Publications

• X-ray structure of Arthrobacter globiformis M30 ketose 3-epimerase for the production of D-allulose from D-fructose.

  Yoshida H., Yoshihara A., Gullapalli P.K., Ohtani K., Akimitsu K., Izumori K., Kamitori S.

  The X-ray structure of ketose 3-epimerase from Arthrobacter globiformis M30, which was previously reported to be a D-allulose 3-epimerase (AgD-AE), was determined at 1.96 Å resolution. The crystal belonged to the hexagonal space group P6₅22, with unit-cell parameters a = b = 103.98, c = ... >> More

  The X-ray structure of ketose 3-epimerase from Arthrobacter globiformis M30, which was previously reported to be a D-allulose 3-epimerase (AgD-AE), was determined at 1.96 Å resolution. The crystal belonged to the hexagonal space group P6₅22, with unit-cell parameters a = b = 103.98, c = 256.53 Å. The structure was solved by molecular replacement using the structure of Mesorhizobium loti L-ribulose 3-epimerase (MlL-RE), which has 41% sequence identity, as a search model. A hexagonal crystal contained two molecules in the asymmetric unit, and AgD-AE formed a homotetramer with twofold symmetry. The overall structure of AgD-AE was more similar to that of MlL-RE than to the known structures of D-psicose (alternative name D-allulose) 3-epimerases (D-PEs or D-AEs), although AgD-AE and MlL-RE have different substrate specificities. Both AgD-AE and MlL-RE have long helices in the C-terminal region that would contribute to the stability of the homotetramer. AgD-AE showed higher enzymatic activity for L-ribulose than D-allulose; however, AgD-AE is stable and is a unique useful enzyme for the production of D-allulose from D-fructose. << Less

  Acta Crystallogr. F Struct. Biol. Commun. 74:669-676(2018) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Overexpression of D-psicose 3-epimerase from Ruminococcus sp. in Escherichia coli and its potential application in D-psicose production.

  Zhu Y., Men Y., Bai W., Li X., Zhang L., Sun Y., Ma Y.

  The D-psicose 3-epimerase (DPE) gene from Ruminococcus sp. was cloned and overexpressed in Escherichia coli. The recombinant protein was purified and characterized. It was optimally active at pH 7.5-8.0 and 60 °C. Activity was not dependent on the presence of metal ions; however, it became more th ... >> More

  The D-psicose 3-epimerase (DPE) gene from Ruminococcus sp. was cloned and overexpressed in Escherichia coli. The recombinant protein was purified and characterized. It was optimally active at pH 7.5-8.0 and 60 °C. Activity was not dependent on the presence of metal ions; however, it became more thermostable with added Mn(2+). The K (m) of the enzyme for D-psicose (48 mM) was lower than that for D-tagatose (230 mM), suggesting that D-psicose is the optimum substrate. More importantly, the thermostability of the novel DPE from Ruminococcus is the strongest among all of the D-psicose and D-tagatose 3-epimerases and may be suitable for the industrial production of D-psicose from fructose. << Less

  Biotechnol Lett 34:1901-1906(2012) [PubMed] [EuropePMC]

• Cloning, expression, and characterization of a D-psicose 3-epimerase from Clostridium cellulolyticum H10.

  Mu W., Chu F., Xing Q., Yu S., Zhou L., Jiang B.

  The noncharacterized protein ACL75304 encoded by the gene Ccel_0941 from Clostridium cellulolyticum H10 (ATCC 35319), previously proposed as the xylose isomerase domain protein TIM barrel, was cloned and expressed in Escherichia coli . The expressed enzyme was purified by nickel-affinity chromatog ... >> More

  The noncharacterized protein ACL75304 encoded by the gene Ccel_0941 from Clostridium cellulolyticum H10 (ATCC 35319), previously proposed as the xylose isomerase domain protein TIM barrel, was cloned and expressed in Escherichia coli . The expressed enzyme was purified by nickel-affinity chromatography with electrophoretic homogeneity and then characterized as d-psicose 3-epimerase. The enzyme was strictly metal-dependent and showed a maximal activity in the presence of Co(2+). The optimum pH and temperature for enzyme activity were 55 °C and pH 8.0. The half-lives for the enzyme at 60 °C were 6.8 h and 10 min when incubated with and without Co(2+), respectively, suggesting that this enzyme was extremely thermostable in the presence of Co(2+) but readily inactivated without metal ion. The Michaelis-Menten constant (K(m)), turnover number (k(cat)), and catalytic efficiency (k(cat)/K(m)) values of the enzyme for substrate d-psicose were estimated to be 17.4 mM, 3243.4 min(-1), and 186.4 mM min(-1), respectively. The enzyme carried out the epimerization of d-fructose to d-psicose with a conversion yield of 32% under optimal conditions, suggesting that the enzyme is a potential d-psicose producer. << Less

  J. Agric. Food Chem. 59:7785-7792(2011) [PubMed] [EuropePMC]

• Characterization of a D-psicose-producing enzyme, D-psicose 3-epimerase, from Clostridium sp.

