JPH04218378A - Gene coding alcohol dehydrogenase - Google Patents

Abstract

PURPOSE:To make it possible to efficiently obtain an alcohol by culturing transformant transformed with a manifestation vector containing a specific gene. CONSTITUTION:A bacterial cell obtained by culturing Bacillus stearothermophilusu NCA 1503 strain is extracted to afford chromosome DAN (A). Then the ingredient A is subjected to restriction enzyme treatment to produce a plasmid vector pTBAD35 (B) exhibiting restriction enzyme-cut map having 3.5Kb BamHI fragment and containing a gene coding alcohol dehydrogenase (ANH). Then the ingredient B is introduced into the above- mentioned NCA 1503 strain or B. subtilis strain to afford a transformant (C). Then the transformant C is cultured to produce ADH. On the one hand, a transformant obtained by introducing DNA for changing an amino acid constituting ADH is cultured to produce a modified ADH capable of shifting optimum pH to a basic side. As necessary the transformant C is cultured to produced an alcohol.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention relates to a structural gene encoding alcohol dehydrogenase; an expressible gene having the structural gene, an expression control region and a terminator; The present invention relates to a vector; a transformant having the expression vector; and a method for producing alcohol dehydrogenase and a method for producing alcohol using the transformant. Furthermore, the present invention
A modification enzyme of the alcohol dehydrogenase having an optimum pH on the basic side; a transformant having an expression vector encoding the modification enzyme; and a method for producing alcohol dehydrogenase using the transformant, and alcohol Relating to a manufacturing method.

0002

[Prior Art] Alcohol dehydrogenase (ADH) reversibly catalyzes the reaction of oxidizing alcohol to produce aldehyde and the reaction of reducing aldehyde to produce alcohol, as shown in Chemical Formula 1. :

0003

[Chemical formula 1]

[0004] Here, R is an alkyl group.

[0005] Therefore, ADH is an industrially useful enzyme that can be used for qualitative and quantitative determination of alcohol or aldehyde, production of alcohol or aldehyde, and the like. ADH is classified into four types depending on the availability of coenzymes. One of them is ADH that uses NAD as a coenzyme, and ADH derived from animals and microorganisms are known. Examples of animal-derived ADH include human and horse-derived ADH.
DH is known. Examples of ADH derived from microorganisms include Bacillus stearothermophilus (Bacillus stearothermophilus).
llus stearothermophilus)
ADH derived from Saccharomyces cerevisiae, and Saccharomyces cerevisiae
) derived ADH is known. In particular, Bacillus stearothermophilus strain NCA1503 [Biotec
hnology and bioengineering
g, 17, 1375-1377 (1975)]
DH has a wide range of uses because it is heat resistant.

[0006] This enzyme is, for example, FEBS let
ers, 92, 365-367 (1987). starotherm
It is obtained by culturing the bacterial cells of S. ophillus NCA1503 strain, and isolating and purifying the cytoplasmic fraction of the bacterial cells. However, since the enzyme content in the bacterial cells of this strain is small and the purification procedure is complicated, the enzyme cannot be obtained in good yield. Therefore, it has been difficult to prepare a large amount of ADH.

[0007]

[Problem to be solved by the invention] The present invention solves the above-mentioned conventional problems, and its purpose is to
DH, especially Bacillus stearotherm
An object of the present invention is to provide a gene encoding ADH derived from S. ophillus NCA1503 strain. Another object of the present invention is to provide an expression vector containing the above gene and a transformant containing the expression vector. Still another object of the present invention is to provide a method for efficiently producing ADH using the above transformant, and a method for producing alcohol using the transformant.

[0008] Still another object of the present invention is to
The purpose of this invention is to investigate the active center of ADH derived from S. llus stearothermophilus and to provide a gene encoding ADH whose optimal pH has shifted to the more basic side. Still other objects of the present invention are to provide an expression vector having the above gene, a transformant having the expression vector, and a method for producing ADH or alcohol using the transformant.

[0009]

[Means for Solving the Problems] The ADH structural gene of the present invention is an ADH shown in the amino acid sequence of Sequence Listing 1, or in which His at position 43 of the amino acid sequence is replaced with another basic amino acid, or It encodes a modified ADH represented by an amino acid sequence in which the His residue is modified so that the basicity of the His residue is increased.

[0010] The expressible gene of the present invention encoding ADH or modified ADH is the above-mentioned structural gene;
It includes an expression control region consisting of the nucleotide sequence shown by C at position 9; and a terminator which is present in the 3' downstream region of the structural gene and consists of the nucleotide sequence from A at position 1317 to T at position 1342.

The expression vector of the present invention has the above-mentioned structural gene; or an expressible gene containing the above-mentioned structural gene, an expression control region, and a terminator.

The transformant of the present invention can be obtained by introducing the above expression vector into a host cell.

The method for producing ADH of the present invention includes the steps of culturing the above transformant and collecting alcohol dehydrogenase or modified alcohol dehydrogenase from the culture.

The method for producing alcohol of the present invention includes the step of culturing the above transformant in a medium containing a carbon source.

