ABSTRACT
BACKGROUND: D-psicose 3-epimerase (DPEase) is a potential catalytic enzyme for D-psicose production. D-psicose, also known as D-allulose, is a low-calorie sweetener that has gained considerable attention as a healthy alternative sweetener due to its notable physicochemical properties. This research focused on an in-depth investigation of the expression of the constructed DPEase gene from Agrobacterium tumefaciens in Escherichia coli for D-psicose synthesis. Experimentally, this research created the recombinant enzyme, explored the optimization of gene expression systems and protein purification strategies, investigated the enzymatic characterization, and then optimized the D-psicose production. Finally, the produced D-psicose syrup underwent acute toxicity evaluation to provide scientific evidence supporting its safety. RESULTS: The optimization of DPEase expression involved the utilization of Mn2+ as a cofactor, fine-tuning isopropyl ß-D-1-thiogalactopyranoside induction, and controlling the induction temperature. The purification process was strategically designed by a nickel column and an elution buffer containing 200 mM imidazole, resulting in purified DPEase with a notable 21.03-fold increase in specific activity compared to the crude extract. The optimum D-psicose conversion conditions were at pH 7.5 and 55 °C with a final concentration of 10 mM Mn2+ addition using purified DPEase to achieve the highest D-psicose concentration of 5.60% (w/v) using 25% (w/v) of fructose concentration with a conversion rate of 22.42%. Kinetic parameters of the purified DPEase were Vmax and Km values of 28.01 mM/min and 110 mM, respectively, which demonstrated the high substrate affinity and efficiency of DPEase conversion by the binding site of the fructose-DPEase-Mn2+ structure. Strategies for maintaining stability of DPEase activity were glycerol addition and storage at -20 °C. Based on the results from the acute toxicity study, there was no toxicity to rats, supporting the safety of the mixed D-fructose-D-psicose syrup produced using recombinant DPEase. CONCLUSIONS: These findings have direct and practical implications for the industrial-scale production of D-psicose, a valuable rare sugar with a broad range of applications in the food and pharmaceutical industries. This research should advance the understanding of DPEase biocatalysis and offers a roadmap for the successful scale-up production of rare sugars, opening new avenues for their utilization in various industrial processes.
Subject(s)
Escherichia coli , Fructose , Recombinant Proteins , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Fructose/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Agrobacterium tumefaciens , Carbohydrate Epimerases/genetics , Carbohydrate Epimerases/metabolism , Carbohydrate Epimerases/isolation & purification , Animals , Racemases and Epimerases/metabolism , Racemases and Epimerases/genetics , Rats , Bacterial Proteins/genetics , Bacterial Proteins/metabolismABSTRACT
BACKGROUND: D-Allulose is an ultra-low calorie sugar of multifarious health benefits, including anti-diabetic and anti-obesity potential. D-Allulose 3-epimerase family enzymes catalyze biosynthesis of D-allulose via epimerization of D-fructose. RESULTS: A novel D-allulose 3-epimerase (DaeB) was cloned from a plant probiotic strain, Bacillus sp. KCTC 13219, and expressed in Bacillus subtilis cells. The purified protein exhibited substantial epimerization activity in a broad pH spectrum, 6.0-11.0. DaeB was able to catalyze D-fructose to D-allulose bioconversion at the temperature range of 35 °C to 70 °C, exhibiting at least 50 % activity. It displaced excessive heat stability, with the half-life of 25 days at 50 °C, and high turnover number (kcat 367 s- 1). The coupling of DaeB treatment and yeast fermentation of 700 g L- 1 D-fructose solution yielded approximately 200 g L- 1 D-allulose, and 214 g L- 1 ethanol. CONCLUSIONS: The novel D-allulose 3-epimerase of Bacillus sp. origin discerned a high magnitude of heat stability along with exorbitant epimerization ability. This biocatalyst has enormous potential for the large-scale production of D-allulose.
