Method for obtaining active insoluble xylose isomerase

Abstract

The invention is in the field of biotechnology and involves recombinant DNA technology. It provides means and methods for obtaining an insoluble active fusion protein comprising xylose isomerase activity. More in particular, the invention relates to a method for obtaining active insoluble xylose isomerase, comprising the expression in a host organism of a recombinant gene encoding a fusion protein comprising a xylose isomerase in combination with a PPIase, thereby obtaining the active insoluble xylose isomerase. It also provides recombinant fusion proteins comprising xylose isomerase activity as well as their use in converting xylose to xylulose and glucose to fructose.

Claims

1. A method for obtaining an active insoluble xylose isomerase, the method comprising: expressing, in E. coli, a recombinant gene encoding a fusion protein; wherein the fusion protein comprises a xylose isomerase linked to SEQ ID NO: 22, thereby obtaining the active insoluble xylose isomerase.

2. The method according to claim 1 wherein SEQ ID NO: 22 is linked to the N-terminus of the xylose isomerase.

3. The method according to claim 1, wherein SEQ ID NO: 22 is linked to the C-terminus of the xylose isomerase.

4. The method according to claim 1, further comprising lysing the host organism after expression of the recombinant gene.

5. The method according to claim 1, further comprising heat treating the expressed fusion protein.

6. The method according to claim 1, wherein the xylose isomerase comprises an amino acid sequence of any one of SEQ ID NOs: 1-18, or an amino acid sequence that is at least 90% identical to an amino acid sequence of any one of SEQ ID NOs: 1-18.

7. The method according to claim 1, wherein the xylose isomerase also has glucose isomerase activity.

8. A recombinant active insoluble xylose isomerase fusion protein, the fusion protein comprising a xylose isomerase linked to SEQ ID NO: 22 produced by the method according to claim 1.

9. The recombinant fusion protein of claim 8, wherein the xylose isomerase also has glucose isomerase activity produced by the method according to claim 1.

10. A method of converting xylose into xylulose, the method comprising: contacting xylose with the recombinant fusion protein of claim 8.

11. A method of converting glucose into fructose, the method comprising: contacting glucose with the recombinant fusion protein of claim 9.

Description

LEGEND TO THE FIGURES

(1) FIG. 1: Diagram showing the relative enzymatic activity recovered from the pellet or supernatant of lysed recombinant cells. After cell lysis, 1 ml of cell lysate of each sample was centrifuged for 2 minutes at 14 000 g in a table centrifuge. Supernatant (soluble proteins) and pellet (insoluble proteins) were collected and used to measure activity of the laccase, pectinase or xylose isomerase fusion proteins.

(2) FIG. 2: Diagram showing the fraction of the total xylose isomerase activity found in the pellet of heated and none heated cell lysates. After cell lysis, 1 ml of cell lysate was heated in a water bath for 30 minutes at 70 degrees Celsius. Thereafter, the heated and non-heated samples were processed as described in example 7.

(3) FIG. 3: Diagram showing the ability of the insoluble enzyme fractions to be reused. Three consecutive rounds of incubation were performed with a xylose solution as the substrate. After one hour of reaction time, the enzymes were recovered by centrifugation and after removal of the supernatant, subjected to a second and third round of incubation with the xylose substrate as described in example 8.

(4) FIG. 4: Phylogenetic tree showing the relationship between the xylose isomerases exemplified herein. The number near the microorganism name is so called branch length, which indicates the average number of changes per aminoacid position in relation to the closest common ancestor depicted by the closest branching point on the diagram.

SUMMARY OF THE INVENTION

(5) We surprisingly observed that xylose isomerases could be obtained in an active and insoluble form when they were produced as a fusion protein with a peptidylprolyl isomerase (PPIase). The invention therefore relates to a method for obtaining active insoluble xylose isomerase, comprising the expression in a host organism of a recombinant gene encoding a fusion protein comprising a xylose isomerase in combination with a PPIase, thereby obtaining the active insoluble xylose isomerase.

(6) The invention also relates to a recombinant fusion protein obtainable by a method as described herein and its use in converting xylose to xylulose or converting glucose to fructose.

DETAILED DESCRIPTION OF THE INVENTION

(7) Peptidylprolyl isomerase (also known as Prolyl isomerase or PPIase) is an enzyme (EC 5.2.1.8) found in both prokaryotes and eukaryotes that interconverts the cis and trans isomers of peptide bonds with the amino acid proline (Fischer G, Schmid F X, Biochemistry 29: 2205-2212 (1990)). Proline has an unusual conformationally restrained peptide bond due to its cyclic structure with its side chain bonded to its secondary amine nitrogen. Most amino acids have a strong energetic preference for the trans peptide bond conformation due to steric hindrance, but proline's unusual structure stabilizes the cis form so that both isomers are populated under biologically relevant conditions. Proteins with prolyl isomerase activity include cyclophilin, FKBPs, and parvulin, although larger proteins can also contain prolyl isomerase domains.

(8) Prolyl isomerase activity was first discovered using a chymotrypsin-based assay. The proteolytic enzyme chymotrypsin has a very high substrate specificity for the four-residue peptide Ala-Ala-Pro-Phe only when the proline peptide bond is in the trans state. Adding chymotrypsin to a solution containing a reporter peptide with this sequence results in the rapid cleavage of about 90% of the peptides, while those peptides with cis proline bondsabout 10% in aqueous solutionare cleaved at a rate limited by uncatalyzed proline isomerization. The addition of a potential prolyl isomerase will accelerate this latter reaction phase if it has prolyl isomerase activity.

(9) Protease-free assays for PPIase activity have also been reported (Zhang et al. J. Bacteriol. 189: 7942-7944 (2007), Janowski et al., Anal. Biochem. 252:299-307 (1997)). Functional PPIase activity assays are commercially available from Selcia, Fyfield business & Research Park, Fyfield Road, Ongar, Essex CM5 0GS United Kingdom.

(10) We constructed recombinant genes, each encoding a fusion protein comprising and consisting essentially of a PPIase polypeptide covalently attached to a protein with xylose isomerase activity. Upon expression of the fusion proteins in a recombinant host organism, the xylose isomerase activity was found in the insoluble pellet obtained after lysis of the cells (FIG. 1).

(11) Hence, the invention relates to a method for obtaining active insoluble xylose isomerase, comprising the expression in a host organism of a recombinant gene encoding a fusion protein comprising a recombinant xylose isomerase and a PPIase.

(12) It appeared that the PPIase part of the fusion protein could be attached to the amino-terminus as well as the carboxy-terminus of the xylose isomerase. Both had the same effect with respect to the recovery of the active insoluble fusion protein. The PPIase part of the fusion protein is also referred to herein as the PPI-tag or the PPIase tag. This is shown in FIG. 1.

(13) This finding was the more surprising since PPIase is known to enhance the solubility of polypeptides, co-expressed in a recombinant host like E. coli (de Marco, A., Microbial Cell Factories 2009, 8:26 doi:10.1186/1475-2859-8-26). It has also been described to increase the solubility of scFvs and Fab fragments (Bothmann H, Plcktuhn A: Nat Biotechnol 1998, 16:376-380, Hayhurst A, Harris WJ. Prot. Expr. Purif. 1999, 15: 336-343, Lin et al., in E. coli. Prot Expr Purif 2008, 59:55-63.)

(14) The term insoluble in this context relates to the solubility of the fusion protein in an aqueous solution, such as phosphate buffered saline. More in particular it relates to solubility in lysis buffer (50 mM Tris-HCl pH7.4, 1% Triton X100, 1 mM CoCl2). A protein is defined herein as being insoluble if 60% or more, such as 70, 80, 85, 90 or even more than 95% of the protein is found in the pellet after incubation at room temperature for 30 minutes and centrifugation at 14.000 g for 2 minutes.

(15) The term active as used herein refers to the enzymatic activity of xylose isomerases or glucose isomerases. Assays for determining such activity are described herein and well known in the art.

(16) In a preferred embodiment, the fusion protein is produced in a bacterium, such as Bacillus or Escherichia. Bacillus subtilis and E. coli are preferred. Production of the fusion protein in other hosts is also possible; yeast and fungal hosts are therein preferred.

(17) In a control experiment, a xylose isomerase gene was expressed in E. coli with and without the PPIase tag. It was found that xylose isomerase activity could be obtained in both experiments. However, whereas the active xylose isomerase expressed without the PPIase tag was exclusively found in the soluble fraction, Xylose isomerase activity of the fusion protein comprising the PPIase tag was predominantly present in the insoluble pellet after lysis of the cells. We repeated this experiment for 18 different xylose isomerases (FIG. 4, Table 1, SEQ ID NO: 1-18)) and found that this was true for all of them.

(18) In another control experiment, we showed that this effect was specific for xylose isomerases. When laccases or a pectinase (SEQ ID NO: 19-21) were expressed with and without a PPIase tag, the laccase or pectinase activity was always found exclusively in the soluble fraction (FIG. 1).

(19) Hence, the invention relates to a method for obtaining active insoluble xylose isomerase, comprising the expression in a host organism of a recombinant gene encoding a fusion protein comprising a xylose isomerase in combination with a PPIase, thereby obtaining the active insoluble xylose isomerase.

(20) We also showed that the effect of the PPIase tag to yield insoluble xylose isomerases, was independent of whether the PPIase tag was attached to the N-terminus or C-terminus of the xylose isomerase. In a representative example we compared the xylose isomerase activity in the soluble and insoluble fractions obtained from a recombinant host, expressing a fusion protein comprising a xylose isomerase according to SEQ ID NO: 1 with an N-terminal and a C-terminal PPIase tag. We found that for both the N-terminal and C-terminal recombinant fusion proteins, xylose isomerase activity was predominantly found in the insoluble fraction (FIG. 1).

(21) Hence, the invention relates to a method as described above wherein the PPIase is attached to either the N-terminus and/or the C-terminus of the xylose isomerase.

(22) We also showed that the effect of obtaining active, insoluble xylose isomerase is independent from the host organism used to produce the fusion protein. Rather, this is an inherent property of the fusion protein, as was shown in the experiments described in example 9. Therein, the supernatant of the cell lysates, containing the residual soluble and active xylose isomerase, was heated to 70 degrees Celsius for 30 minutes at different pH values. It was found that the formation of enzymatically active aggregates can occur in vitro, in the absence of cells, at various pH values and without the initial presence of already existing aggregates.

(23) However, for ease of handling and for economical reasons, Escherichia coli is preferred as the host organism in the present invention.

(24) In another preferred embodiment, the recombinant cells are lysed after expression of the recombinant fusion protein, to recover the active, insoluble fusion protein. Also preferred is when the step of recovering the active, insoluble fusion protein comprises a heat treatment step.

(25) A suitable heat treatment for most of the xylose isomerases exemplified herein is the treatment at 70 degrees Celsius, for 30 minutes. Other temperatures and times may be suitable as well, for example, fusion proteins comprising a xylose isomerase moiety according to SEQ ID NO: 15-18 were best recovered in a method employing a heat treatment step of 50 degrees Celsius for 60 minutes. Using his common technical knowledge and the present disclosure, a person skilled in the art will know how to determine and apply the optimal temperature, time and other conditions for the heat treatment step in obtaining active, insoluble xylose isomerases.

(26) Any PPIase may advantageously be used in the present invention, since all PPIases have a comparable and specific enzymatic activity. In a preferred embodiment, the PPIase is derived from bacterial origine, such as from an enterobacteria. In a particularly preferred embodiment according to the invention, a cyclophilin-type PPIase according to SEQ ID NO: 22 is used, or a PPIase that comprises an amino acid sequence that is at least 90% identical to the amino acid sequence according to SEQ ID NO: 22.

(27) As used herein, the degree of identity between two or more amino acid sequences is equivalent to a function of the number of identical positions shared by the sequences; i.e., % identity=number of identical positions divided by the total number of aligned positions100, excluding gaps, which need to be introduced for optimal alignment of the two sequences, and overhangs. The alignment of two sequences is to be performed over the full length of the polypeptides.

(28) The comparison (aligning) of sequences is a routine task for the skilled person and can be accomplished using standard methods known in the art. For example, a freeware conventionally used for this purpose is Align tool at NCBI recourse http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq &LINK_LOC=align2seq. Other commercial and open software such as Vector NTI are also suitable for this purpose.

