MXPA99005412A - Gene of quinol oxidase of the cytochrome type bd of brevibacterium lactofermen - Google Patents
Gene of quinol oxidase of the cytochrome type bd of brevibacterium lactofermenInfo
- Publication number
- MXPA99005412A MXPA99005412A MXPA/A/1999/005412A MX9905412A MXPA99005412A MX PA99005412 A MXPA99005412 A MX PA99005412A MX 9905412 A MX9905412 A MX 9905412A MX PA99005412 A MXPA99005412 A MX PA99005412A
- Authority
- MX
- Mexico
- Prior art keywords
- leu
- amino acid
- dna
- cytochrome
- gli
- Prior art date
Links
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Abstract
The present invention relates to the oligonucleotides based on the amino acid sequences of the N terminus of the I subunit, and the N terminus of the quinol oxidase subunit II of the cytochrome bd type of brevibacterium flavum, PCR is performed using the oligonucleotides as promoters, and B. flavum chromosome DNA as a template, and a gene encoding quinol oxidase of the cytochrome bd type of B. flavum is obtained from a chromosome DNA library of Brevibacterium lactofermentum using the amplification fragment obtained as a
Description
0UIN0L GENE QXIDASE OF THE CYTOCHROME TYPE BD DE Brevibacterium Lactofermentun DESCRIPTION OF THE INVENTION The present invention relates to a quino oxidase of the cytochrome bd type of Brevibacterium lactofermentu and a DNA encoding the same. Most organisms acquire energy needed for life activity by breathing. In higher organisms, carbohydrates, proteins and aliphatic acids are degraded in acetyl-CoA by the glycolytic path and ß-oxidation in cytoplasm, and acetyl-CoA is degraded by the citric acid cycle in mitochondria. The resulting energy is saved as a reduction energy of NADH and FADH2. Finally, oxidized NADH is completely oxidized to water by the subsequent electron transport system that is present in the inner membranes of mitochondria, and a proton concentration gradient is formed in a form coupled to oxidation, and serves as a force of the synthesis of ATP. Since the bacterial respiratory chain generally comprises several functional enzyme complexes depending on the species and growth circumstance, the efficiency of energy conservation may vary to a greater degree. For example, Escherichia coli contains at least two quinol oxidase types, bo type and bd type, which function as terminal oxidases in the respiratory chain. When comparing a strain of the wild type that carries the enzymes of both types, a mutant strain carrying only the bo type, and a mutant strain carrying only the bd type are compared for the growth performance observed in aerobic culture, the field growth is lower in the mutant carrying only the bd-type enzyme, and depends on the type of terminal oxidases and their energy conservation efficiency (Lecture Abstract for the Conference of the Society for Bioscience and Bioengineering, Japan, 1995, Subject No. 357). Coryneform bacteria such as Brevibacterium lactofermentum and Brevijbacterium flavum are Gram-positive and aerobic bacteria that are used industrially for amino acid producers. Although the terminal oxidases of the respiratory chain have been well researched for those of Proteobacterium, which are very phylogenetically far from the Coryneform bacteria, and those of Bacillus subtilis and the thermophilic Bacillus, which are also Gram-positive bacteria similar to Coryneform bacteria. but phylogenetically but phylogenetically somewhat different from them, the electron transport system of the respiratory chain in the Coryneform bacteria has not been investigated in detail. It is considered important to elucidate the electron transport system of the respiratory chain, which is the key to energy metabolism, in Coryneform bacteria in order to collect the fundamental data to improve the productivity of useful substances. Furthermore, if the enzymes involved in the electron transport system of the respiratory chain in Corineform bacteria and genes for them are identified, they may be useful, for example, by creating strains with higher energy efficiency. To date, it has been reported that Brevibacterium lactofrumum respiration is coupled to proton transport, and this involves cytochromes a, b, and c (Kawahara, Y., et al. (1988) Agrie. Biol. Chem., 52 (8), 1979-1983). The quinol oxidase of the cytochrome bd type of Brevibacterium flavum was also purified and characterized (Kusumoto, Soné and Sakamoto, "Respiratory Chain of Amino Acid Fermenting Bacterium, Brevibacterium flavum, and Characteristics of Its Cytochrome bd Type Menaquinol Oxidase", Abstracts of th 23th Symposium of Bioenergy Study Group, 1997). However, there has not been a reported in relation to the genes that code for quinol oxidase of the cytochrome bd type of Coryneform bacteria. The present invention has been made from the aforementioned point of view, and its object is to obtain a quinol oxidase gene of the cytochrome bd type of Coryneform bacteria, and to elucidate its structure. In the present invention, oligonucleotides are synthesized based on amino acid sequences of the N terminus of subunit I, and the N terminus of subunit II of the quinol oxidase of the cytochrome bd type of Brevibacterium flavum, and preformed PCR using the oligonucleotides as promoters. , and a chromosomal DNA of Brevibacterium flavum as annealed to obtain an amplified fragment. In addition, they screen a chromosomal DNA library of the wild-type Brevibacterium lactofermentum strain using the amplified fragment as a probe, and a gene encoding a quinol oxidase of the cytochrome bd type of Brevibacterium lactofermentum is successfully obtained. In this way, the present invention has been completed. That is, the present invention provides: (1) a DNA fragment encoding a polypeptide defined in the following (A) or (B); (A) a polypeptide which has an amino acid sequence shown in SEQ ID NO: 2 of the sequence listing, (B) a polypeptide which has an amino acid sequence shown in SEQ ID NO: 2 of the Sequence Listing which it comprises substitution, elimination, insertion, addition or inversion of one or a plurality of amino acid residues in the amino acid sequence, and may constitute a protein exhibiting quinol oxidase activity of the cytochrome bd type together with a subunit II of l quinol oxidase of the cytochrome bd type having an amino acid sequence shown in SED I NO: 4, (2) a DNA fragment encoding a polypeptide defined in the following (C) or (D); (C) a polypeptide which has a d amino acid sequence shown in SEQ ID NO: 4 of Sequence Listing, (D) a polypeptide which has a d amino acid sequence shown in SEQ ID NO: 4 of Sequence Listing comprising substitution, elimination, insertion, addition inversion of one or a plurality of residues d amino acids in the amino acid sequence, can constitute a protein that exhibits quinol oxidase activity of the cytochrom bd type together with a subunit I of the quino oxidase of the cytochrome bd type having an amino acid sequence shown in Seq I NO: 2, (3) a DNA fragment encoding a polypeptide defined in the following (A) or (B), and a polypeptide defined in the following (C) or ( D);
(A) a polypeptide which has an amino acid sequence shown in SEQ ID NO: 2 of the sequence listing, (B) a polypeptide having an amino acid sequence shown in Seq Id NO: 2 of the sequence listing comprising , substitution, deletion, insertion, addition or inversion of one or a plurality of amino acid residues in the amino acid sequence, and
may be a protein exhibiting quinol oxidase activity of the cytochrome bd type together with a subunit II of the cytochrome bd quinol oxidase having an amino acid sequence shown in Seq ID
NO: 4, (C) a polypeptide which has an amino acid sequence shown in SEQ Id NO: 4 of the sequence listing, (D) a polypeptide which has a sequence of
amino acids shown in SED ID NO: 4 of the Sequence Listing comprising substitution, deletion, insertion, addition or inversion of one or a plurality of amino acid residues in the amino acid sequence, and
may constitute a protein exhibiting quinol oxidase activity of the cytochrome bd type together with an I subunit of the cytochrome bd quinol oxidase having an amino acid sequence shown in SEQ ID NO: 2, (4) The DNA of (1) ) above, which is a DNA defined in the following (a) or (b): (a) a DNA having a nucleotide sequence corresponding to nucleotide numbers 933 to 2483 in the nucleotide sequence depicted in SEQ ID NO. : 1 in the Sequence Listing; or