Subject(s)
Bacillus/enzymology , Carbohydrate Epimerases/chemistry , Carbohydrate Epimerases/metabolism , Fructose/biosynthesis , Bacillus/genetics , Biocatalysis , Carbohydrate Epimerases/genetics , Carbohydrate Epimerases/isolation & purification , Enzyme Stability , Ethanol/metabolism , Fermentation , Hot Temperature , Hydrogen-Ion Concentration , Kinetics , Models, Molecular , Phylogeny , Protein Structure, Secondary , Protein Structure, Tertiary , Saccharomyces cerevisiae/metabolism , Substrate SpecificityABSTRACT
d-Psicose (d-ribo-2-hexulose or d-allulose) is the Carbon-3 epimer of d-fructose sugar and considered as an unnatural (rare) sugar found in low amount in nature. It has about 70% of the relative sweetness but 0.3% of the energy of sucrose, which is suggested as the most suitable sucrose substitute for food additives. Enzymatic biosynthesis using ketose 3-epimerases is a necessary procedure for the production of d-Psicose from d-fructose. However, significant drawbacks in the application of ketose 3-epimerases at industrial scale observe lower thermal stability as well as bioconversion efficiency, reusability and recovery of the enzyme. We have attempted immobilization of ketose 3-epimerases from Agrobacterium tumefaciens (agtu) d-psicose 3-epimerase (DPEase) on titanium dioxide. Further, Scanning electron microscopy (SEM), inverted microscopy, Fourier transform infrared spectroscopy (FTIR) and UV-vis spectroscopy showed that the enzyme was successfully immobilized on the titanium dioxide (TiO2) surface. Titanium dioxide immobilized agtu-DPEase (TiO2-agtu-DPEase) shows pH optima at 6.0 and 60⯰C as a higher working temperature. TiO2-agtu-DPEase showed a half-life of 180â¯min at 60⯰C, which is higher as compared to Agrobacterium tumefaciens (agtu) DPEase (3.99â¯min at 50⯰C). At equilibrium, 36:64 (D-psicose: d-fructose), the bioconversion efficiency was accounted for titanium dioxide immobilized DPEase, which is higher than the agtu-DPEase. Titanium dioxide immobilized DPEase showed bioconversion efficiency up to 9 cycles of reusability.
Subject(s)
Agrobacterium tumefaciens/enzymology , Carbohydrate Epimerases/metabolism , Enzymes, Immobilized/metabolism , Titanium/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Biotransformation , Carbohydrate Epimerases/chemistry , Carbohydrate Epimerases/isolation & purification , Enzyme Stability , Enzymes, Immobilized/chemistry , Fructose/biosynthesis , Fructose/chemistry , Hydrogen-Ion Concentration , TemperatureABSTRACT
BACKGROUND AND OBJECTIVE: The DPEase enzyme from Agrobacterium tumefaciens is more efficient and has a high activity in D-fructose. The dpe gene has been successfully cloned to Escherichia coli BL21 (DE3) pET-21b dpe but the enzyme has not been purified and its character is unknown. The intent of this study was to purify and assign of DPEase enzyme by recombinant E. coli. MATERIALS AND METHODS: The enzyme was clarified by affinity chromatography and then characterized by following pH, temperature, co-factor parameters. Analysis of molecular weight proteins was done by SDS-PAGE. RESULTS: Through purification, the purified DPEase activity was increased 1,01 times than crude and with 84.2% of yield. The DPEase had an the maximum temperature is 40°C and pH was 8.5. The presence of Mg2+, Mo2+, Cu2+, Ca2+ and Zn2+ inhibited the activity of the enzyme while of Co2+, Mn2+, Fe2+, Ni2+ enhanced the activity. Estimation of molecular weight through SDS-PAGE revealed that weight of DPEase was 32 kDa. CONCLUSION: Purified DPease enzymes shows clear bands that demonstrate successful purification using affinity chromatography. It is expected that after pure enzymes are obtained the character of the enzymes working will be maximized.
Subject(s)
Carbohydrate Epimerases/pharmacology , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Fructose/metabolism , Carbohydrate Epimerases/isolation & purification , Chromatography, Affinity , Enzyme Stability , Escherichia coli Proteins/isolation & purification , Hydrogen-Ion Concentration , Substrate Specificity , TemperatureABSTRACT
The chemoenzymatic synthesis of heparin, through a multienzyme process, represents a critical challenge in providing a safe and effective substitute for this animal-sourced anticoagulant drug. D-glucuronyl C5-epimerase (C5-epi) is an enzyme acting on a heparin precursor, N-sulfoheparosan, catalyzing the reversible epimerization of D-glucuronic acid (GlcA) to L-iduronic acid (IdoA). The absence of reliable assays for C5-epi has limited elucidation of the enzymatic reaction and kinetic mechanisms. Real time and offline assays are described that rely on 1D 1H NMR to study the activity of C5-epi. Apparent steady-state kinetic parameters for both the forward and the pseudo-reverse reactions of C5-epi are determined for the first time using polysaccharide substrates directly relevant to the chemoenzymatic synthesis and biosynthesis of heparin. The forward reaction shows unusual sigmoidal kinetic behavior, and the pseudo-reverse reaction displays nonsaturating kinetic behavior. The atypical sigmoidal behavior of the forward reaction was probed using a range of buffer additives. Surprisingly, the addition of 25 mM each of CaCl2 and MgCl2 resulted in a forward reaction exhibiting more conventional Michaelis-Menten kinetics. The addition of 2-O-sulfotransferase, the next enzyme involved in heparin synthesis, in the absence of 3'-phosphoadenosine 5'-phosphosulfate, also resulted in C5-epi exhibiting a more conventional Michaelis-Menten kinetic behavior in the forward reaction accompanied by a significant increase in apparent Vmax. This study provides critical information for understanding the reaction kinetics of C5-epi, which may result in improved methods for the chemoenzymatic synthesis of bioengineered heparin.