The ADH structural gene of the present invention is derived from the chromosomal DNA of a suitable microorganism that produces ADH, such as B.
stearothermophilus NCA150
It can be obtained from the chromosomal DNA of three strains. For example, from cultured cells of the strain, Journal of Bacteri
It can be prepared according to the method described in Biochemistry, 147, 776-786 (1981). An expression vector is constructed by cutting this chromosomal DNA with an appropriate restriction enzyme and inserting it into the vector DNA cut with the restriction enzyme, or inserting it into the vector DNA cut with the appropriate restriction enzyme via a linker. be done. For example, a 5 to 7 kb DNA fragment obtained by cutting the above chromosomal DNA with the restriction enzyme Sau3AI and a BamHI
Vector DNA pTB524 [Appl
iedandEnvironmentalMicr
biology, 55, 3208-3213 (1
989)] using T4 DNA ligase, an expression vector is constructed.

[0016] The expression vector thus constructed is introduced into host cells. The above pTB52 as a vector
4, transformants can be selected using tetracycline (Tc) resistance as an indicator. Bacillus subtilis is preferred as the host cell, and B. subtilis is preferred. subtilisMI1
13 strains are preferably used. This Bacillus subtilis is B. st
Since the growth rate is higher than that of the Aerothermophilus NCA1503 strain, by using this strain as a host, the intracellular concentration of ADH can be increased and ADH can be produced with high efficiency. The transformation of Bacillus subtilis is described in the Journal of Bacteriology
y, 146, 1091-1097 (1981).

[0017] From among the obtained transformants, the desired A
To detect clones having the DH gene, basically the Journal of Bacteriology,
169, 2591-2597 (1987). That is, ethanol (substrate) and pararoseaniline (indicator) are present in the complete medium.
The above transformant is cultured. Through this culture, the transformant having the target gene is AD.
To produce H, aldehydes are generated from ethanol. Since aldehyde forms a Schiff base with pararose aniline and exhibits a red color, positive clones containing the gene of interest can be obtained by isolating red-stained colonies.

[0018] This positive clone was cultured and plasmid D
When NA was extracted and examined, D derived from the plasmid vector was detected.
It can be seen that a DNA fragment of about 7.0 kb was inserted in addition to NA. This approximately 7.0 kb DNA fragment was converted into Ba
The approximately 3.5 kb BamHI fragment obtained by cutting with mHI was added to the BamH fragment of the above plasmid vector pTB524.
By inserting into the I site, the expression plasmid pTBA
D35 is constructed. Using this pTBAD35, B.
When the S. subtilis MI113 strain was transformed,
Since it was positive on the aldehyde indicator plate containing the above-mentioned pararose aniline, it was found that pTBAD35 contains the target gene. This pTBAD3
A restriction enzyme cleavage map of the 3.5 kb BamHI fragment contained in No. 5 is shown in FIG.

[0019] The base sequence of this 3.5 kb BamHI fragment can be determined, for example, by the dideoxy method using M13 phage [
Methods in Enzymology, 10
, p20, Academic Press, Ne
W york (1983)]. This base sequence is shown in Sequence Table 1. A at position 290 of this base sequence
An open reading frame exists at position 1,300 from T (1,011 bp), and the amino acid sequence (337 amino acids) shown at the bottom of the base sequence in Sequence Listing 1 can be translated from this open reading frame. The molecular weight of the protein predicted from the amino acid sequence in Sequence Listing 1 is 36,098, which agrees well with the molecular weight of ADH, 35,000, obtained by SDS-PAGE. Furthermore, the sequence near the N-terminus of the amino acid sequence was obtained by the Edman degradation method [FEBS letters, 3
3, 1-3 (1973)]. From this, it can be concluded that this amino acid sequence is that of ADH, and that the base sequence indicated by A at position 290 to T at position 1,300 in Sequence Listing 1 is the structural gene of ADH.

Furthermore, when examining the base sequence of Sequence Listing 1, an SD sequence consisting of AAGGAGG (positions 274 to 280) is present upstream of the translation start codon (ATG) of the above open reading frame, and the SD sequence consists of AAGGAGG (positions 274 to 280). Upstream is (CA AAA AG)-3bp-(CTTT
TTG) inverted repeat sequence (positions 252 to 26
8th place) exists. This inverted repeat sequence is thought to act as an operator for ADH expression. Further upstream, (-35,-10)
There are two sets of sequences homologous to the consensus sequence of the region. For this (-35,-10) region, Molecule
ar & General Genetics, 18
6, 339-346 (1982), AD
It is thought to function as a promoter of the H gene. In this way, from G at position 1 of the base sequence in Sequence Listing 1 to 2
The sequence indicated by C at position 89 is considered to be the expression control region of ADH. Furthermore, from position 1317 to position 1342 downstream of the stop codon (TAA) (AA GAA G
An inverted repeat sequence consisting of GC G)-8bp-(CGC CTT CTT) is present and is thought to function as a terminator.

[0021] The inventor further investigated the possibility of obtaining a modified ADH in which the optimum pH was shifted to basicity by modifying the above ADH. To this end, we investigated the mechanism of ADH enzymatic activity and its active center.

