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Applied and Environmental Microbiology, June 2004, p. 3298-3304, Vol. 70, No. 6
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.6.3298-3304.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Cloning, Nucleotide Sequence, and Overexpression of the L-Rhamnose Isomerase Gene from Pseudomonas stutzeri in Escherichia coli

Khim Leang, Goro Takada, Akihiro Ishimura, Masashi Okita, and Ken Izumori^*

Department of Biochemistry and Food Science, Faculty of Agriculture and Rare Sugar Research Center, Kagawa University, Miki-cho, Kagawa 761-0795, Japan

Received 8 October 2003/ Accepted 23 February 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
The gene encoding L-rhamnose isomerase (L-RhI) from Pseudomonas stutzeri was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the L-RhI gene revealed an open reading frame of 1,290 bp coding for a protein of 430 amino acid residues with a predicted molecular mass of 46,946 Da. A comparison of the deduced amino acid sequence with sequences in relevant databases indicated that no significant homology has previously been identified. An amino acid sequence alignment, however, suggested that the residues involved in the active site of L-RhI from E. coli are conserved in that from P. stutzeri. The L-RhI gene was then overexpressed in E. coli cells under the control of the T5 promoter. The recombinant clone, E. coli JM109, produced significant levels of L-RhI activity, with a specific activity of 140 U/mg and a volumetric yield of 20,000 U of soluble enzyme per liter of medium. This reflected a 20-fold increase in the volumetric yield compared to the value for the intrinsic yield. The recombinant L-RhI protein was purified to apparent homogeneity on the basis of three-step chromatography. The purified recombinant enzyme showed a single band with an estimated molecular weight of 42,000 in a sodium dodecyl sulfate-polyacrylamide gel. The overall enzymatic properties of the purified recombinant L-RhI protein were the same as those of the authentic one, as the optimal activity was measured at 60°C within a broad pH range from 5.0 to 11.0, with an optimum at pH 9.0.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Unusual carbohydrates, simply termed "rare sugars," are referred to as sugars that exist in an extremely scanty amount in nature; they include, for instance, D-/L-allose, D-/L-altrose, L-glucose, D-/L-psicose, D-/L-tagatose, L-fructose, D-sorbose, D-tagatose, allitol, and others. Rare sugars have been proven to be of paramount significance not only in food industries but also in pharmaceutical and nutritional industries on account of their multipurpose applications, such as their use as reduced-calorie sweeteners, inhibitors of microbial growth, bulking agents, and memory enhancers (4, 22-25). Yet the unavailability and scarcity of these uncommon sugars result in their high cost and limitations in usage as starting materials or research reagents. Consequently, there has been a dearth of literature reporting on the mass production of rare sugars. In recent years, however, microbial and enzymatic reactions have become increasingly significant and have played pivotal roles in the production of unusual sugars.

