Abstract:
This invention provides a mutant Sphingomonas microorganism which produces a polysaccharide polymer comprising repeating tetramer units having a D-glucose:D-glucuronic acid:L-rhamnose ratio of about 2:1:1, wherein the D-glucose moieties are linked in a β-[1,4] configuration to the D-glucuronic acid moiety. One of the D-glucose moieties is linked to the L-rhamnose moiety in an α-[1,3] configuration, the other glucose moiety is linked to the L-rhamnose moiety in a β-[1,4] configuration, wherein the polysaccharide polymer is substantially non-acetylated. This invention also provides the substantially non-acetylated polymer, as well as process for obtaining the substantially non-acetylated polymer.

Description:
[0001]    This application claims the benefit of U.S. Provisional Application No. 60/302,787, filed on Jul. 3, 2001. 
     
    
     
       BACKGROUND OF THE INVENTION  
         [0002]    1. Field of the Invention  
           [0003]    The present invention relates to a mutant strain of  Sphingomonas elodea  which produces non-acetylated gellan gum. The invention also relates to non-acetylated gellan gum.  
           [0004]    2. Discussion of the Related Art  
           [0005]    Polysaccharides or gums are primarily used to thicken or gel water and are frequently classified into two groups: thickeners and gelling agents. Typical thickeners include starches, xanthan gum, guar gum, carboxymethylcellulose, alginate, methylcellulose, gum karaya and gum tragacanth. Common gelling agents include gelatin, gellan gum, starch, alginate, pectin, carrageenan, agar and methylcellulose.  
           [0006]    Gelling agents are used by the food industry in a variety of applications, including confectionery jellies, jams and jellies, dessert gels, icings and dairy products. Additionally, gelling agents may be used as components of microbiological media. Gelling agents differ in the conditions under which they can be used as well as in the texture of the gels they form. These distinctive properties of gels have led to the exclusive use of certain gelling agents in a number of products (e.g., starch in confectionery jellies; gelatin in capsules; agar in icings; and alginate in pimento strips).  
           [0007]    Gellan gum (S-60) is produced by the microorganism  Sphingomonas elodea  (ATCC 31461) as disclosed in U.S. Pat. Nos. 4,377,636, 4,326,053, 4,326,052 and 4,385,123, the contents of which herein are incorporated by reference. Commercially, the gum is formed by inoculating a carefully formulated fermentation medium with a Sphingomonas organism. The fermentation medium contains a carbon source, phosphate, organic and inorganic nitrogen sources, and appropriate trace elements. The fermentation is carried out under sterile conditions with strict control of aeration, agitation, temperature and pH. When the fermentation is complete, the viscous broth is pasteurized to kill viable cells prior to recovery of the gum.  
           [0008]    The gum can be recovered in several ways. Direct recovery from the broth yields the gum in its native or high acyl form. Recovery after deacylation by treatment with alkali provides the gum in its low acyl form. The acyl groups have a profound influence on gel characteristics. The gel characteristics of gellan gum have been altered by reducing the level of acyl group substitutions by chemical deacylation. The glyceryl group is believed to have a greater effect on gel propoerties than the acetyl group. Baird et al. (1992)  Gellan Gum: Effect of Composition On Gel Properties,  Phillips et al. (Eds.),  Gums and Stabilizers for the Food Industry  6 (pp. 479-487), Oxford, Permagon Press.  
           [0009]    The constituent sugars of gellan gum are D-glucose, D-glucuronic acid and L-rhamnose in the molar ratio of 2:1:1, which are linked together to give a primary structure consisting of a linear tetrasaccharide repeat unit in the following order: D-glucose:D-glucuronic acid:D-glucose:L-rhamnose. Jannson et al. (1983)  Carbohydr. Res.  124:135-139; O&#39;Neill et al. (1983)  Carbohydr. Res.  124:123-133. In the native or high acyl form of gellan gum, two acyl substituents, acetate and glycerate, are present. Both substituents are located on the same glucose residue, and on average, there is one glycerate per repeat unit and one acetate for every two repeat units as shown below. Kuo et al. (1986)  Carbohydr. Res.  156:173-187.  
                         