(b) a DNA which is hybridizable to the nucleotide sequence of (a) above under a severe condition, and which encodes a polypeptide which may constitute a protein exhibiting quinol oxidase activity of the cytochrome bd type together with a subunit II of the cytochrome bd quinol oxidase having an amino acid sequence shown in SEQ Id NO: 4. (5) The DNA of (2) above, which is a DNA defined in the following (c) or (d): (c) a DNA having a nucleotide sequence corresponding to the nucleotide numbers 2476 to 3498 in the nucleotide sequence shown in SEQ ID NO: 3 in the Sequence Listing; or (d) a DNA which is hybridizable with the nucleotide sequence of (c) above under severe condition, and the eral encodes a polypeptide which may constitute a protein exhibiting quinol oxidase activity of the cytochrome bd type together with a subunit I of the quinol oxidase of the cytochrome bd type having an amino acid sequence shown in SED Id NO: 2. (6) The DNA of (3) above, which comprises a DNA defined in the following (a) or (b) ), and a DNA defined in the following (c) or (d): (a) a DNA having a nucleotide sequence corresponding to nucleotide numbers 933 to 2483 in the nucleotide sequence depicted in SEQ ID NO: 1 in the Sequence Listing; or (b) a DNA which is hybridizable to the nucleotide sequence with the nucleotide sequence of (a) above under severe condition, and which encodes a polypeptide which may constitute a protein exhibiting cyclochrome bd quinol oxidase activity together with a subunit II of the quinol oxidase of the cytochrome bd type having an amino acid sequence shown in SEQ ID NO: 4; and (c) a DNA having a nucleotide sequence corresponding to nucleotide numbers 2476 to 3498 in the nucleotide sequence depicted in SEQ ID NO: 3 in the
Sequence Listing; or (d) a DNA that is hybridizable with the nucleotide sequence of (c) above with the nucleotide sequence of
(c) above under severe condition, and which encodes a polypeptide which may constitute a protein exhibiting quinol oxidase activity of the cytochrome bd type together with an I subunit of the quinol or idase of the cytochrome bd type having an amino acid sequence shown in SEQ ID NO: 2. (7) A DNA fragment defined in (1) above, which has a nucleotide sequence comprising nucleotides of nucleotide numbers 933 to 2483 in the nucleotide sequence shown in SEQ ID NO: 1, (8) A DNA fragment defined in (2) above which has a sequence of nucleotides comprising nucleotides of nucleotide numbers 2476 to 3498 in the nucleotide sequence shown in SEQ ID NO: 1, and (9) a DNA fragment defined in (3) above, which has a nucleotide sequence comprising nucleotides of the nucleotide numbers 933 to 3498 in the nucleotide sequence shown in SEQ ID NO: l. In the present description, the term quinol oxidase activity of the cytochrome bd type means activity exhibiting differential absorption spectra of cytochrome b cytochrome d oxidoreduction, which is to oxidize reduced-type quinone compounds (quinols) with oxygen consumption . A DNA fragment encoding quinol oxidase of the cytochrome bd type or a subunit thereof will be referred to as "DNA of the present invention" as may be the case. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 represents the results of the hydropathy analysis of subunits I of the quinol oxidases of the cytochrome bd type of Brevibacterium lactofermentum, Bacillus stearoth.ermoph.ilus and Escherichia coli. The symbol "*" indicates an amino acid residue shared by three oxidases. Figure 2 depicts amino acid sequence alignment of I subunits of the cyclochrome bd quinol oxidases of Brevibacterium lactofermentum, Bacillus stearothermophilus and Escherichia coli. Figure 3 depicts amino acid sequence alignment of subunits II of the quinol oxidases of the cytochrome bd type of Brevibacterium lactofermentum, Bacillus stearothermophilus and Escherichia coli. The present invention will be explained in more detail later herein. The DNA of the present invention can be obtained from B chromosomal DNA. Lactofermentum based on partial amino acid sequences of quinol oxidase of the cytochrome bd type of B. Flavum. Specifically, PCR is pre-performed using oligonucleotides synthesized on the basis of amino acid sequences as promoters, and chromosomal DNA of B. Flavum as annealed to obtain a partial sequence of the quinol oxidase gene of the cytochrome bd type of b. Flavum Next, screening a chromosomal DNA library of B. Lactofermentum using the partial sequence obtained as a probe, a gene encoding a quinol oxidase of the cytochrome bd type of b can be obtained. Lactofermentum. The chromosomal DNA of B. flavum and B. lactofermentum can be prepared by, for example, the method of Saito and Miura (Biochem. Biophys. Acta., 72, 619, (1963)), and the method of KS Kirby (Biochem. J., 64, 405, (1956). )). A chromosome DNA library can be obtained by partially digesting chromosomal DNA with a suitable restriction enzyme, by ligating each of the DNA fragments obtained to a vector DNA replicable autonomously in Escherichia coli cells to prepare a recombinant DNA, and introducing the DNA in E. coli. The vector is not particularly limited, as long as this vector is usually used for genetic cloning, and plasmid vectors such as pUC19, pUC18, pUC118 and puC119, phage vectors such as lambda phage DNA and the like can be used. The promoter used for PCR can be, for example, an oligonucleotide having a nucleotide sequence shown in SEQ ID NO: 7 or SEQ ID NO: 8. In order to confirm that a PCR product obtained has a desired sequence, it can be confirmed that it contains a sequence corresponding to the promoter by nucleotide sequencing, or confirming that the deduced amino acid sequence of the nucleotide sequence contains a partial amino acid sequence of the quinol oxidase of the cytochrome bd type of B. flavum. It can be performed the screening of a library of
B chromosome DNA. lactofermentum using the DNA fragment obtained in PCR as a probe by colony hybridization when using plasmid vectors for library preparation, or plate hybridization when phage vectors are used for library preparation. A positive clone of hybridization can be confirmed to contain a quinol oxidase gene of the cytochrome bd type by DNA nucleotide sequencing prepared from the clone. It is also possible preliminary Southern analysis for a positive clone of hybridization using the probe. A nucleotide sequence of the quinol oxidase gene of the cytochrome bd type of the B. lactofermentum strain ATCC 13869 obtained in the working example is shown in such form as described above in SEQ ID NO: 1. Expected coding regions are shown and amino acid sequences of proteins encoded by them in SEQ ID NO: 1-4. The estimation of coding regions and operon structure and analysis of homology to quinol oxidases of the cytochrome bd type of Bacillus stearothermophilus K1041 and Escherichia coli using GENETYX-Homology Version 2.2.2 (Software Development Co., Ltd.) is performed. The quinol oxidase gene of the cytochrome bd type contains two open reading structures (cydA and cydB reading from the 5"end), and encode I subunit of the cytochrome bd quinol oxidase (also referred to simply as" I subunit "subsequently in the present invention) and subunit II thereof (also referred to simply as "subunit II" later in the present invention), respectively.It is estimated that cydA and cydB comprise 1551 bp and 1023 bp respectively, subunit I consists of 517 residues of amino acids, and subunit II consists of 341 amino acid residues.A sequence similar to an upstream promoter of cydA is presented, a sequence similar to SD is presented upstream of each of cydA and cydB, and a terminator-like sequence is It is considered that cydA and cydB form an operon c and d, while the codon of the N-terminal amino acid residue of subunit I it is indicated as GTG, and the corresponding amino acid as Val in the Sequence Listing, is actually Met. This is considered to be triggered since GTG is recognized as a start methionine. Such cases have been reported elsewhere in the present. Figures 1 and 2 represent the results of the hydropathy analysis performed for comparison of quinol oxidase structures of the cytochrome bd type of the present invention and subunits I of Bacillus stearothermophilus and E. coli, and alignment of the amino acid sequences. Indications I-VII represent regions of transmembrane helix, and therefore it is confirmed that there are at least seven transmembrane helices. It can be understood from the patterns shown in the graphs that they resemble each other. In addition, a region containing a quinol binding site called a Q circuit is presented between V and VI of the I subunit of E. coli, while there is no region that exhibits homology with the last average portion of the Q circuit in B. lactofermentum similar to cydA of B. stearothermophilus, and therefore the region of the Q circuit is shortened. Considering this point, quinol oxidase of the cytochrome bd type of B is expected. lactofermentum has a structure more similar to that of quinol oxidase of the cytochrome bd type of B. stearothermophilus more than E. coli. The comparison of amino acid sequences of subunit I shows that B. lactofermentum has approximately 24.7% homology to B.
