactured by Lot 14482, adjusted to pH 6.8 with sodium hydroxide solution) and 1.4 ml of deionized water, incubated at 30 ° C. for 5 minutes, and then 0.1 ml of an appropriately diluted enzyme solution ( Dilution was carried out with 50 mM Tris-HCl buffer containing 1 mM calcium chloride, pH 7.5) to start the reaction. After incubating at 30 ° C. for 10 minutes, 2 ml of 50 mM hydrochloric acid was added to stop the reaction. The amount of the unsaturated polygalacturonide produced was determined by measuring the absorbance at 235 nm and using the molecular extinction coefficient of unsaturated digalacturonide 4600 (Hasegawa & Nagel, J. Food Sci., 31, 838-845, 1966). In the blind test, 2 ml of 50 mM hydrochloric acid was added to the reaction solution treated without adding the enzyme solution, and then 0.1 ml of the enzyme solution was added. One unit (1 U) of enzyme was defined as an amount that produced unsaturated polygalacturonide equivalent to 1 μmol of unsaturated digalacturonide per minute under the above reaction conditions.
[0026]
(1) N-terminal amino acid sequence When the N-terminal amino acid sequences of these enzymes were determined in the same manner as in Example 2, sequences containing ANFNQQGFSTLNGGTTTGG were detected for both enzymes.
(2) pH-activity curve The pH-activity curves of P103 and P7 recombinant pectate lyase were examined. When polygalacturonic acid was used as a substrate, the pH-activity curve of P103 recombinant pectate lyase was almost completely identical to the pH-activity curve of pectate lyase derived from Bacillus sp. KSM-P103 obtained in Example 2. The optimum reaction pH was around pH 10 in glycine-sodium hydroxide buffer (FIG. 5). Similar results were obtained with the P7 recombinant enzyme.
[0027]
(3) Molecular weight The molecular weight of the obtained recombinant pectate lyase was measured by SDS-polyacrylamide electrophoresis (15% acrylamide). The molecular weight of P7 and P103 recombinant pectate lyase was in the range of 31 to 33 kDa. Turned out to be. This value almost coincided with the molecular weight calculated from the deduced amino acid sequence. As a standard protein, SDS-PAGE Molecular Weight Standards, Low Range manufactured by Biorat was used.
(4) 0.05 M glycine containing 0.4 mM CaCl ₂ of 10 mg of cotton fiber pieces obtained by finely cutting work gloves for quality control (cotton with 40-smus bleached fingers, manufactured by Hisayoshi Nakata) as a pectin release substrate from cotton fibers -Suspended in sodium hydroxide buffer (pH 10.5), added with the two recombinant pectate lyases obtained in Example 4, and reacted at 30 ° C for 1 hour. The reaction completed solution was centrifuged, and pectin released in the supernatant was quantified by the carbazole sulfate method (Sakai, Methods Enzymol., 161, 335-350, 1988). As a result, when 0.1 U of enzyme was used, a pectin release amount of 2.3 to 3.6 mg as galacturonic acid per 1 g of cotton fiber was recognized in any enzyme. This means that both recombinant pectate lyases are acting on protopectin on the surface of cotton fibers, and it was also found that the absorbance at 235 nm of the supernatant was also increased. It is judged that the protopectinase activity is.
[0028]
【The invention's effect】
Using the pectate lyase gene of the present invention, a pectate lyase useful as a detergent, food processing agent, fiber treatment agent, etc. can be produced in a single and large amount.
[0029]
[Sequence Listing]

[0030]

[0031]

[0032]

[Brief description of the drawings]
FIG. 1 shows the nucleotide sequences of primers 1 and 2 for cloning pectate lyase gene.
FIG. 2 is a diagram showing the DNA sequence between primer 1 and primer 2, the deduced amino acid sequence, and the positions of primer 3 and primer 4.
FIG. 3 is a diagram showing primer 5 and primer 6 for amplifying the entire region of pectate lyase of the present invention. The amplified fragment (about 1 kbp) produced with the PCR amplification primers shown in Example 4 is digested with Xho I and then ligated to pHSP64 digested with Sal I and Sma I.
FIG. 4 shows the introduction of a pectate lyase gene (P103-derived pal) into a secretion vector (pHSP64) and the created pectate lyase expression secretion vector pHSP-103PAL.
Amp: Ampicillin resistance marker gene Tet: Tetracycline resistance marker gene FIG. 5 shows the pH-activity curve of P103 recombinant pectate lyase (Example 4). Relative activity was expressed with a value of about pH 10 showing maximum activity in 50 mM glycine-sodium hydroxide buffer as 100%.
FIG. 6 shows the homology between the amino acid sequences of P103 recombinant pectate lyase and other enzymes.
Amino acid numbers were matched with other amino acid sequences based on 94 to 228 of SEQ ID NO: 1. * Indicates a common amino acid residue.

Claims (5)

  A pectate lyase gene encoding the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence in which one or several amino acids of the amino acid sequence are deleted, substituted, or added. Pectate lyase gene, one or several bases are deleted in the nucleotide sequence or the base sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4, pectin according to claim 1 wherein is made of substituted or added in the nucleotide sequence Acid lyase gene.   A recombinant vector containing the gene according to claim 1 or 2.   A transformant comprising the recombinant vector according to claim 3.   The transformant according to claim 4, wherein the host is a microorganism.

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