(29) We have also shown herein that the effect underlying the invention is not restricted to a particular xylose isomerases according to a particular sequence. The xylose isomerases as exemplified herein show a great diversity in their amino acid sequence (FIG. 4). The most distant examples having only 19% sequence identity to each other. Nevertheless, they all exhibited the same effect of yielding an active and insoluble xylose isomerase after expression as a fusion protein with a PPIase tag.

(30) Accordingly, in a preferred method according to the invention, the xylose isomerase comprises an amino acid sequence selected from the group consisting of a sequence according to SEQ ID NO: 1-18, or an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of a sequence according to SEQ ID NO: 1-18.

(31) Many xylose isomerases also have glucose isomerase activity. Hence, the invention also relates to a method as described above wherein the xylose isomerase also has glucose isomerase activity. Some of the xylose isomerases exemplified herein also have profound glucose isomerase activity, for example XI1, XI2, XI4, XI9 and XI13.

(32) The invention also relates to recombinant fusion proteins obtainable by any of the methods described above. More in particular, the invention relates to a recombinant fusion protein as described above, comprising a recombinant xylose isomerase and a PPIase.

(33) The invention also relates to a recombinant fusion protein as described above, that has xylose isomerase activity and is insoluble.

(34) The invention also relates to a recombinant fusion protein as described above, wherein the PPIase is attached to the N-terminus of the xylose isomerase.

(35) The invention also relates to a recombinant fusion protein as described above, wherein the PPIase is attached to the C-terminus of the xylose isomerase.

(36) The invention also relates to a recombinant fusion protein as described above, produced in E. coli.

(37) The invention also relates to a recombinant fusion protein as described above, wherein the PPIase comprises an amino acid sequence according to SEQ ID NO: 22, or a sequence that is at least 90% identical to the amino acid sequence according to SEQ ID NO: 22.

(38) The invention also relates to a recombinant fusion protein as described above, wherein the xylose isomerase comprises an amino acid sequence selected from the group consisting of sequences according to SEQ ID NO: 1-18, or an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of a sequence according to SEQ ID NO: 1-18.

(39) The invention also relates to a recombinant fusion protein as described above, wherein the xylose isomerase also has glucose isomerase activity.

(40) The active, insoluble xylose isomerases as described herein may advantageously be used in any application wherein the interconversion of xylose to xylulose is advantageous. The same is true for the glucose isomerases described herein.

EXAMPLES

Example 1: Preparation of a Polypeptide Comprising a Polypeptide According to SEQ ID NO: 1-21

(41) The DNA constructs encoding the polypeptides according to SEQ ID NO: 1-21 were designed using codon frequencies optimized for expression in E. coli and commercially synthesized and cloned into a standard plasmid vector pET28a+ for cytoplasmic expression. The resulting recombinant xylose isomerases are referred to as XI1-XI18, Lac1, Lac2, Pect1 respectively.

(42) The same DNA fragments were also cloned into another plasmid vector based on a standard pET28a+ additionally containing a nucleotide sequence encoding peptidyl-prolyl isomerase (PPIase, SEQ ID NO: 22). This resulted in a nucleotide sequence encoding an N-terminal tag to the expressed xylose isomerase protein. The recombinant proteins comprising the PPIase tag at their N-terminus are referred to as PPI-XI1-PPI-XI18 respectively.

(43) Also, DNA encoding SEQ ID NO: 1 or SEQ ID NO: 2 were cloned into a plasmid vector based on a standard pET28a+ additionally containing a nucleotide sequence encoding peptidyl-prolyl isomerase (PPIase, SEQ ID NO: 22) in such a way that this nucleotide sequence encodes an C-terminal tag to the expressed xylose isomerase. The recombinant proteins comprising the PPIase tag at the C-terminus are referred to as XI1-PPI and XI2-PPI respectively.

(44) As a control, a COT A laccase from B. subtilis, a laccase (CuEO) from E. coli and a pectate lyase from B. subtilis (SEQ ID NO: 19-21 resp.) were cloned into a plasmid vector based on a standard pET28a+, additionally containing a nucleotide sequence encoding peptidyl-prolyl isomerase (PPIase, SEQ ID NO: 22) in such a way that this nucleotide sequence encodes an N-terminal tag to the expressed laccases or pecate lyase.

(45) All the recombinant genes were expressed in Escherichia coli BL21(DE3) under the control of the T7-RNA-polymerase promoter. Nucleotide sequences encoding the xylose isomerases according to SEQ ID NO: 1-SEQ ID NO: 18 are provided herein as SEQ ID NO: 23-40 respectively (Table 1).

Example 2: Heterologous Expression of Recombinant Polypeptides

(46) Protein production was carried out in E. coli BL21(DE3) strain according to the plasmid manufacturer protocol available at http://richsingiser.com/4402/Novagen%20pET%20system%20manual.pdf. The incubation temperature for protein production was 30 degrees Celsius, which was found optimal for maximum yield of the active xylose isomerase. Cells were lysed using lysis buffer (50 mM Tris-HCl pH7.4, 1% Triton X100, 1 mM CoCl2) thereby obtaining a cell lysate. The cell lysate was optionally heated at 70 degrees Celsius for 30 min as described herein.

(47) Cell lysates comprising combinant laccases were prepared the same way except that the lysis buffer contained 1 mM CuCl2 instead of 1 mM CoCl2. Recombinant pectinases were prepared the same way except that the lysis buffer contained 1 mM CaCl2) instead of 1 mM CoCl2.

Example 3: Xylose Isomerase Activity Assay

(48) Xylose isomerase activity was determined in a xylose solution (100 mM xylose, 10 mM MOPS pH 8.0, 1 mM MgCl2). Approximately 0.1 activity units/mL of xylose isomerase activity was used. The amount of enzyme was selected so that during the reaction time the product formation remains linear. Reaction was incubated at 70 C for 15 min, unless specified otherwise in the example.

(49) Xylose isomerase activity (isomerization reaction rate) was determined by measuring xylulose level in the reaction mixture according to the protocol described in Schenk and Bisswanger; (A microplate assay for D-xylose/D-glucose isomerase. Enzyme and Microbial Technology (Elsevier Science Inc, N Y, 1998), V22, pp. 721-723.)

(50) Measurement was performed in the linear stage of the reaction course wherein product accumulation is linear with time. Ten-microliter aliquots of the reaction mixture were taken and pipetted into a 96-well plate, 40 ul of water was added resulting in 50 ul sample. In some cases, higher dilution of the reaction mixture with water was used to prepare 50 ul of the diluted sample to match the dynamic range of the method. 150 ul of a freshly prepared 1:1 mixture (v/v) of solution A (0.05% resorcinol in ethanol) and solution B (0.216 g FeNH4(SO4)2*12 H2O in 1 l concentrated HCl) were added. For color development, the plate was incubated at 80 C. for 40 min. The absorbance was measured with a microplate reader (Thermo) at 630 nM.

Example 4: Pectinase Activity Assay

(51) Pectinase (synonym: pectate lyase) activity assay was carried out essentially as described in Takao M, Nakaniwa T, Yoshikawa K, Terashita T, Sakai T., Purification and characterization of thermostable pectate lyase with protopectinase activity from thermophilic Bacillus sp. TS 47. Biosci Biotechnol Biochem. 2000 64:2360-7. In more detail, pectate lyase activity was assayed by measuring the increase in absorbance at 235 nm of the reaction mixture. Polygalacturonic acid (PGA) sodium salt from de-methylated citrus pectin (purchased from MegaZyme) was used as substrate. A reaction mixture containing 1 ml of 0.1% PGA in 10 mM Tris-HCl buffer, pH 8.0 and 0.5 mM CaCl2), and an appropriate amount of enzyme solution was incubated for 30 minutes at 60 degrees C.

(52) The reaction was stopped by placing the mixture in 100 degrees C. (boiling water bath) for 5 min. Pectate lyase activity was calculated from the difference in absorption of the reaction mixture at 235 nm at the start and at the end of the reaction.

(53) One unit of pectate lyase activity was defined as the enzyme amount oxidizing 1 micro mole of substrate per minute. Using absorption coefficient of the unsaturated bond at the 4-5 position of the uronic acid residue at 235 nm 4 600 mol-1cm-1.

Example 5: Laccase Activity Measurement

(54) The term laccase activity is used herein to mean the capability to act as a laccase enzyme, which may be expressed as the maximal initial rate of the specific oxidation reaction. Relative activity was measured by oxidation of syringaldazine. Reaction course was monitored by change in absorbance at 526 nM (extinction coefficient of syringaldazine at 526 nm is 65 000 M-1 cm-1). The appropriate reaction time was determined to provide initial rates of oxidation when color development is linear in time. Syringaldazine concentration in the reaction mixture was 1 mM to provide maximum initial rates (substrate saturation conditions).

(55) Typically, reactions were carried out in 1 ml volume of 50 mM Tris-HCl buffer pH 8, the substrate was preheated to the desired temperature (60 degrees Celsius) and reaction was initiated by the addition of the enzyme. After the reaction time has elapsed, absorbance at 526 nm of the reaction mixtures was determined by a spectrophotometer, and the absorbence of the blank sample (containing no enzyme) was subtracted.

(56) One unit of laccase activity was defined as the enzyme amount oxidising 1 micro mole of substrate per minute.

Example 6: Relative Xylose Isomerase Activity in Supernatant and Pellet of Cell Lysates

(57) After cell lysis, 1 ml of cell lysate comprising xylose isomerases comprising an amino acid according to SEQ ID NO: 1-14 was heated in a water bath for 30 min at 70 C. One ml of cell lysate comprising enzymes comprising an amino acid according to SEQ ID NO: 15-18 was heated in a water bath for 30 min at 50 C. One ml of cell lysate comprising enzymes comprising an amino acid according to SEQ ID NO: 19-21 was heated in a water bath for 30 min at 70 C.

(58) Thereafter, each sample was centrifuged for 2 min at 14.000 g in a table centrifuge. The supernatant (sup 1) was collected and stored at room temperature. The pellet was resuspended in 1 ml of lysis buffer and centrifuged again (to wash away any soluble protein trapped in the pellet). The supernatant was added to the supernatant obtained in the first centrifugation step (sup1) and used to measure activity of the soluble protein. The pellet was re-suspended in 2 ml lysis buffer (the same volume as the total volume of the soluble fraction); this suspension was used to measure insoluble fraction activity.

(59) It was found that the majority of the PPIase-tagged xylose isomerase activity resided in the insoluble fraction, whereas PPIase-tagged other enzymes such as laccases and pectinases were found in the soluble fraction. This is shown in FIG. 1. In a control experiment using the same 21 enzymes without a PPIase tag, all activity was found in the supernatant (soluble fraction).

Example 7: Relative Activity of PPI-Tagged Xylose Isomerases, Laccases and a Pectinase in the Insoluble Fractions of Heated and Non-Heated Cell Lysate

(60) In this example, the effect of heat treatment of the cell lysate was investigated. After cell lysis, a sample of 1 ml of cell lysate comprising PPIase-tagged xylose isomerases, a laccase or a pectinase was heated in a water bath for 30 min at 70 C. After centrifugation for 2 min at 14.000 g, the enzymatic activity in the insoluble fraction was compared to the activity of the insoluble fraction of same sample without the heat treatment.

(61) We observed that heating effectively promotes aggregates formation. Incubation for 30 min in 70 C was sufficient to convert essentially all enzymatic activity to the insoluble fraction in XI samples, whereas in laccase and pectinase samples all activity remained in solution (FIG. 2). No loss of total activity was observed upon aggregation.

Example 8: Repeated Use of Immobilized Xylose Isomerases

(62) To test the ability of the insoluble aggregates of the enzymes to be reused, three consecutive rounds of incubation were performed with xylose solution (100 mM xylose, 10 mM MOPS pH 8.0, 1 mM MgCl2) as the substrate. After one hour of reaction time at 70 C, the enzymes were recovered by centrifugation (2 min at 14000 g) and after removal of the supernatant, subjected to a second round of incubation with the xylose substrate.

(63) For this purpose, fresh xylose substrate was added to the pellets containing the enzymes, pellets were re-suspended and reactions were allowed to continue for another hour. After that, enzymes were recovered again and a third round of incubation with xylose substrate was carried out the same way. Supernatants from all three reactions with each enzyme were analyzed for xylulose concentration to determine enzyme activity.