  Mu W., Zhang W., Fang D., Zhou L., Jiang B., Zhang T.

  The gene coding for D-psicose 3-epimerase (DPEase) from Clostridium sp. BNL1100 was cloned and expressed in Escherichia coli. The recombinant enzyme was purified by Ni-affinity chromatography. It was a metal-dependent enzyme and required Co(2+) as optimum cofactor. It displayed catalytic activity ... >> More

  The gene coding for D-psicose 3-epimerase (DPEase) from Clostridium sp. BNL1100 was cloned and expressed in Escherichia coli. The recombinant enzyme was purified by Ni-affinity chromatography. It was a metal-dependent enzyme and required Co(2+) as optimum cofactor. It displayed catalytic activity maximally at pH 8.0 and 65 °C (as measured over 5 min). The optimum substrate was D-psicose, and the K m, turnover number (k cat), and catalytic efficiency (k cat/K m) for D-psicose were 227 mM, 32,185 min(-1), and 141 min(-1 )mM(-1), respectively. At pH 8.0 and 55 °C, 120 g D-psicose l(-1) was produced from 500 g D-fructose l(-1) after 5 h. << Less

  Biotechnol Lett 35:1481-1486(2013) [PubMed] [EuropePMC]

• Characterization of an Agrobacterium tumefaciens D-psicose 3-epimerase that converts D-fructose to D-psicose.

  Kim H.J., Hyun E.K., Kim Y.S., Lee Y.J., Oh D.K.

  The noncharacterized gene previously proposed as the D-tagatose 3-epimerase gene from Agrobacterium tumefaciens was cloned and expressed in Escherichia coli. The expressed enzyme was purified by three-step chromatography with a final specific activity of 8.89 U/mg. The molecular mass of the purifi ... >> More

  The noncharacterized gene previously proposed as the D-tagatose 3-epimerase gene from Agrobacterium tumefaciens was cloned and expressed in Escherichia coli. The expressed enzyme was purified by three-step chromatography with a final specific activity of 8.89 U/mg. The molecular mass of the purified protein was estimated to be 132 kDa of four identical subunits. Mn2+ significantly increased the epimerization rate from D-fructose to D-psicose. The enzyme exhibited maximal activity at 50 degrees C and pH 8.0 with Mn2+. The turnover number (k(cat)) and catalytic efficiency (k(cat)/Km) of the enzyme for D-psicose were markedly higher than those for d-tagatose, suggesting that the enzyme is not D-tagatose 3-epimerase but D-psicose 3-epimerase. The equilibrium ratio between D-psicose and D-fructose was 32:68 at 30 degrees C. D-Psicose was produced at 230 g/liter from 700-g/liter D-fructose at 50 degrees C after 100 min, corresponding to a conversion yield of 32.9%. << Less

  Appl. Environ. Microbiol. 72:981-985(2006) [PubMed] [EuropePMC]

• Directed divergent evolution of a thermostable D-tagatose epimerase towards improved activity for two hexose substrates.

  Bosshart A., Hee C.S., Bechtold M., Schirmer T., Panke S.

  Functional promiscuity of enzymes can often be harnessed as the starting point for the directed evolution of novel biocatalysts. Here we describe the divergent morphing of an engineered thermostable variant (Var8) of a promiscuous D-tagatose epimerase (DTE) into two efficient catalysts for the C3 ... >> More

  Functional promiscuity of enzymes can often be harnessed as the starting point for the directed evolution of novel biocatalysts. Here we describe the divergent morphing of an engineered thermostable variant (Var8) of a promiscuous D-tagatose epimerase (DTE) into two efficient catalysts for the C3 epimerization of D-fructose to D-psicose and of L-sorbose to L-tagatose. Iterative single-site randomization and screening of 48 residues in the first and second shells around the substrate-binding site of Var8 yielded the eight-site mutant IDF8 (ninefold improved kcat for the epimerization of D-fructose) and the six-site mutant ILS6 (14-fold improved epimerization of L-sorbose), compared to Var8. Structure analysis of IDF8 revealed a charged patch at the entrance of its active site; this presumably facilitates entry of the polar substrate. The improvement in catalytic activity of variant ILS6 is thought to relate to subtle changes in the hydration of the bound substrate. The structures can now be used to select additional sites for further directed evolution of the ketohexose epimerase. << Less

  ChemBioChem 16:592-601(2015) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Characterization of a novel metal-dependent D-psicose 3-epimerase from Clostridium scindens 35704.

