JPH0691818B2 - Method for producing neopullulanase by transformed Bacillus subtilis - Google Patents

Description

Detailed Description of the Invention

[0001]

This invention relates to a neopullulanase having the ability to cleave the α-1,4 and α-1,6 glucoside bonds of the polysaccharide pullulan and starch (in the present application, neopullulanase is The present invention relates to a method for efficiently producing neopullulanase using a plasmid having a DNA fragment encoding a pullulanase-like enzyme produced by Bacillus thermophilus TRS40.

[0002]

[Prior Art and Problems to be Solved by the Present Invention]
Conventionally, as the enzyme acting on pullulan, glucoamylase, which acts on glucose unit from the end of pullulan, α
A pullulanase that cleaves only 1,6-glucosidic bonds,
Isopururanaze give isopanose cleaves alpha-l, 4-glucosidic bonds in pullulan, panose (6 ^2-.alpha.
-Glucosyl maltose-producing Thermoactinomyces
Amylase (TVA) of vulgaricus) is known.

The enzyme of the present invention is similar to TVA in that it acts on pullulan to produce panose, but at the same time, the enzyme is completely TVA in that it produces glucose and maltose.
It is a novel enzyme with a new action specificity. This enzyme also acts on starch and can be said to be a type of α-amylase. Since α-amylase is an enzyme widely used in the starch saccharification industry and the like, it is expected that various uses will be developed by using this enzyme alone or in combination with other various enzymes.

[0004]

The present invention relates to a method for producing thermostable neopullulanase using Bacillus subtilis having a novel plasmid.

The present inventors separated Bacillus stearothermophilus TRS40, which secretes a thermostable neopullulanase that decomposes pullulan and mainly produces panose, from compost in Tsurumi-ku, Osaka City. The activity is different from any previously reported pullulanase, and it is an enzyme named neopullulanase by the present inventors. Bacillus stearothermophilus TRS40 has the following mycological properties.

1. Morphological properties 1) Cell shape and size (0.6-1.0) × (2
~ 3.5) μ bacilli. There are few chains with no linkage. The sweetened broth agar shows the same bacillus form as the broth agar. 2) Motility ... Yes. 3) Spores ... (0.4 to 0.5) x (0.5 to 0.6)
It has an oval shape of μ, exists near the end, and has a thin membrane wall. 60 ° C
Many are formed in 16 hours. Swelling of the sporangium is observed. 4) Gram stainability: Positive.

2. Growth condition 1) Meat broth agar plate culture: Good growth. The surface is slightly dry and white. 2) Meat broth agar slope culture: Spreading and no surface gloss. 3) Liquid culture of broth .... Good growth at pH 7.0. The liquid becomes cloudy. 4) Gelatin stab culture: liquefy gelatin. 5) Salt broth liquid culture: Does not grow at 7% saline concentration. 6) Glucose / asparagine agar medium: poor growth. 7) Glucose / Nitrate agar medium ... Slightly grown.

3. Physiological properties 1) Reduction of nitrate: Positive. 2) Hydrolysis of starch ... Iodine coloration is negative when grown on sialet containing starch. 3) Fermentability of citric acid: Positive. 4) Catalase production: Negative. 5) Growth range: Grow up to 65 ° C in a medium containing trypsin, yeast extract, and salt. The optimum growth temperature is 60 ° C. 6) Fermentability of sugar: An acid is formed from glucose, lactose and sucrose. However, no gas is generated. 7) Acetylmethylcarbinol formation: Positive. 8) Degradation of gelatin: Positive.

[0009] The above results are based on Bargey's Manual of Systematic Bacteriodeterm, Volume 2 of the Manual of Systematic Bacteriotermination.
ination vol 2. (1986)), this bacterium is Bacillus stearothermophilus
Was identified.

Next, the enzyme produced by this strain will be described in detail. 1. Action and substrate specificity This enzyme acts on pullulan and finally produces panose, glucose and maltose. In terms of quantity, panose is the most common.

When starch is acted on, maltose and glucose are mainly produced, but in the latter stage of the reaction, panose and isomaltosyl maltose (isom) as branched dextrins are produced.
altosyl maltose (abbreviation IMM) is generated. It is considered to be a kind of α-amylase, because the iodine coloration decreases more rapidly than the production of reducing sugars. Generally, α-amylase is roughly classified into a saccharified type that hydrolyzes starch relatively well and a liquefied type that has a high starch liquefaction rate but a low saccharification degree. And, when the former is allowed to act on starch, the minimum low molecular weight limit dextrin obtained is IMM. The enzyme of the present invention produces glucose and maltose from the beginning when it acts on starch, and belongs to a saccharified group, but the minimum limit dextrin is panose rather than IMM. In this respect, it is a unique α-amylase. Furthermore, when this enzyme acts on pullulan and starch at the same concentration and under the same conditions,
Works as well or better on pullulan as on starch. TVA has a significantly greater effect on starch than on pullulan. In this sense, TVA is considered as a type of α-amylase, whereas this enzyme is a type of pullulanase, and was named neopullulanase to distinguish it from conventional pullulanase.