(64) FIG. 3 shows the enzymatic activities of aggregates of XI1 with N-terminally and C-terminally attached PPI tag as well as XI2-XI4 with N-terminal tag in the three consecutive rounds.

(65) This demonstrates that enzyme aggregates remain fully insoluble under the reaction conditions and can be quantitatively recovered from the mixtures by centrifugation and reused.

Example 9: Formation of Insoluble Aggregates from Soluble Fraction of the Lysate

(66) Soluble fraction of PPI-XI1 lysate containing approximately 30% of the total enzymatic activity was obtained by centrifuging the sample 2 min 14000 g and collecting the supernatant. Aliquots of this sample were diluted five-fold with the following buffers at increasing pH values:

(67) 100 mM sodium succinate buffer pH 5.0 or

(68) 100 mM sodium acetate buffer pH 6.0 or

(69) 100 mM phosphate buffer pH 7.0 or

(70) 100 mM MOPS buffer pH 8.0 or

(71) 100 mM MOPs buffer pH 9.0.

(72) Aliquots of the diluted samples were collected for activity measurement (before heating soluble samples), rest of the volume of the diluted samples (1 mL) was incubated for 30 min at 70 C and centrifuged for 2 min at 14000 g. Supernatant was collected to measure activity in soluble fraction (after heating soluble), pellet was suspended in 1 ml of the same buffer and used to measure activity in the insoluble fraction (after heating insoluble). Virtually all the activity was found in insoluble fraction. Total amount of activity (soluble+insoluble) remained the same after heating as before heating.

(73) This experiment shows that formation of aggregates can occur in vitro, in the absence of cells, at various pH values and without the initial presence of already existing aggregates.