Regarding ADH, as described in the section of the prior art, B. stearothermophilu
In addition to enzymes derived from S. ADHs derived from E. cerevisiae, humans and horses are known, and their amino acid sequences have been revealed, as shown in FIGS. 2-4 [respectively, J. Biol. Chem. , 258
, 2674 (1983); Proc. Natl.
Acad. Sci. , USA, 82, 2703
(1985); and Eur. J. Biochem
．． 16, 25 (1970)]. Among these, the mechanism of action of horse-derived ADH has been clarified [The Enzyme, 11, 104-19
0, Academic Press, Inc. (19
75)]. FIG. 5 shows the detailed mechanism of the reaction represented by the chemical formula 1 described in the prior art section. This ADH is Zn
It combines with one molecule of water and performs an enzymatic reaction via NAD+. In FIG. 5, ADH (indicated by E), Zn and H2O first combine to form an enzyme-NAD+ complex, H+ is released from this complex, and isomerization of the complex occurs. Alcohol (R-CH2OH) is added to this,
ADH-NAD+-alcohol ternary complex is formed,
The rearrangement reaction forms an ADH-NADH-aldehyde complex. Then the dissociation of the aldehyde and H2O
is added to Zn, an ADH-Zn-NADH complex is formed, and NADH is dissociated by isomerization, and the original A
Return to the form of DH-Zn-H2O. This first stage (N
Figure 6 shows the mechanism of proton transfer at a portion considered to be the active center of the enzyme (AD+ is added and protons are liberated). As shown in Figure 6, Cys at position 46, His at position 61, and Cys at position 174 of horse-derived ADH coordinate with Zn as ligands, and one molecule of water is added to Zn, and this water molecule AD through hydrogen bond to
It is thought that Ser at position 48 and His at position 51 of H bind together. In this way, Zn and the above Cys
(46th place), His (61st place), Cys (174th place),
It is believed that Ser (position 48) and His (position 51) form the active site of this enzyme. These amino acids constituting the active site are relatively well preserved in various ADHs, as shown in FIGS. 2-4.

[0023] The inventors have discovered that the above-mentioned S. steam
The active site of ADH derived from the ADH strain NCA1503 was predicted from the active site of the horse-derived ADH mentioned above, and by changing the amino acids that form this active site, it was possible to vary the optimal pH and activity. Thought. The inventors used the following procedure to discover that the amino acids involved in the active site of ADH, shown in Sequence Listing 1, include Cys at position 38 (one of the ligands for Zn), Thr at position 40, and His at position 43. We have confirmed this and found that by changing at least one of these, the optimal pH and/or activity of ADH can be changed. More specifically, by replacing His at position 43 in the amino acid sequence of Sequence Listing 1 with another basic amino acid, or by modifying the His residue so that the basicity of the His residue is increased, Appropriate pH
We succeeded in obtaining ADH in which ADH was shifted to the basic side.

[0024] The inventors first identified Cys at position 38, Thr at position 40, and Thr at position 43 of the amino acid sequence shown in Sequence Listing 1.
The His position was changed as shown in Table 1 below. this is,
The above site of the DNA sequence shown in Sequence Listing 1 is changed by site-directed mutagenesis to form an expression vector in the same manner as pTBAD35 was constructed, and a transformant is created as described below. This was done by culturing to express the enzyme protein.

[0025]

[Table 1]

[0026] When examining the presence or absence of ADH activity (measured by the method described below) in each of the obtained culture solutions, it was found that T40S had about 60% of the original ADH activity, but C38S and T
No activity was observed for 40A and H43A. In this way, Thr at position 40 (corresponding to position 48 in horse-derived ADH) is replaced by Ser, which also has an OH group.
has some activity when converted to 40
When Thr at position 43 and His at position 43 are converted to Ala, there is no activity at all, so B. steam
It was found that ADH derived from Rothermophilus NCA 1503 strain also has the same active site and activation mechanism as ADH derived from horses.

Based on the above findings, the inventors discovered that H at position 43, which is one of the amino acids that forms the active center of ADH,
The modified ADH of the present invention was created by modifying is. In this modified ADH, the above-mentioned His is substituted with another basic amino acid, or the His residue is modified so that the basicity of the His residue is increased. For example, this H
is is replaced with Arg or Lys. For example, Ar
The DNA sequence encoding ADH obtained by substitution with g can be obtained by changing the codon CAT corresponding to the His site to CGT by site-specific mutation. The pK value of the imidazole ring in the side chain of His is 6.0, and the pK value of the guanidyl group in the side chain of Arg is 12.5.
It is. Therefore, the original ADH is as shown in Figure 7.
While it has an optimum pH near neutrality (about pH 8.0), ADH (H43R) converted to Arg has an optimum pH on the basic side (about pH 9.0).

[0028] The structural gene of the modified ADH of the present invention in which the codon corresponding to His at position 43 has been modified is also integrated into an appropriate vector and used as an expression vector, in the same manner as the structural gene of the unmodified ADH. It is said that For example, an expressible gene consisting of a modified ADH structural gene, an expression control region, and a terminator is transferred to the vector pTB524.
The expression vector pTBAD35H43R is obtained. For example, B. subtilis M
When introduced into strain I113, a transformant B. subtil
is MI113 (pTBAD35H43R) is obtained.

ADH or modified ADH is produced by culturing the transformant of the present invention. For example, the above B
．． subtilis MI113 (pTBAD35)
When cultured, the parent strain B. star other
mophilus strain NCA1503, ADH can be produced more than 10 times more efficiently. ADH produced by this transformant can be purified using a conventional enzyme purification method. Furthermore, the above recombinant plasmid pTBAD35 or pTBAD35
H43R to the original B. stearothermoph
By introducing A. illus NCA1503 strain, A.
A transformant with increased DH production is obtained. The obtained ADH can be used as various reagents or as an enzyme for alcohol production. Alternatively, it is also recommended to directly produce alcohol by culturing the transformant in a medium containing a carbon source.