Further, when the present enzyme acts on maltosyl-β-cyclodextrin in which maltose is bound to β-cyclodextrin through α-1,6 glucosyl bond,
First, the α-1,6 bond is cleaved to produce maltose and β-cyclodextrin. The β-cyclodextrin then undergoes re-degradation to give glucose, maltose and maltotriose.

That is, this enzyme mainly recognizes maltose, and even if maltose is bound by α-1,4 bond, α-
It can be considered as an enzyme having a new action specificity that cleaves the bond even if it is bound by 1,6 bond.

2. Action PH PH 5.0 to 7.5. The measurement is 0.05 M sodium acetate-acetic acid buffer (PH4 to 6) or disodium phosphate-
Using a 1 potassium phosphate buffer (PH6-8), 0.5
% Reaction in pullulan at 60 ° C. for 15 minutes. The reducing sugar produced is measured by the DNS method.

3. Optimum action PH Under the conditions of the above two, approximately PH 6.0.

4. Working temperature In 0.1 M phosphate buffer (PH 6.0) and 0.5% pullulan, the reaction was carried out at various temperatures for 15 minutes, and the reaction was carried out up to 75 ° C.

V. Optimum temperature of action 60 to 65 ° C. under the above conditions.

6. Thermostability 60 at various temperatures in 0.2M acetate buffer (PH 6.0)
When it was heated for minutes and the residual activity was measured, 60% of the activity remained at 65 ° C. Furthermore, 1 mM, E
When DTA was added, 90% or more of the activity remained.

7. PH stability Stable between pH 5.5 and 9.0 (P using various buffers
After adjusting H and leaving it at 60 ° C. for 60 minutes, it was adjusted to pH 7.0 and the residual activity was measured).

(8) Purification method After the culture supernatant was subjected to ammonium sulfate fractionation (35-60% saturation), the precipitate was dissolved in a small amount of water, dialyzed, and then DEA at pH 8.0.
Adsorbed on E-cellulose. Then, the salt concentration was gradually increased to 0.6 M to elute the enzyme. Further, column chromatography of Sephadex G-200 was repeated twice, and then affinity chromatography was carried out using a Sepharose 6B column to which α-cyclodextrin was bound.

The protein absorption of the eluate showed one peak, which was completely in agreement with the peak of enzyme activity. Electrophoresis of this active fraction by SDS-PAGE showed a single band.

9, molecular weight 62000 dalton (SDS-PAGE method)

(10) Products Glucose and maltose as well as panose were given from pullulan. Also, glucose, maltose, panose, and IMM were given from starch.

Eleven, 0.25 ml of enzyme solution was added to 0.25 ml of 1% pullulan (dissolved in 0.2 M acetate buffer (PH 6.0)) at 60 ° C.
-The reaction was carried out for 15 minutes, and the resulting increase in reducing power was quantified by the DNS method. In the above reaction, the enzyme power to generate 1 μmol of reducing sugar per minute was defined as 1 unit.

From the above physicochemical properties, it can be said that the enzyme of the present invention is a novel pullulanase that has never been reported.

In the present invention, the thermostable neopullulanase gene is expressed in Bacillus stearothermophilus, particularly Bacillus stearothermophilus TRS40.
Cleaved with the restriction enzyme Hind III. Hind III is used because it gives a fragment of a gene that effectively retains the molecular weight of the enzyme and the genetic information (signal sequence) corresponding to the carrier protein required for extracellular secretion of the produced protein. Is. The cutting method is a conventional method.

The cleaved fragments are shotgun in a pecter plasmid such as pTB522 (Journal of Gen).
eral Microbiology 131, 1757, 19
(Described in 1985), and prepare a recombinant plasmid that can be replicated and maintained using Bacillus subtilis as a host. The target plasmid has a molecular weight of about 2.2 Md and a restriction enzyme Eco.
It is a recombinant plasmid containing two DNA cleavage sites and DNA fragments having molecular weights of 0.8, 0.4 and 1.0 (Md) when treated with EcoRI. More specifically, the DNA fragment was incorporated into the pTB522, and the present inventors et al.
The recombinant plasmid designated as 0 has the desired properties (copy number, type of drug resistance, cleavage sites of various restriction enzymes, etc.) to enable production of the target substance by a general genetic engineering technique. The restriction map of this plasmid pPP10 is shown in the attached FIG.