  Zhang W., Fang D., Xing Q., Zhou L., Jiang B., Mu W.

  The noncharacterized protein CLOSCI_02528 from Clostridium scindens ATCC 35704 was characterized as D-psicose 3-epimerase. The enzyme showed maximum activity at pH 7.5 and 60°C. The half-life of the enzyme at 50°C was 108 min, suggesting the enzyme was relatively thermostable. It was strictly meta ... >> More

  The noncharacterized protein CLOSCI_02528 from Clostridium scindens ATCC 35704 was characterized as D-psicose 3-epimerase. The enzyme showed maximum activity at pH 7.5 and 60°C. The half-life of the enzyme at 50°C was 108 min, suggesting the enzyme was relatively thermostable. It was strictly metal-dependent and required Mn²⁺ as optimum cofactor for activity. In addition, Mn²⁺ improved the structural stability during both heat- and urea-induced unfolding. Using circular dichroism measurements, the apparent melting temperature (T m) and the urea midtransition concentration (C m) of metal-free enzyme were 64.4°C and 2.68 M. By comparison, the Mn²⁺-bound enzyme showed higher T m and C m with 67.3°C and 5.09 M. The Michaelis-Menten constant (K m), turnover number (k cat), and catalytic efficiency (k cat/K m) values for substrate D-psicose were estimated to be 28.3 mM, 1826.8 s⁻¹, and 64.5 mM⁻¹ s⁻¹, respectively. The enzyme could effectively produce D-psicose from D-fructose with the turnover ratio of 28%. << Less

  PLoS One 8:e62987-e62987(2013) [PubMed] [EuropePMC]

• Characterization of D-tagatose-3-epimerase from Rhodobacter sphaeroides that converts D-fructose into D-psicose.

  Zhang L., Mu W., Jiang B., Zhang T.

  A non-characterized gene, previously proposed as the D-tagatose-3-epimerase gene from Rhodobacter sphaeroides, was cloned and expressed in Escherichia coli. Its molecular mass was estimated to be 64 kDa with two identical subunits. The enzyme specificity was highest with D-fructose and decreased f ... >> More

  A non-characterized gene, previously proposed as the D-tagatose-3-epimerase gene from Rhodobacter sphaeroides, was cloned and expressed in Escherichia coli. Its molecular mass was estimated to be 64 kDa with two identical subunits. The enzyme specificity was highest with D-fructose and decreased for other substrates in the order: D-tagatose, D-psicose, D-ribulose, D-xylulose and D-sorbose. Its activity was maximal at pH 9 and 40 degrees C while being enhanced by Mn(2+). At pH 9 and 40 degrees C, 118 g D-psicose l(-1) was produced from 700 g D-fructose l(-1) after 3 h. << Less

  Biotechnol. Lett. 31:857-862(2009) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Crystal structures of D-psicose 3-epimerase from Clostridium cellulolyticum H10 and its complex with ketohexose sugars.

  Chan H.C., Zhu Y., Hu Y., Ko T.P., Huang C.H., Ren F., Chen C.C., Ma Y., Guo R.T., Sun Y.

  D-psicose 3-epimerase (DPEase) is demonstrated to be useful in the bioproduction of D-psicose, a rare hexose sugar, from D-fructose, found plenty in nature. Clostridium cellulolyticum H10 has recently been identified as a DPEase that can epimerize D-fructose to yield D-psicose with a much higher c ... >> More

