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Molecular cloning and biochemical characterization of a novel erythrose reductase from Candida magnoliae JH110.

Lee DH, Lee YJ, Ryu YW, Seo JH - Microb. Cell Fact. (2010)

Bottom Line: ER has gained interest because of its importance in the production of erythritol, which has extremely low digestibility and approved safety for diabetics.The result suggested that NADPH binding partners are completely conserved in the C. magnoliae JH110 ER.The C. magnoliae JH110 ER with high activity and catalytic efficiency would be very useful for in vitro erythritol production and could be applied for the production of erythritol in other microorganisms, which do not produce erythritol.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea.

ABSTRACT

Background: Erythrose reductase (ER) catalyzes the final step of erythritol production, which is reducing erythrose to erythritol using NAD(P)H as a cofactor. ER has gained interest because of its importance in the production of erythritol, which has extremely low digestibility and approved safety for diabetics. Although ERs were purified and characterized from microbial sources, the entire primary structure and the corresponding DNA for ER still remain unknown in most of erythritol-producing yeasts. Candida magnoliae JH110 isolated from honeycombs produces a significant amount of erythritol, suggesting the presence of erythrose metabolizing enzymes. Here we provide the genetic sequence and functional characteristics of a novel NADPH-dependent ER from C. magnoliae JH110.

Results: The gene encoding a novel ER was isolated from an osmophilic yeast C. magnoliae JH110. The ER gene composed of 849 nucleotides encodes a polypeptide with a calculated molecular mass of 31.4 kDa. The deduced amino acid sequence of ER showed a high degree of similarity to other members of the aldo-keto reductase superfamily including three ER isozymes from Trichosporonoides megachiliensis SNG-42. The intact coding region of ER from C. magnoliae JH110 was cloned, functionally expressed in Escherichia coli using a combined approach of gene fusion and molecular chaperone co-expression, and subsequently purified to homogeneity. The enzyme displayed a temperature and pH optimum at 42 degrees C and 5.5, respectively. Among various aldoses, the C. magnoliae JH110 ER showed high specific activity for reduction of erythrose to the corresponding alcohol, erythritol. To explore the molecular basis of the catalysis of erythrose reduction with NADPH, homology structural modeling was performed. The result suggested that NADPH binding partners are completely conserved in the C. magnoliae JH110 ER. Furthermore, NADPH interacts with the side chains Lys252, Thr255, and Arg258, which could account for the enzyme's absolute requirement of NADPH over NADH.

Conclusions: A novel ER enzyme and its corresponding gene were isolated from C. magnoliae JH110. The C. magnoliae JH110 ER with high activity and catalytic efficiency would be very useful for in vitro erythritol production and could be applied for the production of erythritol in other microorganisms, which do not produce erythritol.

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SDS-PAGE analysis of CmER expressed in recombinant E. coli. (A) Expression of CmER in the transformant E. coli BL21(DE3) harboring pCmER10, (B) Expressed CmER fused to GST at its N-terminus under the control of the tac promoter in E. coli BL21, (C) CmER expressed from the GST-fusion approach in conjunction with co-expression of molecular chaperones; Plasmids encoding the molecular chaperone, pGro7, pKJE7, and pGKJE3, were used to express GroEL-GroES, DnaK-DnaJ-GrpE, and GroEL-GroES-DnaK-DnaJ-GrpE, respectively, (D) SDS-PAGE analysis of CmER expressed and purified from the GST-fusion approach in conjunction with co-expression of molecular chaperones, GroEL-GroES-DnaK-DnaJ-GrpE. Lane M, protein standards; lane T, IPTG-induced total fraction; lane S, soluble fraction; lane I, insoluble fraction. Lane 1, soluble fraction of GST-fused CmER-expressed cells; lane 2, purified GST-CmER fusion protein; lane 3, cleavage of GST from GST-CmER fusion protein by Factor Xa; lane 4, purified intact CmER.
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Figure 3: SDS-PAGE analysis of CmER expressed in recombinant E. coli. (A) Expression of CmER in the transformant E. coli BL21(DE3) harboring pCmER10, (B) Expressed CmER fused to GST at its N-terminus under the control of the tac promoter in E. coli BL21, (C) CmER expressed from the GST-fusion approach in conjunction with co-expression of molecular chaperones; Plasmids encoding the molecular chaperone, pGro7, pKJE7, and pGKJE3, were used to express GroEL-GroES, DnaK-DnaJ-GrpE, and GroEL-GroES-DnaK-DnaJ-GrpE, respectively, (D) SDS-PAGE analysis of CmER expressed and purified from the GST-fusion approach in conjunction with co-expression of molecular chaperones, GroEL-GroES-DnaK-DnaJ-GrpE. Lane M, protein standards; lane T, IPTG-induced total fraction; lane S, soluble fraction; lane I, insoluble fraction. Lane 1, soluble fraction of GST-fused CmER-expressed cells; lane 2, purified GST-CmER fusion protein; lane 3, cleavage of GST from GST-CmER fusion protein by Factor Xa; lane 4, purified intact CmER.