Subject(s)
Carbohydrate Epimerases/metabolism , Glucuronic Acid/metabolism , Iduronic Acid/metabolism , Biocatalysis , Carbohydrate Conformation , Carbohydrate Epimerases/isolation & purification , Glucuronic Acid/chemistry , Humans , Iduronic Acid/chemistry , KineticsABSTRACT
Recently, rare sugars have caused extensively attention due to their beneficial physiological functions and potential applications in food systems and medical fields. Ketose 3-epimerase (KEase) can catalyze reversibly the epimerization between ketoses which is the pivotal enzyme in Izumoring strategy and an effective tool for biological production of rare sugars. In this work, a KEase from Caballeronia fortuita was recombined and characterized as a d-tagatose 3-epimerase (DTEase, EC 5.1.3.31). The recombinant DTEase displayed the highest activity at pH7.5 and 65°C in the presence of Co2+. The recombinant DTEase displayed the relatively high thermostability and the half-life (t1/2) was determined to be 7.13, 5.13, and 1.05h at 50, 55, and 60°C, respectively. The recombinant DTEase had a wide substrate specificity and the specific activities towards d-tagatose, d-allulose, d-fructose and l-sorbose were measured to be 801±2.3, 450±2.7, 270±1.5 and 55±1.8Umg-1, respectively. So far, the recombinant DTEase exhibited the highest specific activity towards d-tagatose compared with other reported KEases. Furthermore, the recombinant DTEase could produce 314.2g/L d-sorbose from 500g/L d-tagatose and 147.0g/L d-allulose from 500g/L d-fructose, with a transformation ratio of 68.2% and 29.4%, respectively. The recombinant DTEase could realize effectively the transformations between various ketoses and was a prominent candidate for production of rare sugars.
Subject(s)
Burkholderiaceae/enzymology , Carbohydrate Epimerases/chemistry , Sugars/metabolism , Biotransformation , Burkholderiaceae/classification , Burkholderiaceae/genetics , Carbohydrate Epimerases/genetics , Carbohydrate Epimerases/isolation & purification , Cloning, Molecular , Enzyme Activation , Fermentation , Gene Expression , Hexoses/chemistry , Hydrogen-Ion Concentration , Kinetics , Phylogeny , Sequence Analysis, DNA , Substrate Specificity , TemperatureABSTRACT
BACKGROUND: Increasing efforts have been made to assess the potential of Escherichia coli strains for the production of complex recombinant proteins. Since a considerable part of therapeutic proteins are glycoproteins, the lack of the post-translational attachment of sugar moieties in standard E. coli expression strains represents a major caveat, thus limiting the use of E. coli based cell factories. The establishment of an E. coli expression system capable of protein glycosylation could potentially facilitate the production of therapeutics with a putative concomitant reduction of production costs. RESULTS: The previously established E. coli strain expressing the soluble form of the functional human-derived glycosyltransferase polypeptide N-acetylgalactosaminyltransferase 2 (GalNAc-T2) was further modified by co-expressing the UDP-GlcNAc 4-epimerase WbgU derived from Plesiomonas shigelloides. This enables the conversion of uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) to the sugar donor uridine 5'-diphospho-N-acetylgalactosamine (UDP-GalNAc) in the bacterial cytoplasm. Initially, the codon-optimised gene wbgU was inserted into a pET-derived vector and a Tobacco Etch Virus (TEV) protease cleavable polyhistidine-tag was translationally fused to the C- terminus of the amino acid sequence. The 4-epimerase was subsequently expressed and purified. Following the removal of the polyhistidine-tag, WbgU was analysed by circular dichroism spectroscopy to determine folding state and thermal transitions of the protein. The in vitro activity of WbgU was validated by employing a modified glycosyltransferase assay. The conversion of UDP-GlcNAc to UDP-GalNAc was shown by capillary electrophoresis analysis. Using a previously established chaperone pre-/co- expression platform, the in vivo activity of both glycosyltransferase GalNAc-T2 and 4-epimerase WbgU was assessed in E. coli, in combination with a mucin 10-derived target protein. Monitoring glycosylation by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS), the results clearly indicated the in vivo glycosylation of the mucin-derived acceptor peptide. CONCLUSION: In the present work, the previously established E. coli- based expression system was further optimized and the potential for in vivo O-glycosylation was shown by demonstrating the transfer of sugar moieties to a mucin-derived acceptor protein. The results offer the possibility to assess the practical use of the described expression platform for in vivo glycosylations of important biopharmaceutical compounds in E. coli.
