JP2003250547A - Novel plasmid vector - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a plasmid vector capable of artificially controlling phenanthrene-decomposing function of a microorganism of Sphingomonas xenophaga AJ1 strain by elucidating the information transmission mechanism of the microorganism. <P>SOLUTION: A plasmid including a group of enzyme genes relating to the decomposition of phenanthrene is found from the Sphingomonas xenophaga AJ1 strain. The plasmid vector is a plasmid vector including a replication protein gene derived from pSXAc2 and an autonomous replication sequence and includes five phenanthrene decomposition enzyme genes, genes encoding a protein having unclear functions and a replication protein, and an autonomous replication sequence. A decomposing ability to phenanthrene or the decomposition or metabolic products can be imparted to the Sphingomonas by introducing the plasmid into the microorganism. The decomposing ability to phenanthrene is artificially controlled by elucidating the information transmission mechanism of the AJ1 strain using a replication protein and an autonomous replication sequence formed by deletion, addition or substitution of an amino acid or a nucleotide. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel plasmid vector possessed by Sphingomonas xenophaga AJ1 strain, and a plasmid vector containing a replication protein gene and a replication origin derived from the vector.

[0002]

2. Description of the Related Art In Gram-negative bacteria, a substance called an autoinducer is rapidly produced from the late logarithmic growth phase, which forms a complex with a protein to initiate the transcription of genes. Exists in. The Sphingomonas xenopherga AJ1 strain is also one of the strains having such a signal transduction mechanism. This strain is a bacterium that decomposes phenanthrene, which is a persistent compound, and the following findings have been obtained regarding the information transmission mechanism. 1) Produces 3-hydroxyoctanoylhomoserine lactone as an auto inducer (Agricultural Chemical Society of Japan 20
Collection of abstracts of the 2000 conference). 2) Destruction of a protein presumed to bind to an autoinducer reduces the autoinducer productivity and phenanthrene resolution (Agricultural Chemistry Society of Japan 2000
Annual Conference Abstracts). 3) When a mutation is introduced into the autoinducer synthetic gene, the autoinducer productivity and the phenanthrene resolution are reduced (Abstracts of Papers Presented at the 2001 Annual Meeting of the Japanese Society of Agricultural Chemistry).

[0003]

[Problems to be Solved by the Invention] As described above, the information transmission mechanism of the Sphingomonas zenofaga AJ1 strain is gradually being clarified, but there are still many unclear points.

It is an object of the present invention to elucidate the information transmission mechanism of Sphingomonas xenophaga AJ1 strain and artificially control the decomposition function of microbial cells for phenanthrene.

[0005]

[Means for Solving the Problems] As a result of intensive studies to solve the above problems, the present inventors have found that a plasmid containing a gene for a group of enzymes involved in the degradation of phenanthrene from Sphingomonas xenophaga AJ1 strain. The present invention has been completed and the present invention has been completed.

That is, the present invention provides a plasmid vector pSXA.
c2.

The present invention also provides a plasmid vector pSXA.
It is a plasmid vector containing a replication protein gene derived from c2 and an origin of replication.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.

The base sequence of the plasmid vector pSXAc2 is
As shown in SEQ ID NO: 1. This plasmid contains 5 phenanthrene-degrading enzyme genes. That is, 1-hydroxy-2-naphthoic acid carboxylase (SEQ ID NO: 2),
1,2-dihydroxynaphthalene dioxygenase (SEQ ID NO: 3), 2-hydroxychromene-2-carboxylic acid isomerase (SEQ ID NO: 4), salicylaldehyde dehydrogenase (SEQ ID NO: 5), catechol-2,3-diochigenase (SEQ ID NO: 6) ) Is included in the gene. In addition, it also includes a gene encoding a protein of unknown function (SEQ ID NO: 7) or a replication protein (SEQ ID NO: 8), a replication origin (SEQ ID NO: 9), and the like.

Since the plasmid vector pSXAc2 is contained in the Sphingomonas xenopherga AJ1 strain, it can be prepared from this strain according to a conventional method (for example, the method described in Example 1). The Sphingomonas zenofaga AJ1 strain has been deposited at the Patent Microorganism Depositary Center, National Institute of Advanced Industrial Science and Technology under the deposit number FERM P-13008 (deposit date: June 17, 1992). The strain was initially deposited under the name "Pseudomonas sp. AJ1-1-1", but after that, the name was changed to "Sphingomonas sp. AJ1".

Since the plasmid vector pSXAc2 contains a phenanthrene-degrading enzyme gene, this plasmid can be introduced into a microorganism of the genus Sphingomonas or the like, and the microorganism can be given the ability to decompose phenanthrene and its degradation metabolites.

Further, since the plasmid vector pSXAc2 is isolated from Sphingomonas xenopherga, it is considered that the plasmid vector pSXAc2 stably replicates and propagates in Sphingomonas xenophaga and other microorganisms of the genus Sphingomonas. Therefore, it is possible to construct an expression vector using a microorganism of the genus Sphingomonas as a host by utilizing the replication protein gene and the replication origin derived from this vector. As the replication protein and the replication origin used here, the proteins and DNA fragments represented by the sequences shown in SEQ ID NO: 8 and SEQ ID NO: 9, respectively, may be used as they are, but within such a range that their functions are not lost. Those in which amino acids or nucleotides are deleted, added or substituted may be used.

[0013]

[Example 1] Preparation of plasmid vector pSXAc2 Sphingomonas zenophaga AJ1 strain harboring plasmid pSXAc2 was inoculated into 5 ml of Marine Broth medium and incubated at 30 ° C for 2
It was shake-cultured for 4 hours. Add 100 ml of Marine Br to this culture.
It was inoculated into oth medium and shake-cultured at 30 ° C for 16 hours. This culture solution was centrifuged at 10,000 G for 15 minutes at 4 ° C to recover the cultured cells, which were washed with 20 mM Tris-HCl (pH 8.0), and the cells were washed with 5 ml GTE [composition] per 2 g wet weight. : 200 mM glucose, 2
5 mM Tris-HCl, 10 mM EDTA] solution was suspended. 0.5 ml (v / v) of alkaline SDS solution I [0.5N NaOH, 10% SDS] was added to this suspension.
v) Added, shaken at room temperature for 5 minutes, and allowed to stand at 0 ° C for 12 hours.
Alkaline SDS solution II [0.25N NaOH, 5% SDS, 5 mM E
0.25 ml (v / v) of DTA], shake at room temperature for 5 minutes, and
Let stand for 0 minutes. This was centrifuged at 0 ° C. and 15,000 G for 20 minutes to obtain a supernatant containing the plasmid. Add 15% (w / v) of polyethylene glycol 8000 to the obtained supernatant and stir at 0 ° C.
After standing for 2 hours, centrifugation at 3200 G for 5 minutes at 4 ° C gave a plasmid-containing precipitate. 5 ml of the obtained precipitate
After denaturing with 5 M potassium acetate and leaving it at 0 ° C for 15 minutes,
A solution of plasmid pSXAc2 was obtained by centrifugation at 15,000 G for 20 minutes.

Example 2 Introduction into T3 Strain and Detection The plasmid pSXAc2 obtained in the above Example 1 was transformed into the homoserine lactone synthesizing strain T3 derived from the Sphingomonas xenophaga AJ1 strain by the following procedure. Introduced. (1) Production of competent cells Lu was selected as a signal transmitter, homoserine lactone synthesis-deficient strain T3 strain.
Culture was carried out with shaking in 5 ml of ria-Bertani (LB) medium at 30 ° C. for 24 hours. This culture was inoculated into 100 ml of LB medium and incubated at 30 ° C.
It was shake-cultured for 4 hours. When the absorbance at 600 nm reached about 0.5, the shaking was stopped and the mixture was allowed to stand at 0 ° C for 30 minutes.
This culture solution was centrifuged at 4 ° C. and 8,000 G for 10 minutes to collect the cultured cells, which were suspended in a 10% glycerol solution to prepare a competent cell of T3 strain. (2) Introduction of plasmid by electroporation The plasmid pSXAc2 obtained in Example 1 was added to a competent cell of the T3 strain in a 0.1 ml cuvette, and GENEPULSER I
Voltage was applied using I (BIO RAD). The solution was added to 1 ml of LB medium, and cultured at 30 ° C. for 2 hours with shaking. The culture solution was applied to LB agar medium and incubated at 30 ° C for 16 hours. Colony hybridization (J. Sambrook, EFFritsch, T. Maniat
is, "Molecular Cloning 2nded.", ColdSpring Hoarbor
Laboratory PRESS, 1989) was performed to examine the presence or absence of the plasmid in the strain obtained after the above plasmid introduction treatment. As a probe for detecting each plasmid, pSXA
The c2 was labeled with a DIG labeling reagent (manufactured by Roche Diagnostics) and hybridized. As a result, all colonies were detected by the above probe, and it was confirmed that the plasmid pSXAc2 replicated and proliferated in the cells and could be introduced into bacteria belonging to Sphingomonas xenopherga. (3) using the transformant T3 (pSXAc2) strain and T3 strain of the plasmid pSXA2c having a degrading enzyme gene group of phenanthrene-degrading metabolic intermediates for the measurement of 1-hydroxy-2-naphthoic acid degradability, 100 mM phenanthrene is N, N -5 ml of BSM medium dissolved in dimethylformamide and containing 0.8% Glucose
To a final concentration of 1 μM.
3-Hydroxyoctanoylhomoserine lactone (3OHC8-HSL) was added so that it became, and the mixture was shake-cultured at 30 ° C. for 276 hours. Collect the culture solution after 276 hours and use M to remove the cells.
An equal amount of etanol was added, and centrifugation was performed at 4 ° C. and 8,000 G for 10 minutes to obtain an HPLC sample. The HPLC analysis conditions are shown below.
Column: ODS (C18), flow rate: 1.0 ml / min, mobile phase: methanol: water = 60/40 to 90/10 (v / v).