We are mainly working on the production and/or mass production of various kinds of rare sugars by using microorganisms screened from a variety of ecological habitats and their enzymes. For example, Pseudomonas cichorii ST-24 and Acinetobacter sp. strain DL-28, which produce D-tagatose 3-epimerase and L-ribose isomerase, respectively, were isolated from soil and utilized for the preparation of a number of uncommon sugars (1, 16-19, 32). In order to improve the productivity of the sugars, the enzymes were later genetically engineered (14, 15, 26). Furthermore, members of our laboratory recently isolated a mutant strain, characterized as Pseudomonas stutzeri, of Pseudomonas sp. strain LL172 that constitutively synthesized L-rhamnose isomerase (L-rhamnose ketol-isomerase [EC 5.3.1.14]) on L-lyxose medium (5), and the enzyme was then exploited for the production of L-mannose, D-allose, and L-talose (6-8). In preliminary work by Wilson and Ajl (36), L-rhamnose isomerase was found to participate in L-rhamnose metabolism in Escherichia coli in which the enzyme catalyzed the reversible isomerization of L-rhamnose to its corresponding ketose, L-rhamnulose. This finding was supported by many extensive studies at molecular levels. Power and Garcia-Martin et al. indicated that four structural genes were involved in L-rhamnose metabolism, and these four genes were found to be rhaA, rhaB, rhaD, and rhaT, encoding rhamnose isomerase, rhamnulose kinase, rhamnulose-1-phosphate aldolase, and rhamnose permease, respectively (12, 29). A gene cluster encoding the enzyme for L-rhamnose metabolism in E. coli was sequenced and characterized (27). L-RhI is also known to be responsible for the degradation of L-lyxose in E. coli, in which L-RhI converts the aldose to its corresponding ketose, L-xylulose (3). However, comparatively little is known about the diversity of molecular properties of L-RhI from Pseudomonas strains. In this study, we report the cloning and sequencing of the L-RhI gene from P. stutzeri. Moreover, to the best of our knowledge, we present for the first time data concerning the overproduction of L-RhI in E. coli JM109 and the purification and some properties of the recombinant enzyme.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains, plasmids, and growth conditions.
The bacterial strains and plasmids used for this study are listed in Table 1. P. stutzeri was grown at 30°C for 48 h in a medium composed of 2% tryptic soy broth supplemented with MnCl₂ (final concentration, 1 mM). E. coli strains DH5, JM103, JM105, JM109, M15, XL1-Blue, XL2-Blue MRF', and BL21(DE3) harboring plasmids were grown in super broth medium with ampicillin (100 µg/ml). For expression experiments, E. coli M15 was cultivated at 37°C in super broth with kanamycin (25 µg/ml). When required, isopropyl-ß-D-thiogalactopyranoside (IPTG; 1 mM) and 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-Gal; 40 µg/ml) from Takara Shuzo (Osu, Japan) were added to the medium.

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TABLE 1. Strains and plasmids used for this study

Chemicals.
Restriction enzymes and modifying enzymes were purchased from Nipongene (Tayama, Japan) or Takara Shuzo. L-Rhamnose was obtained from Wako Pure Chemicals (Tokyo, Japan).

Purification of wild-type L-RhI and N-terminal amino acid sequencing.
Wild-type P. stutzeri was grown at 30°C for 48 h in a medium composed of 2% tryptic soy broth supplemented with MnCl₂ (final concentration, 1 mM). Cells were harvested and washed twice with 50 mM Tris-HCl buffer (pH 8.5). A crude extract was prepared by grinding the washed cells with aluminum oxide, and the cell extract was subjected to Mn²⁺ treatment and polyethylene glycol fractionation. The resultant 10 to 20% polyethylene glycol fraction with high L-RhI activity was applied to a Mono Q HR5/5 (0.5 by 5 cm; Amersham Biosciences) ion-exchange column. The purified enzyme was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Analytical SDS-PAGE was performed on a slab gel (12% polyacrylamide) in Tris-glycine (pH 8.3) at a current of 20 mA for 3 h. The protein bands were visualized by staining with Coomassie brilliant blue R-250 (21). For a determination of the internal amino acid sequence of L-RhI, digestion of purified L-RhI with CNBr was performed and the proteins separated in the gel were electroblotted onto a polyvinylidene difluoride (PVDF) membrane (Hybond-N⁺; Amersham Biosciences). The peptide fragments were eluted from the membrane with 70% formic acid and were sequenced by an automated Edman degradation method.

DNA manipulations.
Genomic DNA preparation and recombinant DNA techniques were performed according to standard procedures (30). DNAs were prepared from agarose gels with a Geneclean II kit (BIO 101, Vista, Calif.). Plasmid DNAs were isolated by the alkaline lysis method of Birnboim and Doly (9). E. coli cells were transformed according to the method of Hanahan (13).