The host Bacillus subtilis transformed with the above-mentioned recombinant plasmid may be any strain of Bacillus subtilis that has lost the amylase-producing ability. Such a defective strain can be easily obtained by a conventional method.

The present invention relates to a method for producing neopullulanase using Bacillus subtilis. That is, as a nutrient medium in which a bacterium that maintains recombinant DNA can grow, for example, 1 to 10% of carbohydrates such as starch, soluble starch, dextrin, and acid-treated starch as a carbon source, soybean meal as a nitrogen source, soybean meal extract , Dry yeast, milk casein, peptone, meat extract, corn stay liquor, peptide-containing substances, amino acids alone or an appropriate admixture are added, and if necessary, a small amount of inorganic salts is added in an appropriate amount. , Inoculated with the bacterial cells, PH 6.5-7.3, 37
Neopullulanase is allowed to grow and accumulate by agitating and culturing at a temperature of 24 to 72 hours. In order to recover and purify neopullulanase from this culture, the culture filtrate may be subjected to ordinary means such as fractionation / precipitation with ammonium sulfate, alcohol precipitation, and membrane concentration method.

[0030]

Example 1 Production of recombinant plasmid and cultivation of transformed Bacillus subtilis First, chromosomal DNA of Bacillus stearothermophilus TRS40 is prepared as follows.

Bacillus stearothermophilus TRS
40 was cultured overnight at 60 ° C. in a 500 ml flask containing 100 ml L medium. Collect this by centrifugation, 20m
l TE buffer (10 mM Tris, 1 mM EDTA,
Washed with PH 8.5), 3 ml of 15% sucrose-50 m
M Tris (PH8.5) -50M EDTA-1mg /
It was suspended in ml lysozyme and allowed to stand in ice for 30 minutes. Add 3 ml of 1% sarcosyl-50 mM Tris (PH
8.5) -50 mM EDTA was added and mixed. Then, 5.4 g CsCl was added, 0.3 ml of ethidium bromide solution (10 mg / ml) was added, and the mixture was separated in an ultracentrifuge with an RP65T rotor (manufactured by Hitachi Ltd.). After the ultracentrifugation was completed, the DNA segment was extracted with an injection needle, and extraction with n-butanol was repeated 3 times to remove ethidium bromide. This DNA sample was treated with 10 mM Tris (PH7.5)-
It was dialyzed in 0.1 mM EDTA and stored at 4 ° C.

Then, a vector plasmid is prepared by treating with restriction enzymes and ligase as follows. That is, on the other hand, a vector plasmid pTB522 (tetracycline resistance) capable of replicating and maintaining Bacillus subtilis as a host was prepared by a conventional method, and then Bacillus stearothermophilus TR
The chromosomal DNA of S40 and pTB522 are cut with the restriction enzyme Hind III. The reaction conditions are as follows.

TRS40 DNA 30 μl 10 × Hind III buffer 10 μl (A) Bovine serum albumin (5 mg / ml) 2 μl water 56 μl Hind III solution 2 μl

PTB522 10
μl 10 × Hind III buffer 10 μl (B) Bovine serum albumin (5 mg / ml) 2 μl water 76 μl Hind III solution 2 μl

In the table, 10 × Hind III buffer means 1
00 mM Tris (PH7.4), 100 mM MgC
1 ₂ , 500 mM NaCl, 10 mM dithiothreitol.

The above solution is reacted at 37 ° C. for 1.5 to 2 hours. The two kinds of DNAs A and B obtained above were mixed, 20 μl of 3M potassium acetate was added, and then 0.45 ml of ethanol was added and centrifuged, and the supernatant was removed. The sediment was washed with ethanol, dehydrated and desalted, and dried in a desiccator. 2 x ligase buffer (132 mM
Tris _{(PH7.6) -13.2M · Mgcl 2)} 10
0 μl, 100 mM dithiothreitol 20 μl, 5 m
After adding 20 μl of M.ATP and 60 μl of water and mixing, D
Add 1 μl of NA ligase and keep at 8 ° C for 16 hours.
A solution was used.

The third step is the step of cloning by introducing the above vector into Bacillus subtilis.