The chromatogram of the culture broth of the T3 strain by HPLC is shown in FIG. 1, and the chromatogram of the culture broth of the T3 (pSXAc2) strain is shown in FIG. As shown in these figures, T3 (pSX
In the culture medium of the Ac2) strain, 1,2-dihydroxynaphthalene, which is not detected in the culture medium of the T3 strain, is detected. Based on this, pSXAc2 was found to contain 1-hydroxy-2-naphthoic acid.
It is considered to have a gene for an enzyme that converts to 1,2-dihydroxynaphthalene.

The phenanthrene decomposition rate of the AJ1 strain, T3 strain, and T3 (pSXAc2) strain was calculated from the peak reduction amount of phenanthrene in the culture solution after separation by HPLC. The results are shown in Table 1.

[0017]

[Table 1]

[Example 3] Determination of nucleotide sequence of pSXAc2 The nucleotide sequence of the plasmid pSXAc2 having a size of about 9,800 bp prepared in Example 2 was determined. Nucleotide sequence determination, dideoxy nucleotide enzyme method using pUC119 (Takara Shuzo) [Dideoxy chain termination method, Sanger,
F. et al., Proc. Natl. Acad. Sci. USA, 74, p5643.
1977]. The nucleotide sequence of plasmid pSXAc2 is shown in SEQ ID NO: 1.

Six ORFs were found in the plasmid pSXAc2. The amino acid sequences encoded by these six ORFs (ORF1 to ORF6) are shown in SEQ ID NOs: 2 to 7 in order. In addition, an ORF (ORF7) in the opposite direction to these ORFs and a sequence presumed to be an origin of replication were also found. The amino acid sequence encoded by the inverted ORF is shown in SEQ ID NO: 8, and the nucleotide sequence of the replication origin is shown in SEQ ID NO: 9. The positions of the above ORF and the replication origin on the plasmid pSXAc2 are shown in FIG. 3 together with the restriction enzyme cleavage sites.

The functions of the proteins encoded by ORF1 to ORF7 were estimated from the homology with known proteins. The results are shown in Table 2.

[0021]

[Table 2]

[0022]

INDUSTRIAL APPLICABILITY The present invention provides a plasmid derived from Sphingomonas xenopherga. By using this plasmid, it is possible to impart a degrading ability to a microorganism having no degrading ability with respect to phenanthrene or its degradation metabolite. Further, since this plasmid is considered to be capable of stable replication and growth in a microorganism belonging to the genus Sphingomonas, it can be used as an expression vector using a microorganism belonging to the genus Sphingomonas as a host.