Subject(s)
Escherichia coli/metabolism , Mucins/metabolism , Amino Acid Sequence , Carbohydrate Epimerases/isolation & purification , Carbohydrate Epimerases/metabolism , Circular Dichroism , Glycosylation , Mucins/chemistry , N-Acetylgalactosaminyltransferases/metabolism , Peptides/chemistry , Peptides/metabolism , Polypeptide N-acetylgalactosaminyltransferaseABSTRACT
The UGlcAE has the capability of interconverting UDP-d-galacturonic acid and UDP-d-glucuronic acid, and UDP-d-galacturonic acid is an activated precursor for the synthesis of pectins in plants. In this study, we identified nine UGlcAE protein-encoding genes in tomato. The nine UGlcAE genes that were distributed on eight chromosomes in tomato, and the corresponding proteins contained one or two trans-membrane domains. The phylogenetic analysis showed that SlUGlcAE genes could be divided into seven groups, designated UGlcAE1 to UGlcAE6, of which the UGlcAE2 were classified into two groups. Expression profile analysis revealed that the SlUGlcAE genes display diverse expression patterns in various tomato tissues. Selective pressure analysis indicated that all of the amino acid sites of SlUGlcAE proteins are undergoing purifying selection. Fifteen stress-, hormone-, and development-related elements were identified in the upstream regions (0.5 kb) of these SlUGlcAE genes. Furthermore, we investigated the expression patterns of SlUGlcAE genes in response to three hormones (indole-3-acetic acid (IAA), gibberellin (GA), and salicylic acid (SA)). We detected firmness, pectin contents, and expression levels of UGlcAE family genes during the development of tomato fruit. Here, we systematically summarize the general characteristics of the SlUGlcAE genes in tomato, which could provide a basis for further function studies of tomato UGlcAE genes.
Subject(s)
Carbohydrate Epimerases/metabolism , Fruit/metabolism , Multigene Family , Solanum lycopersicum/enzymology , Amino Acid Sequence , Carbohydrate Epimerases/chemistry , Carbohydrate Epimerases/genetics , Carbohydrate Epimerases/isolation & purification , Fruit/enzymology , Fruit/genetics , Gene Expression Regulation, Plant , Genomics , Glucuronic Acid/metabolism , Hexuronic Acids/metabolism , Solanum lycopersicum/genetics , Solanum lycopersicum/metabolism , Phylogeny , Plant Proteins/chemistry , Plant Proteins/genetics , Plant Proteins/isolation & purification , Plant Proteins/metabolism , Sequence Alignment , Sequence Analysis, DNA , Sequence Analysis, ProteinABSTRACT
d-Allulose 3-epimerase (DAEase) catalyzes the epimerization between d-fructose and d-allulose. We had PCR-cloned and overexpressed the gene encoding Agrobacterium sp. ATCC 31749 DAEase (AsDAEase) in Escherichia coli. A high yield of active AsDAEase, 35,300U/L or 1350U/g of wet cells, was acquired with isopropyl ß-d-1-thiogalactopyranoside induction at 20°C for 20h. Although only six residues including residue 234 located in tetrameric interface are different between AsDAEase and A. tumefaciens DAEase (AtDAEase), the specific activity of purified AsDAEase is much larger than that of AtDAEase. The optimal pHs and optimal temperatures of the purified recombinant AsDAEase are 7.5-8.0 and 55-60°C, respectively. The half-life of the enzyme is 267min at 55°C in the presence of 0.1mM Co2+, and the equilibrium ratio between d-allulose and d-fructose is 30:70 at 55°C. Besides characterizing AsDAEase, mutation N234D was constructed to assess its influence on activity. The specific activity of the purified N234D AsDAEase is only 25.5% of wild-type's activity, suggesting residue N234 is an important interfacial residue which substantially affects enzyme activity. The high specific activity and high expression yield of AsDAEase suggest its prospect to be applied in d-allulose production.