Mentions: To express CmER in recombinant Escherichia coli, the transformant E. coli BL21(DE3) harboring pCmER10 was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37°C. After IPTG induction for 6 h, a protein of an apparent 33 kDa molecular weight appeared as a major band in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis (Fig. 3A). Even though an overall expression level was quite high, the protein was produced mainly as inclusion bodies. In order to express the soluble and active form of CmER in E. coli, the fusion gene approach was attempted. Glutathione S-transferase (GST) is a naturally-occurring 26 kDa protein which is often used for the soluble expression of heterologous proteins [20]. In addition, the GST fusion tag with integrated protease sites has been used for convenient purification of the proteins of interest. Thus, the CmER fused to GST at its N-terminus was expressed under the control of the tac promoter by 1 mM IPTG induction in E. coli BL21, which also resulted in the formation of inclusion bodies (Fig. 3B). Since it has been reported that the co-expression of molecular chaperones increases the soluble form of several enzymes in E. coli [21,22], their effects on CmER expression were tested in the transformant E. coli cells harboring pGSTCmER. Plasmid pGro7, pKJE7, or pG-KJE3 [23] was used for co-expression of GroEL-GroES, DnaK-DnaJ-GrpE, or GroEL-GroES-DnaK-DnaJ-GrpE, respectively. Plasmid pGro7, pKJE7, or pG-KJE3 was simultaneously transformed with the plasmid pGSTCmER into E. coli BL21 cells. Among the chaperone families co-expressed, only the GroEL-GroES-DnaK-DnaJ-GrpE chaperone family exerted positive effects on soluble expression of CmER as verified by SDS-PAGE (Fig. 3C). After confirming soluble enzyme expression, the GST-removed CmER was purified by using the affinity column chromatography after cleavage with Factor Xa. The enzyme purity was estimated by SDS-PAGE analysis with staining of Coomassie brilliant blue. The final yield of intact CmER was 18 mg (~60 mg/liter of culture) of >95% pure CmER with a molecular mass of ~31 kDa, which corresponds well to the calculated mass (Fig. 3D).


Molecular cloning and biochemical characterization of a novel erythrose reductase from Candida magnoliae JH110.

Lee DH, Lee YJ, Ryu YW, Seo JH - Microb. Cell Fact. (2010)

SDS-PAGE analysis of CmER expressed in recombinant E. coli. (A) Expression of CmER in the transformant E. coli BL21(DE3) harboring pCmER10, (B) Expressed CmER fused to GST at its N-terminus under the control of the tac promoter in E. coli BL21, (C) CmER expressed from the GST-fusion approach in conjunction with co-expression of molecular chaperones; Plasmids encoding the molecular chaperone, pGro7, pKJE7, and pGKJE3, were used to express GroEL-GroES, DnaK-DnaJ-GrpE, and GroEL-GroES-DnaK-DnaJ-GrpE, respectively, (D) SDS-PAGE analysis of CmER expressed and purified from the GST-fusion approach in conjunction with co-expression of molecular chaperones, GroEL-GroES-DnaK-DnaJ-GrpE. Lane M, protein standards; lane T, IPTG-induced total fraction; lane S, soluble fraction; lane I, insoluble fraction. Lane 1, soluble fraction of GST-fused CmER-expressed cells; lane 2, purified GST-CmER fusion protein; lane 3, cleavage of GST from GST-CmER fusion protein by Factor Xa; lane 4, purified intact CmER.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2902421&req=5