[Sequence list]                                SEQUENCE LISTING        <110> MARINE BIOTECHNOLOGY INSTITUTE CO., LTD. <120> A NOVEL PLASMID VECTOR <130> P02-008 <160> 9 <170> PatentIn Ver. 2.1 <210> 1 <211> 9824 <212> DNA <213> Sphingomonas xenophaga <220> <221> CDS <222> (857) .. (1876) <220> <221> CDS <222> (1992) .. (2897) <220> <221> CDS <222> (3054) .. (3659) <220> <221> CDS <222> (3693) .. (5114) <220> <221> CDS <222> (5124) .. (6041) <220> <221> CDS <222> (6528) .. (7340) <400> 1 aaatagtctt gccgctggag ttagcgggtt ctgcccatat gcgcgctcct acttcaactt 60 ctaaataatt cgctacgcct tttttgctaa tactccagcg cgtcgctttt ttaagcccgc 120 ggaaggtttt ataatttctg cacgaacacg gttgtatgct gttgtaactc gtcgaagtgt 180 tggcctttcc gctataatgg ccctaaggct ggccgaaata gctcgaactg gcctacnggc 240 gcccaaaagt cgccttacta gactagtaac agcgtcgggc aggcagacac cctttttgcc 300 gcaaacggaa tttataacgc gttttgcacc ggtggttttg cctactctcg cagcgctata 360 agtcagaact ctacccacgg ctcgtcgaac agtacgcata cgagacgcgg cttcccttat 420 aattgcgggt tttagagtct tgcccattag actgccttct tctaaccccg ttcgactgct 480 accgctaccc ttcggacagt ttgtcgccat agaaatatct actgtggggg ccgtaggcgc 540 agtcgcttta ggcaagtttt acggcgtctg acttcgtcct cttgcctaac ccgtacaaaa 600 ttaaggcgca gccggccaat gttcatgcgc gtgagcctga aaattcttac ccttcgacat 660 agttttttgc cggccaatag gtaccggggt ttgcccacga cgtgtaggcc aaagtagctt 720 attccttttt cgggtagcga ggccccgctt ccggaaaaga atttttccct ggcaataaca 780 aggttccaaa aatacgacta ggtattttac ggttcgaatc tgccccgaca agagaaatcg 840 gcagacgagg gcgaaa atg ttt ggt gaa tgt gct gtt aat att tgt gtt cct 892                   Met Phe Gly Glu Cys Ala Val Asn Ile Cys Val Pro                     1 5 10 tat gat gaa att aaa tgg gct ggt tta cat cct tta tgg gaa gtt att 940 Tyr Asp Glu Ile Lys Trp Ala Gly Leu His Pro Leu Trp Glu Val Ile          15 20 25 cat gct tta atg ttt gct gaa cct gct tct tgg gtt aaa gct cat cgt 988 His Ala Leu Met Phe Ala Glu Pro Ala Ser Trp Val Lys Ala His Arg      30 35 40 tgg aaa caa ggt tgt gtt tat gat aat tta tta cgt tct tct gat att 1036 Trp Lys Gln Gly Cys Val Tyr Asp Asn Leu Leu Arg Ser Ser Asp Ile  45 50 55 60 att act att ttt caa tgg gaa cgt cgt gtt tta cct tta aat aat cct 1084 Ile Thr Ile Phe Gln Trp Glu Arg Arg Val Leu Pro Leu Asn Asn Pro                  65 70 75 ggt tta gaa tgt tct tct gct att act ttt tct tta act gct ggt gct 1132 Gly Leu Glu Cys Ser Ser Ala Ile Thr Phe Ser Leu Thr Ala Gly Ala              80 85 90 caa tta gtt tta cct ggt gaa gtt ggt cct tat cat cgt cat act att 1180 Gln Leu Val Leu Pro Gly Glu Val Gly Pro Tyr His Arg His Thr Ile          95 100 105 tgt gct ttt cgt ttt cct gaa ggt gaa ggt gct caa act gct gtt gat 1228 Cys Ala Phe Arg Phe Pro Glu Gly Glu Gly Ala Gln Thr Ala Val Asp     110 115 120 ggt gaa aaa tta gtt att tct tgg ttt gat tta gtt tgt act cct ggt 1276 Gly Glu Lys Leu Val Ile Ser Trp Phe Asp Leu Val Cys Thr Pro Gly 125 130 135 140 act caa tgg cat gat cat caa aat ggt act gat caa ttt atg att tgg 1324 Thr Gln Trp His Asp His Gln Asn Gly Thr Asp Gln Phe Met Ile Trp                 145 150 155 tta gat ggt tta gat att cct tgt gtt cgt cat ttt gat tta ggt ttt 1372 Leu Asp Gly Leu Asp Ile Pro Cys Val Arg His Phe Asp Leu Gly Phe             160 165 170 gct gaa aaa tgg ttt caa gct tat cat cct gat cct gct cct cct ggt 1420 Ala Glu Lys Trp Phe Gln Ala Tyr His Pro Asp Pro Ala Pro Pro Gly         175 180 185 gaa tct tta gct cat tat gct cat aat tta cgt cct ttt cgt ggt aat 1468 Glu Ser Leu Ala His Tyr Ala His Asn Leu Arg Pro Phe Arg Gly Asn     190 195 200 tct gct gat cgt cgt aat act tct tat cct gct cct cat tat cct tat 1516 Ser Ala Asp Arg Arg Asn Thr Ser Tyr Pro Ala Pro His Tyr Pro Tyr 205 210 215 220 aaa caa gct cgt ttt cct tta gat cgt tta att act tta gat cct cat 1564 Lys Gln Ala Arg Phe Pro Leu Asp Arg Leu Ile Thr Leu Asp Pro His                 225 230 235 gct ggt cat gct tta gat ttt tct aat cct gct act ggt ggt cct gtt 1612 Ala Gly His Ala Leu Asp Phe Ser Asn Pro Ala Thr Gly Gly Pro Val             240 245 250 tgg gaa act tgg tct gct cgt gtt aaa tta att cct cgt ggt cct gaa 1660 Trp Glu Thr Trp Ser Ala Arg Val Lys Leu Ile Pro Arg Gly Pro Glu         255 260 265 act gtt cct cgt cgt tgt act gaa tct act att tta att gtt gtt gaa 1708 Thr Val Pro Arg Arg Cys Thr Glu Ser Thr Ile Leu Ile Val Val Glu     270 275 280 ggt aaa ggt tat gtt gat att gaa ggt gtt tgt act cgt tgg tct tgt 1756 Gly Lys Gly Tyr Val Asp Ile Glu Gly Val Cys Thr Arg Trp Ser Cys 285 290 295 300 gtt gtt ttt tta cct tct tgg aaa cgt aaa cgt cct cat att gaa gat 1804 Val Val Phe Leu Pro Ser Trp Lys Arg Lys Arg Pro His Ile Glu Asp                 305 310 315 gaa gct att att ttt ggt ttt tat gat cat gct act caa caa cat tta 1852 Glu Ala Ile Ile Phe Gly Phe Tyr Asp His Ala Thr Gln Gln His Leu             320 325 330 ggt tta tgg cgt gaa gaa tct tta tgagctagcg gcgcaggaag ctacatattt 1906 Gly Leu Trp Arg Glu Glu Ser Leu         335 340 ctcggcagcc gcggtgtaaa aggtggaagt gtttggtagg caggcgtagt tgtgacccac 1966 aatgacgtat acaaaagtag gaaga atg tct aaa gct gct gtt tgt gaa tta 2018                             Met Ser Lys Ala Ala Val Cys Glu Leu                                             345 ggt tat att ggt att gaa gtt caa gat gct gaa gat tgg cgt gaa tat 2066 Gly Tyr Ile Gly Ile Glu Val Gln Asp Ala Glu Asp Trp Arg Glu Tyr 350 355 360 365 gct tgt tgt att gtt ggt atg gaa att gtt gaa tat ggt gaa gat gat 2114 Ala Cys Cys Ile Val Gly Met Glu Ile Val Glu Tyr Gly Glu Asp Asp                 370 375 380 cgt tta tat tta cgt atg gat tat tgg cat cat cgt att gtt gtt cat 2162 Arg Leu Tyr Leu Arg Met Asp Tyr Trp His His Arg Ile Val Val His             385 390 395 ggt tgt cct ggt gaa gat cct gaa tat gtt ggt tgg cgt gtt gat tct 2210 Gly Cys Pro Gly Glu Asp Pro Glu Tyr Val Gly Trp Arg Val Asp Ser         400 405 410 ttt aat gaa ttt caa gct att gtt caa aaa tta gat gat gct ggt att 2258 Phe Asn Glu Phe Gln Ala Ile Val Gln Lys Leu Asp Asp Ala Gly Ile     415 420 425 tct tgg cgt gaa gct tct gct gat gaa gct caa gct cgt tgt gtt tta 2306 Ser Trp Arg Glu Ala Ser Ala Asp Glu Ala Gln Ala Arg Cys Val Leu 430 435 440 445 gaa tta att aaa act gct gat cct ggt ggt aat cct act gaa att ttt 2354 Glu Leu Ile Lys Thr Ala Asp Pro Gly Gly Asn Pro