Subject(s)
Agrobacterium tumefaciens/enzymology , Amino Acids/metabolism , Carbohydrate Epimerases/metabolism , Recombinant Proteins/metabolism , Carbohydrate Epimerases/chemistry , Carbohydrate Epimerases/isolation & purification , Cobalt/pharmacology , Enzyme Stability/drug effects , Fructose/metabolism , Hydrogen-Ion Concentration , Kinetics , Models, Molecular , Molecular Weight , Mutagenesis, Site-Directed , Structural Homology, Protein , Substrate Specificity/drug effects , TemperatureABSTRACT
Heparin and heparan sulfate (HS) glycosaminoglycans have important roles in anticoagulation, human development, and human diseases. HS C5-epimerase, which catalyzes the epimerization of GlcA to IdoA, is a crucial enzyme involved in the biosynthesis of heparin-related biomolecules. Here, we describe a detailed method for measuring the total activity of HS C5-epimerase that involves the following steps: H/D exchange upon epimerization of the substrate with HS C5-epimerase, low-pH nitrous acid treatment of the substrate, the separation of low-pH nitrous acid-cleaved disaccharides using HPLC, and mass spectrometry analysis. This nonradioactive method is rapid and sensitive and, importantly, allows us to study the reversible nature of HS C5-epimerase.
Subject(s)
Carbohydrate Epimerases/metabolism , Deuterium Exchange Measurement/methods , Enzyme Assays/methods , Heparitin Sulfate/metabolism , Mass Spectrometry/methods , Animals , Biocatalysis , Carbohydrate Epimerases/isolation & purification , Chromatography, Ion Exchange , Chromatography, Liquid , Disaccharides/metabolism , Glucuronic Acid/chemistry , Glucuronic Acid/metabolism , Humans , Iduronic Acid/chemistry , Iduronic Acid/metabolism , Sf9 CellsABSTRACT
Sialic acids are one of the most important carbohydrate classes in biology. Some bacterial pathogens can scavenge sialic acids from their surrounding environment and degrade them as a source of carbon, nitrogen and energy. This sequestration and subsequent catabolism of sialic acid require a cluster of genes known as the `Nan-Nag' cluster. The enzymes coded by these genes are important for pathogen colonization and persistence. Importantly, the Nan-Nag genes have proven to be essential for Staphylococcus aureus growth on sialic acids, suggesting that the pathway is a viable antibiotic drug target. The enzyme N-acetylmannosamine-6-phosphate 2-epimerase is involved in the catabolism of sialic acid; specifically, the enzyme converts N-acetylmannosamine-6-phosphate into N-acetylglucosamine-6-phosphate. The gene was cloned into an appropriate expression vector, and recombinant protein was expressed in Escherichia coli BL21 (DE3) cells and purified via a three-step procedure. Purified N-acetylmannosamine-6-phosphate 2-epimerase was screened for crystallization. The best crystal diffracted to a resolution of beyond 1.84 Å in space group P21212. Understanding the structural nature of this enzyme from methicillin-resistant S. aureus will provide us with the insights necessary for the development of future antibiotics.
Subject(s)
Bacterial Proteins/biosynthesis , Carbohydrate Epimerases/biosynthesis , Cloning, Molecular , Gene Expression Regulation, Enzymologic , Methicillin-Resistant Staphylococcus aureus/enzymology , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Carbohydrate Epimerases/genetics , Carbohydrate Epimerases/isolation & purification , Cloning, Molecular/methods , Crystallization , X-Ray DiffractionABSTRACT
A purified recombinant enzyme from Spirochaeta thermophila, that is suggested to be a cellobiose 2-epimerase, was a 47 kDa monomer with a specific activity of 29.2 U min(-1) for mannobiose. The epimerization activity of the recombinant enzyme for mannobiose was maximal at pH 7.0 and 60 °C with a half-life of 124 h. The enzyme exhibited a higher epimerization activity for mannose or the mannose moiety at the reducing end of ß- and α-1,4-glycosyl-mannose than for glucose or the glucose moiety of ß- and α-1,4-glycosyl-glucose. The enzyme was identified as a mannobiose 2-epimerase by evaluating its substrate specificity with not only glucose-containing sugars but also mannose-containing sugars. The activities of the reported cellobiose 2-epimerases from Caldicellulosiruptor saccharolyticus, Dictyoglomus turgidum and Ruminococcus marinus for mannobiose were higher than those for cellobiose, strongly suggesting that these enzymes are not cellobiose 2-epimerases but are mannobiose 2-epimerases.