Figure 3: SDS-PAGE analysis of CmER expressed in recombinant E. coli. (A) Expression of CmER in the transformant E. coli BL21(DE3) harboring pCmER10, (B) Expressed CmER fused to GST at its N-terminus under the control of the tac promoter in E. coli BL21, (C) CmER expressed from the GST-fusion approach in conjunction with co-expression of molecular chaperones; Plasmids encoding the molecular chaperone, pGro7, pKJE7, and pGKJE3, were used to express GroEL-GroES, DnaK-DnaJ-GrpE, and GroEL-GroES-DnaK-DnaJ-GrpE, respectively, (D) SDS-PAGE analysis of CmER expressed and purified from the GST-fusion approach in conjunction with co-expression of molecular chaperones, GroEL-GroES-DnaK-DnaJ-GrpE. Lane M, protein standards; lane T, IPTG-induced total fraction; lane S, soluble fraction; lane I, insoluble fraction. Lane 1, soluble fraction of GST-fused CmER-expressed cells; lane 2, purified GST-CmER fusion protein; lane 3, cleavage of GST from GST-CmER fusion protein by Factor Xa; lane 4, purified intact CmER.
Mentions: To express CmER in recombinant Escherichia coli, the transformant E. coli BL21(DE3) harboring pCmER10 was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37°C. After IPTG induction for 6 h, a protein of an apparent 33 kDa molecular weight appeared as a major band in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis (Fig. 3A). Even though an overall expression level was quite high, the protein was produced mainly as inclusion bodies. In order to express the soluble and active form of CmER in E. coli, the fusion gene approach was attempted. Glutathione S-transferase (GST) is a naturally-occurring 26 kDa protein which is often used for the soluble expression of heterologous proteins [20]. In addition, the GST fusion tag with integrated protease sites has been used for convenient purification of the proteins of interest. Thus, the CmER fused to GST at its N-terminus was expressed under the control of the tac promoter by 1 mM IPTG induction in E. coli BL21, which also resulted in the formation of inclusion bodies (Fig. 3B). Since it has been reported that the co-expression of molecular chaperones increases the soluble form of several enzymes in E. coli [21,22], their effects on CmER expression were tested in the transformant E. coli cells harboring pGSTCmER. Plasmid pGro7, pKJE7, or pG-KJE3 [23] was used for co-expression of GroEL-GroES, DnaK-DnaJ-GrpE, or GroEL-GroES-DnaK-DnaJ-GrpE, respectively. Plasmid pGro7, pKJE7, or pG-KJE3 was simultaneously transformed with the plasmid pGSTCmER into E. coli BL21 cells. Among the chaperone families co-expressed, only the GroEL-GroES-DnaK-DnaJ-GrpE chaperone family exerted positive effects on soluble expression of CmER as verified by SDS-PAGE (Fig. 3C). After confirming soluble enzyme expression, the GST-removed CmER was purified by using the affinity column chromatography after cleavage with Factor Xa. The enzyme purity was estimated by SDS-PAGE analysis with staining of Coomassie brilliant blue. The final yield of intact CmER was 18 mg (~60 mg/liter of culture) of >95% pure CmER with a molecular mass of ~31 kDa, which corresponds well to the calculated mass (Fig. 3D).

Bottom Line: ER has gained interest because of its importance in the production of erythritol, which has extremely low digestibility and approved safety for diabetics.The result suggested that NADPH binding partners are completely conserved in the C. magnoliae JH110 ER.The C. magnoliae JH110 ER with high activity and catalytic efficiency would be very useful for in vitro erythritol production and could be applied for the production of erythritol in other microorganisms, which do not produce erythritol.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea.

ABSTRACT

Background: Erythrose reductase (ER) catalyzes the final step of erythritol production, which is reducing erythrose to erythritol using NAD(P)H as a cofactor. ER has gained interest because of its importance in the production of erythritol, which has extremely low digestibility and approved safety for diabetics. Although ERs were purified and characterized from microbial sources, the entire primary structure and the corresponding DNA for ER still remain unknown in most of erythritol-producing yeasts. Candida magnoliae JH110 isolated from honeycombs produces a significant amount of erythritol, suggesting the presence of erythrose metabolizing enzymes. Here we provide the genetic sequence and functional characteristics of a novel NADPH-dependent ER from C. magnoliae JH110.

Results: The gene encoding a novel ER was isolated from an osmophilic yeast C. magnoliae JH110. The ER gene composed of 849 nucleotides encodes a polypeptide with a calculated molecular mass of 31.4 kDa. The deduced amino acid sequence of ER showed a high degree of similarity to other members of the aldo-keto reductase superfamily including three ER isozymes from Trichosporonoides megachiliensis SNG-42. The intact coding region of ER from C. magnoliae JH110 was cloned, functionally expressed in Escherichia coli using a combined approach of gene fusion and molecular chaperone co-expression, and subsequently purified to homogeneity. The enzyme displayed a temperature and pH optimum at 42 degrees C and 5.5, respectively. Among various aldoses, the C. magnoliae JH110 ER showed high specific activity for reduction of erythrose to the corresponding alcohol, erythritol. To explore the molecular basis of the catalysis of erythrose reduction with NADPH, homology structural modeling was performed. The result suggested that NADPH binding partners are completely conserved in the C. magnoliae JH110 ER. Furthermore, NADPH interacts with the side chains Lys252, Thr255, and Arg258, which could account for the enzyme's absolute requirement of NADPH over NADH.

Conclusions: A novel ER enzyme and its corresponding gene were isolated from C. magnoliae JH110. The C. magnoliae JH110 ER with high activity and catalytic efficiency would be very useful for in vitro erythritol production and could be applied for the production of erythritol in other microorganisms, which do not produce erythritol.

Show MeSH
Related in: MedlinePlus