PCR amplification of L-RhI probe.
The purified chromosomal DNA (1 µg) was used as a template for PCR. To amplify a part of the L-RhI gene, we synthesized the following degenerate oligonucleotide primers on the basis of the elucidated N-terminal and internal amino acid sequences: forward, F1 (5'-GARTTYMGNATHGCNCARGAYGT-3') and F2 (5'-GAYGTNGTNGCNMGNGARAAYGA-3'); and reverse, R1 (5'-GGNGCRTCNSHRAANGTRTT-3'), R2 (5'-ARDATNGGYTCNACRTCNGT-3'), and R3 (5'-GCYTCNGCNARDATNGGYTC-3') (R = A or G; Y = C or T; N = A, C, or T; V = G, A, or C; B = G, T, or C; M = A or T; S = G or C; H = A, C, or T; K = G or T). The following six pairs of synthetic primers were designed: F1-R1, F2-R1, F1-R2, F1-R3, F2-R2, and F2-R3. The amplification reactions were performed in 25-µl reaction volumes containing PCR buffer, a 0.25 mM concentration of each deoxynucleoside triphosphate, 2.5 mM MgCl₂, 100 pmol of each primer, and 1.25 U of LA Taq DNA polymerase (Takara Shuzo), and the PCR conditions were as follows: holding at 95°C for 15 min; running 30 cycles at 95°C for 1 min, 55°C for 1 min, and 72°C for 2 min; and holding at 72°C for 7 min (iCycler thermal cycler 582BR; Bio-Rad). Under these conditions, the DNA sequence for the specific amplified 300-bp fragment was obtained.

Southern blotting, colony hybridization, and labeling.
Genomic DNA from P. stutzeri was partially digested with several restriction enzymes, separated in an agarose gel (1%), and blotted onto a PVDF membrane by the method of Southern (33). The 300-bp fragment amplified by PCR was labeled with digoxigenin-11-dUTP (DIG-11-dUTP) by use of a DIG labeling kit (Roche Diagnostics, Mannheim, Germany) and was used as probe.

Screening of a genomic DNA library and cloning of L-RhI.
The genomic DNA of P. stutzeri was partially digested with EcoRI to yield fragments predominantly in the range of 2 to 4 kb. The restriction fragments were separated by electrophoresis and visualized by staining with ethidium bromide. The fragments were subjected to Southern hybridization with the 300-bp labeled probe described above. Of several hybridized fragments, a 2.9-kb EcoRI fragment was cloned into an EcoRI-digested pUC119 vector and introduced into E. coli DH5 to construct a genomic library.

Nucleotide sequence analysis.
The DNA sequence was determined by the dideoxy nucleotide chain termination method (31) in a LIC-4200L DNA sequencer (Licor Biotechnology Division). We completely analyzed both directional strands by overlapping them at every junction, using IRD700 and IRD800 dye-labeled primers and a Thermo Sequence cycle sequencing kit (Amersham) according to the manufacturer's instructions. A search for proteins homologous to the L-RhI protein was performed by comparing the nucleotide sequence and the deduced amino acid sequence with the DNA and protein sequences available from the DDBJ and SwissProt databases with the BLAST program (2). Multiple sequence alignments were constructed with the CLUSTALW program available from DDBJ.

Construction of expression plasmid pOI-01.
The L-RhI gene was amplified by a PCR performed under the conditions described above. Synthetic oligonucleotide primers were designed to incorporate an NcoI site in the forward primer and a HindIII site in the multiple cloning site of M13 as follows: forward primer, 5'-GACCATGGCGGAGTTCAGGATCGCGCA-3', with an engineered NcoI site (underlined); and a universal reverse primer, 5'-GAGCGGATAACAATTTCACACAGG-3'. An approximately 1.5-kb NcoI-HindIII fragment containing the complete coding sequence of L-RhI was prepared by digesting the PCR-amplified fragment with NcoI and HindIII. The expression plasmid pOI-01 was constructed by ligating the fragment into plasmid pQE60 (Qiagen, Valencia, Calif.), which had been previously digested with NcoI and HindIII.