Thr Glu Ile Phe                 450 455 460 tat ggt cct cgt caa ttt caa tct aaa cct ttt cat cct ggt cgt cct 2402 Tyr Gly Pro Arg Gln Phe Gln Ser Lys Pro Phe His Pro Gly Arg Pro             465 470 475 tta cat ggt cgt ttt att act ggt tct gaa ggt tta ggt cat tgt att 2450 Leu His Gly Arg Phe Ile Thr Gly Ser Glu Gly Leu Gly His Cys Ile         480 485 490 gtt cgt caa act gat ttt tgg gct gct gct aaa ttt tat gaa act tta 2498 Val Arg Gln Thr Asp Phe Trp Ala Ala Ala Lys Phe Tyr Glu Thr Leu     495 500 505 ggt att aaa ggt gat gtt gaa tat gaa tta cct tta cct aat ggt gct 2546 Gly Ile Lys Gly Asp Val Glu Tyr Glu Leu Pro Leu Pro Asn Gly Ala 510 515 520 525 cct gct caa tta ggt ttt cct cat tgt aat gaa cgt caa cat tct tta 2594 Pro Ala Gln Leu Gly Phe Pro His Cys Asn Glu Arg Gln His Ser Leu                 530 535 540 gct tta ggt aat atg gct att cct aaa cgt ttt aat cat tta atg ttt 2642 Ala Leu Gly Asn Met Ala Ile Pro Lys Arg Phe Asn His Leu Met Phe             545 550 555 gaa tat act gaa gat tta gat gtt ggt tta gct cat gat att gct gtt 2690 Glu Tyr Thr Glu Asp Leu Asp Val Gly Leu Ala His Asp Ile Ala Val         560 565 570 aat gct tgt ttt cat att gct tta caa tta ggt aaa cat gct aat gat 2738 Asn Ala Cys Phe His Ile Ala Leu Gln Leu Gly Lys His Ala Asn Asp     575 580 585 aaa gct tta tct ttt tat gtt tta ggt cct tct ggt tgg tta gtt gaa 2786 Lys Ala Leu Ser Phe Tyr Val Leu Gly Pro Ser Gly Trp Leu Val Glu 590 595 600 605 ttt ggt tgg aaa tct gct aaa gtt cct gat act tgt gtt tat tat act 2834 Phe Gly Trp Lys Ser Ala Lys Val Pro Asp Thr Cys Val Tyr Tyr Thr                 610 615 620 cct gat cct att tgg ggt cat ggt gct gaa gct cct ggt cat ggt tta 2882 Pro Asp Pro Ile Trp Gly His Gly Ala Glu Ala Pro Gly His Gly Leu             625 630 635 gat att gat tta tct tgaccaaagc taaataaata aacaaagggc cgtaagcctt 2937 Asp Ile Asp Leu Ser         640 gccgttttgt gtattgggag aggaggttta tgtccctttc actataaaaa gataagaaaa 2997 agtactcttt accattttac ctcaaaccta taaaaaattc ctctcctgcc acgaat atg 3056                                                               Met tct cgt act att gat ttt tat ttt gat ttt tct tct cct ttt tct tat 3104 Ser Arg Thr Ile Asp Phe Tyr Phe Asp Phe Ser Ser Pro Phe Ser Tyr     645 650 655 tta gct act aat cat tta ggt gat gtt gct tat gct cat ggt att caa 3152 Leu Ala Thr Asn His Leu Gly Asp Val Ala Tyr Ala His Gly Ile Gln 660 665 670 675 tta aaa tat cgt cct att gat gct cct cat gct aaa att gct cct gaa 3200 Leu Lys Tyr Arg Pro Ile Asp Ala Pro His Ala Lys Ile Ala Pro Glu                 680 685 690 aat tta ggt cct tct aat cgt gaa gct gaa cct aaa cgt cat tat tta 3248 Asn Leu Gly Pro Ser Asn Arg Glu Ala Glu Pro Lys Arg His Tyr Leu             695 700 705 aaa gct gat tta gat cgt tgg gct tgt tta tat gaa gtt cct tta att 3296 Lys Ala Asp Leu Asp Arg Trp Ala Cys Leu Tyr Glu Val Pro Leu Ile         710 715 720 ttt cct gct tct aat caa ggt aaa cat tgg aat att ggt tta aat ttt 3344 Phe Pro Ala Ser Asn Gln Gly Lys His Trp Asn Ile Gly Leu Asn Phe     725 730 735 tct cgt gct gaa gtt caa act ggt gct tta tct act tta att ttt cgt 3392 Ser Arg Ala Glu Val Gln Thr Gly Ala Leu Ser Thr Leu Ile Phe Arg 740 745 750 755 gct att tgg ggt gat ggt att gat cct ggt gat gaa act tta gtt gct 3440 Ala Ile Trp Gly Asp Gly Ile Asp Pro Gly Asp Glu Thr Leu Val Ala                 760 765 770 cct tgt gct gat aaa tta ggt ttt gat aaa tct gct tta gaa cgt att 3488 Pro Cys Ala Asp Lys Leu Gly Phe Asp Lys Ser Ala Leu Glu Arg Ile             775 780 785 att tct act gat gct gct act gaa cgt tat gat gaa caa act cat ttt 3536 Ile Ser Thr Asp Ala Ala Thr Glu Arg Tyr Asp Glu Gln Thr His Phe         790 795 800 gct tta caa cgt ggt gtt ttt ggt gct cct act atg ttt tta tct gaa 3584 Ala Leu Gln Arg Gly Val Phe Gly Ala Pro Thr Met Phe Leu Ser Glu     805 810 815 caa att tgg tgg ggt caa gat cgt tta act ttt tta gct aat tat gtt 3632 Gln Ile Trp Trp Gly Gln Asp Arg Leu Thr Phe Leu Ala Asn Tyr Val 820 825 830 835 ggt ggt tta cct cgt aat cct gct tct tgatctggaa gacttactag 3679 Gly Gly Leu Pro Arg Asn Pro Ala Ser                 840 ccgacggcgg cgt atg tat act cgt tta tgg att ggt ggt gct ttt tta 3728                Met Tyr Thr Arg Leu Trp Ile Gly Gly Ala Phe Leu                845 850 855 caa ggt act gat aaa aaa tct att gaa gat aaa aat cct gct act ggt 3776 Gln Gly Thr Asp Lys Lys Ser Ile Glu Asp Lys Asn Pro Ala Thr Gly             860 865 870 gaa gtt tta act cgt gtt cct gct ggt act gtt aaa gat att tta cgt 3824 Glu Val Leu Thr Arg Val Pro Ala Gly Thr Val Lys Asp Ile Leu Arg         875 880 885 gct gtt gct gtt gct aaa gaa gct tat att aaa tgg gct act tct ggt 3872 Ala Val Ala Val Ala Lys Glu Ala Tyr Ile Lys Trp Ala Thr Ser Gly     890 895 900 gct gct gaa cgt aat gat att att att cat gct gct gat cgt tta caa 3920 Ala Ala Glu Arg Asn Asp Ile Ile Ile His Ala Ala Asp Arg Leu Gln 905 910 915 920 act aaa act caa gaa tat att act gtt atg act aaa aat aat ggt gct 3968 Thr Lys Thr Gln Glu Tyr Ile Thr Val Met Thr Lys Asn Asn Gly Ala                 925 930 935 cct att aaa aaa gct tct gtt act gtt tct atg gct att aat gtt tta 4016 Pro Ile Lys Lys Ala Ser Val Thr Val Ser Met Ala Ile Asn Val Leu             940 945 950 gaa gaa ttt gct ggt tta cct tct aat tta caa ggt caa act att cct 4064 Glu Glu Phe Ala Gly Leu Pro Ser Asn Leu Gln Gly Gln Thr Ile Pro         955 960 965 act gat act tct gaa cgt tta ggt atg act cat cgt gaa gct att ggt 4112 Thr Asp Thr Ser Glu Arg Leu Gly Met Thr His Arg Glu Ala Ile Gly     970 975 980 gtt att tta aat att gtt cct tgg aat gtt cct gct att tta gct att 4160 Val Ile Leu Asn Ile Val Pro Trp Asn Val Pro Ala Ile Leu Ala Ile 985 990 995 1000 cgt cat att gct ttt cct tta gct act ggt aat act ttt gtt ttt tta 4208 Arg His Ile Ala Phe Pro Leu Ala Thr Gly Asn Thr Phe Val Phe Leu                1005 1010 1015 aat gct gaa tgg att cct ggt gct gat gct tta att act ttt tta gtt 4256 Asn Ala Glu Trp Ile Pro Gly Ala Asp Ala Leu Ile Thr Phe Leu Val            1020 1025 1030 gaa gaa gct gat gtt cct gat ggt gtt tta aat tat gtt tat ggt aat 4304 Glu Glu Ala Asp Val Pro Asp Gly Val Leu Asn Tyr Val Tyr Gly Asn        1035 1040 1045 ggt gct cct gaa gtt gct gat gct tta act ggt cat aaa gat att cgt 4352 Gly Ala Pro Glu