(74) TABLE-US-00001 TABLE1 DNAandaminoacidsequencesasexemplifiedherein.SEQIDNO:1- 18arexyloseisomerasesfromanumberofbacterialspecies,knownintheartperse.SEQ IDNO:19and20areaCOTAlaccasefromBacillussubtilisandEscherichiacoli respectively,SEQIDNO:21isapectatelyase(synonym:pectinase)fromBacillussubtilis. SEQIDNO:22representsaPPlasefromEcoli.SEQIDNO:s23-44areDNAsequences encodingthepolypeptideswithanaminoacidsequenceaccordingtoSEQIDNO:s1-22. SEQIDNO: Sequence 1 MAEFFPEIPKVQFEGKESTNPLAFKFYDPEEIIDGKPLKDHLKFSVAFWHTFVNEGRDPF 60 GDPTADRPWNRYTDPMDKAFARVDALFEFCEKLNIEYFCFHDRDIAPEGKTLRETNKILD 120 KVVERIKERMKDSNVKLLWGTANLFSHPRYMHGAATTCSADVFAYAAAQVKKALEITKEL 180 GGEGYVFWGGREGYETLLNTDLGFELENLARFLRMAVDYAKRIGFTGQFLIEPKPKEPTK 240 HQYDFDVATAYAFLKSHGLDEYFKFNIEANHATLAGHTFQHELRMARILGKLGSIDANQG 300 DLLLGWDTDQFPTNVYDTTLAMYEVIKAGGFTKGGLNFDAKVRRASYKVEDLFIGHIAGM 360 DTFALGFKVAYKLVKDGVLDKFIEEKYRSFREGIGRDIVEGKVDFEKLEEYIIDKETIEL 420 PSGKQEYLESLINSYIVKTILELR 444 2 MSFQPTPEDRFTFGLWTVGWQGRDPFGDATRPALDPVETVQRLAELGAYGVTFHDDDLIP 60 FGSSDTERESHIKRFRQALDATGMTVPMATTNLFTHPVFKDGGFTANDRDVRRYALRKTI 120 GNIDLAAELGAKTYVAWGGREGAESGGAKDVRDALDRMKEAFDLLGEYVTAQGYDLRFAI 180 EPKPNEPRGDILLPTVGHALAFIERLERPELYGVNPEVGHEQMAGLNFPHGIAQALWAGK 240 LFHIDLNGQSGIKYDQDLRFGAGDLRAAFWLVDLLETAGYEGPRHFDFKPPRTEDFDGVW 300 ASAAGCMRNYLILKDRAAAFRADPEVQEALRAARLDQLAQPTAADGLDALLADRAAFEDF 360 DVDAAAARGMAFEHLDQLAMDHLLGARG 388 3 MNKYFENVSKIKYEGPKSNNPYSFKFYNPEEVIDGKTMEEHLRFSIAYWHTFTADGTDQF 60 GKATMQRPWNHYTDPMDIAKARVEAAFEFFDKINAPYFCFHDRDIAPEGDTLRETNKNLD 120 TIVAMIKDYLKTSKTKVLWGTANLFSNPRFVHGASTSCNADVFAYSAAQVKKALEITKEL 180 GGENYVFWGGREGYETLLNTDMEFELDNFARFLHMAVDYAKEIGFEGQFLIEPKPKEPTK 240 HQYDFDVANVLAFLRKYDLDKYFKVNIEANHATLAFHDFQHELRYARINGVLGSIDANTG 300 DMLLGWDTDQFPTDIRMTTLAMYEVIKMGGFDKGGLNFDAKVRRASFEPEDLFLGHIAGM 360 DAFAKGFKVAYKLVKDRVFDKFIEERYASYKDGIGADIVSGKADFRSLEKYALERSQIVN 420 KSGRQELLESILNQYLFAE 439 4 MPFVDHRAQKIRRSKEELLKHMQTFKLDLKFSVGIWYFTPGGGRFHEPYVEQKSIPERIE 60 MAAEMAKFGVKGIEAHYPAEVNEENLHLYKQLEKEAGIRLVAVPLSLFYDKIFEFGSLSN 120 PYEKYRKVAYERLVNGLKLVKEANADICIIWPGIDGYTYSYGHLYYHMWDTFEELVAQAM 180 DEVPGVQVAIEPKPYEPAPNNIYRTTADGILAARDIEARLKNPENLKLLQEGHALVGLNP 240 EVGHVRMGFEDLPYAYARVAREGRLFHTHWNSQPLGNYDQDLNIGVVDWDSTEALLYTLK 300 MVGYQGYFGIDINPERMPVIKAIEINTKVLQIMNERIERLPHDRIIECYFDPENHRGELE 360 LILAENHK 368 5 MSSYRPEPEDKFSFGLWTVGWRGVNTFGDAVRPPLDPAEAVHRLAGLGAYGITFHDDDLI 60 PPGSSAAERDAILGRFRKALDETGLTVPMATVNLFSHPVFRDGGFTSNSRATRRYAIRKA 120 VRAIDLAAELGARTFVCWGGQDGAETEAGKDDRAALERLREAFNLMCGYVREQGYDLRFA 180 VEPKPNEPRGDVLLPTVGHALAFIGELEHPEMVGVNPEVGHEQMAGLNFAHGVAQALWHG 240 KLFHIDLNGQRGVKYDQDLRFGAGDVKEAFFLVDLLERSGYDGPRHFDFKPPRTEDVDGV 300 WESAAACMRNYLILKEKAAAFRADPEVADALAASRVAELSEPTLGTGESLADLLAEDFDV 360 DAAGERGYHFERLDQLAMDHLFGVR 385 6 MPYFDNISTIAYEGPASKNPLAFKFYNPEEKVGDKTMEEHLRFSVAYWHTFTGDGSDPFG 60 AGNMIRPWNKYSGMDLAKARVEAAFEFFEKLNIPFFCFHDVDIAPEGETLKETYKNLDII 120 VDMIEEYMKTSKTKLLWNTANLFTHPRFVHGAATSCNADVFAYAAAKVKKGLEIAKRLGA 180 ENYVFWGGREGYETLLNTDMKLELDNLARFLHMAVDYAKEIGFDGQFLIEPKPKEPTKHQ 240 YDFDVATALAFLQTYGLKDYFKFNIEANHATLAGHTFEHELRVARIHGMLGSVDANQGDM 300 LLGWDTDEFPTDLYSTTLAMYEILKNGGLGRGGLNFDAKVRRGSFEPEDLFYAHIAGMDS 360 FAVGLKVAHRLIEDRVFDEFIEERYKSYTEGIGREIVEGTADFHKLEAHALQLGEIQNQS 420 GRQERLKTLLNQYLLEVCAAR 441 7 MYEPKPEHRFTFGLWTVGNVGRDPFGDAVRERLDPVYVVHKLAELGAYGVNLHDEDLIPR 60 GTPPQERDQIVRRFKKALDETGLKVPMVTANLFSDPAFKDGAFTSPDPWVRAYALRKSLE 120 TMDLGAELGAEIYVVWPGREGAEVEATGKARKVWDWVREALNFMAAYAEDQGYGYRFALE 180 PKPNEPRGDIYFATVGSMLAFIHTLDRPERFGLNPEFAHETMAGLNFVHAVAQALDAGKL 240 FHIDLNDQRMSRFDQDLRFGSENLKAAFFLVDLLESSGYQGPRHFDAHALRTEDEEGVWA 300 FARGCMRTYLILKERAEAFREDPEVKELLAAYYQEDPAALALLGPYSREKAEALKRAELP 360 LEAKRRRGYALERLDQLAVEYLLGVRG 387 8 MYEPKPEHRFTFGLWTVGNVGRDPFGDAVRERLDPVYVGHKLAELGVHGVNLHDEDLIPR 60 GTPPQERDQIVRRFKRALDETGLKVPMVTGNLFSDPGFKDGGFTSRDPWVRAYAFRKSLE 120 TMDLGAELGAEIYVVWPGREGAEVEATGKARKVWDWVREPLNFMAAYAEDQGYGYRFALE 180 PKPNEPRGDIYFATVGSMLALIHTLERPERFGLNPEFAHETMAGLNFVHAVAQALDAGKL 240 LHIDLNGQRMNRFDQDLRFGSENLKAAFLLVDLLESSGYQGPRHFDAHALRTEDEEGVWA 300 FARGCMRTYLILKERAEAFREDPEVKELLAAYYQEDPAALPLMDPYSHEKAEALKRAELP 360 LEAKRHRGYALERLDQLAVEYLLGVRG 387 9 MSYQPTPEDRFTFGLWTVGWQGRDPFGDATRPALDPVETVQRLAELGAHGVTFHDDDLIP 60 FGSSDTERESHIKRFRQALDATGMTVPMATTNLFTHPVFKDGGFTANDRDVRRYALRKTI 120 RNIDLAVELGAKTYVAWGGREGAESGAAKDVRVALDRMKEAFDLLGEYVTSQGYDIRFAI 180 EPKPNEPRGDILLPTVGHALAFIERLERPELYGVNPEVGHEQMAGLNFPHGIAQALWAGK 240 LFHIDLNGQSGIKYDQDLRFGAGDLRAAFWLVDLLESAGYEGPRHFDFKPPRTEDIDGVW 300 ASAAGCMRNYLILKERAAAFRADPEVQEALRASRLDELAQPTAADGVQELLADRTAFEDF 360 DVDAAAARGMAFERLDQLAMDHLLGAR 387 10 MAYFENVDKVVYEGPASENPLAFKFYNPEEKVGDKTMEEHLRFSVAYWHTFVGDGADPFG 60 VGTAIRPWNRYSGMDLAKARVEAAFELFDKLNIPFFCFHDVDIAPEGATLKETYQNLDTI 120 VDMIEEYMKTSKTKLLWNTANLFTHPRFVHGAATSCNADVFAYAAAKVKKGLEIAKRLGA 180 ENYVFWGGREGYETLLNTNMKLELDNLARFLHMAVDYAKEIGFDGQFLIEPKPKEPTKHQ 240 YDFDVATALAFLQTYGLKDYFKFNIEANHATLAGHTFEHELRVARIHGMLGSVDANQGDP 300 LLGWDTDEFPTDLYSTTLAMYEILQNGGLGKGGLNFDAKVRRGSFEPEDLFYAHIAGMDS 360 FAIGLKVAYRLIEDRVFESVVEERYKSYTEGIGRDIIDGKADFHTLEAYALNLRDISNRS 420 GRQERLKTLLNQYLLEVCVAR 441 11 MSVQPTPADHFTFGLWTVGWTGADPFGVATRKNLDPVEAVHKLAELGAYGITFHDNDLIP 60 FDATEAEREKILGDFNQALKDTGLKVPMVTTNLFSHPVFKDGGFTSNDRSIRRFALAKVL 120 HNIDLAAEMGAETFVMWGGREGSEYDGSKDLAAALDRMREGVDTAAGYIKDKGYNLRIAL 180 EPKPNEPRGDIFLPTVGHGLAFIEQLEHGDIVGLNPETGHEQMAGLNFTHGIAQALWAEK 240 LFHIDLNGQRGIKYDQDLVFGHGDLTSAFFTVDLLENGFPNGGPKYTGPRHFDYKPSRTD 300 GYDGVWDSAKANMSMYLLLKERALAFRADPEVQEAMKTSGVFELGETTLNAGESAADLMN 360 DSASFAGFDAEAAAERNFAFIRLNQLAIEHLLGSR 395 12 MEVFKNVPQTIKYEGKDSDNPLAFKYYNPEEKVGGKTMEEHLRFSVAYWHTLTGDGSDPF 60 GMGTMLRPWDTATDPMELAKARVRAAFEFMSKLGVKYFCFHDRDIAPEGRTLAETNKNLD 120 EIVSLIKELMDETGIKLLWGTANLFSNPRFVHGAATSPNADVFAYAAAQVKKAMEITKEL 180 GGENYVFWGGREGYETLLNTDMELEQENFARFLHMAVDYAREIGFEGQFLIEPKPKEPTK 240 HQYDFDAATVISFLKKYDLDKHFKLNIEANHATLAGHTFQHELHVSRINGMLGSVDANQG 300 DLLLGWDTDQFPTNIYETTLAMYEILKNGGLEPGGLNFDAKVRRASFEPVDLFYAHIAGM 360 DAFARGLKVAHKLLESGELEDFISERYKSYRNGIGEKIVKGEVGFKELEDYALNNGKITN 420 VSGRQELLESIVNKYIIEA 439 13 MMAYFPNVSKITYSGKQLKSGLSFNHYNPKELVGGKTMEEQLRFSVAFWHTFTESGTDPF 60 GAGSKIRPWDRFTGMDLAKARVEAAFEFFEKLGNPYFCFHDRDIAPEGDTLRETNKNLDV 120 IVAMIKDYMKTSKVKLLWNTANMFTNPRFVHGAASSCNADVFAYAAAQVKKGLEVGKELG 180 AENYVFWGGREGYETLLNTDLKLEQDNLARFFHMAVDYAKEIGFDAQFLLEPKPKEPTKH 240 QYDFDAATTIAFLKTYDLDQHFKLNLEANHATLAGHTFEHEIRVARTHGLLGSLDANQGD 300 PLLGWDTDEFPTDLYSTTLAMYEVLKNGGLGRGGLNFDAKTRRASFTDEDLFYAHIAGMD 360 SFALGLKVANRLIEDRVFDAFIEERYSSYKEGIGADIVSGKADFKSLENYILDKKEIINQ 420 SGRLEQLKNTLNHYIVQEAYQSVNA 445 14 MNYQPTPEDRFTFGLWTVGWQGRDPFGDATRRALDPVESVRRLAELGAHGVTFHDDDLIP 60 FGSSDSEREEHVKRFRQALDDTGMKVPMATTNLFTHPVFKDGGFTANDRDVRRYALRKTI 120 RNIDLAVELGAETYVAWGGREGAESGGAKDVRDALDRMKEAFDLLGEYVTSQGYDIRFAI 180 EPKPNEPRGDILLPTVGHALAFIERLERPELYGVNPEVGHEQMAGLNFPHGIAQALWAGK 240 LFHIDLNGQNGIKYDQDLRFGAGDLRAAFWLVDLLESAGYSGPRHFDFKPPRTEDFDGVW 300 ASAAGCMRNYLILKERAAAFRADPEVQEALRASRLDELARPTAADGLQALLDDRSAFEEF 360 DVDAAAARGMAFERLDQLAMDHLLGARG 388 15 MQAYFDQLDRVRYEGSKSSNPLAFRHYNPDELVLGKRMEEHLRFAACYWHTFCWNGADMF 60 GVGAFNRPWQQPGEALALAKRKADVAFEFFHKLHVPFYCFHDVDVSPEGASLKEYINNFA 120 QMVDVLAGKQEESGVKLLWGTANCFTNPRYGAGAATNPDPEVFSWAATQVVTAMEATHKL 180 GGENYVLWGGREGYETLLNTDLRQEREQLGRFMQMVVEHKHKIGFQGTLLIEPKPQEPTK 240 HQYDYDAATVYGFLKQFGLEKEIKLNIEANHATLAGHSFHHEIATAIALGLFGSVDANRG 300 DAQLGWDTDQFPNSVEENALVMYEILKAGGFTTGGLNFDAKVRRQSTDKYDLFYGHIGAM 360 DTMALALKIAARMIEDGELDKRIAQRYSGWNSELGQQILKGQMSLADLAKYAQEHHLSPV 420 HQSGRQEQLENLVNHYLFDK 440 16 MREYFANVPKIKYEGKDSKNPLAFKYYNPDEVVGGKTMKEHLRFTLSYWHTLTGAGSDPF 60 GVGTMLRPWDCAEDEMELAKMRMEANFELMDKLGIEYFAFHDRDIAPEGKTLADTNEKLD 120 EIVAYCKELMQKHGKKLLWGTANMFGNPRFVHGAATTCNADVFAYAAAQTKKAMDVTKEL 180 GGENYVFWGGREGYETLLNTDLGLEQDNLARFFQMAVDYAKKIGFTGQFLIEPKPKEPTK 240 HQYDFDVATVLGFLRKYNLEKYFKMNIEANHATLAQHTFQHEVAVARVNGVLGSLDVNQG 300 DPNLGWDTDQFPTNIYDATMVMYEVLKNGGIAPGGLNFDAKTRRASFEPEDLFLSYIAGM 360 DTMAKGLRVAYSLLDDAVLENNTSERYKTFSEGIGKDIVEGKVDFESLEKYALENSVISN 420 KSGRQEYLESVVNQYIFND 439 17 MAQSHSSSVNYFGSVNKVVFEGKASTNPLAFKYYNPQEVIGGKTMKEHLRFSIAYWHTFT 60 ADGTDVFGAATMQRPWDHYKGMDLARARVEAAFEMFEKLDAPFFAFHDRDIAPEGSTLKE 120 TNQNLDIIVGMIKDYMRDSNVKLLWNTANMFTNPRFVHGAATSCNADVFAYAAAQVKKGL 180 ETAKELGAENYVFWGGREGYETLLNTDLKFELDNLARFMHMAVDYAKEIEYTGQFLIEPK 240 PKEPTTHQYDTDAATTIAFLKQYGLDNHFKLNLEANHATLAGHTFEHELRMARVHGLLGS 300 VDANQGHPLLGWDTDEFPTDLYSTTLAMYEILQNGGLGSGGLNFDAKVRRSSFEPDDLVY 