To ascertain the presence of the 1.5-kb NcoI-HindIII fragment in pQE60, we first transformed plasmid pOI-01 into E. coli DH5. The insert was confirmed after plasmid extraction by a standard method (30). The expression plasmid pOI-01 was then introduced into E. coli DH5, JM103, JM105, JM109, M15, XL1-Blue, XL2-Blue MRF', and BL21(DE3). The transformants were grown at 37°C for 12 h in 3 ml of super broth medium containing 3.5% Bacto tryptone, 2% yeast extract, and 0.5% NaCl supplemented with ampicillin and kanamycin (in the case of M15). L-RhI production was initiated by the addition of 1 mM IPTG. Cells were collected, washed, and disrupted by sonication at 4°C. After centrifugation at 12,000 x g, both the supernatant and pellet were assayed for L-RhI activity via the method described below.

Preparation of crude extract and purification of recombinant L-RhI.
E. coli JM109 cells carrying recombinant L-RhI were cultivated in 2 liters of super broth medium supplemented with ampicillin at a final concentration of 100 µg/ml at 37°C for 12 h in a 2.5-liter jar fermentor. L-RhI production was initiated by the addition of 1 mM IPTG when the culture reached an optical density at 600 nm of 0.7, and growth was then continued for another 4 h. The cells were harvested by centrifugation at 9,000 x g for 10 min at 4°C and washed twice with 50 mM Tris-HCl buffer (pH 7.5), and the washed cells (10 g) were disrupted by grinding with activated aluminum with a mortar and pestle. The ruptured cells were then resuspended in the same buffer. The cell debris was removed by centrifugation at 12,000 x g for 30 min at 4°C. The supernatant fluid (100 ml) obtained was utilized as a crude extract to be purified. Unless otherwise stated, all purification steps were performed at room temperature on an AKTA purifier system (Amersham). The supernatant of the crude extract (100 ml) was first applied to a DEAE-Toyopearl 650 M (1.6 by 10 cm) column that had been previously equilibrated with the buffer described above, and the enzyme was eluted with a linear gradient of KCl (0 to 1.0 M). The fractions with high L-RhI activity were pooled together and then passed through a Q-Sepharose HP (1.6 by 10 cm) column equilibrated with the buffer described above and eluted with a linear gradient of KCl (0 to 1.0 M). The fractions with high L-RhI activity were collected and subsequently loaded onto a Hi-Trap phenyl-Toyopearl (0.7 by 5 cm) column equilibrated with the same buffer. The enzyme was eluted with a linear gradient of (NH₄)₂SO₄ (1.0 to 0 M) in 50 mM Tris-HCl buffer (pH 7.5). The active fractions were collected and the purified enzyme was analyzed by SDS-PAGE under the conditions described above. The purified enzyme was used for further enzymatic characterization.

Enzyme assay and protein determination.
The L-RhI activity was assayed in a 0.5-ml reaction mixture containing 50 mM glycine-NaOH buffer (pH 9.0), 1 mM MnCl₂, and 50 µl of appropriately diluted enzyme. The reaction was initiated by the addition of L-rhamnose (final concentration, 10 mM), the mixture was incubated for 10 min at 30°C, and the reaction was then stopped by the addition of 50 µl of trichloroacetic acid (10%). The formation of L-rhamnulose was determined spectrophotometrically by the method of Dishe and Borenfreund (11). Briefly, 100 µl of a 1.5% cysteine hydrochloride solution, 3 ml of 70% H₂SO₄, and 100 µl of a 0.12% alcoholic solution of carbazole were added successively to the reaction mixture and incubated at 35°C for 20 min. Color development, indicating the presence of ketose, was measured at 540 nm. One unit of enzyme activity represented 1 µmol of L-rhamnulose formed within 1 min under these assay conditions. The protein concentration was measured by using a Bio-Rad (Hercules, Calif.) protein assay kit according to a standard procedure (10).