 
           [0010]    Sphingans are polysaccharides produced by bacteria of the genus Sphingomonas. U.S. Pat. No. 5,854,034 discloses a method for increasing production of sphingans in various strains of Sphingomonas. The disclosed method involves isolating sequences of DNA as segments from sphingan-producing bacteria, cloning the isolated segments, and incorporating multiple copies of the cloned segments into sphingan-producing or non-producing mutants of Sphingomonas bacteria. The patent does not disclose modification of acyl group substitution of gellan gum.  
           [0011]    Chemical mutagenesis of gellan gum producing  Sphingomonas paucimobilis  bacteria has been reported. Jay et al. (1988)  Carbohydr. Polymers  35:179-188. Several mutant strains of  Sphingomonas paucimobilis,  which produce non-acetylated, non-glycerylated and non-acylated gellan gum, respectively, reportedly were obtained. The article does not describe isolation or identification of the genes responsible for acetylation and glyceration of gellan gum in Sphingomonas bacteria. Moreover, experiments conducted by the inventors suggest that the mutant  Sphingomonas paucimobilis  obtained in Jay, et al. do not produce fully non-acetylated or non-glycerylated gellan gum.  
           [0012]    To date, the predominant method utilized for gellan gum deacylation has been by hydrolysis under alkaline conditions. However, it has been found that chemical processes for deacylating gellan gum may result in a number of undesirable side effects that may cause hydrolysis of the polymer backbone, resulting in an irreversible change in the conformation of the molecule and lower molecular weight.  
           [0013]    Modification of gellan gum has been described previously. For example, Baird et al., described methods for preparing chemically deacetylated gellan gum as well as chemically deacylated gellan gum. Baird et al. (1992)  Gellan Gum: Effect of Composition On Gel Properties,  Phillips et al. (Eds.)  Gums and Stabilizers for the Food Industry  6 (pp. 479-487), Oxford, Permagon Press. Chemical deacylation of gellan gum produced by  Sphingomonas elodea  also is described in Kuo et al. (1986)  Carbohydr. Res.  156:173-187.  
           [0014]    It would be highly desirable to avoid chemical deacylation of gellan gum by obtaining and using mutant strains of  Sphingomonas elodea  microorganisms to produce non-acylated or non-acetylated gellan gum.  
         SUMMARY OF THE INVENTION  
         [0015]    The present invention provides a mutant Sphingomonas microorganism which produces a polysaccharide polymer comprising repeating tetramer units having a D-glucose:D-glucuronic acid:L-rhamnose ratio of about 2:1:1, wherein the D-glucose moieties are linked in a β-[1,4] configuration to the D-glucuronic acid moiety. One of the D-glucose moieties is linked to the L-rhamnose moiety in an α-[1,3] configuration, the other glucose moiety is linked to the L-rhamnose moiety in a β-[1,4] configuration, wherein the polysaccharide polymer is substantially non-acetylated.  
           [0016]    The present invention also provides a polysaccharide polymer comprising repeating tetramer units having a D-glucose:D-glucuronic acid:L-rhamnose ratio of about 2:1:1, wherein the D-glucose moieties are linked in a β-[1,4] configuration to the D-glucuronic acid moiety. One of the glucose moieties is linked to the L-rhamnose moiety in an α-[1,3] configuration, the other glucose moiety is linked to the L-rhamnose moiety in a β-[1,4] configuration, wherein the polysaccharide polymer is substantially non-acetylated.  
           [0017]    The present invention further provides a process for preparing a polysaccharide polymer comprising repeating tetramer units having a D-glucose:D-glucuronic acid, L-rhamnose ratio of about 2:1:1, wherein the D-glucose moieties are linked in a β-[1,4] configuration to the D-glucuronic acid moiety. One of the glucose moieties is linked to the L-rhamnose moiety in an α-[1,3] configuration, the other glucose moiety is linked to the L-rhamnose moiety in a β-[1,4] configuration, wherein the polysaccharide polymer is substantially non-acetylated. The process involves: (a) obtaining a mutant Sphingomonas which produces the substantially non-acetylated polysaccharide polymer; and (b) culturing the mutant Sphingomonas under conditions effective to produce the substantially non-acetylated polysaccharide polymer.  