Val Ala Asp Ala Leu Thr Gly His Lys Asp Ile Arg    1050 1055 1060 aaa att gct ttt act ggt tct tct cgt gtt ggt cgt cat att gct caa 4400 Lys Ile Ala Phe Thr Gly Ser Ser Arg Val Gly Arg His Ile Ala Gln 1065 1070 1075 1080 aaa gct tct caa cat att aaa cgt tat act tta gaa tta ggt ggt aaa 4448 Lys Ala Ser Gln His Ile Lys Arg Tyr Thr Leu Glu Leu Gly Gly Lys                1085 1090 1095 tct cct tta gtt att tta gat gat gct gat att gat gct gct gtt aat 4496 Ser Pro Leu Val Ile Leu Asp Asp Ala Asp Ile Asp Ala Ala Val Asn            1100 1105 1110 tct gct act ttt ggt act ttt tta gtt caa ggt caa gtt tgt tta tct 4544 Ser Ala Thr Phe Gly Thr Phe Leu Val Gln Gly Gln Val Cys Leu Ser        1115 1120 1125 tct gat cgt tta att tta cat cgt tct att ggt gat caa ttt gct ttt 4592 Ser Asp Arg Leu Ile Leu His Arg Ser Ile Gly Asp Gln Phe Ala Phe    1130 1135 1140 gct ttt aaa gtt gct gct aaa aat tta aat tct ggt gat cct cgt gaa 4640 Ala Phe Lys Val Ala Ala Lys Asn Leu Asn Ser Gly Asp Pro Arg Glu 1145 1150 1155 1160 ttt act act att tta ggt gct aat gtt tct tta cgt ggt ggt gaa cgt 4688 Phe Thr Thr Ile Leu Gly Ala Asn Val Ser Leu Arg Gly Gly Glu Arg                1165 1170 1175 gtt aat ggt tat att gat gat gct tta gat gaa ggt gct act gtt gat 4736 Val Asn Gly Tyr Ile Asp Asp Ala Leu Asp Glu Gly Ala Thr Val Asp            1180 1185 1190 gct ggt ggt tta gct gaa ggt gct tta att cct tct act att ttt gat 4784 Ala Gly Gly Leu Ala Glu Gly Ala Leu Ile Pro Ser Thr Ile Phe Asp        1195 1200 1205 cat gtt act tct tct atg cgt tta tat cgt gaa gaa act ttt ggt cct 4832 His Val Thr Ser Ser Met Arg Leu Tyr Arg Glu Glu Thr Phe Gly Pro    1210 1215 1220 tta act gtt gtt ttt cgt tgt gat ggt gaa gaa gaa gct att gaa tta 4880 Leu Thr Val Val Phe Arg Cys Asp Gly Glu Glu Glu Ala Ile Glu Leu 1225 1230 1235 1240 gct aat act aat gct tat ggt tta tct gct tct tta ttt ggt cgt gat 4928 Ala Asn Thr Asn Ala Tyr Gly Leu Ser Ala Ser Leu Phe Gly Arg Asp                1245 1250 1255 tta aat aaa gct tta gct gtt ggt atg tct att gaa act ggt act gtt 4976 Leu Asn Lys Ala Leu Ala Val Gly Met Ser Ile Glu Thr Gly Thr Val            1260 1265 1270 cat att aat ggt tct tat gtt caa ggt gaa gtt caa gtt cct tat ggt 5024 His Ile Asn Gly Ser Tyr Val Gln Gly Glu Val Gln Val Pro Tyr Gly        1275 1280 1285 ggt tat aaa gct tct ggt ttt ggt cgt ttt gaa tct cgt gaa gtt tta 5072 Gly Tyr Lys Ala Ser Gly Phe Gly Arg Phe Glu Ser Arg Glu Val Leu    1290 1295 1300 gat cat ttt act gaa aat aaa tat att tct gct ttt gaa ttt tgacgactc 5123 Asp His Phe Thr Glu Asn Lys Tyr Ile Ser Ala Phe Glu Phe 1305 1310 1315 atg gct tta act ggt att tta cgt cct ggt tat gtt caa tta cgt gtt 5171 Met Ala Leu Thr Gly Ile Leu Arg Pro Gly Tyr Val Gln Leu Arg Val    1320 1325 1330 tta gat tta gat gaa gct att caa cat tat cgt gat cgt att ggt tta 5219 Leu Asp Leu Asp Glu Ala Ile Gln His Tyr Arg Asp Arg Ile Gly Leu 1335 1340 1345 1350 aat tta gtt tct gtt gaa ggt ggt cgt gct ttt ttt caa gct ttt gat 5267 Asn Leu Val Ser Val Glu Gly Gly Arg Ala Phe Phe Gln Ala Phe Asp                1355 1360 1365 gaa ttt gat cgt cat tct att att tta cgt gaa gct gat tct gct ggt 5315 Glu Phe Asp Arg His Ser Ile Ile Leu Arg Glu Ala Asp Ser Ala Gly            1370 1375 1380 tta gat cgt atg gct ttt aaa gtt gct aaa gat tct gat tta gat cat 5363 Leu Asp Arg Met Ala Phe Lys Val Ala Lys Asp Ser Asp Leu Asp His        1385 1390 1395 ttt gct gaa cgt tta tta gat gct ggt gtt aat gtt gat gtt att cct 5411 Phe Ala Glu Arg Leu Leu Asp Ala Gly Val Asn Val Asp Val Ile Pro    1400 1405 1410 gct ggt gaa gat cct ggt gtt ggt cgt aaa att cgt ttt aat gct cct 5459 Ala Gly Glu Asp Pro Gly Val Gly Arg Lys Ile Arg Phe Asn Ala Pro 1415 1420 1425 1430 act gct cat gtt ttt gat tta tat gct gaa atg gaa tta tct gaa act 5507 Thr Ala His Val Phe Asp Leu Tyr Ala Glu Met Glu Leu Ser Glu Thr                1435 1440 1445 ggt cct gct gtt cgt aat cct gat gtt tgg att gct gaa cct cgt ggt 5555 Gly Pro Ala Val Arg Asn Pro Asp Val Trp Ile Ala Glu Pro Arg Gly            1450 1455 1460 atg cgt gct act cgt ttt gat cat tgt gct tta aat ggt gtt gat att 5603 Met Arg Ala Thr Arg Phe Asp His Cys Ala Leu Asn Gly Val Asp Ile        1465 1470 1475 tct gct tct gct aaa att ttt gtt gaa gtt tta gat ttt tct gtt act 5651 Ser Ala Ser Ala Lys Ile Phe Val Glu Val Leu Asp Phe Ser Val Thr    1480 1485 1490 gaa gaa tta gtt gat gaa tct act ggt gct cgt tta ggt att ttt tta 5699 Glu Glu Leu Val Asp Glu Ser Thr Gly Ala Arg Leu Gly Ile Phe Leu 1495 1500 1505 1510 tct tgt tct aat aaa gct cat gat gtt gct ttt tta ggt tat cct gaa 5747 Ser Cys Ser Asn Lys Ala His Asp Val Ala Phe Leu Gly Tyr Pro Glu                1515 1520 1525 gat ggt cgt att cat cat act tct ttt aat tta gaa tct tgg aat gat 5795 Asp Gly Arg Ile His His Thr Ser Phe Asn Leu Glu Ser Trp Asn Asp            1530 1535 1540 gtt ggt cat nnn gct gat att att tct cgt tat gat att tct tta gat 5843 Val Gly His Xaa Ala Asp Ile Ile Ser Arg Tyr Asp Ile Ser Leu Asp        1545 1550 1555 att ggt cct act cgt cat ggt att act cgt ggt caa act att tat ttt 5891 Ile Gly Pro Thr Arg His Gly Ile Thr Arg Gly Gln Thr Ile Tyr Phe    1560 1565 1570 ttt gat cct tct ggt aat cgt aat gaa act ttt tct ggt ggt tat act 5939 Phe Asp Pro Ser Gly Asn Arg Asn Glu Thr Phe Ser Gly Gly Tyr Thr 1575 1580 1585 1590 tat tat cct gat aat cct cgt cgt gtt tgg caa gct gaa aat gct ggt 5987 Tyr Tyr Pro Asp Asn Pro Arg Arg Val Trp Gln Ala Glu Asn Ala Gly                1595 1600 1605 aaa gct att ttt tat tat gaa aaa gct tta aat gat cgt tta aat gat 6035 Lys Ala Ile Phe Tyr Tyr Glu Lys Ala Leu Asn Asp Arg Leu Asn Asp            1610 1615 1620 ggt gaa tgactacatt acccctgcct acccaaacat ttccactttt tactccggcg 6091 Gly Glu ggctgccaat aaaatatgta gctttcctgc gccggctagc tctttcgcnt cgtctgccta 6151 atttctcttg gtcgggccaa ttcgaacagt aaatactagt cgtatttttg gtaactttct 6211 tcttgccagg aaaattcttt tcgnaagcgg gctcgctccc gaaaaggnaa taagctactt 6271 tgccttacac gtcgtggcag acgcggtact ctagccggca aaaaactatg tcgaggggta 6331 agaagtttcc gccgcttatg aaagtagtac aggacgttac agacaaagtc atactattta 6391 agcagttaac aggcgtcaag acgccgaaaa aagactatta gtcggtcgaa gtcgacatta 6451 ttgtagttac cgctaacgta ttttttggag ggggtaagac aatatttcgg ggcgaaaagt 6511 ttggaggtaa