360 AHIAGMDAFARGLKVAHKLIEDRVFEDVIQHRYRSFTEGIGLEITEGRANFHTLEQYALN 420 NKTIKNESGRQERLKAILNQYILEV 445 18 MFFKNVGMIEYEGADSENPYAFKYYNPDEYVGGKTMKEHLRFAVAYWHTFDADGKDPFGD 60 GTMFRAWNRLTYPLDKAKARAESAFEFFEKLGVPYFCFHDVDIVDEGATLRETFAYLDQM 120 SSFLKEMMETSRVQLLWNTANMFTHPRYVHGAATSCNADVYAYAAAKVKKGLDIAKELGA 180 ENYVFWGGREGYETLLNTDMKLELENLASFYRMAVEYAREIGFDGQFLIEPKPKEPTKHQ 240 YDFDAATTIAFLETYGLKDHFKLNLEANHATLAGHTFEHELRVAALHDMLGSIDANQGDL 300 LLGWDTDEFPTDLYSAVLAMYEILKAGGFKTGGINFDAKVRRPSFADEDLFHAHIAGMDT 360 YAVGLKVASRLLEDKALDQVIEERYESYTKGIGLEIKEGRTDLKKLAAYALEHDHIENQS 420 GRQERLKATVNRYLLNALREAPRGKETR 448 19 MTLEKFVDALPIPDTLKPVQQTTEKTYYEVTMEECAHQLHRDLPPTRLWGYNGLFPGPTI 60 EVKRNENVYVKWMNNLPSEHFLPIDHTIHHSDSQHEEPEVKTVVHLHGGVTPPDSDGYPE 120 AWFSKDFEQTGPYFKREVYHYPNQQRGATLWYHDHAMALTRLNVYAGLVGAYIIHDPKEK 180 RLKLPSGEYDVPLLITDRTINEDGSLFYPSGPENPSPSLPKPSIVPAFCGDTILVNGKVW 240 PYLEVEPRKYRFRVINASNARTYNLSLDNGGEFIQIGSDGGLLPRSVKLNSFSLAPAERY 300 DIIIDFTAYEGESIILANSEGCGGDANPETDANIMQFRVTKPLAQKDESRKPKYLASYPS 360 VQNERIQNIRTLKLAGTQDEYGRVVQLLNNKRWHDPVTEAPKAGTTEIWSIVNPTQGTHP 420 IHLHLVSFRVLDRRPFDIARYQERGELSYTGPAVPPPPSEKGWKDTIQAHAGEVLRIAVT 480 FGPYSGRYVWHCHILEHEDYDMMRPMDITDPHK 513 20 MQRRDFLKYSVALGVASALPLWNRAVFAAERPTLPIPDLLTTDARNRIQLTIGAGQSTFG 60 GKTATTWGYNGNLLGPAVKLQRGKAVTVDIYNQLTEETTLHWHGLEVPGEVDGGPQGIIP 120 PGGKRSVTLNVDQPAATCWFHPHQHGKTGRQVAMGLAGLVVIEDDEILKLMLPKQWGIDD 180 VPVIVQDKKFSADGQIDYQLDVMTAAVGWFGDILLINGAIYPQHAAPRGWLRLRLLNGCN 240 ARSLNFATSDNRPLYVIASDGGLLPEPVKVSELPVLMGERFEVLVEVNDNKPFDLVTLPV 300 SQMGMAIAPFDKPHPVMRIQPIAISASGALPDTLSSLPALPSLEGLTVRKLQLSMDPMLD 360 MMGMQMLMEKYGDQAMAGMDHSQMMGHMGHGNMNHMNHGGKFDFHHANKINGQAFDMNKP 420 MFAAAKGQYERWVISGVGDMMLHPFHIHGTQFRILSENGKPPAAHRAGWKDTVKVEGNVS 480 EVLVKFNHDAPKEHAYMAHCHLLEHEDTGMMLGFTVSDP 519 21 MKELGHEVLKPYDGWAAYGEGTTGGAMASPQNVFVVTNRTELIQALGGNNHTNQYNSVPK 60 IIYVKGTIDLNVDDNNQPVGPDFYKDPHFDFEAYLREYDPATWGKKEVEGPLEEARVRSQ 120 KKQKDRIMVYVGSNTSIIGVGKDAKIKGGGFLIKNVDNVIIRNIEFEAPLDYFPEWDPTD 180 GTLGEWNSEYDSISIEGSSHIWIDHNIFTDGDHPDRSLGTYFGRPFQQHDGALDIKNSSD 240 FITISYNVFTNHDKVTLIGASDSRMADSGHLRVTLHHNYYKNVTQRLPRVRFGQVHIYNN 300 YYEFSNLADYDFQYAWGVGVFSQIYAQNNYFSFDWDIDPSLIIKVWSKNEESMYETGTIV 360 DLPNGRRYIDLVASYNESNTLQLKKEVTWKPMFYHVIHPTPSVPALVKAKAGAGNLH 417 22 MVTFHTNHGDIVIKTFDDKAPETVKNFLDYCREGFYNNTIFHRVINGFMIQGGGFEPGMK 60 QKATKEPIKNEANNGLKNTRGTLAMARTQAPHSATAQFFINVVDNDFLNFSGESLQGWGY 120 CVFAEVVDGMDVVDKIKGVATGRSGMHQDVPKEDVIIESVTVSEGTSENLYFQGA 175 23 atggccgaattttttcctgagattccgaaaattcagttcgaaggtaaagaaagcaccaat 60 ccgctggcatttcgtttttatgatccgaacgaagtgattgatggtaaaccgctgaaagat 120 cacctgaaatttagcgttgcattttggcatacctttgtgaatgaaggtcgtgatccgttt 180 ggtgatccgaccgcagaacgtccgtggaatcgttttagcgatccgatggataaagcattt 240 gcacgtgttgatgcactgtttgaattttgcgaaaaactgaacatcgagtatttctgcttt 300 cacgatcgcgatattgcaccggaaggtaaaaccctgcgtgaaaccaacaaaattctggat 360 aaagtggtggaacgcatcaaagaacgtatgaaagatagcaatgttaaactgctgtggggc 420 accgcaaacctgtttagccatccgcgttatatgcatggtgcagcaaccacctgtagcgca 480 gatgtttttgcctatgcagcagcacaggttaaaaaagcactggaaatcaccaaagaactg 540 ggtggtgaaggttatgttttttggggtggtcgtgaaggctatgaaacactgctgaatacc 600 gatctgggtctggaactggaaaatctggcacgttttctgcgtatggcagttgaatatgcg 660 aaaaaaatcggttttaccggtcagtttctgattgaaccgaaaccgaaagaaccgaccaaa 720 caccagtatgattttgatgttgcaaccgcctatgcctttctgaaaaatcatggtctggat 780 gagtacttcaaattcaacattgaagcaaatcatgcaaccctggcaggtcatacctttcag 840 catgaactgcgcatggcacgcattctgggtaaactgggtagcattgatgccaatcagggt 900 gatctgctgctgggttgggatacagatcagtttccgaccaacatttatgataccaccctg 960 gcaatgtatgaagtgattaaagccggtggttttaccaaaggtggtctgaattttgatgca 1020 aaagttcgtcgtgccagctataaagttgaggacctgtttattggtcatatcgcaggtatg 1080 gatacctttgcactgggctttaaaatcgcatataaactggcaaaagatggcgtgttcgat 1140 aaattcatcgaggaaaaatatcgcagcttcaaagaaggcattggcaaagaaattgttgag 1200 ggcaaaaccgactttgagaaactggaagaatacatcatcgacaaagaagatattgaactg 1260 ccgagcggcaaacaagaatatctggaaagcctgctgaacagctatatcgttaaaaccatt 1320 gcagaactgcgctaa 1335 24 atgagctttcagccgaccccggaagatcgctttacctttggcctgtggaccgtgggctgg 60 cagggccgcgatccgtttggcgatgcgacccgcccggcgctggatccggtggaaaccgtg 120 cagcgcctggcggaactgggcgcgtatggcgtgacctttcatgatgatgatctgattccg 180 tttggcagcagcgataccgaacgcgaaagccatattaaacgctttcgccaggcgctggat 240 gcgaccggcatgaccgtgccgatggcgaccaccaacctgtttacccatccggtgtttaaa 300 gatggcggctttaccgcgaacgatcgcgatgtgcgccgctatgcgctgcgcaaaaccatt 360 ggcaacattgatctggcggcggaactgggcgcgaaaacctatgtggcgtggggcggccgc 420 gaaggcgcggaaagcggcggcgcgaaagatgtgcgcgatgcgctggatcgcatgaaagaa 480 gcgtttgatctgctgggcgaatatgtgaccgcgcagggctatgatctgcgctttgcgatt 540 gaaccgaaaccgaacgaaccgcgcggcgatattctgctgccgaccgtgggccatgcgctg 600 gcgtttattgaacgcctggaacgcccggaactgtatggcgtgaacccggaagtgggccat 660 gaacagatggcgggcctgaactttccgcatggcattgcgcaggcgctgtgggcgggcaaa 720 ctgtttcatattgatctgaacggccagagcggcattaaatatgatcaggatctgcgcttt 780 ggcgcgggcgatctgcgcgcggcgttttggctggtggatctgctggaaaccgcgggctat 840 gaaggcccgcgccattttgattttaaaccgccgcgcaccgaagattttgatggcgtgtgg 900 gcgagcgcggcgggctgcatgcgcaactatctgattctgaaagatcgcgcggcggcgttt 960 cgcgcggatccggaagtgcaggaagcgctgcgcgcggcgcgcctggatcagctggcgcag 1020 ccgaccgcggcggatggcctggatgcgctgctggcggatcgcgcggcgtttgaagatttt 1080 gatgtggatgcggcggcggcgcgcggcatggcgtttgaacatctggatcagctggcgatg 1140 gatcatctgctgggcgcgcgcggctaa 1167 25 atgaacaaatattttgaaaacgtgagcaaaattaaatatgaaggcccgaaaagcaacaac 60 ccgtatagctttaaattttataacccggaagaagtgattgatggcaaaaccatggaagaa 120 catctgcgctttagcattgcgtattggcatacctttaccgcggatggcaccgatcagttt 180 ggcaaagcgaccatgcagcgcccgtggaaccattataccgatccgatggatattgcgaaa 240 gcgcgcgtggaagcggcgtttgaattttttgataaaattaacgcgccgtatttttgcttt 300 catgatcgcgatattgcgccggaaggcgataccctgcgcgaaaccaacaaaaacctggat 360 accattgtggcgatgattaaagattatctgaaaaccagcaaaaccaaagtgctgtggggc 420 accgcgaacctgtttagcaacccgcgctttgtgcatggcgcgagcaccagctgcaacgcg 480 gatgtgtttgcgtatagcgcggcgcaggtgaaaaaagcgctggaaattaccaaagaactg 540 ggcggcgaaaactatgtgttttggggcggccgcgaaggctatgaaaccctgctgaacacc 600 gatatggaatttgaactggataactttgcgcgctttctgcatatggcggtggattatgcg 660 aaagaaattggctttgaaggccagtttctgattgaaccgaaaccgaaagaaccgaccaaa 720 catcagtatgattttgatgtggcgaacgtgctggcgtttctgcgcaaatatgatctggat 780 aaatattttaaagtgaacattgaagcgaaccatgcgaccctggcgtttcatgattttcag 840 catgaactgcgctatgcgcgcattaacggcgtgctgggcagcattgatgcgaacaccggc 900 gatatgctgctgggctgggataccgatcagtttccgaccgatattcgcatgaccaccctg 960 gcgatgtatgaagtgattaaaatgggcggctttgataaaggcggcctgaactttgatgcg 1020 aaagtgcgccgcgcgagctttgaaccggaagatctgtttctgggccatattgcgggcatg 1080 gatgcgtttgcgaaaggctttaaagtggcgtataaactggtgaaagatcgcgtgtttgat 1140 aaatttattgaagaacgctatgcgagctataaagatggcattggcgcggatattgtgagc 1200 ggcaaagcggattttcgcagcctggaaaaatatgcgctggaacgcagccagattgtgaac 1260 aaaagcggccgccaggaactgctggaaagcattctgaaccagtatctgtttgcggaataa 1320 26 atgccgtttgttgatcatcgtgcacagaaaattcgtcgcagcaaagaagaactgctgaaa 60 catatgcagaccttcaaactggatctgaaatttagcgtgggcatctggtattttacaccg 120 ggtggtggtcgttttcatgaaccgtatgttgaacagaaaagcattccggaacgtattgaa 180 atggcagcagaaatggcaaaatttggcgtgaaaggtattgaagcacattatccggctgaa 240 gtgaatgaagaaaatctgcacctgtataaacagctggaaaaagaagcaggtattcgtctg 300 gttgcagttccgctgagcctgttttatgataaaatctttgaatttggcagcctgagcaac 360 ccgtatgaaaaatatcgtaaagttgcctatgaacgcctggtgaatggtctgaaactggtt 420 aaagaagcaaacgccgatatttgcattatttggcctggtattgatggctatacctatagc 480 tatggtcacctgtattatcacatgtgggatacctttgaagaactggttgcacaggcaatg 540 gatgaagttccgggtgttcaggttgcaattgaaccgaaaccgtatgaaccggcaccgaat 600 aacatttatcgtaccaccgcagatggtattctggcagcacgtgatattgaagcgcgtctg 660 aaaaatccggaaaacctgaaactgctgcaagaaggtcacgcactggttggtctgaatccg 720 gaagttggtcatgttcgtatgggttttgaagatctgccgtatgcatatgcccgtgttgca 780 cgtgaaggtcgtctgtttcatacccattggaatagccagccgctgggtaattatgatcag 840 gatctgaatattggtgtggtggattgggatagcaccgaagcactgctgtataccctgaaa 900 atggttggttatcagggctattttggcatcgatatcaatccggaacgcatgccggttatt 960 aaagccattgaaattaacaccaaagtgctgcagattatgaacgaacgcattgaacgtctg 1020 ccgcatgatcgtattattgagtgttattttgaccctgagaatcatcgtggtgaactggaa 1080 ctgattctggccgaaaatcataaataa 1107 27 atgagcagctatcgtccggaaccggaagataaattcagctttggtctgtggaccgttggt 60 tggcgtggtgttaatacctttggtgatgcagttcgtccgcctctggaccctgcagaagca 120 gttcatcgtctggcaggtctgggtgcatatggtattacctttcatgatgatgatctgatt 180 ccgcctggtagcagcgcagcagaacgtgatgcaattctgggtcgttttcgtaaagcactg 240 gatgaaaccggtctgaccgttccgatggcaaccgttaacctgtttagccatccggttttt 300 