stearothermophilus and, approximately 38.6% to E. coli, and it is considered that, as for the I subunit as a whole, the quinol oxidase of the cytochrome bd type of B. lactofermentum has a structure more similar to quinol oxidase of the cytochrome bd type of B. coli rather than to B. stearothermophilus. has been reported H19, H186 and M393 for E. coli cydA, and H21, H184 and M326 for c and D of B. stearothermophilus as functionally important residues in view of being a ligand of hem b558. These amino acids are also conserved in cydA of B. lactofermentum such as H18, H185, and M350. Figure 3 represents alignment of amino acid sequences of the three types of bacterial subunits II. As for subunit II, B. lactofermentum shows approximately 25.9% homology to B. stearothermophilus, and approximately 34.8% to E. coli. The DNA of the present invention is a DNA encoding subunit I, which is encoded by the nucleotide sequence shown in SEQ ID NO: 2, subunit II, which is encoded by the nucleotide sequence shown in SEQ ID NO. : 4, or quinol oxidase protein of the cytochrome bd type containing this subunit I and subunit II. The subunit I, subunit II or quinol oxidase protein of the cytochrome bd type can be produced by introducing such DNA into a suitable host cell, and cultivating the transformant in such a way that the DNA can be expressed. It can be mentioned DNA having a nucleotide sequence comprising nucleotides of nucleotide numbers 933-2483 as a DNA encoding subunit I, a DNA having a nucleotide sequence comprising nucleotides of nucleotide numbers 2476-3498 as a DNA encoding subunit II, and a DNA having a nucleotide sequence comprising nucleotides of nucleotide numbers 933-3498 as a DNA encoding both. The quinol oxidase protein of the cytochrome bd type produced or a subunit of the msma can be collected and purified from the culture by a method commonly used for the purification of proteins such as sautéing, solvent precipitation, gel filtration chromatography, and exchange chromatography. of ions. The DNA of the present invention encoding subunit I can be either one encoding a polypeptide having an amino acid sequence shown in SEQ ID NO: 2 comprising substitution, deletion, insertion, addition or inversion of one or a plurality of amino acid residues in the amino acid sequence, or a polypeptide that can constitute a protein that exhibits quinol oxidase activity of the cytochrome bd type together with subunit II. The DNA of the present invention encoding the subunit II can be either one encoding a polypeptide having an amino acid sequence shown in SEQ ID NO: 4 comprising substitution, deletion, insertion, addition or inversion of one or a plurality of amino acid residues in the amino acid sequence, or a polypeptide that can constitute a protein that exhibits quinol oxidase activity of cytochrome bd together with subunit I. In addition, a DNA encoding a cytochrome bd quinol oxidase that contains mutations in the subunit I, subunit II or both is also included in the DNA of the present invention. The term "a plurality of amino acid residues" preferably means 1-40, more preferably 1-10 amino acids of residues. DNA, which encodes substantially the same protein as subunit I and / or subunit II as described above, for example, by modifying the nucleotide sequence, for example, by means of the site-directed mutagenesis method such that one or more amino acid residues at a specified site involve substitution, elimination, insertion, addition or inversion. The DNA modified as described above can be obtained by the conventionally known mutation treatment. The mutation treatment includes a method for creating DNA encoding subunit I and / or subunit II in vitro, for example, with hydroxylamine, and a method for treating a microorganism, for example, a bacterium belonging to the genus by harvesting DNA that encodes subunit I and / or subunit II with ultraviolet irradiation or a mutation agent such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and nitrous acid usually used for the mutation treatment. The replacement, deletion, insertion, addition, or inversion of nucleotides as described above also includes mutation (mutant or variant) that occurs naturally, for example, on the basis of the individual difference or the difference in species or genus of coryneform bacteria that harvest quinol oxidase of the cytochrome bd type. DNA, which codes for substantially the same protein as subunit i and / or subunit II, is obtained by expressing DNA having mutation as described above in an appropriate cell, and investigating the activity of an expressed product. DNA, which encodes substantially the same protein as subunit I and / or subunit II, is also obtained by isolating DNA that is hybridizable with DNA having, for example, a nucleotide sequence corresponding to nucleotide numbers from 933 to 2483 of the nucleotide sequence represented in SED ID NO: l / or a nucleotide sequence corresponding to nucleotide numbers from 2476 to 3498 of the nucleotide sequence depicted in SEQ ID NO: in the Sequence Listing under severe condition, and encodes a protein having the activity of subunit I and / or subunit II, of DNA encoding subunit I and / or subunit II that has mutation or of a cell harvesting it. The "severe condition" referred to herein is a condition under which the so-called specific hybrid is formed, and no non-specific hybrid is formed. It is difficult to clearly express this condition using any numerical value. However, for example, the severe condition includes a condition under which DNA having high homology, for example, DNA having homology of not less than 50%, hybridizes with each other, and DNA having less homology than the previous ones does not they hybridize with each other Alternatively, the severe condition is exemplified by a condition under which DNA is hybridized to each other at a salt concentration corresponding to an ordinary wash condition in Southern hybridization, i.e., 60 ° C, 1 x SSC, 0.1% SDS , preferably 0.1 x SSC, 0.1% SDS. The gene, which is hybridizable under the condition as described above, includes those that have a stop codon generated within a coding region of the gene, and those that have no activity due to the mutation of the active site. However, such inconveniences can be easily eliminated by ligating the gene with a commercially available expression expression vector, and investigating cytochrome bd quinol oxidase activity.