[0030] As mentioned above, the optimum pH of ADH produced by the transformant obtained by transformation with pTBAD35H43R is shifted to the basic side;
is approximately 9.0. Furthermore, as shown in the Examples below, the affinity of this modified ADH for ethanol is significantly lower than that of the ADH derived from the wild-type strain. Therefore, this ADH has a reduced ability to oxidize ethanol and is therefore optimal as an enzyme for ethanol production. Since the parent strain NCA1503 strain from which the gene of the present invention has been extracted is heat resistant, the obtained transformant is also heat resistant. Therefore, by using this, alcohol can be produced efficiently and advantageously industrially. be able to.

[0031]

EXAMPLES The present invention will be explained with reference to the following examples.

Example 1 Isolation of a DNA fragment having a structural gene encoding ADH, an expression control region and a terminator, and construction of an expression vector B. stearothermophilus NCA
Strain 1503 was cultured with shaking at 55° C. for 16 hours in a medium (pH 7.3) containing 2% tryptone, 1% yeast extract, and 0.5% NaCl. The obtained culture was centrifuged to collect the bacterial cells, and the cells were collected using a conventional method [Journal
of Bacteriology, 147, 776
786 (1981), supra], chromosomal DNA was prepared. This chromosomal DNA was partially digested with the restriction enzyme Sau3AI to obtain a 5-7 kb DNA fragment.

[0033] Apart from this, plasmid vector pTB
524 with the restriction enzyme BamHI (forms a cut end that can be ligated with the cut end cut by Sau3AI), and this DNA fragment and the above 5 to 7 kb DNA fragment were combined with T4
They were ligated using DNA ligase (BRL) to construct an expression vector.

[0034] Using this expression vector, the Journal
of Bacteriology, 146, 10
Bacillus subtilis B according to 91-1097 (1981) (supra)
．． subtilis MI113 strain was transformed. The transformant contained 1% tryptone, 0.5% yeast extract,
NaCl 0.5%, agar 1.5% and Tc 12.5
Plated on L medium containing ug/ml and selected for Tc resistance. Next, the selected transformants were picked using toothpicks and treated with antibiotic medium (
AM) 3 (manufactured by Difco) 1.75%, parallose aniline 0.005%, sodium bisulfite 0.025
% and aldehyde indicator plates containing 1.5% Bacto Agar (Difco). A clone (transformant having the target gene) that produced aldehyde and turned red was isolated. This isolation method was
l of Bcteriology, 169,259
1-2597 (1987) (supra) was basically used. However, LB medium (tryptone 1.0%, yeast extract 0.5% and NaCl 1.0
%), antibiotic medium (AM) 3, which has a buffering effect at pH 7.0, was used. This is because the color reaction of pararose aniline is stable at pH 7.0 to 8.0, but since LB medium does not have pH buffering capacity, the pH of the medium may change depending on the products of the bacterial cells. This is because in that case, it becomes difficult to judge the coloration. By using antibiotic medium (AM) 3, the color development of pararose aniline was stabilized, making it easy to select positive clones. Approximately 3,000 transformant clones were examined in this manner, and one positive clone was obtained that appeared red on the aldehyde indicator plate.

[0035] This positive clone was treated with Tc (12.5 μg
/ml) in L medium (tryptone 1.0%, yeast extract 0.5% and NaCl 0.5%) at 37°C.
The cells were cultured with shaking overnight. Thereafter, the bacterial cells were collected by centrifugation, and the plasmid DNA was extracted using the standard method [Journal of
Bacteriology, 149, 824-83
0 (1982),]. The plasmid DNA thus obtained contained a DNA fragment of about 7.0 kb inserted in addition to the DNA derived from the plasmid vector. When this approximately 7.0 kb DNA fragment was digested with BamHI, a approximately 3.5 kb BamHI fragment was obtained. This BamHI fragment was transferred to the above plasmid vector pT
It was inserted into the BamHI site of B524 according to the same method as described above. The obtained recombinant plasmid was transformed into pTBAD35.
Using this name, B. subtilis MI
When strain 113 was transformed, it showed positive on the above aldehyde indicator plate. From this, pTBA
It was concluded that D35 contains the ADH gene of interest. 3 contained in this pTBAD35
．． A restriction enzyme cleavage map of the 5 kb BamHI fragment is shown in FIG.

Example 2 Determination of the base sequence and deduced amino acid sequence of the structural gene encoding ADH, expression control region and terminator Recombinant plasmid pTBAD35 obtained in Example 1
The transformant having the following was cultured in L medium containing Tc (12.5 μg/ml) at 37° C. for 16 hours. Thereafter, plasmid DNA was extracted in the same manner as in Example 1, and B
Digestion with amHI yielded a 3.5 kb BamHI fragment. The base sequence of this 3.5 kb BamHI fragment was
Dideoxy method using 3 phages [Methods i
n Enzymology, 10, p20, Aca
demic Press, New York (198
3), supra]. The determined base sequence is shown in Sequence Table 1.

[0037] When this base sequence was examined, it was found that 29
A 1,011 bp open reading frame from A at position 0 to T at position 1,300 was found. The 337 translatable amino acid sequences are shown below the base sequences in Sequence Listing 1. The molecular weight of the protein predicted from the amino acid sequence shown in Sequence Listing 1 is 36,098, and the SDS
- Molecular weight of ADH obtained by PAGE: 35,000
It was a good match. Furthermore, the sequence near the N-terminus of this amino acid sequence was obtained using the Edman degradation method [FEBS
letters, 33, 1-3 (1973), supra]. Therefore, it was confirmed that the amino acid sequence shown in Sequence Listing 1 is that of ADH.