  D-psicose 3-epimerase (DPEase) is demonstrated to be useful in the bioproduction of D-psicose, a rare hexose sugar, from D-fructose, found plenty in nature. Clostridium cellulolyticum H10 has recently been identified as a DPEase that can epimerize D-fructose to yield D-psicose with a much higher conversion rate when compared with the conventionally used DTEase. In this study, the crystal structure of the C. cellulolyticum DPEase was determined. The enzyme assembles into a tetramer and each subunit shows a (β/α)(8) TIM barrel fold with a Mn(2+) metal ion in the active site. Additional crystal structures of the enzyme in complex with substrates/products (D-psicose, D-fructose, D-tagatose and D-sorbose) were also determined. From the complex structures of C. cellulolyticum DPEase with D-psicose and D-fructose, the enzyme has much more interactions with D-psicose than D-fructose by forming more hydrogen bonds between the substrate and the active site residues. Accordingly, based on these ketohexose-bound complex structures, a C3-O3 proton-exchange mechanism for the conversion between D-psicose and D-fructose is proposed here. These results provide a clear idea for the deprotonation/protonation roles of E150 and E244 in catalysis. << Less

  Protein Cell 3:123-131(2012) [PubMed] [EuropePMC]

• Purification and characterization of D-allulose 3-epimerase derived from Arthrobacter globiformis M30, a GRAS microorganism.

  Yoshihara A., Kozakai T., Shintani T., Matsutani R., Ohtani K., Iida T., Akimitsu K., Izumori K., Gullapalli P.K.

  An enzyme that catalyzes C-3 epimerization between d-fructose and d-allulose was found in Arthrobacter globiformis strain M30. Arthrobacter species have long been used in the food industry and are well-known for their high degree of safety. The enzyme was purified by ion exchange and hydrophobic i ... >> More

  An enzyme that catalyzes C-3 epimerization between d-fructose and d-allulose was found in Arthrobacter globiformis strain M30. Arthrobacter species have long been used in the food industry and are well-known for their high degree of safety. The enzyme was purified by ion exchange and hydrophobic interaction chromatographies and characterized as a d-allulose 3-epimerase (d-AE). The molecular weight of the purified enzyme was estimated to be 128 kDa with four identical subunits. The enzyme showed maximal activity and thermostability in the presence of Mg²⁺. The optimal pH and temperature for enzymatic activity were 7.0-8.0 and 70°C, respectively. The enzyme was immobilized to ion exchange resin whereupon it was stable for longer periods than the free enzyme when stored at below 10°C. In the column reaction, the enzyme activity also maintained stability for more than 4 months. Under these conditions, 215 kg of d-allulose produced per liter immobilized enzyme, and this was the highest production yield of d-allulose reported so far. These highly stable properties suggest that this enzyme represents an ideal candidate for the industrial production of d-allulose. << Less

  J. Biosci. Bioeng. 123:170-176(2017) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• A D-psicose 3-epimerase with neutral pH optimum from Clostridium bolteae for D-psicose production: cloning, expression, purification, and characterization.

  Jia M., Mu W., Chu F., Zhang X., Jiang B., Zhou L.L., Zhang T.

  D-Tagatose 3-epimerase family enzymes can efficiently catalyze the epimerization of free keto-sugars, which could be used for D-psicose production from D-fructose. In previous studies, all optimum pH values of these enzymes were found to be alkaline. In this study, a D-psicose 3-epimerase (DPEase) ... >> More

  D-Tagatose 3-epimerase family enzymes can efficiently catalyze the epimerization of free keto-sugars, which could be used for D-psicose production from D-fructose. In previous studies, all optimum pH values of these enzymes were found to be alkaline. In this study, a D-psicose 3-epimerase (DPEase) with neutral pH optimum from Clostridium bolteae (ATCC BAA-613) was identified and characterized. The gene encoding the recombinant DPEase was cloned and expressed in Escherichia coli. In order to characterize the catalytic properties, the recombinant DPEase was purified to electrophoretic homogeneity using nickel-affinity chromatography. Ethylenediaminetetraacetic acid was shown to inhibit the enzyme activity completely; therefore, the enzyme was identified as a metalloprotein that exhibited the highest activity in the presence of Co²⁺. Although the DPEase demonstrated the most activity at a pH ranging from 6.5 to 7.5, it exhibited optimal activity at pH 7.0. The optimal temperature for the recombinant DPEase was 55 °C, and the half-life was 156 min at 55 °C. Using D-psicose as the substrate, the apparent K(m), k(cat), and catalytic efficiency (k(cat)/K(m)) were 27.4 mM, 49 s⁻¹, and 1.78 s⁻¹ mM⁻¹, respectively. Under the optimal conditions, the equilibrium ratio of D-fructose to D-psicose was 69:31. For high production of D-psicose, 216 g/L D-psicose could be produced with 28.8 % turnover yield at pH 6.5 and 55 °C. The recombinant DPEase exhibited weak-acid stability and thermostability and had a high affinity and turnover for the substrate D-fructose, indicating that the enzyme was a potential D-psicose producer for industrial production. << Less

  Appl. Microbiol. Biotechnol. 98:717-725(2014) [PubMed] [EuropePMC]

• Gene cloning and characterization of L-ribulose 3-epimerase from Mesorhizobium loti and its application to rare sugar production.