The host for expression of the DNA of the present invention includes, for example, various types of bacteria including E. coli, corinerform bacteria such as B. lactofermentum, and B. flavum, eukaryotic cells such as saccharomyces cerevisiae and the like. In order to introduce the DNA of the present invention into a host such as those introduced above, the host cell can be transformed with a recombinant vector which is obtained by inserting the DNA of the present invention into a selected vector depending on the type of host in which you get the expression. Those methods can be performed using genetic recombination methods well known to those skilled in the art. The DNA of the present invention and quinol oxidase of the cytochrome bd type of the subunits thereof encoded by them are considered to be useful for elucidating the electron transport system of coryneform bacteria. The DNA of the present invention is also expected to be used by seeding coryneform bacteria producing useful substances with high energy efficiency. The present invention will be explained specifically with reference to the following examples. < 1 > Purification of quinol oxidase of the cytochrome bd type of Brevibacterium flavum. The bacterial cells (approximately 120 g in wet weight) of the B strain. ATCC 14067 flavum that have been cultured by the end of the stationary phase are suspended in 200 ml of a buffer (0.5% NaCl, 10 mM sodium phosphate, pH 7.4), and are immediately altered by stirring at high speed by means of a pearl mixer (Biospec) in the presence of 0.5 mM glass particles. Then this suspension of altered cells is brought to centrifugation at 5,000 rpm for 10 minutes and the resulting supernatant is further subjected to centrifugation at 15,000 rpm for 30 minutes. The precipitates obtained in both centrifugations are combined, and are suspended in the same buffer as mentioned above to obtain a membrane preparation. The above membrane preparation (5 mg / ml, 0.5% NaCl, 10 mM sodium phosphate, pH 7.4) is homogenized by a Teflon homogenizer, and is centrifuged at 40,000 rpm for 20 minutes, and the precipitates are collected. The precipitates are added with 1.5% sodium cholate, 0.5% sodium deoxycholate, 0.1% NaCl, and 10 mM sodium phosphate (pH 7.4), then homogenized and centrifuged at 40,000 rpm for 20 minutes to collect the precipitates . The precipitates are further added with 10 mM sodium phosphate (pH 7.4), homogenized, and centrifuged at 40,000 rpm for 20 minutes to collect the precipitates. The membrane preparation is washed with cholic acid as described above is suspended in a buffer containing surfactants, n-nonanoyl-N-methylglucamide (MEGA-9) and decanoyl-N-methylglucamide (MEGA-10) each to 1% . This suspension is homogenized on ice, sonified, and centrifuged at 40,000 rpm for 20 minutes to obtain a supernatant. The above supernatant obtained by centrifugation is adsorbed on a hydroxyapatitia column equilibrated with 1% MEGA-9, 1% MEGA-10, 10% glycerol, and 10 mM sodium phosphate (pH 7.4), and fractionated by elution with a concentration of sodium phosphate increased in steps (0, 50, 150, 250 and 400 nM). Cytochromes are detected in the fractions by reduced difference spectrum less oxidized. As a result, cytochromes c and b are detected in the fraction eluted at 50 mM sodium phosphate, cytochromes c, b and a in the fraction eluted at 150 mM, and cytochromes b and d in the fraction eluted at 250 mM. The fraction eluted at a sodium phosphate concentration of 250 nM is dialyzed against 10% glycerol and 10 mM sodium phosphate (pH 7.4), after adsorbing on DEAE-Toyopearl (Tohso) column equilibrated with the same buffer, and fractions by elution with an increased NaCl concentration in steps (0, 80, 100, 120, 140 and 300 mM). The cytochromes are detected in the fractions by difference spectrum of reduced less oxidized. As a result, cytochromes b and d are detected in the fraction eluted at a NaCl concentration of 120 mM. This fraction is used as a quinol oxidase enzyme preparation of the cytochrome bd type. The above enzyme preparation is subjected to SDS polyacrylamide gel electrophoresis using 13.5% gel, and blot analysis is carried out on a PVDF membrane. Portions of the membrane corresponding to subunit I and subunit II are subjected to amino acid sequence analysis to determine the N-terminal amino acid sequences. The amino acid sequences are shown in SEQ ID NO: 5 (subunit I) and SEQ ID NO: 6 (subunit II), respectively. <; 2 > Isolation of the quinol oxidase gene of the cytochrome bd type from Brevibacterium lactofermentum. Screening of a chromosome DNA library of B. lactofermentum is performed for clones which contain the quinol oxidase gene of the cytochrome bd type by colony hybridization. Two types of oligonucleotides are synthesized based on the above partial amino acid sequences of quinol oxidase of the cytochrome bd type of B. flavum. One is prepared based on the N-terminal amino acid sequence of the cytochrome bd quinol oxidase subunit I (bbdl: SEQ ID NO: 7), and the other is prepared based on the N-terminal amino acid sequence of subunit II (bbd2: SEQ ID NO: 8). CPR is performed using the above promoters bbdl and bbd2 and the chromosome DNA of strain ATCC 14067 as annealed. As well as for the reaction condition, after denaturation at 94 ° C for one minute, a cycle comprising denaturation at 95 ° C for 45 seconds, destemming at 50 ° C for 60 seconds, and repeating chain extension reaction at 62 ° C for 90 seconds for 35 cycles. As a result, fragments of approximately 1500 p.b., 800 p.b., and 100 p.b. Based on the molecular weight 56.4 kD of the estimated I subunit of the purified protein, and the reported molecular weights of I subunits of quinol oxidases of the cytochrome bd type of other bacteria, the fragment of approximately 1500 p.b. it is considered to be the desired CPR product. Therefore, the PCR product is electrophoresed on a 2% agarose gel, and a portion of the fragment of approximately 1.5 p.b. of the gel to extract the DNA. This fragment of APN is terminated blunt using the Blunting DNA kit (Takara Shuzo), and ligated to the vector pUC118 digested with Smal and treated with alkaline phosphatase using the DNA ligation kit Ver. 2 (Takara Shuzo). The strain of E. coli XL-1 Blue is transformed with the obtained recombinant promoter. The plasmid of the obtained transformant is prepared, and the inserted nucleotide sequence is determined. The nucleotide sequence is performed using the fluorescein-labeled M4 promoter (Takara, Shuzo, SEQ Id NO: 9) as the forward promoter, and the RV-MF fluorescein-labeled promoter (Takara Shuzo, SEQ ID NO: 10) as the promoter in reverse according to the Thermo Sequence fluorescent-labeled promoter cycle sequencing kit (Amersham Life Science). As a result, it is confirmed that the quinol oxidase gene of the cytochrome bd type is contained in the plasmid on the basis of homology with the promoter. This partial clone is designated BD1. This BD1 is amplified by PCR using the M4 and RV-M promoters mentioned above, and a DIG-labeled probe (digoxigenin) is prepared using DIG DNA labeling equipment (Boehringer Mannheim). The chromosomal DNA library of B. lactofermentum is screened using the probe mentioned above. The library is obtained by partially digesting the chromosomal DNA of B. lactofermentum ATCC 13869, inserting the product into the BamHI site of pUC18, and transforming E. coli XL-1 Blue with the obtained recombinant plasmid. Colony hybridization is performed in the colonies of transformants using the DIG-labeled probe mentioned above. The detection of the probe is performed using DIG Detection equipment (Boehringer Mannheim) which uses anticuepro anti DIG labeled with alkaline phosphatase. The colony plasmid is prepared, digested with EcoRI and PstI, and subjected to Southern blotting using BD1 as a probe. As a result, two positive clones are obtained.
The inserted fragments of these positive clones are designated BD21 and BD31, respectively. BD21 comprises approximately 3.8 kp.b. and BD31 comprises approximately 9.0 kp.b. These clones are subcloned, and their nucleotide sequences are determined. The results are shown in SEQ ID NO: 1. The expected coding regions and amino acid sequences of the proteins encoded by them are shown in SED ID NO: 1-4. SEQUENCE LISTING < 110 > Ajimoto Co., Inc.