           [0018]    This invention is also directed to a DNA sequence which expresses the acetyl transferase gene of Sphingomonas and compositions comprising a  Sphingomonas elodea  polysaccharide polymer with substantially reduced levels of acetate.  
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0019]    [0019]FIG. 1 is a genetic and physical map of the DNA region of  Sphingomonas elodea  encoding acetyl transferase.  
         [0020]    [0020]FIG. 2 shows the complete DNA sequence for the acetyl transferase gene. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0021]    As used herein, the term “non-acetylated” refers to a non-native form of a polysaccharide which differs from its native form in that it is produced with a substantially reduced level of acetyl substitution. Thus, non-acetylated gellan gum differs from native gellan gum in that native gellan gum has, on average, about one acetyl group substituent every 2 repeat units, whereas the non-acetylated gellan gum of this invention is produced with substantially no acetyl substitution and no effect on glyceryl substitution. By introducing a null mutation in the acetyl transferase gene, the non-acetylated gellan gum of this invention substantially lacks acetyl groups. As used herein, the term “substantially reduced levels of acetyl substitution” refers to a polysaccharide containing acetyl content reduced by about 85%, preferably reduced by about 90%, and most preferably reduced by about 95%.  
         [0022]    The non-acetylated gellan gum of this invention also differs from de-acetylated gellan gum in that de-acetylated gellan gum is produced by removing the acetyl substituent from native gellan gum, whereas non-acetylated gellan gum is produced such that the acetyl substituent is never added because the acetyl transferase gene is inactivated.  
         [0023]    As used herein, the term “acetyl transferase deficient” refers to a non-native form of a strain of bacteria which differs from its native form in that it lacks a functional acetyl transferase gene. Thus, an acetyl transferase deficient strain of  Sphingomonas elodea  differs from naturally occurring  Sphingomonas elodea  in that naturally occurring  Sphingomonas elodea  has a functional acetyl transferase gene, whereas an acetyl transferase deficient  Sphingomonas elodea  does not have a functional acetyl transferase gene. A naturally occurring strain of Sphingomonas may be rendered “acetyl transferase deficient” by disabling, inactivating, removing or partially removing the acetyl transferase gene.  
         [0024]    Significantly, the DNA sequence of  Sphingomonas elodea  that encodes acetyl transferase production has been identified. Acetyl transferase is responsible for acetylation of gellan gum. Accordingly, acetylation of the gellan gum in  Sphingomonas elodea  may be eliminated through mutations of the acetyl transferase gene using methods well known to those skilled in the art, such as, for example, point mutations, transposon mutagenesis, deletions, insertions, and the like. These procedures may be used to obtain the mutant strains of  Sphingomonas elodea  of this invention, which produce non-acetylated gellan gum.  
         [0025]    The mutant  Sphingomonas elodea  of this invention produce non-acetylated gellan gum. The mutant  Sphingomonas elodea  of this invention can be grown under conditions generally known in the art for growth of wild type  Sphingomonas elodea.  For example, the mutants of this invention can be grown on suitable assimilable carbon sources, such as glucose, sucrose, maltose, starch, complex carbohydrates, such as molasses or corn syrup, various organic acids and the like. Mixtures of carbon sources may also be employed. The concentration of carbon sources supplied is often between 10 and 60 grams per liter (g/l). An assimilable source of organic or inorganic nitrogen also is necessary for growth, and is generally between about 0.1 and 10.0 g/l. Examples of suitable nitrogen sources are ammonia, ammonium salts, nitrate, urea, yeast extract, peptone or other hydrolyzed proteinaceous materials or mixtures thereof. Minerals also are necessary for growth. Examples of suitable minerals include phosphorus, sulfur, potassium, sodium, iron and magnesium.  