cgtgatggtggttttaccagcaatagccgtgcaacccgtcgttatgcaattcgcaaagca 360 gttcgtgcaattgatctggcagccgaactgggtgcacgtacctttgtttgttggggtggt 420 caggatggtgcagaaaccgaagcaggtaaagatgatcgtgcagcactggaacgtctgcgt 480 gaagcatttaatctgatgtgtggttatgttcgtgaacagggttatgatctgcgttttgca 540 gttgaaccgaaaccgaatgaaccgcgtggtgatgtgctgctgccgaccgtgggtcatgca 600 ctggcatttattggtgaactggaacatccggaaatggttggtgtgaatccggaagttggt 660 catgagcagatggcaggcctgaattttgcacatggtgttgcacaggcactgtggcatggt 720 aaactgtttcatattgatctgaatggtcagcgtggcgtgaaatatgatcaggatctgcgc 780 tttggtgccggtgatgttaaagaagcattttttctggttgatctgctggaacgtagcggt 840 tatgatggtccgcgtcattttgattttaaaccgcctcgtaccgaagatgttgatggtgtt 900 tgggaaagcgcagccgcatgtatgcgtaattatctgattctgaaagaaaaagccgcagca 960 tttcgtgccgatcctgaagttgcagatgccctggcagcaagccgtgttgcagaactgagc 1020 gaaccgaccctgggcaccggtgaaagcctggcagacctgctggcggaagattttgatgtg 1080 gatgcagccggtgaacgtggttatcattttgaacgtctggatcagctggcaatggatcac 1140 ctgtttggtgttcgttaa 1158 28 atgccgtattttgataacattagcaccattgcgtatgaaggcccggcgagcaaaaacccg 60 ctggcgtttaaattttataacccggaagaaaaagtgggcgataaaaccatggaagaacat 120 ctgcgctttagcgtggcgtattggcatacctttaccggcgatggcagcgatccgtttggc 180 gcgggcaacatgattcgcccgtggaacaaatatagcggcatggatctggcgaaagcgcgc 240 gtggaagcggcgtttgaattttttgaaaaactgaacattccgtttttttgctttcatgat 300 gtggatattgcgccggaaggcgaaaccctgaaagaaacctataaaaacctggatattatt 360 gtggatatgattgaagaatatatgaaaaccagcaaaaccaaactgctgtggaacaccgcg 420 aacctgtttacccatccgcgctttgtgcatggcgcggcgaccagctgcaacgcggatgtg 480 tttgcgtatgcggcggcgaaagtgaaaaaaggcctggaaattgcgaaacgcctgggcgcg 540 gaaaactatgtgttttggggcggccgcgaaggctatgaaaccctgctgaacaccgatatg 600 aaactggaactggataacctggcgcgctttctgcatatggcggtggattatgcgaaagaa 660 attggctttgatggccagtttctgattgaaccgaaaccgaaagaaccgaccaaacatcag 720 tatgattttgatgtggcgaccgcgctggcgtttctgcagacctatggcctgaaagattat 780 tttaaatttaacattgaagcgaaccatgcgaccctggcgggccatacctttgaacatgaa 840 ctgcgcgtggcgcgcattcatggcatgctgggcagcgtggatgcgaaccagggcgatatg 900 ctgctgggctgggataccgatgaatttccgaccgatctgtatagcaccaccctggcgatg 960 tatgaaattctgaaaaacggcggcctgggccgcggcggcctgaactttgatgcgaaagtg 1020 cgccgcggcagctttgaaccggaagatctgttttatgcgcatattgcgggcatggatagc 1080 tttgcggtgggcctgaaagtggcgcatcgcctgattgaagatcgcgtgtttgatgaattt 1140 attgaagaacgctataaaagctataccgaaggcattggccgcgaaattgtggaaggcacc 1200 gcggattttcataaactggaagcgcatgcgctgcagctgggcgaaattcagaaccagagc 1260 ggccgccaggaacgcctgaaaaccctgctgaaccagtatctgctggaagtgtgcgcggcg 1320 cgctaa 1326 29 atgtatgaaccgaaaccggaacatcgctttacctttggcctgtggaccgtgggcaacgtg 60 ggccgcgatccgtttggcgatgcggtgcgcgaacgcctggatccggtgtatgtggtgcat 120 aaactggcggaactgggcgcgtatggcgtgaacctgcatgatgaagatctgattccgcgc 180 ggcaccccgccgcaggaacgcgatcagattgtgcgccgctttaaaaaagcgctggatgaa 240 accggcctgaaagtgccgatggtgaccgcgaacctgtttagcgatccggcgtttaaagat 300 ggcgcgtttaccagcccggatccgtgggtgcgcgcgtatgcgctgcgcaaaagcctggaa 360 accatggatctgggcgcggaactgggcgcggaaatttatgtggtgtggccgggccgcgaa 420 ggcgcggaagtggaagcgaccggcaaagcgcgcaaagtgtgggattgggtgcgcgaagcg 480 ctgaactttatggcggcgtatgcggaagatcagggctatggctatcgctttgcgctggaa 540 ccgaaaccgaacgaaccgcgcggcgatatttattttgcgaccgtgggcagcatgctggcg 600 tttattcataccctggatcgcccggaacgctttggcctgaacccggaatttgcgcatgaa 660 accatggcgggcctgaactttgtgcatgcggtggcgcaggcgctggatgcgggcaaactg 720 tttcatattgatctgaacgatcagcgcatgagccgctttgatcaggatctgcgctttggc 780 agcgaaaacctgaaagcggcgttttttctggtggatctgctggaaagcagcggctatcag 840 ggcccgcgccattttgatgcgcatgcgctgcgcaccgaagatgaagaaggcgtgtgggcg 900 tttgcgcgcggctgcatgcgcacctatctgattctgaaagaacgcgcggaagcgtttcgc 960 gaagatccggaagtgaaagaactgctggcggcgtattatcaggaagatccggcggcgctg 1020 gcgctgctgggcccgtatagccgcgaaaaagcggaagcgctgaaacgcgcggaactgccg 1080 ctggaagcgaaacgccgccgcggctatgcgctggaacgcctggatcagctggcggtggaa 1140 tatctgctgggcgtgcgcggctaa 1164 30 atgtatgaaccgaaaccggaacatcgctttacctttggcctgtggaccgtgggcaacgtg 60 ggccgcgatccgtttggcgatgcggtgcgcgaacgcctggatccggtgtatgtgggccat 120 aaactggcggaactgggcgtgcatggcgtgaacctgcatgatgaagatctgattccgcgc 180 ggcaccccgccgcaggaacgcgatcagattgtgcgccgctttaaacgcgcgctggatgaa 240 accggcctgaaagtgccgatggtgaccggcaacctgtttagcgatccgggctttaaagat 300 ggcggctttaccagccgcgatccgtgggtgcgcgcgtatgcgtttcgcaaaagcctggaa 360 accatggatctgggcgcggaactgggcgcggaaatttatgtggtgtggccgggccgcgaa 420 ggcgcggaagtggaagcgaccggcaaagcgcgcaaagtgtgggattgggtgcgcgaaccg 480 ctgaactttatggcggcgtatgcggaagatcagggctatggctatcgctttgcgctggaa 540 ccgaaaccgaacgaaccgcgcggcgatatttattttgcgaccgtgggcagcatgctggcg 600 ctgattcataccctggaacgcccggaacgctttggcctgaacccggaatttgcgcatgaa 660 accatggcgggcctgaactttgtgcatgcggtggcgcaggcgctggatgcgggcaaactg 720 ctgcatattgatctgaacggccagcgcatgaaccgctttgatcaggatctgcgctttggc 780 agcgaaaacctgaaagcggcgtttctgctggtggatctgctggaaagcagcggctatcag 840 ggcccgcgccattttgatgcgcatgcgctgcgcaccgaagatgaagaaggcgtgtgggcg 900 tttgcgcgcggctgcatgcgcacctatctgattctgaaagaacgcgcggaagcgtttcgc 960 gaagatccggaagtgaaagaactgctggcggcgtattatcaggaagatccggcggcgctg 1020 ccgctgatggatccgtatagccatgaaaaagcggaagcgctgaaacgcgcggaactgccg 1080 ctggaagcgaaacgccatcgcggctatgcgctggaacgcctggatcagctggcggtggaa 1140 tatctgctgggcgtgcgcggctaa 1164 31 atgagctatcagccgaccccggaagatcgctttacctttggcctgtggaccgtgggctgg 60 cagggccgcgatccgtttggcgatgcgacccgcccggcgctggatccggtggaaaccgtg 120 cagcgcctggcggaactgggcgcgcatggcgtgacctttcatgatgatgatctgattccg 180 tttggcagcagcgataccgaacgcgaaagccatattaaacgctttcgccaggcgctggat 240 gcgaccggcatgaccgtgccgatggcgaccaccaacctgtttacccatccggtgtttaaa 300 gatggcggctttaccgcgaacgatcgcgatgtgcgccgctatgcgctgcgcaaaaccatt 360 cgcaacattgatctggcggtggaactgggcgcgaaaacctatgtggcgtggggcggccgc 420 gaaggcgcggaaagcggcgcggcgaaagatgtgcgcgtggcgctggatcgcatgaaagaa 480 gcgtttgatctgctgggcgaatatgtgaccagccagggctatgatattcgctttgcgatt 540 gaaccgaaaccgaacgaaccgcgcggcgatattctgctgccgaccgtgggccatgcgctg 600 gcgtttattgaacgcctggaacgcccggaactgtatggcgtgaacccggaagtgggccat 660 gaacagatggcgggcctgaactttccgcatggcattgcgcaggcgctgtgggcgggcaaa 720 ctgtttcatattgatctgaacggccagagcggcattaaatatgatcaggatctgcgcttt 780 ggcgcgggcgatctgcgcgcggcgttttggctggtggatctgctggaaagcgcgggctat 840 gaaggcccgcgccattttgattttaaaccgccgcgcaccgaagatattgatggcgtgtgg 900 gcgagcgcggcgggctgcatgcgcaactatctgattctgaaagaacgcgcggcggcgttt 960 cgcgcggatccggaagtgcaggaagcgctgcgcgcgagccgcctggatgaactggcgcag 1020 ccgaccgcggcggatggcgtgcaggaactgctggcggatcgcaccgcgtttgaagatttt 1080 gatgtggatgcggcggcggcgcgcggcatggcgtttgaacgcctggatcagctggcgatg 1140 gatcatctgctgggcgcgcgctaa 1164 32 atggcctattttgagaacgtggataaagtggtttatgaaggtccggcaagcgaaaatccg 60 ctggcctttaaattctataacccggaagaaaaagtgggcgacaaaacaatggaagaacat 120 ctgcgttttagcgttgcatattggcatacctttgttggtgatggtgcagatccgtttggt 180 gttggcaccgcaattcgtccgtggaatcgttatagcggtatggatctggcaaaagcacgt 240 gttgaagcagcatttgaactgttcgataaactgaacatcccgtttttttgcttccacgat 300 gttgatattgcaccggaaggtgcaaccctgaaagaaacctatcagaatctggataccatc 360 gtggatatgatcgaagagtatatgaaaaccagcaaaaccaaactgctgtggaataccgca 420 aacctgtttacccatccgcgttttgttcatggtgcagcaaccagctgtaatgcagatgtt 480 tttgcctatgcagcagccaaagttaaaaaaggtctggaaattgcaaaacgtctgggtgcc 540 gaaaactatgttttttggggtggtcgtgaaggttatgaaaccctgctgaataccaatatg 600 aaactggaactggataatctggcacgttttctgcacatggcagttgattatgcaaaagaa 660 attggtttcgatggccagtttctgattgaaccgaaaccgaaagaaccgaccaaacaccag 720 tatgattttgatgttgcaaccgcactggcatttctgcagacctatggtctgaaagattac 780 ttcaaatttaacatcgaagccaaccatgccaccctggcaggtcatacatttgaacatgaa 840 ctgcgtgttgcacgtattcatggtatgctgggtagcgttgatgcaaatcagggtgatccg 900 ctgctgggttgggataccgatgaatttccgaccgatctgtatagcaccacactggcaatg 960 tatgaaattctgcaaaatggtggtctgggtaaaggtggtctgaattttgatgccaaagtt 1020 cgtcgtggtagctttgaacctgaggacctgttttatgcacatattgcaggtatggatagc 1080 tttgcaattggcctgaaagttgcatatcgcctgattgaagatcgtgtttttgaaagcgtt 1140 gtggaagaacgctataaaagctataccgaaggtattggtcgcgatattattgatggtaaa 1200 gccgattttcataccctggaagcctatgcactgaatctgcgtgatatttcaaatcgtagc 1260 ggtcgtcaagaacgtctgaaaacactgctgaaccagtatctgctggaagtttgtgttgcc 1320 cgttaa 1326 33 atgagcgtgcagccgaccccggcggatcattttacctttggcctgtggaccgtgggctgg 60 accggcgcggatccgtttggcgtggcgacccgcaaaaacctggatccggtggaagcggtg 120 cataaactggcggaactgggcgcgtatggcattacctttcatgataacgatctgattccg 180 tttgatgcgaccgaagcggaacgcgaaaaaattctgggcgattttaaccaggcgctgaaa 240 gataccggcctgaaagtgccgatggtgaccaccaacctgtttagccatccggtgtttaaa 300 gatggcggctttaccagcaacgatcgcagcattcgccgctttgcgctggcgaaagtgctg 360 cataacattgatctggcggcggaaatgggcgcggaaacctttgtgatgtggggcggccgc 