Example 3 Production of ADH using Bacillus subtilis transformant The transformant containing the recombinant plasmid pTBAD35 obtained in Example 1 was grown in L medium containing Tc (12.5 μg/ml). The cells were cultured overnight at 37° C. and the cells were collected by centrifuging the culture. Add to this lysozyme (1mg/ml)
1/25 of the above culture was added to 50 mM phosphate buffer (pH 7.8) containing 50 mM phosphate buffer (pH 7.8), thoroughly suspended, and then incubated at 37° C. for 30 minutes. Thereafter, when this suspension was observed using a microscope, it was confirmed that bacteriolysis by lysozyme had occurred. 30,000 ml of this lysate
The supernatant obtained by ultracentrifugation at 0 rpm for 30 minutes was used as a crude enzyme solution, and ADH activity was measured. As a control, B. stearothermophil
The us NCA1503 strain was cultured overnight at 55°C in a 2L medium, and the ADH activity of the crude enzyme solution prepared in the same manner as described above was measured.

[0039] ADH activity can be measured using Methods i.
n Enzymology, I, 500-503,
Academic Press, New York (1
955). In other words, using a reaction system containing ethanol (substrate) and NAD+ (prosthetic group), the NA associated with the oxidation of ethanol by ADH is
The reduction of D+ to NADH is carried out by the NADH-specific 340
It was measured by a spectrophotometer as a change in absorbance in nm. The results are shown in Table 2.

[0040]

[Table 2]

From Table 2, the transformants are B. ste
It can be seen that this strain produces ADH at a rate 10 times or more compared to the Allothermophilus NCA1503 strain.

Reference Example 1 From the recombinant plasmid obtained in Example 1, A of the present invention was
The gene encoding DH was excised and subjected to site-specific mutagenesis (using Amersham's mutation introduction kit).
DNA fragment in which G at position 402 of Sequence Listing 1 was converted to C, 4
A DNA fragment in which A at position 07 was converted to G, a DNA fragment in which A at position 407 was converted to T, and a DNA fragment in which C at position 416 was converted to G and A at position 417 was converted to C. Obtained. These four types of DNA fragments were added to the plasmid vector pTB using the same method as in Example 1.
524 into plasmid vector pTBAD35
C38S, pTBAD35T40A, pTBAD35T
40S and pTBAD35H43A were obtained. These plasmid vectors were used as B. subtili
s MI113 strain, and transformant B. subt
ilis MI113 (pTBAD35C38S), B
．． subtilis MI113 (pTBAD35T
40A), B. subtilis MI113 (pT
BAD35T40S) and B. subtilis
MI113 (pTBAD35H43A) was obtained.

[0043] 50 ml of tetracycline-containing L medium (L-Tc medium; tetracycline content 25 μg/ml) was placed in a 500 ml Erlenmeyer flask and sterilized (4 pieces were prepared), and 1 piece of the above transformant was added to each of these. Each platinum loop was inoculated and cultured with shaking at 37°C overnight. This is 1
Collect bacteria by centrifugation at 0,000 rpm for 5 minutes, and
After washing with M phosphate buffer (pH 7.8), the cells were centrifuged again to collect bacteria. Next, the above bacterial cells were suspended in 10 ml of phosphate buffer containing 1 mg/ml of lysozyme.
After incubation for 30 minutes at ℃, 15,000 rpm
Centrifuged for 20 minutes at m. The resulting supernatant was used as a crude enzyme solution, and the enzyme activity of ADH was measured at pH 7.8 and 55°C. ADH activity was measured by measuring the amount of NADH produced upon oxidation of ethanol by measuring the increase in absorption at 340 nm. This method is
n Enzymology, I, 500-503,
Academic Press. New York
(1955). ADH activity is shown in Table 3. In the section of mutant ADH in Table 3, in C38S, Cys at position 38 is changed to Ser, and in T40A, Thr at position 40 is changed to Al.
In a, T40S has 40th Thr in Ser, and
H43A indicates that His at position 43 has been converted to Ala.

[0044]

[Table 3]

[0045] In Table 3, since C38S, T40A, and H43A have no activity, Cys at position 38, Thr at position 40, and His at position 43 of ADH are likely to be involved in forming the active center of the enzyme. Recognize. This assumption is also supported by the fact that T40S, in which Thr at position 40 is converted to Ser having an OH group like Thr, has an activity of about 60%.

Example 4 Based on the findings of the above reference example, in this example, a modified ADH was created in which His at position 43 was modified, particularly by converting it to Arg, which has a higher degree of basicity than His.

The gene encoding the ADH of the present invention was excised from the recombinant plasmid obtained in Example 1, and the ADH at position 417 of Sequence Listing 1 was mutated by site-specific mutagenesis (using Amersham's mutation introduction kit). D converted to G
An NA fragment was obtained. This DNA fragment was integrated into plasmid vector pTB524 by the same method as in Example 1 to obtain plasmid vector pTBAD35H43R. This plasmid vector was transferred to B. subtilis
It was introduced into MI113 strain and transformant B. subtil
is MI113 (pTBAD35H43R) was obtained.