         [0026]    Optimal temperatures for growth of the mutant  Sphingomonas elodea  of this invention generally are between about 25° C. and about 37° C., preferably between about 30° C. and about 36° C. The mutant  Sphingomonas elodea  cells may be grown aerobically by supplying sufficient air or oxygen so that an adequate level of dissolved oxygen is maintained. The pH generally is maintained at about 6 to about 8 and, preferably at about 6.5 to about 7.5.  
         [0027]    The non-acetylated polysaccharides of the present invention may be recovered from fermentation broths by any suitable means. Such methods are known to those skilled in the art. For example, precipitation with isopropanol, ethanol or other suitable alcohol readily yields the substantially non-acetylated polysaccharides of this invention. Alternatively, the polymers may be recovered from the fermentation broth by ultra-filtration.  
         [0028]    The non-acetylated polysaccharides of the present invention may be used as gelling agents in a variety of fluid food products including confectionery jellies, jams and jellies, dessert gels, icings and dairy products, such as, for example, ice cream, frozen yogurt, cottage cheese, sour cream, non-dairy frozen toppings and bakery fillings.  
         [0029]    The present invention also provides compositions comprising a substantially non-acetylated gellan gum, water, a gelling salt and a sequestrant. The concentration of gelling salt in the compositions will vary depending upon the particular gelling salt used. For example, sodium and potassium gelling salts generally are used at concentrations ranging from about 0.020M to about 0.200M, while calcium and magnesium gelling salts typically are used at concentrations ranging from about 0.002M to about 0.015M. The amount of sequestrant used in the compositions typically ranges from about 0.05 percent to about 0.25 percent by weight.  
         [0030]    When fully hydrated, the non-acetylated gellan gums of the present invention will form gels with many different ions. Preferably, the gelling salt is a calcium salt, a sodium salt or a potassium salt. Most preferably, the gelling salt is CaCl 2 . Sodium citrate is the preferred sequestrant.  
         [0031]    Yet another embodiment of this invention is directed to the DNA sequence encoding acetyl transferase of  Sphingomonas elodea . The DNA sequence of this invention may be isolated from Sphingomonas strains using methods that are well known to those skilled in the art. Typically, the bacteria are cultured to produce a fermentation broth. The bacterial cells are centrifuged and suspended for DNA extraction. The DNA extraction process generally involves removing the proteins from the fermentation broths and then precipitating the DNA using a solvent such as, for example, isopropanol. The precipitated DNA fragments may then be treated with a restriction enzyme, for example, EcoRI or PstI, to produce smaller DNA fragments, which then may be cloned into appropriate vectors according to conventional methods that are well known to those skilled in the art.  
         [0032]    The examples which follow are intended to illustrate some of the preferred embodiments of the present invention, and no limitation is implied.  
       EXAMPLE 1  
     Preparation of a Low Acetyl Mutant of  Sphingomonas elodea    
       [0033]    LAM-1 is a Low Acetyl Mutant of  Sphingomonas elodea  produced by chemical mutagenesis which blocks acetylation of gellan gum. LAM-1 produces gellan gum which is deficient in acetyl group substitution.  
         [0034]    LAM-1 was produced by chemical mutagenesis of  Sphingomonas elodea  strain S-60wtc (S-60wtc is a derivative of strain  S. elodea,  ATCC 31461, which was selected as a spontaneous isolate with increased ability to take up plasmid DNA) under the following conditions:  
                                                       Buffer   TRIS           pH   8           EMS   15 μl/ml           Time   30 min.           Temp   30° C.                      
 