420 gaaggcagcgaatatgatggcagcaaagatctggcggcggcgctggatcgcatgcgcgaa 480 ggcgtggataccgcggcgggctatattaaagataaaggctataacctgcgcattgcgctg 540 gaaccgaaaccgaacgaaccgcgcggcgatatttttctgccgaccgtgggccatggcctg 600 gcgtttattgaacagctggaacatggcgatattgtgggcctgaacccggaaaccggccat 660 gaacagatggcgggcctgaactttacccatggcattgcgcaggcgctgtgggcggaaaaa 720 ctgtttcatattgatctgaacggccagcgcggcattaaatatgatcaggatctggtgttt 780 ggccatggcgatctgaccagcgcgttttttaccgtggatctgctggaaaacggctttccg 840 aacggcggcccgaaatataccggcccgcgccattttgattataaaccgagccgcaccgat 900 ggctatgatggcgtgtgggatagcgcgaaagcgaacatgagcatgtatctgctgctgaaa 960 gaacgcgcgctggcgtttcgcgcggatccggaagtgcaggaagcgatgaaaaccagcggc 1020 gtgtttgaactgggcgaaaccaccctgaacgcgggcgaaagcgcggcggatctgatgaac 1080 gatagcgcgagctttgcgggctttgatgcggaagcggcggcggaacgcaactttgcgttt 1140 attcgcctgaaccagctggcgattgaacatctgctgggcagccgctaa 1188 34 atggaagtgtttaaaaacgttccgcagaccatcaaatatgagggtaaagatagcgataat 60 ccgctggcctttaaatactataacccggaagaaaaagtgggtggcaaaacaatggaagaa 120 catctgcgttttagcgttgcatattggcataccctgaccggtgatggtagcgatccgttt 180 ggtatgggcaccatgctgcgtccgtgggataccgcaaccgatccgatggaactggcaaaa 240 gcacgtgttcgtgcagcatttgaattcatgagcaaactgggtgtgaaatacttttgcttt 300 catgatcgtgatattgcaccggaaggtcgtaccctggcagaaaccaataaaaacctggat 360 gaaattgtgagcctgatcaaagaactgatggatgaaaccggtattaaactgctgtggggc 420 accgcaaacctgtttagcaatccgcgttttgttcatggtgcagcaaccagcccgaatgca 480 gatgtttttgcatatgccgcagcacaggttaaaaaagcaatggaaatcacaaaagaactg 540 ggtggcgaaaactatgttttttggggtggtcgtgaaggttatgaaaccctgctgaatacc 600 gatatggaactggaacaagaaaactttgcacgctttctgcatatggcagttgattatgca 660 cgtgaaattggttttgaaggccagtttctgattgaaccgaaaccgaaagaaccgaccaaa 720 caccagtatgattttgatgcagccaccgttattagctttctgaaaaaatacgatctggac 780 aaacacttcaaactgaacattgaagcaaatcatgcaacactggcaggtcatacctttcag 840 catgaactgcatgttagccgtattaatggtatgctgggtagcgttgatgcaaatcagggt 900 gatctgctgctgggttgggatacagatcagtttccgaccaatatctatgaaaccaccctg 960 gccatgtatgagatcctgaaaaatggtggtctggaaccgggtggtctgaactttgatgcc 1020 aaagttcgtcgtgccagctttgaaccggttgacctgttttatgcacatattgcaggtatg 1080 gatgcatttgcacgcggtctgaaagttgcacacaaactgctggaaagcggtgaactggaa 1140 gattttatcagcgaacgctataaaagctatcgcaatggtattggcgagaaaattgttaaa 1200 ggtgaggtgggctttaaagagctggaagactacgcactgaataacggcaaaatcaccaat 1260 gttagcggtcgtcaagagctgctggaatcaattgtgaacaaatatatcatcgaagcctaa 1320 35 atgatggcgtattttccgaacgtgagcaaaattacctatagcggcaaacagctgaaaagc 60 ggcctgagctttaaccattataacccgaaagaactggtgggcggcaaaaccatggaagaa 120 cagctgcgctttagcgtggcgttttggcatacctttaccgaaagcggcaccgatccgttt 180 ggcgcgggcagcaaaattcgcccgtgggatcgctttaccggcatggatctggcgaaagcg 240 cgcgtggaagcggcgtttgaattttttgaaaaactgggcaacccgtatttttgctttcat 300 gatcgcgatattgcgccggaaggcgataccctgcgcgaaaccaacaaaaacctggatgtg 360 attgtggcgatgattaaagattatatgaaaaccagcaaagtgaaactgctgtggaacacc 420 gcgaacatgtttaccaacccgcgctttgtgcatggcgcggcgagcagctgcaacgcggat 480 gtgtttgcgtatgcggcggcgcaggtgaaaaaaggcctggaagtgggcaaagaactgggc 540 gcggaaaactatgtgttttggggcggccgcgaaggctatgaaaccctgctgaacaccgat 600 ctgaaactggaacaggataacctggcgcgcttttttcatatggcggtggattatgcgaaa 660 gaaattggctttgatgcgcagtttctgctggaaccgaaaccgaaagaaccgaccaaacat 720 cagtatgattttgatgcggcgaccaccattgcgtttctgaaaacctatgatctggatcag 780 cattttaaactgaacctggaagcgaaccatgcgaccctggcgggccatacctttgaacat 840 gaaattcgcgtggcgcgcacccatggcctgctgggcagcctggatgcgaaccagggcgat 900 ccgctgctgggctgggataccgatgaatttccgaccgatctgtatagcaccaccctggcg 960 atgtatgaagtgctgaaaaacggcggcctgggccgcggcggcctgaactttgatgcgaaa 1020 acccgccgcgcgagctttaccgatgaagatctgttttatgcgcatattgcgggcatggat 1080 agctttgcgctgggcctgaaagtggcgaaccgcctgattgaagatcgcgtgtttgatgcg 1140 tttattgaagaacgctatagcagctataaagaaggcattggcgcggatattgtgagcggc 1200 aaagcggattttaaaagcctggaaaactatattctggataaaaaagaaattattaaccag 1260 agcggccgcctggaacagctgaaaaacaccctgaaccattatattgtgcaggaagcgtat 1320 cagagcgtgaacgcgtaa 1338 36 atgaactatcagccgaccccggaagatcgctttacctttggcctgtggaccgtgggctgg 60 cagggccgcgatccgtttggcgatgcgacccgccgcgcgctggatccggtggaaagcgtg 120 cgccgcctggcggaactgggcgcgcatggcgtgacctttcatgatgatgatctgattccg 180 tttggcagcagcgatagcgaacgcgaagaacatgtgaaacgctttcgccaggcgctggat 240 gataccggcatgaaagtgccgatggcgaccaccaacctgtttacccatccggtgtttaaa 300 gatggcggctttaccgcgaacgatcgcgatgtgcgccgctatgcgctgcgcaaaaccatt 360 cgcaacattgatctggcggtggaactgggcgcggaaacctatgtggcgtggggcggccgc 420 gaaggcgcggaaagcggcggcgcgaaagatgtgcgcgatgcgctggatcgcatgaaagaa 480 gcgtttgatctgctgggcgaatatgtgaccagccagggctatgatattcgctttgcgatt 540 gaaccgaaaccgaacgaaccgcgcggcgatattctgctgccgaccgtgggccatgcgctg 600 gcgtttattgaacgcctggaacgcccggaactgtatggcgtgaacccggaagtgggccat 660 gaacagatggcgggcctgaactttccgcatggcattgcgcaggcgctgtgggcgggcaaa 720 ctgtttcatattgatctgaacggccagaacggcattaaatatgatcaggatctgcgcttt 780 ggcgcgggcgatctgcgcgcggcgttttggctggtggatctgctggaaagcgcgggctat 840 agcggcccgcgccattttgattttaaaccgccgcgcaccgaagattttgatggcgtgtgg 900 gcgagcgcggcgggctgcatgcgcaactatctgattctgaaagaacgcgcggcggcgttt 960 cgcgcggatccggaagtgcaggaagcgctgcgcgcgagccgcctggatgaactggcgcgc 1020 ccgaccgcggcggatggcctgcaggcgctgctggatgatcgcagcgcgtttgaagaattt 1080 gatgtggatgcggcggcggcgcgcggcatggcgtttgaacgcctggatcagctggcgatg 1140 gatcatctgctgggcgcgcgcggctaa 1167 37 atgcaggcgtattttgatcagctggatcgcgtgcgctatgaaggcagcaaaagcagcaac 60 ccgctggcgtttcgccattataacccggatgaactggtgctgggcaaacgcatggaagaa 120 catctgcgctttgcggcgtgctattggcataccttttgctggaacggcgcggatatgttt 180 ggcgtgggcgcgtttaaccgcccgtggcagcagccgggcgaagcgctggcgctggcgaaa 240 cgcaaagcggatgtggcgtttgaattttttcataaactgcatgtgccgttttattgcttt 300 catgatgtggatgtgagcccggaaggcgcgagcctgaaagaatatattaacaactttgcg 360 cagatggtggatgtgctggcgggcaaacaggaagaaagcggcgtgaaactgctgtggggc 420 accgcgaactgctttaccaacccgcgctatggcgcgggcgcggcgaccaacccggatccg 480 gaagtgtttagctgggcggcgacccaggtggtgaccgcgatggaagcgacccataaactg 540 ggcggcgaaaactatgtgctgtggggcggccgcgaaggctatgaaaccctgctgaacacc 600 gatctgcgccaggaacgcgaacagctgggccgctttatgcagatggtggtggaacataaa 660 cataaaattggctttcagggcaccctgctgattgaaccgaaaccgcaggaaccgaccaaa 720 catcagtatgattatgatgcggcgaccgtgtatggctttctgaaacagtttggcctggaa 780 aaagaaattaaactgaacattgaagcgaaccatgcgaccctggcgggccatagctttcat 840 catgaaattgcgaccgcgattgcgctgggcctgtttggcagcgtggatgcgaaccgcggc 900 gatgcgcagctgggctgggataccgatcagtttccgaacagcgtggaagaaaacgcgctg 960 gtgatgtatgaaattctgaaagcgggcggctttaccaccggcggcctgaactttgatgcg 1020 aaagtgcgccgccagagcaccgataaatatgatctgttttatggccatattggcgcgatg 1080 gataccatggcgctggcgctgaaaattgcggcgcgcatgattgaagatggcgaactggat 1140 aaacgcattgcgcagcgctatagcggctggaacagcgaactgggccagcagattctgaaa 1200 ggccagatgagcctggcggatctggcgaaatatgcgcaggaacatcatctgagcccggtg 1260 catcagagcggccgccaggaacagctggaaaacctggtgaaccattatctgtttgataaa 1320 taa 1323 38 atgcgcgaatattttgcgaacgtgccgaaaattaaatatgaaggcaaagatagcaaaaac 60 ccgctggcgtttaaatattataacccggatgaagtggtgggcggcaaaaccatgaaagaa 120 catctgcgctttaccctgagctattggcataccctgaccggcgcgggcagcgatccgttt 180 ggcgtgggcaccatgctgcgcccgtgggattgcgcggaagatgaaatggaactggcgaaa 240 atgcgcatggaagcgaactttgaactgatggataaactgggcattgaatattttgcgttt 300 catgatcgcgatattgcgccggaaggcaaaaccctggcggataccaacgaaaaactggat 360 gaaattgtggcgtattgcaaagaactgatgcagaaacatggcaaaaaactgctgtggggc 420 accgcgaacatgtttggcaacccgcgctttgtgcatggcgcggcgaccacctgcaacgcg 480 gatgtgtttgcgtatgcggcggcgcagaccaaaaaagcgatggatgtgaccaaagaactg 540 ggcggcgaaaactatgtgttttggggcggccgcgaaggctatgaaaccctgctgaacacc 600 gatctgggcctggaacaggataacctggcgcgcttttttcagatggcggtggattatgcg 660 aaaaaaattggctttaccggccagtttctgattgaaccgaaaccgaaagaaccgaccaaa 720 catcagtatgattttgatgtggcgaccgtgctgggctttctgcgcaaatataacctggaa 780 aaatattttaaaatgaacattgaagcgaaccatgcgaccctggcgcagcatacctttcag 840 catgaagtggcggtggcgcgcgtgaacggcgtgctgggcagcctggatgtgaaccagggc 900 gatccgaacctgggctgggataccgatcagtttccgaccaacatttatgatgcgaccatg 960 gtgatgtatgaagtgctgaaaaacggcggcattgcgccgggcggcctgaactttgatgcg 1020 aaaacccgccgcgcgagctttgaaccggaagatctgtttctgagctatattgcgggcatg 1080 gataccatggcgaaaggcctgcgcgtggcgtatagcctgctggatgatgcggtgctggaa 1140 aacaacaccagcgaacgctataaaacctttagcgaaggcattggcaaagatattgtggaa 1200 ggcaaagtggattttgaaagcctggaaaaatatgcgctggaaaacagcgtgattagcaac 1260 aaaagcggccgccaggaatatctggaaagcgtggtgaaccagtatatttttaacgattaa 1320 39 atggcgcagagccatagcagcagcgtgaactattttggcagcgtgaacaaagtggtgttt 60 gaaggcaaagcgagcaccaacccgctggcgtttaaatattataacccgcaggaagtgatt 120 ggcggcaaaaccatgaaagaacatctgcgctttagcattgcgtattggcatacctttacc 