< 120 > Quinol oxidase gene of the cytochrome bd type of Brevibacterium lactofermentum
< 130 > OP852
< 140 > < 141 >
< 150 > JP 10-164019 < 151 > 1998-06-11
< 160 > 10
< 170 > Patent In Ver. 2.0 < 210 > 1 < 211 > 3936 < 212 > DNA < 213 > Brevibacterium Lactofermentum
< 220 > < 221 > CDS < 222 > (933) .. (2483)
< 400 > 1 ggatcctctc tgttcaaaac agcacctact cttttactcc cgagttccga cgtgccctcg 60 acgaaagcct agaagtgacg gaccgagatg aggctgctca gaattttaag tttcacgtcc 120 aagacatcat cgaaactggg ttgtttatcg ccagaaataa tggattctgg caaggaaacc 180 tcgtcgttgg cgaaagatat tcccgacgag atgtctgccg aattctcaat tgggaacgaa 240 accaatgagag cacgatttat ggttacaaag tggacagcta cacatcgacg tgcccaatct 300 ttgtgaccta tcacaaggct gatgatgtat ccgaagtact cgttaccagg atgaactcgt 360 cgatccgaat acccttcatt ggtattcccg cggcaaccga aagatcacgt ctaatgagat 420 caagcccatc gctgcgaatg tgtggatctt catgtttttg tgaagaagga cgatgccgaa 480 ggccttgatt tcttctcaat tggtcaagcg cattcagaaa acagcaaaca gtcatcgatg 540 cccggaaaca aaggagttgt gcaaccggtg gtcacaatgg atctacagtt cgacacaccc 600 gcctgtttga gtcgaacaaa gtacctgagc acaaatctcg ccgtaacgga gtaaccaccg 660 tcgaaaagca caaccaagcg aaatcttttc gagtttttgg tgacttgtca acaagggggg 720 gtcattgasa agcaaaatca gggaaaggtt gaccacaatc ggggttagcc tttctaaagt 780 taagctgtga gcgggaactt aggaataaac ttcaacgaca acctttaaga agctcttatt 840 ggttcttcgt tttgtat cga taaatacaat cggtttcctg gctcaataag gctgttcctg 900 tcaatctgta aagaagagga aggggaccta ge gtg gat gtc gtt gac ate gcg 953 Val Asp Val Val Asp lie Ala 1 5 cgg tgg cag ttc gga att acc gtc tat fall ttc att ttt gtc cea 1001 Arg Trp Gln Fen Gli lie Tre Tre Val Tir His Fen lie Fen Val Pro 10 15 20 ctg acc att ggc tta gca ceg ctg gtc gcg ate atg caa acg ttt tgg 1049 Leu Tre lie Gli Leu Ala Pro Leu Val Ala lie Met Gln Tre Fen Trp 25 30 35 ca gtt acc ggc aaa gag falls tgg tat cgg gct acg aga ttt ttt ggc 1097 Gln Val. Tre Gli Lis Glu His Trp Tir Arg Ala Tre Arg Fen Fen Gli 40 45 50 55 act gtc ctg etc ate aac ttc gcg gtt ggt gta cga acg ggc att gtg 1145 Tre Val Leu Leu lie Asn Fen Ala Val Gli Val Ala Tre Gli lie Val 60 65 70 cag gag ttc cag ttc ggt atg aac tgg teg gaa tat teg cgt ttc gtc 1193 Gln Glu Fen Gln Fen Gli Met Asn Trp Ser Glu Tir Ser Arg Fen Val 75 80 85 ggt gat gtt ttc ggc gga ceg ctg gct ttg gag ggg gcg gtc gtg 1241 Gli Asp Val Fen Gli Gli Pro Leu Wing Lau Gli Gli Leu lie Fen 90 95 100 ttc ctt gag tet ttc gtg ttc tgc ctg gt tgg att gt tgg tgg ggg aga 1289 Fen Leu Glu Ser Val Fen Leu Gli Leu Trp lie Fen Gli Trp Gli Lis 105 110 115 att ect gga tgg ctg cat act gcg tec tgg ate gtt gct att gcg 1337 lie Pro Gli Trp Leu His Tre Wing Ser He Trp He Val Wing He Wing 120 125 130 135 acg aat att tet gee tat ttc ate ate gtg gee aac teg ttt atg cag 1385 Tre Asn He Be Wing Tir Fen He He Vrl Wing Asn Ser Met Met Gln 140 145 150 cat ceg gtg ggt gct gag tat aac ect gag act ggt cgg gcg gag ctt 143 3 His Pro Val Gli Wing Glu Tir Asn Pro Glu Tre Gli Arg Wing Glu Leu 155 160 165 act gat ttc tgg gct ctt etc here aac tec gcg ctg cctg gct gcg ttc 1481 Tre Asp Fen Trp Wing Leu Leu Tre Asn Ser Tre Wing Leu Ala Ala Fen 170 175 180 ceg cat gct gtt cgg ggt gtt ttt tta here gct gga act ttc gtc etc 1529
Pro His Wing Val Wing Gli Gli Fen Leu Thr Wing Gli Tre Fen Val Leu 185 190 195 gga att ceg ggt tgg tgg att att cgt gcg falls cgc cag gcg aag aag 1577 Gli He Ser Gli Trp Trp He He Arg Ala His Arg Gln Ala Lis Lis 200 205 210 215 gct gag gcg gaa ate gag teg aag cat tea atg falls agg ceg gcg ttg 1625 Wing Glu Wing Glu He Glu Ser Lis His Met Met His Arg Pro Wing Leu 220 225 230 tgg gtt tgg tgg tgg acc gtt gtc gtc tet tec gtg gca ctg ttc ate 1673 Trp Val Gli Trp Trp Tre Tre Val Val Ser Ser Val Wing Leu Fen He 235 240 245 Act ggc gat here cag gcg aag etc atg ttc gtg cag cag ceg atg aag 1721 Tre Gli Asp Tre Gln Ala Lis Leu Met Fen Val Gln Gln Pro Met Lis 250 2 25555 260 atg gcg teg gcg gaa tec ttg tgt gaa acc gee here gat cea aac tcc 1769 Met Wing Wing Wing Glu Ser Leu Cis Glu Tre Wing tre Asp Pro Asn Fen 265 270 275 tec att ctg here att ggt acg falls aac aac tgc gat acg gta acc falls 1817 Ser He Leu Tre He Gli Tre His Asn Asn Cis Asp Tre Val Tre His 280 285 290 295 ctg ate gat gtt ceg ttt gtg ctt cea ttc ttg gct gaa gga aaa ttc 1865 Leu He Asp Val Pro Fen Val Leu Pro Ftn Leu Ala Glu Gli Lis Fen 300 305 310 acc ggt gtg act ttg cag ggt gta aac cag cta ca gct gca gcg gag 1913
Tre Gli Val Tre Leu Gln Gli Val Asn Gln Leu Gln Ala Ala Ala Glu 315 320 325 ca gca tac ggt ect ggc aac tac tec ect aac ttg ttt gtc acc tac 1961