         [0035]    Several colonies from fresh plates were resuspended in 5.0 ml sterile deionized water. The suspension was shaken vigorously, centrifuged, and then suspended in 10 ml of buffer. Ethyl Methane Sulfonate (15 μl/ml) was added and the suspension was incubated on a roller drum for 30 minutes at 30° C. After incubation, the mutated cultures were centrifuged, washed once in buffer and resuspended. Aliquots were dispensed to YM flasks for expression. LAM-1 was isolated from the mutated cultures using screening procedures well known by those skilled in the art. The amount of O-acyl substitution was determined by a calorimetric assay described in McComb et al. (1957)  Anal. Chem.  29:819-821. A low acyl mutant (LAM-1) was fermented in 100 ml salts medium in 500 ml shake flasks and gellan gum recovered. Neutral sugars and organic acid content were determined by HPLC analysis of trifluoroacetic acid hydrolysates. Results show that the mutant is deficient in addition of acetyl to gellan, while the glyceryl level is comparable to that of the control (see Exp. 1 below in Table 1). A subsequent experiment (Exp. 2 in Table 1) showed that the low level of acetyl was similar to that of a chemically deacylated purified gellan gum sample available under the tradename KELCOGEL (CP Kelco, San Diego, Calif.).  
                                                                                                                                                                           TABLE 1                       LAM-1 Acyl Analysis.                                        Percent   Percent   Percent   Percent           Strain   Rhamnose   Glucose   Glycerate   Acetate                        Exp. 1                S-60wtc   13   21   3.4   2.8           LAM-1   13   21   4.0   0.4                        Exp. 2                S-60wtc   12   27   7.9   2.9           LAM-1   12   26   8.7   0.2           KELCOGEL   18   28   0.3   0.2                                Glycerate per   Acetate per           Strain   Repeat Unit   Repeat Unit                        Exp. 1                S-60wtc   0.4   0.6           LAM-1   0.5   0.1                        Exp. 2                S-60wtc   0.80   0.51           LAM-1   0.84   0.03           KELCOGEL   0.02   0.02                      
 