One platinum loop of the above transformant was inoculated into 50 ml of L medium containing tetracycline (25 μg/ml).
Shaking culture was performed at 37°C for 17 hours. This is 8000r
Collect bacteria by centrifuging at pm for 10 minutes, wash with 20mM potassium phosphate buffer (pH 7.8), and centrifuge again at 8000 r.
Centrifugation was performed for 10 minutes at pm. In addition, lysozyme 1
20mM phosphate buffer (pH 7.8) containing mg/ml
5 ml (1/10 volume of the culture solution) was added and incubated at 37°C for 30 minutes to disrupt the bacterial cells. After cooling on ice for 10 minutes, the mixture was centrifuged at 30,000 rpm for 30 minutes, and the resulting supernatant was used as a crude enzyme solution.

[0049] The above crude enzyme solution was heated at 60°C for 10 minutes,
Denatured proteins were removed by centrifugation at 15,000 rpm for 10 minutes. Since the target enzyme was derived from a thermophilic bacterium, it had high heat resistance and was stable even after heat treatment, but host-derived proteins were denatured and removed. The obtained protein was treated with an ion exchange column as follows. DEAE as an ion exchanger
Using cellulose (DE52 manufactured by Whatman),
The buffer solution is 20mM potassium phosphate buffer (pH 7).
．． 8) was used. KCl (potassium chloride) as elution salt
Elution was performed using a linear gradient of 0M to 0.37M. The fraction with high ADH activity,
The purified enzyme was obtained by dialysis against 20mM potassium phosphate buffer (pH 7.8) at 4°C for 20 hours.

[0050] Using the purified enzyme and the enzyme (purified product) produced by the transformant obtained in Example 1, various p.
The activity was measured under H conditions and the optimum pH was determined. When measuring activity, at pH 6.0 to 8.5, 50mM phosphate buffer (indicated by a white blank in Figure 7) was
50mM glycine-KOH for 8.5-10.5
A buffer solution (indicated by the black mark in FIG. 7) was used. The measurement results are shown in FIG. From Figure 7, the optimal pH of ADH (wild type) produced by the transformant having pTBAD35 is in the neutral range of 7.0 to 8.5, whereas the optimal pH of ADH produced by the transformant having pTBAD35H43R is It can be seen that the optimum pH of (H43R) is in the basic region of about 9.0. Similar results were obtained when crude enzyme was used instead of purified enzyme.

Example 5 Enzyme produced by the transformant having pTBAD35 obtained in Example 1 (wild type), pTBAD obtained in Reference Example
The enzyme produced by the transformant having D35T40S (T
40S), and pTBAD35H obtained in Example 4
Enzyme produced by a transformant having 43R (H43R)
(purified products were used for each), the initial reaction rate was measured at various ethanol concentrations (0.1mM to 1.0M) (carried out at pH 7.8 and 9.0), and Lin
Rate constants were determined by eweaver-Burk plot. The reciprocal of Km (mM) indicates the affinity of the enzyme for the substrate (ethanol), and kcat (s-1) indicates the affinity of the enzyme for the substrate (ethanol).
Maximum reaction rate per mole is shown. The results are shown in Table 4.

[0052]

[Table 4]

[0053] kcat (pH 9.0)/kcat (pH
7.8) was larger at pH 7.8 than at pH 9.0 for the wild-type enzyme and the T40S mutant enzyme, indicating that the optimum pH was in the neutral region. In contrast, the H43R mutant enzyme was kcat (pH 9.0
)/kcat (pH 7.8) value is as large as 1.91,
It was shown that the enzyme activity was higher at pH 9.0.

The Km value of H43R is more than 50 times greater than that of the wild-type enzyme, indicating a significantly lower affinity for ethanol. In other words, the oxidizing power of ethanol is quite low. ADH is the final step enzyme for ethanol production and catalyzes the reaction that produces ethanol from aldehydes. It also has the ability to oxidize ethanol to aldehydes as a reverse reaction. As mentioned above, H43R has a low ability to oxidize ethanol, and therefore is found to be optimal as an enzyme for ethanol production.

[0055]

[Effects of the Invention] According to the present invention, as described above, B. s
tearothermophilus NCA1503
A structural gene encoding a strain-derived ADH or a modified ADH in which the amino acids forming the active site of the ADH have been changed;
An expressible gene having the structural gene, an expression control region, and a terminator; a recombinant plasmid having the structural gene or the expressible gene; a transformant into which the recombinant plasmid has been introduced; A was there
A method for producing DH; and a method for producing alcohol using the transformant are provided. The transformant of the present invention is
B. stearothermophilus NCA
Since the ADH production amount is more than 10 times higher than that of the 1503 strain, by using this transformant, a larger amount of ADH can be produced than before.
ADH can be obtained at low cost. Furthermore, it is also possible to industrially and efficiently produce alcohol using this transformant or ADH. Modified ADH in which the amino acids that form the active site of ADH have been changed, especially modified ADH in which His at position 43 has been converted to a more basic amino acid, have an optimal pH that is higher than that of the original ADH. Shifted to the basic side. Additionally, it has a lower affinity for alcohol, making it suitable for producing alcohol.