Nucleotide sequence accession number.
The nucleotide sequence reported in this paper has been deposited in the DDBJ nucleotide sequence database under accession number AB121136.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cloning and sequence analysis of the gene encoding L-RhI.
Purified L-RhI was subjected to SDS-PAGE and electroblotted onto a PVDF membrane for N-terminal amino acid sequence determination. Moreover, for the determination of internal amino acid sequences, the purified enzyme was also digested with CNBr. Digestion of the enzyme with CNBr rendered four fragments, of 13, 12, 11, and 8 kDa. The digests were separated by SDS-PAGE and transferred to a PVDF membrane filter. The N-terminal and four internal amino acid sequences were identified to be AEFRIAQDVVARENDRRASARK, IDQSHNVTDP, AEFR, SNTFSDAP, and ATETLKRAYRTDVEPILAEARRRTG, respectively. The analysis revealed that the deduced amino acids of the 12-kDa fragment of the internal sequence exactly matched the first four N-terminal residues. The resultant 300-bp DNA fragment, which was the only distinct product amplified by a PCR with the F1-R1 pair of primers, was subcloned into HincII-digested pUC119 to produce plasmid pKL-1, which was subsequently introduced into E. coli DH5. White colonies were isolated on X-Gal plates containing ampicillin, and the plasmid DNA was sequenced to ensure that the desired fragment was inserted in the correct orientation. The same 300-bp PCR product labeled with DIG-11-dUTP was used as a probe for Southern blot hybridization and the following colony hybridization.

The chromosomal DNA from P. stutzeri was digested with several restriction enzymes (SacI, SalI, SalI-EcoRI, EcoRI, EcoRI-HindIII, HindIII, PstI, PstI-BamHI, and BamHI) and subjected to Southern hybridization with the 300-bp probe. A 2.9-kb EcoRI fragment (Fig. 1) was found to hybridize with the probe, and this fragment was subcloned into the corresponding site in the polylinker of plasmid pUC119 which had previously been digested with EcoRI, generating plasmid pAI-1, which was subsequently transformed into E. coli DH5. The transformants were analyzed by colony hybridization. Twenty positive clones were obtained from 5,000 transformants. All 20 colonies contained the same 2.9-kb EcoRI fragment (data not shown).

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FIG. 1. Nucleotide sequence and deduced amino acid sequence of L-RhI gene. A putative ribosome-binding site is boxed and marked "SD." The amino acid sequences of L-RhI determined by Edman degradation and the sequences which are consistent with the N-terminal and internal peptide sequences are underlined and in italics, respectively. Internal amino acid sequences are marked with broken lines. An asterisk indicates the translation stop codon.

The 2.9-kb EcoRI-digested fragment was sequenced on both strands, and the complete nucleotide sequence of the insert in pAI-1, together with the deduced amino acid sequence of the L-RhI protein, is demonstrated in Fig. 1. The nucleotide sequence corresponds to an open reading frame of 1,290 bp, starting with an ATG initiation codon at position 202 and ending with a TGA termination codon at position 1,492. A putative ribosome-binding site (AGGGAG) is located six nucleotides upstream from the initiation codon. The L-RhI gene encodes a protein consisting of 430 amino acid residues with a predicted molecular mass of 46,946 Da. The deduced N-terminal amino acid sequence of L-RhI is consistent with that of the N terminus of L-RhI as determined by automated Edman degradation. The G+C content in the coding region is 65.55%.

The deduced amino acid sequence of the L-RhI protein demonstrated the highest amino acid homology (25%) to that from Bacillus subtilis (DDBJ accession number Z93938-5) (28), while it was 24% identical to L-RhI from Bacillus halodurans (DDBJ accession number AP001512-139) (34) and 17% identical to that from E. coli K-12 (Swiss-Prot accession number P32170) (27), as shown in Fig. 2.