  Uechi K., Takata G., Fukai Y., Yoshihara A., Morimoto K.

  A gene encoding L-ribulose 3-epimerase (L-RE) from Mesorhizobium loti, an important enzyme for rare sugar production by the Izumoring strategy, was cloned and overexpressed. The enzyme showed highest activity toward L-ribulose (230 U/mg) among keto-pentoses and keto-hexoses. This is the first repo ... >> More

  A gene encoding L-ribulose 3-epimerase (L-RE) from Mesorhizobium loti, an important enzyme for rare sugar production by the Izumoring strategy, was cloned and overexpressed. The enzyme showed highest activity toward L-ribulose (230 U/mg) among keto-pentoses and keto-hexoses. This is the first report on a ketose 3-epimerase showing highest activity toward keto-pentose. The optimum enzyme reaction conditions for L-RE were determined to be sodium phosphate buffer (pH 8.0) at 60 °C. The enzyme showed of higher maximum reaction a rate (416 U/mg) and catalytic efficiency (43 M(-1) min(-1)) for L-ribulose than other known ketose 3-epimerases. It was able to produce L-xylulose efficiently from ribitol in two-step reactions. In the end, 7.2 g of L-xylulose was obtained from 20 g of ribitol via L-ribulose at a yield of 36%. << Less

  Biosci. Biotechnol. Biochem. 77:511-515(2013) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Crystal structures of D-tagatose 3-epimerase from Pseudomonas cichorii and its complexes with D-tagatose and D-fructose.

  Yoshida H., Yamada M., Nishitani T., Takada G., Izumori K., Kamitori S.

  Pseudomonas cichoriiid-tagatose 3-epimerase (P. cichoriid-TE) can efficiently catalyze the epimerization of not only d-tagatose to d-sorbose, but also d-fructose to d-psicose, and is used for the production of d-psicose from d-fructose. The crystal structures of P. cichoriid-TE alone and in comple ... >> More

  Pseudomonas cichoriiid-tagatose 3-epimerase (P. cichoriid-TE) can efficiently catalyze the epimerization of not only d-tagatose to d-sorbose, but also d-fructose to d-psicose, and is used for the production of d-psicose from d-fructose. The crystal structures of P. cichoriid-TE alone and in complexes with d-tagatose and d-fructose were determined at resolutions of 1.79, 2.28, and 2.06 A, respectively. A subunit of P. cichoriid-TE adopts a (beta/alpha)(8) barrel structure, and a metal ion (Mn(2+)) found in the active site is coordinated by Glu152, Asp185, His211, and Glu246 at the end of the beta-barrel. P. cichoriid-TE forms a stable dimer to give a favorable accessible surface for substrate binding on the front side of the dimer. The simulated omit map indicates that O2 and O3 of d-tagatose and/or d-fructose coordinate Mn(2+), and that C3-O3 is located between carboxyl groups of Glu152 and Glu246, supporting the previously proposed mechanism of deprotonation/protonation at C3 by two Glu residues. Although the electron density is poor at the 4-, 5-, and 6-positions of the substrates, substrate-enzyme interactions can be deduced from the significant electron density at O6. The O6 possibly interacts with Cys66 via hydrogen bonding, whereas O4 and O5 in d-tagatose and O4 in d-fructose do not undergo hydrogen bonding to the enzyme and are in a hydrophobic environment created by Phe7, Trp15, Trp113, and Phe248. Due to the lack of specific interactions between the enzyme and its substrates at the 4- and 5-positions, P. cichoriid-TE loosely recognizes substrates in this region, allowing it to efficiently catalyze the epimerization of d-tagatose and d-fructose (C4 epimer of d-tagatose) as well. Furthermore, a C3-O3 proton-exchange mechanism for P. cichoriid-TE is suggested by X-ray structural analysis, providing a clear explanation for the regulation of the ionization state of Glu152 and Glu246. << Less

  J. Mol. Biol. 374:443-453(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.