       EXAMPLE 2  
       
       [0036]    Identification and Inactivation of the Gene for Acetyl Transferase  
         [0037]    This example demonstrates that there is a specific gene encoding the protein that catalyzes acetylation of gellan gum.  
         [0038]    The gene for acetyl transferase was identified by complementation of the LAM-1 mutant. A gene library of  Sphingomonas elodea  DNA was constructed by ligating a partial PstI digest of genomic DNA into the PstI site of pLAFR3, a broad host range cosmid vector, conferring tetracycline resistance. Staskawicz et al. (1987)  J. Bacteriol.  169:5789-94. This ligation mixture was transformed into  E. coli  strain DH5αMCR (Life Technologies Gibco BRL, Rockville, Md.). The library was then transferred into LAM-1 by triparental conjugal mating. The vector used for gene library construction was mobilizable but not self-transmissible. Transfer functions were provided by a second plasmid pRK2013 in  E. coli  strain JZ279. Ditta et al. (1980)  Proc. Natl. Acad. Sci. USA  77:77347-7351. Strains were grown overnight in selective media: S-60 gene library in  E. coli  in 5 ml LB medium with tetracycline (10 μg/ml); JZ279/pRK2013 in 5 ml LB medium with kanamycin (50 μg/ml); and LAM-1 in 15 ml YEME medium. LB media contains 10 g/l tryptone, 5 g/l yeast extract and 10 g/l NaCl; YEME media contains 2.5 g/l yeast extract and 0.25 g/l malt extract.  E. coli  strains were concentrated two-fold and LAM-1 ten-fold. Then, 1 ml of each strain was mixed and collected on a sterile filter membrane. This membrane was transferred to a LB plate and incubated for 7 hours at 36° C. Cells were then scraped off the filter and stored in distilled water and glycerol. This culture yielded about 10 6  cells/ml when plated on selective medium: YM medium with streptomycin (25 μg/ml, to counterselect  E. coli ) and tetracycline (5 μg/ml to select for plasmid containing strains). YM medium contains 3 g/l yeast extract, 3 g/l malt extract, 5 g/l peptone and 10 g/l glucose.  
         [0039]    The LAM-1 plasmid-containing strains were then tested for acetyl composition. Each isolate was run through a three stage fermentation protocol. A colony of each test strain was inoculated into 1 ml of YM media in a 24 well Costar dish and incubated overnight at 30° C. with shaking at 250 rpm. Then 50 μl of each culture was transferred to 1 ml of salts media in a Costar dish and incubated for about 24 hours with shaking at 250 rpm at 30° C. A 0.1 ml aliquot of these cultures was used to inoculate 2.5 ml of salts media in four dram shell vials, containing ceramic balls to facilitate mixing. These were shaken at 350 rpm for about 72 hours at 36° C. The fermentation broth was hydrolyzed with 2ml of 1M trifluoroacetic acid at 90° C. for about 16 hours. A 1 ml aliquot of hydrolyzed broth was mixed with 4.5 ml of 0.137 mg/ml propionic acid (internal standard). Acyl composition was determined by high performance ion-exclusion chromatography with chemically suppressed conductivity detection, using a Dionex BioLC system. (Dionex). Salts media contains 0.229 g/l NaCl, 0.165 g/l CaCl 2 .2H 2 O, 2.8 g/l K 2 HPO 4 , 1.2 g/l KH 2 PO 4 , 1.9 g/l NaNO 3 , 1.0 g/l NZAmine (EKC), 36.46 g/l Star Dri corn syrup, 2.5 mg/l FeSO 4 .7H 2 O, 24 μg/l Co 2 Cl.6H 2 O, and 0.101 g/l MgSO 4 .7H 2 O.  
         [0040]    From a screen of 1398 plasmid-containing strains, four plasmids were obtained that restored acetyl substitution to gellan gum. These plasmids had 11 kb of DNA in common, as shown in FIG. 1. The sequence of this region was determined. On the 2.2 kb and 5.2 kb BamHI fragments, a gene was located which had homology to other known acetyl transferases. The gene sequence (SEQ ID NO: 1) and the protein sequence (SEQ ID NO: 2) of acetyl transferase are shown in FIG. 2.  
         [0041]    The putative acetyl transferase gene was inactivated. Primers were designed to amplify an internal portion of the putative acetyl transferase gene. Nucleotides encoding restriction sites XbaI and SacI (underlined) were added to the ends of the PCR primers:  
                                       p43Actr5′→TTG  GAG  CTC  TCT GGA CCT ATC TGC T                           p44Actr3′→GTT  TCT  AGA  CTT CAG GAG CCG ACT G          
 
         [0042]    Primers P43Actr5′ (SEQ ID NO: 3) and P44Actr3′ (SEQ ID NO: 4), plasmid pRC311 as a template, and Ampli Taq DNA polymerase were employed in a PCR reaction to amplify the 377 base pair internal fragment of the putative acetyl transferase gene. Thirty-five PCR cycles consisting of denaturation at 96° C., annealing at 68° C. and extension at 72° C. were used to amplify the expected DNA sequence. The PCR product was digested with XbaI and SacI and ligated into similarly digested plasmid pLO2. This plasmid confers kanamycin resistance, has a site for mobilization and can replicate in  E. coli  but not  S. elodea.  Lenz et al.(1994)  J. Bacteriol.  176:4385-4393.  
         [0043]    The plasmid was transferred to  S. elodea  by conjugation using triparental matings. Since the plasmid cannot replicate in  S. elodea,  selection for kanamycin (7.5 μg/ml) resistance selects for those colonies in which the plasmid has integrated into the homologous region of the chromosome. This results in insertion of the plasmid into the putative acetyl transferase gene. Kanamycin resistant colonies were selected, purified and tested in fermentation. Analysis of the composition of gellan gum by HPLC assay of fermentation broth samples after hydrolysis with trifluoroacetic acid showed that acetyl substitution was substantially reduced, thus confirming that this is the gene that controls acetylation of the gellan polysaccharide.  
                                 TABLE 2                           Gellan Broth O-Acyl Analysis.                strain   % glycerate   % acetate                       S-60wtc   5.7   3.8           S-60wtc   5.8   3.9           LAM-1   6.6   0.3           LAM-1   6.1   0.2           S-60wtc::pLO2AT-1   5.5   0.2           S-60wtc::pLO2AT-1   5.9   0.4                      
 