ggaaac atg gtt cgt tgt act act ttt gaa cgt ggt aaa tct 6563                   Met Val Arg Cys Thr Thr Phe Glu Arg Gly Lys Ser                  1625 1630 1635 tgt gaa tta aaa aat cgt cgt caa gct caa gct act tta tct aaa act 6611 Cys Glu Leu Lys Asn Arg Arg Gln Ala Gln Ala Thr Leu Ser Lys Thr            1640 1645 1650 tct ttt tta tct ttt cct cct tta ttt tct ggt ggt ttt tgt act tct 6659 Ser Phe Leu Ser Phe Pro Pro Leu Phe Ser Gly Gly Phe Cys Thr Ser        1655 1660 1665 ggt cgt cgt cgt cgt act aat cgt ggt gct caa aaa gct gct tct tct 6707 Gly Arg Arg Arg Arg Thr Asn Arg Gly Ala Gln Lys Ala Ala Ser Ser    1670 1675 1680 cgt ttt gct cct att att caa att ggt aaa gct cgt gct cat cct ggt 6755 Arg Phe Ala Pro Ile Ile Gln Ile Gly Lys Ala Arg Ala His Pro Gly 1685 1690 1695 1700 tat cgt tgt cgt act tgt tct tat tct tgt ttt ttt cgt cgt cgt ggt 6803 Tyr Arg Cys Arg Thr Cys Ser Tyr Ser Cys Phe Phe Arg Arg Arg Gly                1705 1710 1715 tta gtt tct cgt cgt ttt gtt cct gaa gaa ttt cgt tct tta gct gtt 6851 Leu Val Ser Arg Arg Phe Val Pro Glu Glu Phe Arg Ser Leu Ala Val            1720 1725 1730 cgt cct cgt tct gtt cgt act gct gct ggt gct ttt gaa cct gct cgt 6899 Arg Pro Arg Ser Val Arg Thr Ala Ala Gly Ala Phe Glu Pro Ala Arg        1735 1740 1745 ttt ttt cct gaa gaa cgt tgt cgt cct tta cct ggt tgt tct ttt cct 6947 Phe Phe Pro Glu Glu Arg Cys Arg Pro Leu Pro Gly Cys Ser Phe Pro    1750 1755 1760 gaa gct gct tgt cct tct tta tta cat cat gct ttt tct gct ggt cat 6995 Glu Ala Ala Cys Pro Ser Leu Leu His His Ala Phe Ser Ala Gly His 1765 1770 1775 1780 gct gct gtt aaa gct cgt cgt tta cct caa cct ttt cgt cct gtt att 7043 Ala Ala Val Lys Ala Arg Arg Leu Pro Gln Pro Phe Arg Pro Val Ile                1785 1790 1795 tct gaa tgt cat tta caa cat ggt gtt tgt tta gct gct gct cat att 7091 Ser Glu Cys His Leu Gln His Gly Val Cys Leu Ala Ala Ala His Ile            1800 1805 1810 ggt gct gct cgt tgt ggt aaa ggt att aat ggt tgt cgt cct cgt aat 7139 Gly Ala Ala Arg Cys Gly Lys Gly Ile Asn Gly Cys Arg Pro Arg Asn        1815 1820 1825 gaa aaa cct caa gaa aat ggt cgt aaa cgt tta cgt aaa act gtt ttt 7187 Glu Lys Pro Gln Glu Asn Gly Arg Lys Arg Leu Arg Lys Thr Val Phe    1830 1835 1840 tta cgt gaa ggt tta gaa gct nnn att aaa cgt tct caa caa gct ggt 7235 Leu Arg Glu Gly Leu Glu Ala Xaa Ile Lys Arg Ser Gln Gln Ala Gly 1845 1850 1855 1860 gct cct cgt cgt cct gct tat ttt tat ttt aaa gct aaa cct act ggt 7283 Ala Pro Arg Arg Pro Ala Tyr Phe Tyr Phe Lys Ala Lys Pro Thr Gly                1865 1870 1875 aaa ggt gaa act aaa gat ttt cct ggt ttt act ggt tta aaa cct ggt 7331 Lys Gly Glu Thr Lys Asp Phe Pro Gly Phe Thr Gly Leu Lys Pro Gly            1880 1885 1890 cct ggt aat tgagggaatg ctatctaaag actaaaatca tgttatttct 7380 Pro Gly Asn        1895 ttgccttcct cactagtgta ctttggcgac gtttaaaggc ggctttgccg cgttttcgag 7440 cgtcttagtg acttatacgg cgaactctct tagtacgtgt acggagtcgt gtaagacgaa 7500 cggtttaatc gaagttcgaa ctggtcctcg tcctcgattt ccaaatattt gtactcgtct 7560 aaatcgttaa ttttgttgtc ctgctggtgg ttgtgctgca agttaatttg caagggttag 7620 tcttcaattt catgctggaa caaatcgatt aagaaatcgt ctaactcatt gtcatagatt 7680 tgctacttta actctttaaa aaagtagtat tcttttaact aatcctggtg ctagtggaaa 7740 atttctggta aaaaattatg gtaatagtcg aagtcttcga ttggttgctg gtcatcttag 7800 ttgtcatcct ttaactgcat tatttcgtgc tcgatttgct ggttgtagtc gatttcgtat 7860 aaatccttga actggtcaat tttttcaatt tattccttat catcgtctat ttctttttgg 7920 tgttcatcgt gttggtgcat taaattatca tagttgggtt catggtcatt atcatcttcc 7980 tgcaaatgga aatggaaatt gtatatttgc aagtcttcgt aaaaattaaa caaaatttaa 8040 tggtcttcta agtcgtggta ttattgctcg atttcgtgca tttcatggta atggaagaaa 8100 tagttataga ttttataaaa ctcatgttta tcgattaaat gcttatacta ttaatcttta 8160 tcttactggt ggtagattat ttattgttca tgctcattgt gcatttccaa ataatagtcc 8220 tcggtattta ggtttttctc ttgctagtgt ttatttccgg cgagagagta tatatacacg 8280 ccctccgcct tcgctaccga aaggcggaga aaaaaagcgc ttcttttaaa accattttgt 8340 gccctgtgac agtgagacgc ctgtatacgg ctgaatatcg atattgaaca tcagtttttt 8400 cttaatggtt gtgtgtgatg caatgagcat aaaaagcaga aagccggaag cacgatgtag 8460 gcctgcatca ttttcatatt aaaggcaaga ccgacaagtg cgaatgcgga taagagccaa 8520 atgattttgc cgcgcttcac cgcgcgcatc aaacaccagg aaccgagaag cagtgtaaag 8580 acgagcatgc tgtcgagatt attcgtccgg ctgacggccg ccgcaatcgg cgtcagcgcc 8640 atgacaaaag cggcgattct ggccgcgccc gtgccgaacg aaggctttat catgaaatac 8700 agcagcagaa cggaaccgag ccccgccaaa gcctgaggaa gaatcacgct ccatgtatgt 8760 acgccgaaga ccaaagcgaa aatcgtttga atccataaag cgacaggcgg tttgtctacc 8820 gtgacaaagc cggacgggtc aaaggatgcg tagaagaagt tgtgaaagct ttgcgtcatg 8880 cttttgacgg cagccaaata atactggttg gcggcatcat cctgccaaat gccgtacata 8940 ttcaaagccg ccgctgctga ttaaaatacc gataagaacg atgtccagtc gttttgtttt 9000 caatgggttt cacctctttt gtgccttccc attgtaataa aataaatatg attaaagctc 9060 tgaaattaaa cgataatcga ttttaaagca atgctttacc aaaattttct ttccggaaaa 9120 aagagggcgg gaagccgcct cttccgcctc ccgccgtgtt gattcaacag agcgggcctt 9180 tcgttcggga acgcttcttc tttttggatt ttcgatatag aacagggagg tagaaagttg 9240 aaaactatga taaaataaag tggacttagc aggggaaatt tgggacttag gaggggaaat 9300 gggagttagc aggggaaaat gggacttaga aggggaaatt tgtgaatata atgaatatta 9360 tttatcattt ttttaggttt ttggattttg ccttttgttt attttctcgt atccggagga 9420 cgggccatca agctagcaga cgaccgtagc ctagcgaggg cttttcctta ttcgatgaaa 9480 cggatgtgca gcaccgtctg cgccatgaga tcggccgttt tttgatacag ctcccattct 9540 tcaaaggcgg cgaatacttt catcatgtcc tgcaatgtct gtttcagtat gataaattcg 9600 tcaattgtcc gcagttctgc ggcttttttc tgataatcag ccagcttcag ctgtaataac 9660 atcaatggcg attgcataaa aaacctcccc cattctgtta taaagccccg cttttcaaac 9720 ctccattcct ttgtcatatc cccttacgat agatttctga tttttagtac aataaagaaa 9780 cggaaggagt gatcacatga aaccgctgca aatttccgcc gaaa 9824    <210> 2 <211> 340 <212> PRT <213> Sphingomonas xenophaga <400> 2 Met Phe Gly Glu Cys Ala Val Asn Ile Cys Val Pro Tyr Asp Glu Ile   1 5 10 15 Lys Trp Ala Gly Leu His Pro Leu Trp Glu Val Ile His Ala Leu Met              20 25 30 Phe Ala Glu Pro Ala Ser Trp Val Lys Ala His Arg Trp Lys Gln Gly          35 40 45 Cys Val Tyr Asp Asn Leu Leu Arg Ser Ser Asp Ile Ile Thr Ile Phe      50 55 60 Gln Trp Glu Arg Arg Val Leu Pro Leu Asn Asn Asn Pro Gly Leu Glu Cys  