Bacillus subtilis NA1 is one of the amylase-deficient strains obtained by ordinary mutation treatment (announced at the Kyoto Conference of the Japan Society for Agricultural Chemistry, 1986, Kyoto Conference, Lecture No. 2A-22), but this was used in L medium. After culturing overnight, 1 ml of the TFI medium in a 100 ml volumetric flask was added to 20 m.
inoculate l.

This was cultured at 37 ° C., and 1 hour after the logarithmic growth phase was over, 4 ml of TFII medium in 36 ml of a 500 ml flask was inoculated and cultured for 1.5 hours to obtain competent cells.

The TFI and TFII media are as follows, respectively.

1 ml of this competent cell was mixed with the DNA solution obtained above, and after vigorous shaking culture at 37 ° C. for 30 minutes, the cells were collected by centrifugation, and 1 ml of L medium was added to 37.
Incubate with shaking at 1.5 ° C for 1.5 hours. 100 μl of the culture
Each was spread on an agar plate containing 25 µg / ml of tetracycline and cultured overnight at 37 ° C to obtain a transformant.

This transformant was subjected to PLL agar plate (1% pullulan, 0.2% tryptone, 0.2% yeast extract, 0.2% Nacl, 1.5% agar (PH7.3)).
After culturing at 37 ° C. overnight, ethanol was poured onto the plate and left for several hours to obtain halo-forming colonies. The recombinant plasmid harbored by this clone strain was
It was named PP10.

Example 2 (Production of neopullulanase) Bacillus subtilis NA1 retaining pPP10 obtained in Example 1 was cultured in L medium containing 2.5% soluble starch (containing 2.5 μg / ml tetracycline).
Culturing was carried out at a temperature of 24 hours. The culture solution was centrifuged and the supernatant was collected and heat-treated at 60 ° C for 15 minutes. At this stage, it was confirmed by SDS polyacrylamide electrophoresis that the protein in the culture supernatant was almost exclusively thermostable neopullulanase.

When the related strains were measured by the neopullulanase activity measuring method, they were as follows.

Neopullulanase obtained from Bacillus subtilis NA1 carrying the recombinant plasmid pPP10 was heat treated at 60 ° C. and 65 ° C. for 60 minutes respectively,
It was confirmed that the enzyme activity was maintained at 90% or more and about 60% or more, respectively, and the heat resistance was excellent.

[0046]

EFFECTS OF THE INVENTION Generally, Bacillus stearothermophilus, which is a thermophilic bacterium, has a lower final cell concentration in culture than Bacillus subtilis, and it is relatively difficult to obtain a large amount of cell product. Therefore, in the present invention, a large amount of neopullulanase can be obtained by incorporating the gene of neopullulanase, which is a thermostable enzyme in Bacillus stearothermophilus, into Bacillus subtilis. In particular, hind the DNA of Bacillus stearothermophilus TRS40
The DNA fragment obtained by treatment with III was designated as pTB522.
The Bacillus subtilis NA1 incorporated in the present invention effectively realizes the present invention. However, according to the present invention, since Bacillus subtilis NA1 which is a mutant strain of α-amylase and protease deficiency is used as the host bacterium, most of the extracellular secretory enzyme contained in the culture filtrate is heat-resistant cloned in this strain. Since only the active neopullulanase is produced and the gene thereof is amplified, the productivity of the enzyme can be remarkably improved, and the thermophilic bacterium gene has been cloned into mesophilic bacterium.
Since proteins other than the thermostable neopullulanase in the culture filtrate are mostly denatured and precipitated by heat treatment at ℃ for 15 minutes, the target enzyme was concentrated and purified by culturing Bacillus stearothermophilus TRS40. It can be performed much more simply and efficiently than when the enzyme is concentrated and purified.

[0047]

[Brief description of drawings]

FIG. 1 is a restriction map of the recombinant plasmid pPP10 prepared using the vector plasmid pTB522. In the figure, the portion corresponding to the 2.2 Md Hind III fragment indicated by the bold line is Bacillus stearothermophilus TRS4.
0 is a thermostable neopullulanase gene. However, the numbers in the circle represent the molecular size in Md.

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Claims (1)

[Claims] 1. Bacillus stearothermophilus (Ba
cillus stearothermophilus) TRS40 (Deposit No. 9609 of the Institute for Microbiology Research Institute) is a restriction enzyme hind III
(Hind III) -treated neopullulanase-encoding DNA fragment and the vector fragment obtained by treating with Hind III were ligated to Bacillus subtilis replicatively maintained with a recombinant DNA having a molecular weight of 2.2 Md. Cultured in
A method for producing neopullulanase by transformed Bacillus subtilis, which comprises recovering neopullulanase from the culture.