[0056]

[Sequence list] Sequence number: 1 Sequence length: 1506 Sequence type: Number of nucleic acid strands: Double stranded Topology: Linear Sequence type: Genomic DNA origin Organism name: Hycilus stearothermophilus (
Bacillus stearothermophil
us) Strain name: NCA1503 Sequence characteristics Symbol representing characteristics: -35signal Location: 83. ．． 88 and 188. ．． 193 How the feature was determined: S Symbol representing the feature: -10signal Location: 106. ．． 111; and 211,216 How the feature was determined: S Symbol representing the feature: CDS Location: 290. ．． 1303 Method for determining features: S Symbol representing features: terminator Location: 13
17. ．． 1342 How the characteristics were determined: S GGCTCCATGT CAAGCGTCAT CGC
TCCCCAC TCTTAAAGGG GCAGGA
ACTT AGTAAAAAT 60TAA
CTTTTTTT GTGTCCAAAA ATTTGA
CGTA AATCCATTTA CACTATATG
A CTAGAACAGA 120AACTTT
ATAT GATGTCAACT CCCGAACCA
A ATTTTTAACT TTTTATCCAA A
AATATTTTTT 180CATTTTTTT
G AACATTTTAT TTGTGATATT T
TTCACAAGT TAAATGTATG CTAC
ACTACA 240TATGTACAGA T
CAAAAAGTC CCTTTTTGCC TAGA
AGGAGG ATTATAATC ATG AAA
GCT 298

Met Lys Ala


1GCA GTT GTG GAA CAA
TTT AAA AAG CCG TTA CAA
GTG AAA GAA GTG GAA
346Ala Val Val Glu Gln Ph
e Lys Lys Pro Leu Gln Val
Lys Glu Val Glu 5
10
15AAA CCT AAG
ATC TCA TAC GGG GAA GTA T
TA GTG CGC ATC AAA GCG TG
T 394Lys Pro Lys Ile
Ser Tyr Gly Glu Val Leu
Val Arg Ile Lys Ala Cys 2
0 25
30
35GGG GTA TGC CA
T ACA GAC TTG CAT GCC GCA
CAT GGC GAC TGG CCT GTA
442Gly Val Cys His T
hr Asp Leu His Ala Ala Hi
s Gly Asp Trp Pro Val
40
45
50AAG CCT AAA CTG CCT
CTC ATT CCT GGC CAT GAA G
GC GTC GGT GTA ATT 4
90Lys Pro Lys Leu Pro Leu
Ile Pro Gly His Glu Gly
Val Gly Val Ile
55 60
65GAA G
AA GTA GGT CCT GGG GTA AC
A CAT TTA AAA GTT GGA GAT
CGC GTA 538Glu Glu
Val Gly Pro Gly Val Thr H
is Leu Lys Val Gly Asp Ar
g Val 70
75
80GGT ATC CCT TGG CTT
TAT TCG GCG TGC GGT CAT
TGT GAC TAT TGC TTA
586Gly Ile Pro Trp Leu Ty
r Ser Ala Cys Gly His Cys
Asp Tyr Cys Leu 85
90
95AGC GGA CAA
GAA ACA TTA TGC GAA CGT C
AA CAA AAC GCT GGC TAT TC
C 634Ser Gly Gln Glu
Thr Leu Cys Glu Arg Gln
Gln Asn Ala Gly Tyr Ser10
0 105
110
115GTC GAT GGT GG
T TAT GCT GAA TAT TGC CGT
GCT GCA GCC GAT TAT GTC
682Val Asp Gly Gly T
yr Ala Glu Tyr Cys Arg Al
a Ala Ala Asp Tyr Val
120
125
130GTA AAA ATT CCT GAT
AAC TTA TCG TTT GAA GAA G
CC GCT CCA ATC TTT 7
30Val Lys Ile Pro Asp Asn
Leu Ser Phe Glu Glu Ala
Ala Pro Ile Phe
135 140
145TGC G
CT GGT GTA ACA ACA TAT AA
A GCG CTC AAA GTA ACA GGC
GCA AAA 778Cys Ala
Gly Val Thr Thr Tyr Lys A
la Leu Lys Val Thr Gly Al
a Lys 150
155
160CCA GGT GAA TGG GTA
GCC ATT TAC GGT ATC GGC
GGG CTT GGA CAT GTC
826Pro Gly Glu Trp Val Al
a Ile Tyr Gly Ile Gly Gly
Leu Gly His Val 165
170
175GCA GTC CAA
TAC GCA AAG GCG ATG GGG T
TA AAC GTC GTT GCT GTC GA
T 874Ala Val Gln Tyr
Ala Lys Ala Met Gly Leu
Asn Val Val Ala Val Asp18
0 185
190
195TTA GGT GAT GA
A AAA CTT GAG CTT GCT AAA
CAA CTT GGT GCA GAT CTT
922Leu Gly Asp Glu L
ys Leu Glu Leu Ala Lys Gl
n Leu Gly Ala Asp Leu
200
205
210GTC GTC AAT CCG AAA
CAT GAT GAT GCA GCA CAA T
GG ATA AAA GAA AAA 9
70Val Val Asn Pro Lys His
Asp Asp Ala Ala Gln Trp
Ile Lys Glu Lys
215 220
225GTG G
GC GGT GTG CAT GCG ACT GT
C GTC ACA GCT GTT TCA AAA
GCC GCG 1018Val Gly
Gly Val His Ala Thr Val V
al Thr Ala Val Ser Lys Al
a Ala 230
235
240TTC GAA TCA GCC TAC
AAA TCC ATT CGT CGC GGT
GGT GCT TGC GTA CTC 1
066Phe Glu Ser Ala Tyr Ly
s Ser Ile Arg Arg Gly Gly
Ala Cys Val Leu 245
250
255GTC GGA TTA
CCG CCG GAA GAA ATA CCT A
TT CCA ATT TTC GAT ACA GT
A 1114Val Gly Leu Pro
Pro Glu Glu Ile Pro Ile
Pro Ile Phe Asp Thr Val26
0 265
270
275TTA AAT GGA GT
A AAA ATT ATT GGT TCT ATC
GTT GGT ACG CGC AAA GAC
1162Leu Asn Gly Val L
ys Ile Ile Gly Ser Ile Va
l Gly Thr Arg Lys Asp
280
285
290TTA CAA GAG GCA CTT
CAA TTT GCA GCA GAA GGA A
AA GTA AAA ACA ATT 12
10Leu Gln Glu Ala Leu Gln
Phe Ala Ala Glu Gly Lys
Val Lys Thr Ile
295 300
305GTC G
AA GTG CAA CCG CTT GAA AA
C ATT AAC GAC GTA TTC GAT
CGT ATG 1258Val Glu
Val Gln Pro Leu Glu Asn I
le Asn Asp Val Phe Asp Ar
g Met 310
315
320TTA AAA GGG CAA ATT
AAC GGC CGC GTC GTG TTA
AAA GTA GAT 1
300Leu Lys Gly Gln Ile As
n Gly Arg Val Val Leu Lys
Val Asp 325
330
335 337TAAAAAAGTAG A
TTAAAAAAGA AGGCGTCTGA GGGC
GCCTTC TTATTTTACT TCAACGG
AAA 1360ATACTTGATCA
TGAAGC TCTTCCTTAT TTACGTC
CCA CAAAACGTCC GATACGGTCG
1420ATCAGACGGCTCAGGAG
GTA TAGCATATTA CCCGTGGTGC
TAGATAAAAACT CAAACAAGCA
1480TAAAAATAGCCCTTGCATGA
GGATCC
150
6