65 70 75 80 Ser Ser Ala Ile Thr Phe Ser Leu Thr Ala Gly Ala Gln Leu Val Leu                  85 90 95 Pro Gly Glu Val Gly Pro Tyr His Arg His Thr Ile Cys Ala Phe Arg             100 105 110 Phe Pro Glu Gly Glu Gly Ala Gln Thr Ala Val Asp Gly Glu Lys Leu         115 120 125 Val Ile Ser Trp Phe Asp Leu Val Cys Thr Pro Gly Thr Gln Trp His     130 135 140 Asp His Gln Asn Gly Thr Asp Gln Phe Met Ile Trp Leu Asp Gly Leu 145 150 155 160 Asp Ile Pro Cys Val Arg His Phe Asp Leu Gly Phe Ala Glu Lys Trp                 165 170 175 Phe Gln Ala Tyr His Pro Asp Pro Ala Pro Pro Gly Glu Ser Leu Ala             180 185 190 His Tyr Ala His Asn Leu Arg Pro Phe Arg Gly Asn Ser Ala Asp Arg         195 200 205 Arg Asn Thr Ser Tyr Pro Ala Pro His Tyr Pro Tyr Lys Gln Ala Arg     210 215 220 Phe Pro Leu Asp Arg Leu Ile Thr Leu Asp Pro His Ala Gly His Ala 225 230 235 240 Leu Asp Phe Ser Asn Pro Ala Thr Gly Gly Pro Val Trp Glu Thr Trp                 245 250 255 Ser Ala Arg Val Lys Leu Ile Pro Arg Gly Pro Glu Thr Val Pro Arg             260 265 270 Arg Cys Thr Glu Ser Thr Ile Leu Ile Val Val Glu Gly Lys Gly Tyr         275 280 285 Val Asp Ile Glu Gly Val Cys Thr Arg Trp Ser Cys Val Val Phe Leu     290 295 300 Pro Ser Trp Lys Arg Lys Arg Pro His Ile Glu Asp Glu Ala Ile Ile 305 310 315 320 Phe Gly Phe Tyr Asp His Ala Thr Gln Gln His Leu Gly Leu Trp Arg                 325 330 335 Glu Glu Ser Leu             340    <210> 3 <211> 302 <212> PRT <213> Sphingomonas xenophaga <400> 3 Met Ser Lys Ala Ala Val Cys Glu Leu Gly Tyr Ile Gly Ile Glu Val   1 5 10 15 Gln Asp Ala Glu Asp Trp Arg Glu Tyr Ala Cys Cys Ile Val Gly Met              20 25 30 Glu Ile Val Glu Tyr Gly Glu Asp Asp Arg Leu Tyr Leu Arg Met Asp          35 40 45 Tyr Trp His His Arg Ile Val Val His Gly Cys Pro Gly Glu Asp Pro      50 55 60 Glu Tyr Val Gly Trp Arg Val Asp Ser Phe Asn Glu Phe Gln Ala Ile  65 70 75 80 Val Gln Lys Leu Asp Asp Ala Gly Ile Ser Trp Arg Glu Ala Ser Ala                  85 90 95 Asp Glu Ala Gln Ala Arg Cys Val Leu Glu Leu Ile Lys Thr Ala Asp             100 105 110 Pro Gly Gly Asn Pro Thr Glu Ile Phe Tyr Gly Pro Arg Gln Phe Gln         115 120 125 Ser Lys Pro Phe His Pro Gly Arg Pro Leu His Gly Arg Phe Ile Thr     130 135 140 Gly Ser Glu Gly Leu Gly His Cys Ile Val Arg Gln Thr Asp Phe Trp 145 150 155 160 Ala Ala Ala Lys Phe Tyr Glu Thr Leu Gly Ile Lys Gly Asp Val Glu                 165 170 175 Tyr Glu Leu Pro Leu Pro Asn Gly Ala Pro Ala Gln Leu Gly Phe Pro             180 185 190 His Cys Asn Glu Arg Gln His Ser Leu Ala Leu Gly Asn Met Ala Ile         195 200 205 Pro Lys Arg Phe Asn His Leu Met Phe Glu Tyr Thr Glu Asp Leu Asp     210 215 220 Val Gly Leu Ala His Asp Ile Ala Val Asn Ala Cys Phe His Ile Ala 225 230 235 240 Leu Gln Leu Gly Lys His Ala Asn Asp Lys Ala Leu Ser Phe Tyr Val                 245 250 255 Leu Gly Pro Ser Gly Trp Leu Val Glu Phe Gly Trp Lys Ser Ala Lys             260 265 270 Val Pro Asp Thr Cys Val Tyr Tyr Thr Pro Asp Pro Ile Trp Gly His         275 280 285 Gly Ala Glu Ala Pro Gly His Gly Leu Asp Ile Asp Leu Ser     290 295 300    <210> 4 <211> 202 <212> PRT <213> Sphingomonas xenophaga <400> 4 Met Ser Arg Thr Ile Asp Phe Tyr Phe Asp Phe Ser Ser Pro Phe Ser   1 5 10 15 Tyr Leu Ala Thr Asn His Leu Gly Asp Val Ala Tyr Ala His Gly Ile              20 25 30 Gln Leu Lys Tyr Arg Pro Ile Asp Ala Pro His Ala Lys Ile Ala Pro          35 40 45 Glu Asn Leu Gly Pro Ser Asn Arg Glu Ala Glu Pro Lys Arg His Tyr      50 55 60 Leu Lys Ala Asp Leu Asp Arg Trp Ala Cys Leu Tyr Glu Val Pro Leu  65 70 75 80 Ile Phe Pro Ala Ser Asn Gln Gly Lys His Trp Asn Ile Gly Leu Asn                  85 90 95 Phe Ser Arg Ala Glu Val Gln Thr Gly Ala Leu Ser Thr Leu Ile Phe             100 105 110 Arg Ala Ile Trp Gly Asp Gly Ile Asp Pro Gly Asp Glu Thr Leu Val         115 120 125 Ala Pro Cys Ala Asp Lys Leu Gly Phe Asp Lys Ser Ala Leu Glu Arg     130 135 140 Ile Ile Ser Thr Asp Ala Ala Thr Glu Arg Tyr Asp Glu Gln Thr His 145 150 155 160 Phe Ala Leu Gln Arg Gly Val Phe Gly Ala Pro Thr Met Phe Leu Ser                 165 170 175 Glu Gln Ile Trp Trp Gly Gln Asp Arg Leu Thr Phe Leu Ala Asn Tyr             180 185 190 Val Gly Gly Leu Pro Arg Asn Pro Ala Ser         195 200    <210> 5 <211> 474 <212> PRT <213> Sphingomonas xenophaga <400> 5 Met Tyr Thr Arg Leu Trp Ile Gly Gly Ala Phe Leu Gln Gly Thr Asp   1 5 10 15 Lys Lys Ser Ile Glu Asp Lys Asn Pro Ala Thr Gly Glu Val Leu Thr              20 25 30 Arg Val Pro Ala Gly Thr Val Lys Asp Ile Leu Arg Ala Val Ala Val          35 40 45 Ala Lys Glu Ala Tyr Ile Lys Trp Ala Thr Ser Gly Ala Ala Glu Arg      50 55 60 Asn Asp Ile Ile Ile His Ala Ala Asp Arg Leu Gln Thr Lys Thr Gln  65 70 75 80 Glu Tyr Ile Thr Val Met Thr Lys Asn Asn Gly Ala Pro Ile Lys Lys                  85 90 95 Ala Ser Val Thr Val Ser Met Ala Ile Asn Val Leu Glu Glu Phe Ala             100 105 110 Gly Leu Pro Ser Asn Leu Gln Gly Gln Thr Ile Pro Thr Asp Thr Ser         115 120 125 Glu Arg Leu Gly Met Thr His Arg Glu Ala Ile Gly Val Ile Leu Asn     130 135 140 Ile Val Pro Trp Asn Val Pro Ala Ile Leu Ala Ile Arg His Ile Ala 145 150 155 160 Phe Pro Leu Ala Thr Gly Asn Thr Phe Val Phe Leu Asn Ala Glu Trp                 165 170 175 Ile Pro Gly Ala Asp Ala Leu Ile Thr Phe Leu Val Glu Glu Ala Asp             180 185 190 Val Pro Asp Gly Val Leu Asn Tyr Val Tyr Gly Asn Gly Ala Pro Glu         195 200 205 Val Ala Asp Ala Leu Thr Gly His Lys Asp Ile Arg Lys Ile Ala Phe     210 215 220 Thr Gly Ser Ser Arg Val Gly Arg His Ile Ala Gln Lys Ala Ser Gln 225 230 235 240 His Ile Lys Arg Tyr Thr Leu Glu Leu Gly Gly Lys Ser Pro Leu Val                 245 250 255 Ile Leu Asp Asp Ala Asp Ile Asp Ala Ala Val Asn Ser Ala Thr Phe             260 265 270 Gly Thr Phe Leu Val Gln Gly Gln Val Cys Leu Ser Ser Asp Arg Leu         275 280 285 Ile Leu His Arg Ser Ile Gly Asp Gln Phe Ala Phe Ala Phe Lys Val     290 295 300 Ala Ala Lys Asn Leu Asn Ser Gly Asp Pro Arg Glu Phe Thr Thr Ile 305 310 315 320 Leu Gly Ala Asn Val Ser Leu Arg Gly Gly Glu Arg Val Asn Gly Tyr                 325 330 335 Ile Asp Asp Ala Leu Asp Glu Gly Ala Thr Val Asp Ala Gly Gly Leu             340 345 350 Ala