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FIG. 2. Multiple sequence alignment of L-RhI proteins. Abbreviations: P.s., P. stutzeri; B.s., B. subtilis (28); B.h., B. halodurans (34); E.c., E. coli (27). The consensus residues are boxed and the residues involved in the active site of L-RhI from E. coli are in bold italics (20).

Expression, purification, and properties of recombinant L-RhI.
A coding region of the L-RhI gene was digested as an NcoI-HindIII fragment and ligated to the multiple cloning site of plasmid pQE60, which was previously excised with NcoI-HindIII in order to not incorporate the six histidines, resulting in plasmid pOI-01. The expression plasmid pOI-01, containing the full-length coding sequence of L-RhI, was cloned into several E. coli expression strains as described in Materials and Methods. As shown in Table 2, strain JM109 had the highest enzyme activity, followed by JM105, XL2-Blue MRF', XL1-Blue, M15, and DH5, in that order. On the other hand, no activity was observed for JM103 and BL21(DE3) transformants. The enzyme production reached its maximum after 4 to 5 h of induction by IPTG at 37°C. A maximal volumetric yield of 20,000 U of soluble L-RhI per liter of medium was obtained after 12 h of cultivation and 4 to 5 h of induction by IPTG. This suggests a 20-fold increase in the volumetric yield compared to the expression level in wild-type P. stutzeri.

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TABLE 2. Productivity of recombinant L-RhI in various E. coli strains

Strain JM109 harboring pOI-01 was then cultured in super broth medium, and the enzyme was extracted by grinding with aluminum oxide as mentioned in Materials and Methods. The recombinant L-RhI protein was purified from the crude extract by a series of chromatographic procedures based on anion exchange and hydrophobicity. The results of the overall purification procedure are summarized in Table 3. Up to the final purification step, the enzyme was purified 6.2-fold, with a recovery yield of 20.7%. Proteins from each purification step were separated and the purified enzyme was confirmed by SDS-PAGE analysis. Electrophoresis of the SDS-treated purified L-RhI protein revealed a single band with an estimated molecular mass of 42,000 Da, which was the same as that of the authentic enzyme from P. stutzeri, as illustrated in Fig. 3. The recombinant L-RhI protein produced by E. coli JM109 showed almost the same enzymatic characteristics as intrinsic L-RhI. However, the kinetic properties of the enzyme were satisfactorily improved, as demonstrated in Table 4. Table 5 shows the Michaelis constants of various aldose substrates as determined by Lineweaver-Burk plots of velocity versus substrate concentration. L-Rhamnose, L-mannose, L-lyxose, D-ribose, and D-allose were kinetically favored substrates, followed by D-xylose, D-altrose, D-arabinose, and D-glucose.

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TABLE 3. Summary of purification of recombinant L-RhI from E. coli JM109

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FIG. 3. SDS-PAGE analysis of recombinant L-RhI from each purification step. Lanes: 1 and 6, molecular standard marker (DAIICHI, Tokyo, Japan) containing myosin (200 kDa), ß-galactosidase (116 kDa), bovine serum albumin (66 kDa), aldolase (42 kDa), and carbonic anhydrase (30 kDa); 2, crude extract; 3 to 5, recombinant L-RhI purified by DEAE-Toyopearl 650 M, Q-Sepharose HP, and phenyl-Toyopearl 650 M chromatography, respectively. The arrow indicates purified recombinant L-RhI from the phenyl-Toyopearl 650 M fraction.