[Brief explanation of the drawing]

[Figure 1] Contains the structural gene of ADH of the present invention, a gene having an expression control region and a terminator, and pTBAD
Fig. 3 is a restriction enzyme cleavage map of a 3.5 kb BamHI fragment derived from No. 35.

[Figure 2]B. stearothermophilus
ADH derived from NCA1503 strain, S. cerevi
FIG. 2 is a comparison diagram showing the amino acid sequences of ADH derived from S. siae, ADH derived from human, and ADH derived from horse.

FIG. 3 is a continuation of the arrangement of FIG. 2; staro
A derived from thermophilus NCA1503 strain
D.H., S. FIG. 2 is a comparison diagram showing the amino acid sequences of ADH derived from S. cerevisiae, ADH derived from human, and ADH derived from horse.

FIG. 4 is a continuation of the arrangement of FIGS. 2 and 3; s
tearothermophilus NCA1503
ADH derived from the strain S. A from A. cerevisiae
FIG. 2 is a comparison diagram showing the amino acid sequences of DH, human-derived ADH, and horse-derived ADH.

FIG. 5 is a flowchart showing the mechanism of the reversible reaction of alcohol to aldehyde or aldehyde to alcohol using ADH as a catalyst.

[Figure 6] Shows the structure of the active center of ADH, showing the structure of the active center of ADH.
FIG. 2 is a mechanism diagram showing the exchange of protons from

[Figure 7]B. stearothermophilus
It is a graph showing the optimal pH of ADH derived from NCA1503 strain and H43R.

Claims (17)

[Claims] 1. A structural gene encoding alcohol dehydrogenase shown by the amino acid sequence shown in Sequence Listing 1. 2. The structural gene according to claim 1, which has a base sequence represented by A at position 290 to T at position 1,300 in Sequence Listing 1. [Claim 3] Bacillus starother
The structural gene according to claim 1, which is derived from Mophilus NCA1503 strain. Claim 4: An alcohol dehydrogenase having the amino acid sequence shown in Sequence Listing 1, wherein the 43rd H of the amino acid sequence is
An alcohol dehydrogenase in which is has been substituted with another basic amino acid, or the His residue has been modified so that the basicity of the His residue is increased. 5. Claim 4, wherein the His is replaced with Arg.
The modified alcohol dehydrogenase described in . 6. A structural gene encoding the alcohol dehydrogenase of claim 4. 7. The structural gene according to claim 6, which has a base sequence represented by A at position 290 to T at position 1300 in Sequence Listing 1, with A at position 417 being replaced with G. 8. The structural gene according to claim 1 or 4, which is present in the 5' upstream region of the structural gene and is located at position 1 in Sequence Listing 1.
An expression control region consisting of the base sequence shown by C at position 289 from
An expressible gene that includes a terminator consisting of the base sequence shown by A at position 7 to T at position 1342; and encodes alcohol dehydrogenase or modified alcohol dehydrogenase. 9. An expression vector comprising the structural gene according to claim 1 or 4. 10. An expression vector comprising the gene according to claim 8. 11. The expression vector according to claim 10, which is pTBAD35. 12. The expression vector according to claim 10, which is pTBAD35H43R. 13. A transformant obtained by introducing the expression vector according to claim 9 or 10 into a host cell. 14. The host cell is Bacillus s.
The transformant according to claim 13, which is P. ubtilis. 15. The host cell is Bacillus st.
Claim 1, which is Aerothermophilus.
3. The transformant according to 3. 16. A method for producing alcohol dehydrogenase, which comprises the steps of culturing the transformant according to claim 13 and collecting alcohol dehydrogenase from the culture. 17. A method for producing alcohol, which comprises the step of culturing the transformant according to claim 15 in a medium containing a carbon source.