Gln Ala Tir Gli Pro Gli Asn Tir Ser Pro Asn Leu Fen Val Tre Tir 330 335 340 tgg tea tcc cgc gca atg ate ggc cta atg ctt ggt tet ttg gct ate 2009 Trp Ser Fen Arg A a Met He Gli Leu Met Leu Gli Ser Leu Ala He 345 350 355 gct gcg att gcg tgg ctg ttg ctg cgt aag aag cgc aca ce act gga 2057 Ala Ala He Ala Trp Leu Leu Arg Lis Lis Arg Tre Pro Tre Gli 360 365 370 375 aag att gct cgt etc ttc ca ate ggc age etc att gee att cea ttc 2105 Lis He Wing Arg Leu Fen Gln He Gli Ser Leu He Wing Pro Fen 380 385 390 cea ttc ttg gct aac tet gctt gggt tgg ttc acc acc gag atecgc cgc 2153 Pro Fen Leu Ala Asn Ser Wing Gli Trp He Fen Tre Glu Met Gli Arg 395 400 405 cag ect tgg gtg gta falls ceg aat ect gaa tet gee ggc gat gee cga 2201 Gln Pro Trp Val Val His Pro Asn Pro Glu Ser Wing Gli Asp Ala Arg 410 415 420 here gag atg ate cgg atg act gtt gat atg ggt gtg tet gat cat gcg 2249 Tre Glu Met He Arg Met Tre Val Asp Met Gli Val Ser Asp His Wing 425 430 435 ceg tgg ca gtc tgg ctg act cta att ggc ttc acg a tt etc tat etc. 2297 Pro Trp Gln Val Trp Leu Tre Leu He Gli Fen Tre He Leu Tir Leu 440 445 450 455 ate ttg ttc gtg gtg tgg gtg tgg ctg att cgc cgc gca gtt ctg ate 2345 He Leu Fen Val Val Trp Val Trp Leu He Arg Arg Ala Leu Leu He 460 465 470 gga cea cea gaa gag ggc gct ce gtg tec gag gca aag act gga ceg 2393 Gli Pro Pro Glu Glu Gli Ala Pro Ser Val Glu Ala Lis Tre Gli Pro 475 480 485 gca acc ceg att ggt tea gat atg ecc atg here ceg ctg cat ttt acc 2441 Wing Tre Pro He Gli Being Asp Met Pro Met Tre Pro Leu Gln Fen Tre 490 495 500 gtg ceg ecc ca ce cea cae gre aaa agg aar aac car gga tet 2483 Val Pro Pro Gln Lys Pro Val His Asn Asn Arg Gly His Ser 505,510,515 taataccttt tggtttattc tcatcgcatt tttgtttgcg ggatactttc tectegaagg 2543 attcgacttc ggtgtcggaa ttttagcgcc gatcctcggt aaagattecg ccgctaaaaa 2603 cgcgatcgtc cgcaccgtcg gccctgtgtg ggacggaaat gaagtgtggc tgatcctggc 2723 tctgccgctg ttcctcgtgc ttgtgtcgtt gatcatgcgc gtggtgggcc ttgaatggcg 2783 caagaaagtc gatgatcctc gtggtctgac gttggcaaaa cgggcca tct ttattggttc 2843 ttggactcca ccgctgatgt ggggattcat cttcgccaat attttcaagc ttgcatgccc 2903 atcaaggcgg atcacaccat cgatgctgca gtggctctgc tgtgcaatgt tcaacgtctt 2963 cgccatcctg ggtgcacttg cattcactgc gctgttcgct cttcatggcc ttgcattcat 3023 ccgcctgaaa actgctggtc gggtgcgcac cgatgcggcg aaggcagctc cagtagtcgc 3083 acttcttgct gcggtgactg gtggaccttt cgtgttgtgg gctgccatcg catacggccg 3143 ttcctggtcc tggatcctcg cagtgctgat catcgcagcg gttctcggtg gagetttege 3203 actgatcaaa gaccgcgatg gattaagctt cctgtccact tccgtcgctg tcatcggtgt 3263 agttgcactg ctgtttagtt ccctattccc caacgtcatg ccaacaacgc ttgccgatgg 3323 cgtgactgga tatttggaac gcctccgcaa gccactacgc attgaccatc ctgacttgga 3383 ccgccactgt gatcgcaccg ctggttgtcc tctaccaagg ctggacctac tgggtgttcc 3443 gcaaacgact tcacgccgag ccagtgtctg cctaaaagtt ggaaaaattg agtactaaat 3503 ctgacgctcc ggctagtcgc cgcacaggcc ccgtcgatcc gcggcttttg cgcctatccc 3563 ctgctacccg ccgttgggtg ataatcgcag gtgttctcac actctcgcga cgcgttgaaa 3623 cagtcgcaat gggcttgctc atcggccaga tggcagcggg catcattgag gt ttcgggaa 3683 gttctttgcc ccgaatggcc ctcatcgcgc tcgccatcac ggtggttgtg cgcggacttc 3743 ttgcgtgggc acaggatcgg ttcggagcgc gcatcgtccc aggtgactgt ggatcttcgg 3803 gagaaaaccc tgcggcacct ggcacaaagc gatccccgca ccatcgatca agccttgtgg 3863 cgcacccgtt tgacctctgg ccttgatggt ttggggcctt acctcaccgg atttttgccg 3923 cactggccgc falls 3936 < 210 > < 211 > 517 < 212 > PRT < 213 > Brevibacterium lactofermentum
< 400 > 2 Val Asp Val Val Asp He Ala Arg Trp Gln Fen Gli He Tre Tre Val
1 5 10 15
Tir His Fen He Fen Val Pro Leu Tre He Gli Leu Pro Pro Leu Val 20 25 30 Wing He Met Gln Tre Fen Trp Gln Val Tre Gli Lis Glu His Trp Tir
40 45 Arg Ala Tre Arg Fen Fen Gli Tre Val Leu Leu He Asn Fen Ala Val
50 55 60 Gli Val Ala Tre Gli He Val Gln Glu Ftn Gln Fen Gli Met Asn Trp
65 70 75 80
Ser Glu Tir Being Arg Fen Val Gli Asp Val Fen Gli Gli Pro Leu Ala 85 90 95
Leu Glu Gli Leu He Fen Fen Fen Leu Glu Ser Val Fen Leu Gli Leu 100 105 110
Trp He Fen Gli Trp Gli Lis He Pro Gli Trp Leu His Tre Ala Ser
115 120 125 He Trp He Val Wing He Wing Wing Tre Asn He Being Wing Tir Fen He He 130 135 140 Val Wing Asn Being Fen Met Gln His Pro Val Gli Wing Glu Tir Asn Pro 145 150 155 160
Glu Tre Gli Arg Wing Glu Leu Tre Asp Fen Trp Wing Leu Leu Tre Asn 165, 170 175
Ser Tre Ala Leu Ala Ala Fen Pro His Ala Val Ala Gli Gli Fen Leu 180 185 190 Tre Ala Gli Tre Fen Val Leu Gli He Ser Gli Trp Trp He He Arg
195 200 205 Ala His Arg Gln Ala Lis Lis Ala Glu Ala Glu He Glu Ser Lis His 210 215 220 Ser Met His Arg Pro Ala Leu Trp Val Gli Trp Trp Tre Tre Val Val 225 230 235 240
Ser Ser Val Ala Leu Fen He Tre Gli Asp Tre Gln Ala Lis Leu Met
245 250 255
Fen Val Gln Bln Pro Met Met Met Wing Being Wing Glu Ser Leu Cis Glu 260 265 270 Tre Wing Tre Asp Pro Asn Fen Being He Leu Tre He Gli Tre His Asn
275 280 285 Asn Cis Asp Tre Val Tre His Leu He Asp Val Pro Fen Val Leu Pro
290 295 300 Fen Leu Ala Glu Gli Lis Fen Tre Gli Val Tre Leu Gln Gli Val Asn
305 310 315 320
Gln Leu Gln Ala Ala Ala Glu Gln Ala Tir Gli Pro Gli Asn Tir Ser 325 330 335
Pro Asn Leu Fen Val Tre Tre Trp Ser Fen Arg Ala Met He Gli Leu 340 345 350 \ Met Leu Gli Ser Leu Ala He Ala Wing He Wing Trp Leu Leu Leu Arg
355 360 365 Lis Lis Arg Tre Pro Tre Gli Lis He Ala Arg Leu Fen Gln He Gli
370 375 380 Ser Leu He Wing Pro Fen Fen Pro Fen Leu Wing Asn Ser Wing Gli Trp
385 390 395 400
He Fen Tre Glu Met Gli Arg Gln Pro Trp Val Val his Pro Asn Pro 405 410 415
Glu Be Wing Gli Asp Wing Arg Tre Glu Met He Arg Met Tre Val Asp 420 425 430
Met Gli Val Ser Asp His Wing Pro Trp Gln Val Trp Leu Tre Leu He
435 440 445 Gli Fen Tre He Leu Tir Leu He He Fen Val Val Trp Val Trp Leu
