Activator for methanol dehydrogenase and gene thereof

Details Activator for methanol dehydrogenase and gene thereof Activator for methanol dehydrogenase and gene thereof

1999-09-01

2001-08-28

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S006120, C435S320100, C536S023100, C530S350000

Reexamination Certificate

active

06280972

ABSTRACT:

TECHNICAL FIELD
The present invention relates to a novel activator of methanol dehydrogenase, DNA coding for it, cell expressing the DNA, and method for producing the activator.
BACKGROUND ART
The
Bacillus methanolicus
strain C1, which is a methanol assimilating bacterium belonging to the genus Bacillus, has been known to have methanol dehydrogenase (also referred to with an abbreviation “MDH” hereinafter), which oxidizes methanol used as a carbon source into formaldehyde (
Arch. Microbiol
., 152, 280-288, 1989). MDH may be used for measurement of methanol content in a sample. In such a purpose, it is important to increase the specific activity of the enzyme, and means for achieving it have long been desired.
Arfman et al. has recently reported that a factor promoting this enzyme activity of MDH is contained in the
Bacillus methanolicus
C1. They purified a protein constituting the factor, and determined a partial amino acid sequence of the N-terminus of this protein (
The Journal of Biological Chemistry
, 266, 3955-3960, 1991). However, neither the whole structure of this factor and nor the genetic structure therefor has not been known at all.
By the way,
Bacillus subtilis
could not grow by utilizing methanol as an only carbon source, and therefore it has not been thought at all that this microorganism has methanol dehydrogenase, which is used for the first reaction of the methanol assimilation. In fact, when the present inventors searched known chromosome DNA sequences of
Bacillus subtilis
for a gene product or gene which has significant homology with the amino acid sequence and the nucleotide sequence of MDH of the
Bacillus methanolicus
C1, such a gene product or gene has not been found. Therefore, in microorganism not assimilating methanol like
Bacillus subtilis
, any activator of MDH has not been known, and its existence has not been expected.
SUMMARY OF THE INVENTION
The object of the present invention is to provide a novel means for promoting the MDH activity.
While the present inventors studied various gene sequences on the genome of
Bacillus subtilis
which is not a methanol assimilating bacterium, they accidentally found that the bacterium had a gene which could code for a protein homologous to the methanol dehydrogenase activator (MDH activator). When this gene was forcibly expressed in
Escherichia coli
, activity that promoted methanol dehydrogenase activity was successfully detected. Thus, the present invention has been accomplished.
That is, the present invention relates to a protein defined in the following (A) or (B):
(A) a protein which has an amino acid sequence of SEQ ID NO: 2;
(B) a protein which has an amino acid sequence of SEQ ID NO: 2 including substitution, deletion, insertion, addition, or inversion of one or several amino acids, and has an activity to promote methanol dehydrogenase activity.
The present invention also provides a DNA which codes for a protein defined in the following (A) or (B):
(A) a protein which has an amino acid sequence of SEQ ID NO: 2;
(B) a protein which has an amino acid sequence of SEQ ID NO: 2 including substitution, deletion, insertion, addition, or inversion of one or several amino acids, and has an activity to promote methanol dehydrogenase activity.
Specific examples of the above DNA include a DNA defined in the following (a) or (b):
(a) a DNA which comprises a nucleotide sequence of SEQ ID NO: 1;
(b) a DNA which is hybridizable with a nucleotide sequence of SEQ ID NO: 1 or a probe prepared from the nucleotide sequence under a stringent condition, and codes for a protein having an activity to promote methanol dehydrogenase activity.
The above stringent condition is exemplified by the condition in which in which washing is performed at 60 C, and at a salt concentration corresponding to 1×SSC and 0.1% SDS
The present invention also provides a cell to which the aforementioned DNA is introduced in such a manner that a protein encoded by the DNA can be expressed.
The present invention further provides a method for producing a protein having an activity to promote methanol dehydrogenase activity, which comprises culturing the aforementioned cell in a medium to produce and accumulate the protein, and collecting the protein.
For the purpose of the present invention, “methanol dehydrogenase activity” means an activity catalyzing the reaction in which methanol is oxidized to generate formaldehyde. The “activity to promote methanol dehydrogenase activity” means an activity that significantly increases specific activity of methanol dehydrogenase, when a factor which has this activity coexists with the methanol dehydrogenase.
A protein which has the activity to promote the methanol dehydrogenase activity according to the present invention will be referred to as “MDH activator” hereinafter.
According to the present invention, there is provided a novel activator for methanol dehydrogenase. This MDH activator can be utilized for basic researches for the reaction mechanism analysis of methanol dehydrogenase as well as industrially important processes such as detection of alcohol concentration in objective samples. Moreover, according to the present invention, DNA that encodes the alcohol dehydrogenase activator is obtained, and it enables efficient production of the factor. Therefore, this factor can be provided in a large scale at a low cost.
DETAILED DESCRIPTION OF THE INVENTION
Hereafter, the present invention will be explained in detail.
The DNA of the present invention can be obtained through PCR (polymerase chain reaction) utilizing chromosome DNA of
Bacillus subtilis
, for example, the
Bacillus subtilis
strain 168, as a template, as well as a primer having the nucleotide sequence of SEQ ID NO: 5 shown in Sequence Listing (BsYqk-G1) and a primer having the nucleotide sequence of SEQ ID NO: 6 shown in Sequence Listing (BsYqk-G2). Because both of these primers have restriction enzyme ClaI or BamHI recognition sites in their 5′ sequences, the amplification product digested with these restriction enzymes can be inserted into a vector having ClaI and BamHI digested ends.
The nucleotide sequences of the aforementioned primers were designed based on the nucleotide sequence of yqkG gene (SEQ ID NO: 2), of which function is unknown, and the amino acid sequence of the gene product thereof (SEQ ID NO: 1) of a
Bacillus subtilis
, which had been found as one of the genes having an amino acid sequence highly homologous to that of the MDH activator derived from methanol assimilating
Bacillus methanolicus
strain C1 through database searching based on partial amino acid sequence information of that sequence (SEQ ID NO: 7). By using these primers, a DNA fragment containing the coding region of the yqkg gene and its flanking region (3′ non-translation region of about 390 bases) can be obtained.
The nucleotide sequence of the coding region of the DNA of the present invention obtained as described above and an amino acid sequence which may be encoded by the sequence are shown in SEQ ID NO: 1. The amino acid sequence alone is shown in SEQ ID NO: 2. These sequences were identical to the known sequences of yqkg.
At first, it was scarcely expected that
Bacillus subtilis
not assimilating methanol retained on a genome a gene for an enzyme which participated in the methanol assimilation and metabolism like the MDH activator, and, moreover, it had evolved up to now while retaining the gene in such a form that it can express an active enzyme therefrom. This is because it is generally considered that, even if such a gene of enzyme that had become unnecessary for the growth of
Bacillus subtilis
is retained, it had suffered random mutations during the evolution of the microorganism, and lost its activity.
However, the present inventors dared to clone the open reading frame (also abbreviated as ORF hereinafter), and expressed it in a forced expression system utilizing
Escherichia coli
as a host in order to confirm whether the yqkG gene, of which function is unknown, actually codes for an alcohol dehydrogenase activator whi

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