180 gcggatggcaccgatgtgtttggcgcggcgaccatgcagcgcccgtgggatcattataaa 240 ggcatggatctggcgcgcgcgcgcgtggaagcggcgtttgaaatgtttgaaaaactggat 300 gcgccgttttttgcgtttcatgatcgcgatattgcgccggaaggcagcaccctgaaagaa 360 accaaccagaacctggatattattgtgggcatgattaaagattatatgcgcgatagcaac 420 gtgaaactgctgtggaacaccgcgaacatgtttaccaacccgcgctttgtgcatggcgcg 480 gcgaccagctgcaacgcggatgtgtttgcgtatgcggcggcgcaggtgaaaaaaggcctg 540 gaaaccgcgaaagaactgggcgcggaaaactatgtgttttggggcggccgcgaaggctat 600 gaaaccctgctgaacaccgatctgaaatttgaactggataacctggcgcgctttatgcat 660 atggcggtggattatgcgaaagaaattgaatataccggccagtttctgattgaaccgaaa 720 ccgaaagaaccgaccacccatcagtatgataccgatgcggcgaccaccattgcgtttctg 780 aaacagtatggcctggataaccattttaaactgaacctggaagcgaaccatgcgaccctg 840 gcgggccatacctttgaacatgaactgcgcatggcgcgcgtgcatggcctgctgggcagc 900 gtggatgcgaaccagggccatccgctgctgggctgggataccgatgaatttccgaccgat 960 ctgtatagcaccaccctggcgatgtatgaaattctgcagaacggcggcctgggcagcggc 1020 ggcctgaactttgatgcgaaagtgcgccgcagcagctttgaaccggatgatctggtgtat 1080 gcgcatattgcgggcatggatgcgtttgcgcgcggcctgaaagtggcgcataaactgatt 1140 gaagatcgcgtgtttgaagatgtgattcagcatcgctatcgcagctttaccgaaggcatt 1200 ggcctggaaattaccgaaggccgcgcgaactttcataccctggaacagtatgcgctgaac 1260 aacaaaaccattaaaaacgaaagcggccgccaggaacgcctgaaagcgattctgaaccag 1320 tatattctggaagtgtaa 1338 40 atgttttttaaaaacgtgggcatgattgaatatgaaggcgcggatagcgaaaacccgtat 60 gcgtttaaatattataacccggatgaatatgtgggcggcaaaaccatgaaagaacatctg 120 cgctttgcggtggcgtattggcatacctttgatgcggatggcaaagatccgtttggcgat 180 ggcaccatgtttcgcgcgtggaaccgcctgacctatccgctggataaagcgaaagcgcgc 240 gcggaaagcgcgtttgaattttttgaaaaactgggcgtgccgtatttttgctttcatgat 300 gtggatattgtggatgaaggcgcgaccctgcgcgaaacctttgcgtatctggatcagatg 360 agcagctttctgaaagaaatgatggaaaccagccgcgtgcagctgctgtggaacaccgcg 420 aacatgtttacccatccgcgctatgtgcatggcgcggcgaccagctgcaacgcggatgtg 480 tatgcgtatgcggcggcgaaagtgaaaaaaggcctggatattgcgaaagaactgggcgcg 540 gaaaactatgtgttttggggcggccgcgaaggctatgaaaccctgctgaacaccgatatg 600 aaactggaactggaaaacctggcgagcttttatcgcatggcggtggaatatgcgcgcgaa 660 attggctttgatggccagtttctgattgaaccgaaaccgaaagaaccgaccaaacatcag 720 tatgattttgatgcggcgaccaccattgcgtttctggaaacctatggcctgaaagatcat 780 tttaaactgaacctggaagcgaaccatgcgaccctggcgggccatacctttgaacatgaa 840 ctgcgcgtggcggcgctgcatgatatgctgggcagcattgatgcgaaccagggcgatctg 900 ctgctgggctgggataccgatgaatttccgaccgatctgtatagcgcggtgctggcgatg 960 tatgaaattctgaaagcgggcggctttaaaaccggcggcattaactttgatgcgaaagtg 1020 cgccgcccgagctttgcggatgaagatctgtttcatgcgcatattgcgggcatggatacc 1080 tatgcggtgggcctgaaagtggcgagccgcctgctggaagataaagcgctggatcaggtg 1140 attgaagaacgctatgaaagctataccaaaggcattggcctggaaattaaagaaggccgc 1200 accgatctgaaaaaactggcggcgtatgcgctggaacatgatcatattgaaaaccagagc 1260 ggccgccaggaacgcctgaaagcgaccgtgaaccgctatctgctgaacgcgctgcgcgaa 1320 gcgccgcgcggcaaagaaacccgctaa 1347 41 atgaccctggaaaaatttgtggatgcgctgccgattccggataccctgaaaccggtgcag 60 cagaccaccgaaaaaacctattatgaagtgaccatggaagaatgcgcgcatcagctgcat 120 cgcgatctgccgccgacccgcctgtggggctataacggcctgtttccgggcccgaccatt 180 gaagtgaaacgcaacgaaaacgtgtatgtgaaatggatgaacaacctgccgagcgaacat 240 tttctgccgattgatcataccattcatcatagcgatagccagcatgaagaaccggaagtg 300 aaaaccgtggtgcatctgcatggcggcgtgaccccgccggatagcgatggctatccggaa 360 gcgtggtttagcaaagattttgaacagaccggcccgtattttaaacgcgaagtgtatcat 420 tatccgaaccagcagcgcggcgcgaccctgtggtatcatgatcatgcgatggcgctgacc 480 cgcctgaacgtgtatgcgggcctggtgggcgcgtatattattcatgatccgaaagaaaaa 540 cgcctgaaactgccgagcggcgaatatgatgtgccgctgctgattaccgatcgcaccatt 600 aacgaagatggcagcctgttttatccgagcggcccggaaaacccgagcccgagcctgccg 660 aaaccgagcattgtgccggcgttttgcggcgataccattctggtgaacggcaaagtgtgg 720 ccgtatctggaagtggaaccgcgcaaatatcgctttcgcgtgattaacgcgagcaacgcg 780 cgcacctataacctgagcctggataacggcggcgaatttattcagattggcagcgatggc 840 ggcctgctgccgcgcagcgtgaaactgaacagctttagcctggcgccggcggaacgctat 900 gatattattattgattttaccgcgtatgaaggcgaaagcattattctggcgaacagcgaa 960 ggctgcggcggcgatgcgaacccggaaaccgatgcgaacattatgcagtttcgcgtgacc 1020 aaaccgctggcgcagaaagatgaaagccgcaaaccgaaatatctggcgagctatccgagc 1080 gtgcagaacgaacgcattcagaacattcgcaccctgaaactggcgggcacccaggatgaa 1140 tatggccgcgtggtgcagctgctgaacaacaaacgctggcatgatccggtgaccgaagcg 1200 ccgaaagcgggcaccaccgaaatttggagcattgtgaacccgacccagggcacccatccg 1260 attcatctgcatctggtgagctttcgcgtgctggatcgccgcccgtttgatattgcgcgc 1320 tatcaggaacgcggcgaactgagctataccggcccggcggtgccgccgccgccgagcgaa 1380 aaaggctggaaagataccattcaggcgcatgcgggcgaagtgctgcgcattgcggtgacc 1440 tttggcccgtatagcggccgctatgtgtggcattgccatattctggaacatgaagattat 1500 gatatgatgcgcccgatggatattaccgatccgcataaataa 1542 42 atgcagcgccgcgattttctgaaatatagcgtggcgctgggcgtggcgagcgcgctgccg 60 ctgtggaaccgcgcggtgtttgcggcggaacgcccgaccctgccgattccggatctgctg 120 accaccgatgcgcgcaaccgcattcagctgaccattggcgcgggccagagcacctttggc 180 ggcaaaaccgcgaccacctggggctataacggcaacctgctgggcccggcggtgaaactg 240 cagcgcggcaaagcggtgaccgtggatatttataaccagctgaccgaagaaaccaccctg 300 cattggcatggcctggaagtgccgggcgaagtggatggcggcccgcagggcattattccg 360 ccgggcggcaaacgcagcgtgaccctgaacgtggatcagccggcggcgacctgctggttt 420 catccgcatcagcatggcaaaaccggccgccaggtggcgatgggcctggcgggcctggtg 480 gtgattgaagatgatgaaattctgaaactgatgctgccgaaacagtggggcattgatgat 540 gtgccggtgattgtgcaggataaaaaatttagcgcggatggccagattgattatcagctg 600 gatgtgatgaccgcggcggtgggctggtttggcgataccctgctgaccaacggcgcgatt 660 tatccgcagcatgcggcgccgcgcggctggctgcgcctgcgcctgctgaacggctgcaac 720 gcgcgcagcctgaactttgcgaccagcgataaccgcccgctgtatgtgattgcgagcgat 780 ggcggcctgctgccggaaccggtgaaagtgagcgaactgccggtgctgatgggcgaacgc 840 tttgaagtgctggtggaagtgaacgataacaaaccgtttgatctggtgaccctgccggtg 900 agccagatgggcatggcgattgcgccgtttgataaaccgcatccggtgatgcgcattcag 960 ccgattgcgattagcgcgagcggcgcgctgccggataccctgagcagcctgccggcgctg 1020 ccgagcctggaaggcctgaccgtgcgcaaactgcagctgagcatggatccgatgctggat 1080 atgatgggcatgcagatgctgatggaaaaatatggcgatcaggcgatggcgggcatggat 1140 catagccagatgatgggccatatgggccatggcaacatgaaccatatgaaccatggcggc 1200 aaatttgattttcatcatgcgaacaaaattaacggccaggcgtttgatatgaacaaaccg 1260 atgtttgcggcggcgaaaggccagtatgaacgctgggtgattagcggcgtgggcgatatg 1320 atgctgcatccgtttcatattcatggcacccagtttcgcattctgagcgaaaacggcaaa 1380 ccgccggcggcgcatcgcgcgggctggaaagataccgtgaaagtggaaggcaacgtgagc 1440 gaagtgctggtgaaatttaaccatgatgcgccgaaagaacatgcgtatatggcgcattgc 1500 catctgctggaacatgaagataccggcatgatgctgggctttaccgtgagcgatccgtaa 1560 43 atgaaagaactgggccatgaagtgctgaaaccgtatgatggctgggcggcgtatggcgaa 60 ggcaccaccggcggcgcgatggcgagcccgcagaacgtgtttgtggtgaccaaccgcacc 120 gaactgattcaggcgctgggcggcaacaaccataccaaccagtataacagcgtgccgaaa 180 attatttatgtgaaaggcaccattgatctgaacgtggatgataacaaccagccggtgggc 240 ccggatttttataaagatccgcattttgattttgaagcgtatctgcgcgaatatgatccg 300 gcgacctggggcaaaaaagaagtggaaggcccgctggaagaagcgcgcgtgcgcagccag 360 aaaaaacagaaagatcgcattatggtgtatgtgggcagcaacaccagcattattggcgtg 420 ggcaaagatgcgaaaattaaaggcggcggctttctgattaaaaacgtggataacgtgatt 480 attcgcaacattgaatttgaagcgccgctggattattttccggaatgggatccgaccgat 540 ggcaccctgggcgaatggaacagcgaatatgatagcattagcattgaaggcagcagccat 600 atttggattgatcataacacctttaccgatggcgatcatccggatcgcagcctgggcacc 660 tattttggccgcccgtttcagcagcatgatggcgcgctggatattaaaaacagcagcgat 720 tttattaccattagctataacgtgtttaccaaccatgataaagtgaccctgattggcgcg 780 agcgatagccgcatggcggatagcggccatctgcgcgtgaccctgcatcataactattat 840 aaaaacgtgacccagcgcctgccgcgcgtgcgctttggccaggtgcatatttataacaac 900 tattatgaatttagcaacctggcggattatgattttcagtatgcgtggggcgtgggcgtg 960 tttagccagatttatgcgcagaacaactattttagctttgattgggatattgatccgagc 1020 ctgattattaaagtgtggagcaaaaacgaagaaagcatgtatgaaaccggcaccattgtg 1080 gatctgccgaacggccgccgctatattgatctggtggcgagctataacgaaagcaacacc 1140 ctgcagctgaaaaaagaagtgacctggaaaccgatgttttatcatgtgattcatccgacc 1200 ccgagcgtgccggcgctggtgaaagcgaaagcgggcgcgggcaacctgcattaa 1254 44 atggttacgtttcacactaatcacggcgacattgttatcaagaccttcgacgacaaagca 60 ccggaaacggtgaagaatttcctggattattgtcgcgagggtttttacaacaataccatt 120 ttccatcgtgtcattaatggttttatgatccagggtggcggtttcgagccgggcatgaag 180 cagaaagccaccaaagaaccgattaagaacgaagcgaataatggcctgaagaacacccgt 240 ggcacgctggcgatggcgcgtacccaggcaccacatagcgcgaccgctcaattctttatc 300 aacgttgttgataacgatttcctgaacttttccggtgagagcttgcaaggctggggttac 360 tgcgttttcgccgaggttgtggacggtatggacgtggtcgacaaaatcaaaggtgtcgcg 420 acgggtcgcagcggtatgcaccaagatgtgccgaaagaagatgtgattatcgagtctgtc 480 accgtgagcgagggcacatctgaaaacttgtatttccagggcgcc 525