450 455 460 He Arg Arg Ala Val Leu He Gli Pro Pro Glu Glu Gli Ala Pro Ser
465 470 475 480
Val Glu Ala Lis Tre Gli Pro Ala Tre Pro He Gli Ser Asp Met Pro 485 490 495
Met Tre Pro Leu Gln Fen Tre Val Pro Pro Gln Pro His Val Lis Arg 500 505 510 Asn Asn His Gli Ser 515
< 210 > 3 < 211 > 3936 < 212 > DNA < 213 > Brevibacterium lactofermentum
< 220 > < 221 > CDS < 222 > (2476) .. (3498)
< 400 > 3 ggatcctctc tgttcaaaac agcacctact ctttLactcc cgagttccga cgtcgggtcg 60 acgaaagcct agaagtgacg gaccgagatg aggctgctca gaattttaag tttcacgtcc 120 aagacatcat cgaaactggg ttgtttatcg ccagaaataa tggattctgg caaggaaacc 180 tcgtcgttgg cgaaagatat tcccgacgag atgtctgccg aattctcaat tgggaacgaa 240 acaatgagag cacgatttat ggttacaaag tggacagcta cacatcgacg tgcccaatct 300 ttgtgaccta tcacaaggct gatgatgtat ccgaagtact cgttaccagg atgaactcgt 360 cgatccgaat acccttcatt ggtattcccg cggcaaccga aagatcacgt ctaatgagat 420 caagcccatc gctgcgaatg tgtggatctt catgtttttg tgaagaagga cgatgccgaa 480 ggccttgatt tcttctacct tggtcaagcg catteagaaa acagcaaaca gtcatcgatg 540 cccggaaaca aaggagttgt gcaaccggtg gtcacaatgg atetacagtt cgacacaccc 600 gcctgtttga gtcgaacaaa gtacctgagc acaaatctcg ccgtaacgga gtaaccaccg 660 tcgaaaagca caaccaagcg aaatcttttc gagtttttgg tgacttgtca acaagggggg 720 gtcattgaca agcaaaatca gggaaaggtt gaccacaatc ggggttagcc tttctaaagt 780 gcgggaactt taagctgtga aggaataaac ttcaaegaca acetttaaga agetettatt 840 ggttcttcgt tttgtctc ga taaatacaat cggtttcctg gctcaataag gctgttcctg 900 aagaagagga tcaatctgta ag g accta gcgtggatgt cgttgacatc gcgcggtggc 960 aattcggaat taccaccgtc tatcagttea tttttgtccc actgaccatt ggcttagcac 1020 cgctggtcgc gatcatgcaa acgttttggc aagttaccgg caaagagcac tggtatcggg 1080 ctacgagatt ttttggcact gtgctgctca tcaacttcgc gcaacgggca ggttggtgta 1140 ttgtgcagga gttccagttc ggtatgaact ggtcggaata ttcgcgtttc gtcggtgatg 1200 ttttcggcgg accgctggct ttggaggggc tcatcgcgtt cttccttgag tctgtgttct 1260 taggtctgtg gattttcgga tgggggaaga ttcctggatg gctgcatact gcgtccattt 1320 ggatcgttgc tattgcgacg aatatttctg cctatttcat catcgtggcc aactcgttta 1380 tgcagcatcc ggt9"9" g gct gagtataacc ctgagactgg tcgggcggag cttactgatt 1440 tctgggctct tctcacaaac tccaccgccg tggctgcgtt cccgcatgct gttgccggtg 1500 gttttttaac agetggaact ttcgtcctcg gaattteggg ttggtggatt attcgtgcgc 1560 accgccaggc gaagaaggct gaggcggaaa tcgagtcgaa gcattcaatg cacaggccgg 1620 cgttgtgggt tggttggtgg accacagttg tctcttccgt ggcactgttc atcactggcg 1680 atacacaggc gaagctcatg ttcgtgcagc agccgatgaa gatggcgtcg gcggaatcct 1740 tgtgtgaaac cgccacagat ccaaacttct ccattctgac aattggtacg cacaacaact 1800 gcgatacggt aacccacctg atcgatgttc CGTTs gtgct tecattettg gctgaaggaa 1860 aattcaccgg tgtgactttg cagggtgtaa accagctcca agctgcagcg gagcaagcat 1920 acggtcctgg caactactcc cctaacttgt ttgtcaccta ctggtcattc cgcgcaatga 1980 tcggcctaat gcttggttct ttggctatcg ctgcgattgc gtggctgttg ctgcgtaaga 2040 agcgcacacc aactggaaag attcgtcgtc tcttccaaat cggcagcctc attgccattc 2100 cattcccatt cttggctaac tctgctggtt ggatcttcac cgagatgggc cgccagcctt 2160 gggtggtaca cccgaatcct gaatctgccg gcgatgcccg aacagagatg atccggatga 2220 ctgttgatat gggtgtgtct gatcatgege cgtggcaagt ctggctgact ctaattggct 2280 tcacgattct ctatctcatc ttgttcgtgg tgtgggtgtg gctgattcgc cgcgcagttc 2340 tgatcggacc accagaggcc ggcgct ccat ccgtggaggc aaagactgga ccggcaaccc 2400 cgattggttc agatatgece atgacaccgc tgcaatttac cgtgccgccc caaccacacg 2460 tgaaaaggaa taacc atg gat ctt aat acc ttt tgg ttt tt att att gca 2511 Met Asp Leu Asn Tre Fen Trp Fen He Leu He Wing 1 5 10 ttt ttg ttt gcg gga tac ttt etc etc gaga gga ttc gac ttc ggt gtc 2559 Fen Leu Fen Wing Gli Tir Fen Leu Leu Glu Gli Fen Asp Fen Gli Val 15 20 25 gga att tta gcg ceg ate ate ggt aaa gat tec gee gct aaa aac aac acg 2607 Gli He Leu Ala Pro He He Gli Lis Asp Ser Ala Ala Lis Asn Tre 30 35 40 ate ate cgc acc ate ggc ect gtc tgg gac gga aat gaa gtg tgg ctg 2655 He He Arg Tre He Gli Pro Val Trp Asp Gli Asn Glu Val Trp Leu 45 50 55 60 ate gtg gca ggt ggt ggc gct ttg ttt gct gtc tct ect gag tgg tac gca 2703 He Val Wing Gli Gli Ala Leu Fen Wing Wing Pro Pro Glu Trp Tir Wing 65 70 75 acg ate ttc tec gga atg tat ctg ceg ctg ttc etc gtg ctt gtg teg 2751 Tre Met Fen Ser Gli Met Tir Leu Pro Leu Fen Leu Val Leu Val Ser 80 85 90 ttg ate atg cgc gtg gtg ggc ctt gaa tgg cgc aag aaa gtc gat gat 2799 Leu He Met Arg Val Val Gli Leu Glu Ti Arg Lis Lis Val Asp Asp 95 100 105 ect cgt tgg cag agg tgg tet gac cgg gee ate ttt att ggt tet tgg 2847 Pro Arg Trp Gln Lis Trp Ser Asp Arg Ala He Fen He Gli Ser Trp
110 115 120 act cea ceg atg tgg gga ttc ate ttc gee aat att ttc aag ctt 2895 Tre Pro Pro Leu Met Trp Gli Fen He Fen Ala Asn He Fen Lis Leu 125 130 135 140 gca tgc cea tea agg cgg ate ac cea teg atg ctg cag tgg etc tgc 2943 Wing Cis Pro Ser Arg Arg He Fre Pro Ser Met Leu Gln Trp Leu Cis 145 150 155 tgt gca atg ttc aac gtc ttc gee ate ctg ggt gca ctt gca tcc act 2991 Cis Ala Met Fen Asn Val Fen Ala He Leu Gli Ala Leu Ala Fen Tre 160 165 170 gcg ctg ttc gct ctt cat ggc ctt gca ttc ate cgc ctg aaa act gct 3039 Ala Leu Fen Ala Leu His Gli Leu Ala Fen He Arg Leu Lis Tre Ala 175 180 185 ggt cgg gtg cgc acc gat gcg gcg aag gca gct cea gta gtc gca ctt 3087 Gli Arg Val Arg Tre Asp Ala Ala Ala Ala Ala Pro Val Val Ala Leu