         [0044]    LAM-1 and S-60wtc::pL02AT-1 were placed on deposit with the American Type Culture Collection under Accession Nos. PTA-4386 and PTA-4387.  
         [0045]    Other variations and modifications of this invention will be obvious to those skilled in the art. This invention is not limited except as set forth in the claims.  
     
       
       
         1 
         
           
             4  
           
           
             1  
             1245  
             DNA  
             Sphingomonas elodea  
           
            1 

atggaaccgg agaccatcct catgtcggac accaccgcag tcgaccgatc tccggtaaag     60 

tcaggcctac gtttttcggc cctagacagc ctgcgcggca tctgcgcatg catgatcgtt    120 

ctgttccacc ttcgctccac tggcgtcgtc acgaactcgc atctggtccg aaacagctgg    180 

atgttcgtcg acttcttttt cgtcctcagc ggcttcgtca tcgcgtgcgg ctatctggag    240 

cgattgcggg agggctattc cgtgcggcag ttcatgctgc tgcgcctggg ccgggtctat    300 

ccgttgcacc tggccgtcct cctcctgttc gtggtgatcg agctagcggg ggccatgctc    360 

ggtaccgccg ggctcagcgc ccgcgccgcc ttttcggagc cgcgaacccc tgcggagctc    420 

gccggcacgc tcgcgctggt ccagatcttc tgcggcttcc cctcgatcgt ctggaacggc    480 

ccgagctgga gcatcgctgc ggaggtctgg acctatctgc tggtcgcgct cgtcgtgcgc    540 

gcgctgcccg ggcgaaccgc atgggctgcc acgggtctgg cgcttgccgc cttcgccacg    600 

ctcgcgctcg ccggtgcggc cgcctgggac ccggcgacgg gctttgcctt tgtccgctgc    660 

gtcctgggct tctcggtggg cgtgctgtgc tggatcctgt tctcggcaat ggggcggccg    720 

aggatgggaa ccgcgatcgc aacgatcttg gagctggtgg cggtcgcatc gtgctgcgcg    780 

ctggtggctt cgggaagcct gccgctggcg gcgccgatcg tgttcgccgg cacggtgctg    840 

ttgttcgcgg ccgagcaggg catggtcagt cggctcctga agctcgcgcc cttcctcgcg    900 

ctcggcaccc tctcctactc gatctacatg gtgcacacgc tggtgatcgc acgcagtctg    960 

gacgtgctct cactcgcggg caggctgttg catcacccgc tggtggagac acggctcggc   1020 

agcggtggta cgatcaaggt gctggtgttc gcgccggacg caatggcgtt cgcggtgctc   1080 

ggcggcatcg tgctgtgttc ggcgctcacc tatcgctgga tcgaggcgcc cgcgcgggac   1140 

ctgtcgcgcg cactggtccg ccagagcggg cggcgcggca gcttggcggc cgccccggac   1200 

gcagcacgcg accccgaggc cctgccggca accgccacga gctga                   1245 

 
           
             2  
             414  
             PRT  
             Sphingomonas elodea  
           
            2 

Met Glu Pro Glu Thr Ile Leu Met Ser Asp Thr Thr Ala Val Asp Arg 
1               5                   10                  15 

Ser Pro Val Lys Ser Gly Leu Arg Phe Ser Ala Leu Asp Ser Leu Arg 
            20                  25                  30 