Glu Gly Ala Leu Ile Pro Ser Thr Ile Phe Asp His Val Thr Ser         355 360 365 Ser Met Arg Leu Tyr Arg Glu Glu Thr Phe Gly Pro Leu Thr Val Val     370 375 380 Phe Arg Cys Asp Gly Glu Glu Glu Ala Ile Glu Leu Ala Asn Thr Asn 385 390 395 400 Ala Tyr Gly Leu Ser Ala Ser Leu Phe Gly Arg Asp Leu Asn Lys Ala                 405 410 415 Leu Ala Val Gly Met Ser Ile Glu Thr Gly Thr Val His Ile Asn Gly             420 425 430 Ser Tyr Val Gln Gly Glu Val Gln Val Pro Tyr Gly Gly Tyr Lys Ala         435 440 445 Ser Gly Phe Gly Arg Phe Glu Ser Arg Glu Val Leu Asp His Phe Thr     450 455 460 Glu Asn Lys Tyr Ile Ser Ala Phe Glu Phe 465 470    <210> 6 <211> 306 <212> PRT <213> Sphingomonas xenophaga <400> 6 Met Ala Leu Thr Gly Ile Leu Arg Pro Gly Tyr Val Gln Leu Arg Val   1 5 10 15 Leu Asp Leu Asp Glu Ala Ile Gln His Tyr Arg Asp Arg Ile Gly Leu              20 25 30 Asn Leu Val Ser Val Glu Gly Gly Arg Ala Phe Phe Gln Ala Phe Asp          35 40 45 Glu Phe Asp Arg His Ser Ile Ile Leu Arg Glu Ala Asp Ser Ala Gly      50 55 60 Leu Asp Arg Met Ala Phe Lys Val Ala Lys Asp Ser Asp Leu Asp His  65 70 75 80 Phe Ala Glu Arg Leu Leu Asp Ala Gly Val Asn Val Asp Val Ile Pro                  85 90 95 Ala Gly Glu Asp Pro Gly Val Gly Arg Lys Ile Arg Phe Asn Ala Pro             100 105 110 Thr Ala His Val Phe Asp Leu Tyr Ala Glu Met Glu Leu Ser Glu Thr         115 120 125 Gly Pro Ala Val Arg Asn Pro Asp Val Trp Ile Ala Glu Pro Arg Gly     130 135 140 Met Arg Ala Thr Arg Phe Asp His Cys Ala Leu Asn Gly Val Asp Ile 145 150 155 160 Ser Ala Ser Ala Lys Ile Phe Val Glu Val Leu Asp Phe Ser Val Thr                 165 170 175 Glu Glu Leu Val Asp Glu Ser Thr Gly Ala Arg Leu Gly Ile Phe Leu             180 185 190 Ser Cys Ser Asn Lys Ala His Asp Val Ala Phe Leu Gly Tyr Pro Glu         195 200 205 Asp Gly Arg Ile His His Thr Ser Phe Asn Leu Glu Ser Trp Asn Asp     210 215 220 Val Gly His Xaa Ala Asp Ile Ile Ser Arg Tyr Asp Ile Ser Leu Asp 225 230 235 240 Ile Gly Pro Thr Arg His Gly Ile Thr Arg Gly Gln Thr Ile Tyr Phe                 245 250 255 Phe Asp Pro Ser Gly Asn Arg Asn Glu Thr Phe Ser Gly Gly Tyr Thr             260 265 270 Tyr Tyr Pro Asp Asn Pro Arg Arg Val Trp Gln Ala Glu Asn Ala Gly         275 280 285 Lys Ala Ile Phe Tyr Tyr Glu Lys Ala Leu Asn Asp Arg Leu Asn Asp     290 295 300 Gly Glu 305    <210> 7 <211> 271 <212> PRT <213> Sphingomonas xenophaga <400> 7 Met Val Arg Cys Thr Thr Phe Glu Arg Gly Lys Ser Cys Glu Leu Lys   1 5 10 15 Asn Arg Arg Gln Ala Gln Ala Thr Leu Ser Lys Thr Ser Phe Leu Ser              20 25 30 Phe Pro Pro Leu Phe Ser Gly Gly Phe Cys Thr Ser Gly Arg Arg Arg          35 40 45 Arg Thr Asn Arg Gly Ala Gln Lys Ala Ala Ser Ser Arg Phe Ala Pro      50 55 60 Ile Ile Gln Ile Gly Lys Ala Arg Ala His Pro Gly Tyr Arg Cys Arg  65 70 75 80 Thr Cys Ser Tyr Ser Cys Phe Phe Arg Arg Arg Gly Leu Val Ser Arg                  85 90 95 Arg Phe Val Pro Glu Glu Phe Arg Ser Leu Ala Val Arg Pro Arg Ser             100 105 110 Val Arg Thr Ala Ala Gly Ala Phe Glu Pro Ala Arg Phe Phe Pro Glu         115 120 125 Glu Arg Cys Arg Pro Leu Pro Gly Cys Ser Phe Pro Glu Ala Ala Cys     130 135 140 Pro Ser Leu Leu His His Ala Phe Ser Ala Gly His Ala Ala Val Lys 145 150 155 160 Ala Arg Arg Leu Pro Gln Pro Phe Arg Pro Val Ile Ser Glu Cys His                 165 170 175 Leu Gln His Gly Val Cys Leu Ala Ala Ala His Ile Gly Ala Ala Arg             180 185 190 Cys Gly Lys Gly Ile Asn Gly Cys Arg Pro Arg Asn Glu Lys Pro Gln         195 200 205 Glu Asn Gly Arg Lys Arg Leu Arg Lys Thr Val Phe Leu Arg Glu Gly     210 215 220 Leu Glu Ala Xaa Ile Lys Arg Ser Gln Gln Ala Gly Ala Pro Arg Arg 225 230 235 240 Pro Ala Tyr Phe Tyr Phe Lys Ala Lys Pro Thr Gly Lys Gly Glu Thr                 245 250 255 Lys Asp Phe Pro Gly Phe Thr Gly Leu Lys Pro Gly Pro Gly Asn             260 265 270    <210> 8 <211> 237 <212> PRT <213> Sphingomonas xenophaga <400> 8 Met Ala Pro Asp Asn Lys Pro Leu Arg Val Thr Arg Thr Cys Tyr Leu   1 5 10 15 Leu Asp Gly Gly His Ser Ile Ser Asn Tyr His Ile Arg Lys Leu Ala              20 25 30 Ile Cys Thr Gln Asn Ile Leu Asp Ile Asp Lys Glu Gly Asn Gly Thr          35 40 45 Leu Arg Ala Leu Ala Arg Tyr Tyr Gly Ala Glu Ser Ser Gly Asn Leu      50 55 60 Lys Gln Ile Lys Asn Ala Ser Glu Arg Leu Tyr Val Lys Gly Lys Gly  65 70 75 80 Lys Arg Pro Ser Thr Ile Thr Gly Thr Trp Val Asp Thr Ile Lys Leu                  85 90 95 Arg Gly Cys Ala Thr Cys Gly Phe Ser Leu Ser Ala Thr Ile Pro Tyr             100 105 110 Leu Thr Phe Leu Thr Gly Gln Val Pro Lys Tyr Ala Leu Ala Asp Val         115 120 125 Gly Arg Leu Ala Arg Ala Leu Leu Arg Gln Phe Pro Thr Val Asp Ser     130 135 140 Thr Gly Arg Trp Leu Ala Ser Glu Ala Asp Asn Gly Ile Lys Lys Trp 145 150 155 160 Ser Leu Lys Gly Asp Arg Gly Pro Asn Gln Phe Ser Tyr Asp Glu Lys                 165 170 175 Ile Ser Gln Phe His Arg Leu Asp Thr Val Thr Gln Ser Ala Lys Glu             180 185 190 Leu Ala Lys Gln Gly Arg Thr Leu Thr Ser Asp Trp Glu Arg Leu Ile         195 200 205 Glu Arg Arg Val Gly Gly Arg Pro Val Val Leu Ile Ala Lys Ser Ala     210 215 220 His Val Tyr Lys Pro Leu Ala Pro Ala Pro Gly Gln Ala 225 230 235 <210> 9 <211> 204 <212> DNA <213> Sphingomonas xenophaga <220> <221> rep_origin <222> (1) .. (204) <400> 9 atccgatgcc agcagacgat cgaactaccg ggcaggaggc ctatgctctt ttatttgttt 60 tccgttttag gtttttggat ttttttacta tttattataa gtaatataag tgtttaaagg 120 ggaagattca gggtaaaagg ggacgattga gggtaaaggg gaggattcag ggtttaaagg 180 ggacgattca ggtgaaataa aata 204

[Brief description of drawings]

FIG. 1 is a chromatogram of T3 strain culture broth by HPLC.

FIG. 2 is a chromatogram obtained by HPLC of a culture solution of the T3 (pSXAc2) strain.

FIG. 3 is a view showing ORFs and restriction enzyme cleavage sites contained in plasmid pSXAc2.

   ─────────────────────────────────────────────────── ─── Continued front page    F-term (reference) 4B024 AA20 BA07 BA08 CA04 CA09                       DA05 EA04 GA11 GA19 HA03                       HA14

Claims (2)

[Claims] 1. A plasmid vector pSXAc2. 2. A plasmid vector containing a replication protein gene derived from the plasmid vector pSXAc2 and a replication origin.