190 195 200 ctt gct gcg gtg act ggt gga ect ttc gtg ttg tgg gct gee ate gca 3135 Leu Ala Wing Val Tre Gli Gli Pro Fen Val Leu Trp Wing Ala He Wing 205. 210 215 220 tac ggc cgt tec tgg tec tgg ate etc gca gtg ctg ate ate gca gcg 3183 Tir Gli Arg Ser Trp Ser Trp He Leu Ala Val Leu He He Ala Ala 225 225 235 gtt etc ggt gga gct tcc gca ctg ate aaa gac cgc gat gga tta age 3231 Val Leu Gli Gli Ala Fen Ala Leu He Lis Asp Arg Asp Gli Leu Ser 240 245 250 ttc ctg tec act tec gtc gct gtc ate ggt gta gtt gca ctg ctg ttt 3279 Fen Leu Ser Tre Ser Val Val Val He G] i Val Val Ala Leu Leu Fen 255 260 265 agt tec cta ttc ecc aac gtc atg cea acg ctt gcc gat ggc gtg 3327 Ser Ser Leu Fen Pro Asn Val Met Pro Tre Tre Leu Ala Asp Gli Val
270 275 280 act gga tat ttg gcc cgc etc cgc aag cea cta cgc att gac cat ect 3375 Tre Gli Tir Leu Glu Arg Leu Arg Lis Pro Leu Arg He Asp His Pro 285 290 295 300 gac ttg gac cgc falls tgt gat cgc acc gct ggt tgt ect cta cea agg Asp Leu Asp Arg His Cis Asp Arg Tre Wing Gli Cis Pro Leu Pro Arg 305 310 315 ctg gac cta ggt gtt ceg caa acg act tea cgc cga gcc agt gtc 3471 Leu Asp Leu Leu Gli Val Pro Gln Tre Tre Ser Arg Arg Ala Ser Val 320 325 330 tgc cta aaa gtt gga aaa att gag tac taaatctgac gctccggcta 3518
Cis Leu Lys Val Gly Lys He Glu Tyr 335 340 gtcgccgcac aggccccgtc gatccgcggc ttttgcgcct atcccctgct acccgccgtt 3578 gggtgataat egeaggtgtt ctcaggtgtt ctgaccgcgt cgcgacagtc gcaatgggct 3638 ccagatggca tgctcatcgg gcgggcatca ttgaggtttc gggaagttct ttgccccgaa 3698 tggaactcat cgcgctcgcc atcacggtgg ttgtgcgcgg acttcttgcg tgggcacagg 3758 atcggttcgg agcgcgcatc gtcccaggtg actgtggatc ttcgggagaa aaccctgcgg 3818 cacctggcac aaagcgatcc ccgcaccatc gatcaagect tgtggcgcac ccgtttgacc 3878 tctggccttg atggtttggg gccttacctc accggattt tgccgcactg gccgccac 3936
< 210 > 4 < 211 > 341 < 212 > PRT < 13 > Brevibacterium lactofermentum
< 400 > 4 Met Asp Leu Asn Tre Fen Trp Fen He Leu He Wing Fen Leu Fen Wing 1 5 10 15
Gli Tir Fen Leu Leu Glu Gli Fen Asp Fen Gli Val Gli He Leu Ala
25 30 Pro He He Gli Lis Asp Be Ala Ala Lis Asn Tre He He Arg Tre 35 40 45 He Gli Pro Val Trp Asp Gli Asn Glu Val Trp Leu He Val Ala Gli
50 55 60 Gli Ala Leu Fen Ala Ala Fen Pro Glu Trp Tir Ala Tre Met Fen Ser 65 70 75 80 Gli Met Tir Leu Pro Leu Fen Leu Val Leu Val Ser Leu He Met Arg 85 90 95
Val Val Gli Leu Glu Trp Arg Lis Lis V? L Asp Aso Pro Arg Trp Gln
100 105 110 Lis Trp Ser Asp Arg Wing He Fen He Gli Ser Trp Tre Pro Pro Leu 115 120 125 Met Trp Gli Fen He Fen Wing Asn He Fen Lis Leu Wing Cis Pro Ser
130 135 140 Arg Arg He Tre Pro Ser Met Leu Gln Trp Leu Cis Cis Ala Met Fen 145 150 155 160 Asn Val Fen Wing He Leu Gli Wing Leu Wing Fen Tre Wing Leu Fen Wing 165 170 175
Leu His Gli Leu Ala Fen He Arg Leu Lis Tre Ala Gli Arg Val Arg
180 185 190 Tre Asp Ala Ala Ala Ala Ala Pro Val Val Ala Ala Leu Ala Ala Ala 195 200 205 Tre Gli Gli Pro Fen Val Leu Trp Ala Ala Ala Ala Tir Gli Arg Ser
210 215 220 Trp Ser Trp He Leu Wing Val Leu He He Wing Wing Val Leu Gli Gli 225 230 235 240 Wing Wing Ala Leu He Lis Asp Arg Asp Gli Leu Ser Fen Leu Ser Tre 245 250 255
Ser Val Ala Val He Gli Val Val Ala Leu Leu Fen Ser Ser Leu Fen
260 265 270 Pro Asn Val Met Pro Tre Tre Leu Ala Asp Gli Val Tre Gli Tir Leu 275 280 285 Glu Arg Leu Arg Lis Pro Leu Arg He Asp His Pro Asp Leu Asp Arg
290 295 300 His Cis Asp Arg Tre Ala Gli Cis Pro Leu Pro Arg Leu Asp Leu Leu 305 310 315 320 Gli Val Pro Gln Tre Tre Ser Arg Arg Ala Ser Val Cis Leu Lis Val 325 330 335
Gli Lis He Glu Tir 340 < 210 > 5 < 211 > 19 < 212 > PRT < 213 > Brevibacterium lactofermentum
< 220 > < 221 > UNSURE < 222 > (18)
< 400 > 5 Met Asp Val Val Asp He Wing Arg Trp Gln Fen Gli He Tre Wing Val
Tir Xaa Fen
< 210 > 6 < 211 > 20 < 212 > PRT < 213 > Brevibacterium lactofermentum
< 220 > < 221 > UNSURE < 222 > (16, 17)
< 400 > 6 Met Asp Leu Asn Tre Fen Trp Fen He Leu He Wing Fen Leu Fen Xaa l 5 10 15 Xaa Tir Fen Leu 20
< 210 > 7 < 211 > 20 < 212 > DNA < 213 > Artificial sequence
< 220 > < 223 > Description of the artificial sequence: promoter for PCR
< 220 > < 221 > misc_feature < 222 > (9, 12) < 223 > n = a or c or g or t
< 400 > 7 atggaygtng tngayatygc 20 < 210 > 8 < 211 > 20 < 212 > DNA < 213 > Artificial sequence
< 220 > < 223 > Description of the artificial sequence: promoter for RCP < 400 > 8 caraargtrt tvarrtccat 20
< 210 > 9 < 211 > 24 < 212 > adn < 213 > Artificial sequence
< 220 > < 223 > Description of the artificial sequence: promoter for PCR
< 400 > 9 cgccagggtt ttcccagtca cgac 24
< 210 > 10 < 211 > 24 < 212 > aDN < 213 > Artificial sequence
< 220 > < 223 > Description of the artificial sequence: promoter for PCR
< 400 > 10 gageggataa caattteaca cagg 24
Claims (9)
1.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10-164019 | 1998-06-11 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| MXPA99005412A true MXPA99005412A (en) | 2000-08-01 |
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