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TABLE 4. Comparison between properties of L-RhI from P. stutzeri and those of L-RhI from E. coli JM109

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TABLE 5. Kinetic parameters of L-RhI from E. coli JM109 for various aldosesa

Top Abstract Introduction Materials and Methods Results Discussion References

 
We previously reported the isolation and characterization of L-RhI from a constitutive mutant strain of P. stutzeri. In this paper, we described for the first time the cloning, sequencing, overproduction, and characterization of the L-RhI gene from this strain. It has been demonstrated that the recombinant enzyme can be successfully expressed in E. coli. The recombinant L-RhI protein was overproduced in a soluble form in E. coli, but not in the inactive fraction of inclusion bodies (Fig. 4). The overproduction of the soluble enzyme in E. coli resulted in simplified purification steps. Furthermore, the lower K_m and higher V_max values of recombinant L-RhI, as shown in Table 4, suggest that the enzyme is practically suitable for application on industrial levels. Moreover, the molecular mass determined by SDS-PAGE indicates that the recombinant L-RhI protein is correctly expressed in E. coli and that the cloned and wild-type enzymes are really the same. A plausible rationale, however, for the change in the kinetic values thus remains an open question at present. Similarly, an increase in the specific activity of recombinant D-tagatose 3-epimerase was reported, but the reason was unknown (14). It has also been reported that the specific activity of chitinase III of an Aeromonas sp. was improved when it was expressed in E. coli and that the reason was the presence of the sugar chain (35). With the results of the enzyme assays, we demonstrated that L-RhI gene expression was entirely dependent on the lac promoter rather than the intrinsic one and on IPTG induction for expression. Regarding the low and undetectable activities from the DH5, JM103, and BL21(DE3) strains, it can be rationalized that the promoters of the hosts were not suitable for expression and that the expressed protein had a toxic effect to a certain extent on the host cells.

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FIG. 4. SDS-PAGE analysis of the effect of IPTG on L-RhI induction from E. coli JM 109 harboring pOI-01. Lanes: 1 and 6, molecular standard marker (see the legend to Fig. 3 for details); 2 and 3, soluble and insoluble fractions, respectively, with IPTG; 4 and 5, soluble and insoluble fractions, respectively, without IPTG.

It has been reported that L-RhI expressed from E. coli requires Mn²⁺ and Zn²⁺ for its activity (20), although it appeared that the enzyme reported herein did require Mn²⁺ for its maximum activity, but not any other metallic ions, including Zn²⁺. It is interesting that the L-RhI protein reported herein showed no significantly homology with any other protein sequence deposited in various databases. In addition, the enzyme exhibited the potential ability to catalyze not only the isomerization reactions of L-rhamnose, L-mannose, and L-lyxose, but also those of D-psicose, L-tagatose, and D-sorbose to D-allose, L-talose, and D-gulose, respectively, even though L-rhamnose was the preferred substrate (Table 5). These results clearly demonstrate that L-RhI from the Pseudomonas strain is a genetically novel enzyme.

In particular, it is worth noting that the recombinant enzyme is a promising and indispensable candidate for the mass production of diverse rare sugars, such as D-allose, D-gulose, L-talose, and L-fructose (6-8), which are commercially costly and unavailable on a large scale. Also, we optimistically expect that rare sugars will be made affordable at a moderate cost and thereby be exploited for various purposes in the not-too-distant future. Our future investigations will focus on the structural genes required for the metabolism of L-rhamnose as well as L-lyxose by the Pseudomonas strain. Finally, it is important to point out that we have also successfully overexpressed the recombinant enzyme fused to a six-His tag and that studies on the properties of the His-tagged recombinant enzyme are currently under way (K. Leang et al., unpublished data). Furthermore, as we have recently succeeded in crystallizing the His-tagged enzyme, its structure-function relationship is under study in our laboratory.

 
* Corresponding author. Mailing address: Department of Biochemistry and Food Science, Faculty of Agriculture and Rare Sugar Research Center, Kagawa University, Miki-cho, Kagawa 761-0795, Japan. Phone: 81-087-891-3106. Fax: 81-087-891-3021. E-mail: izumori{at}ag.kagawa-u.ac.jp.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, June 2004, p. 3298-3304, Vol. 70, No. 6
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.6.3298-3304.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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