Gly Ile Cys Ala Cys Met Ile Val Leu Phe His Leu Arg Ser Thr Gly 
        35                  40                  45 

Val Val Thr Asn Ser His Leu Val Arg Asn Ser Trp Met Phe Val Asp 
    50                  55                  60 

Phe Phe Phe Val Leu Ser Gly Phe Val Ile Ala Cys Gly Tyr Leu Glu 
65                  70                  75                  80 

Arg Leu Arg Glu Gly Tyr Ser Val Arg Gln Phe Met Leu Leu Arg Leu 
                85                  90                  95 

Gly Arg Val Tyr Pro Leu His Leu Ala Val Leu Leu Leu Phe Val Val 
            100                 105                 110 

Ile Glu Leu Ala Gly Ala Met Leu Gly Thr Ala Gly Leu Ser Ala Arg 
        115                 120                 125 

Ala Ala Phe Ser Glu Pro Arg Thr Pro Ala Glu Leu Ala Gly Thr Leu 
    130                 135                 140 

Ala Leu Val Gln Ile Phe Cys Gly Phe Pro Ser Ile Val Trp Asn Gly 
145                 150                 155                 160 

Pro Ser Trp Ser Ile Ala Ala Glu Val Trp Thr Tyr Leu Leu Val Ala 
                165                 170                 175 

Leu Val Val Arg Ala Leu Pro Gly Arg Thr Ala Trp Ala Ala Thr Gly 
            180                 185                 190 

Leu Ala Leu Ala Ala Phe Ala Thr Leu Ala Leu Ala Gly Ala Ala Ala 
        195                 200                 205 

Trp Asp Pro Ala Thr Gly Phe Ala Phe Val Arg Cys Val Leu Gly Phe 
    210                 215                 220 

Ser Val Gly Val Leu Cys Trp Ile Leu Phe Ser Ala Met Gly Arg Pro 
225                 230                 235                 240 

Arg Met Gly Thr Ala Ile Ala Thr Ile Leu Glu Leu Val Ala Val Ala 
                245                 250                 255 

Ser Cys Cys Ala Leu Val Ala Ser Gly Ser Leu Pro Leu Ala Ala Pro 
            260                 265                 270 

Ile Val Phe Ala Gly Thr Val Leu Leu Phe Ala Ala Glu Gln Gly Met 
        275                 280                 285 

Val Ser Arg Leu Leu Lys Leu Ala Pro Phe Leu Ala Leu Gly Thr Leu 
    290                 295                 300 

Ser Tyr Ser Ile Tyr Met Val His Thr Leu Val Ile Ala Arg Ser Leu 
305                 310                 315                 320 

Asp Val Leu Ser Leu Ala Gly Arg Leu Leu His His Pro Leu Val Glu 
                325                 330                 335 

Thr Arg Leu Gly Ser Gly Gly Thr Ile Lys Val Leu Val Phe Ala Pro 
            340                 345                 350 

Asp Ala Met Ala Phe Ala Val Leu Gly Gly Ile Val Leu Cys Ser Ala 
        355                 360                 365 

Leu Thr Tyr Arg Trp Ile Glu Ala Pro Ala Arg Asp Leu Ser Arg Ala 
    370                 375                 380 

Leu Val Arg Gln Ser Gly Arg Arg Gly Ser Leu Ala Ala Ala Pro Asp 
385                 390                 395                 400 

Ala Ala Arg Asp Pro Glu Ala Leu Pro Ala Thr Ala Thr Ser 
                405                 410 

 
           
             3  
             25  
             DNA  
             Artificial Sequence  
             
               primer_bind  
               (1)..(25)  
               PCR primer  
             
           
            3 

ttggagctct ctggacctat ctgct                                           25 

 
           
             4  
             25  
             DNA  
             Artificial Sequence  
             
               primer_bind  
               (1)..(25)  
               PCR primer  
             
           
            4 

gtttctagac ttcaggagcc gactg                                           25