Subject(s)
Carbohydrate Epimerases/metabolism , Mannans/metabolism , Spirochaeta/enzymology , Carbohydrate Epimerases/chemistry , Carbohydrate Epimerases/genetics , Carbohydrate Epimerases/isolation & purification , Cellobiose/metabolism , Enzyme Stability , Hydrogen-Ion Concentration , Molecular Weight , Phylogeny , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Spirochaeta/genetics , Substrate Specificity , TemperatureABSTRACT
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 Mn²âº as optimum cofactor for activity. In addition, Mn²âº 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 Mn²âº-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⻹, and 64.5 mM⻹ s⻹, respectively. The enzyme could effectively produce D-psicose from D-fructose with the turnover ratio of 28%.
Subject(s)
Carbohydrate Epimerases/metabolism , Clostridium/enzymology , Fructose/metabolism , Metals/metabolism , Amino Acid Sequence , Carbohydrate Epimerases/chemistry , Carbohydrate Epimerases/genetics , Carbohydrate Epimerases/isolation & purification , Cloning, Molecular , Clostridium/genetics , Enzyme Stability , Gene Expression , Hydrogen-Ion Concentration , Kinetics , Manganese/metabolism , Molecular Sequence Data , Recombinant Proteins , Sequence Alignment , Substrate Specificity , TemperatureABSTRACT
Lactobacillus casei strains 64H and BL23, but not ATCC 334, are able to ferment D-ribitol (also called D-adonitol). However, a BL23-derived ptsI mutant lacking enzyme I of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) was not able to utilize this pentitol, suggesting that strain BL23 transports and phosphorylates D-ribitol via a PTS. We identified an 11-kb region in the genome sequence of L. casei strain BL23 (LCABL_29160 to LCABL_29270) which is absent from strain ATCC 334 and which contains the genes for a GlpR/IolR-like repressor, the four components of a mannose-type PTS, and six metabolic enzymes potentially involved in D-ribitol metabolism. Deletion of the gene encoding the EIIB component of the presumed ribitol PTS indeed prevented D-ribitol fermentation. In addition, we overexpressed the six catabolic genes, purified the encoded enzymes, and determined the activities of four of them. They encode a D-ribitol-5-phosphate (D-ribitol-5-P) 2-dehydrogenase, a D-ribulose-5-P 3-epimerase, a D-ribose-5-P isomerase, and a D-xylulose-5-P phosphoketolase. In the first catabolic step, the protein D-ribitol-5-P 2-dehydrogenase uses NAD(+) to oxidize D-ribitol-5-P formed during PTS-catalyzed transport to D-ribulose-5-P, which, in turn, is converted to D-xylulose-5-P by the enzyme D-ribulose-5-P 3-epimerase. Finally, the resulting D-xylulose-5-P is split by D-xylulose-5-P phosphoketolase in an inorganic phosphate-requiring reaction into acetylphosphate and the glycolytic intermediate D-glyceraldehyde-3-P. The three remaining enzymes, one of which was identified as D-ribose-5-P-isomerase, probably catalyze an alternative ribitol degradation pathway, which might be functional in L. casei strain 64H but not in BL23, because one of the BL23 genes carries a frameshift mutation.
Subject(s)
Bacterial Proteins/metabolism , Lacticaseibacillus casei/enzymology , Ribitol/metabolism , Aldehyde-Lyases/genetics , Aldehyde-Lyases/isolation & purification , Aldehyde-Lyases/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Base Sequence , Biological Transport , Carbohydrate Epimerases/genetics , Carbohydrate Epimerases/isolation & purification , Carbohydrate Epimerases/metabolism , Fermentation , Gene Expression , Lacticaseibacillus casei/genetics , Mannose/metabolism , Metabolic Networks and Pathways , Molecular Sequence Data , Mutation , NAD/metabolism , Operon , Pentosephosphates/metabolism , Phosphoenolpyruvate/metabolism , Recombinant Fusion Proteins , Sequence Analysis, DNA , Species Specificity , Sugar Alcohol Dehydrogenases/genetics , Sugar Alcohol Dehydrogenases/isolation & purification , Sugar Alcohol Dehydrogenases/metabolismABSTRACT
Cellobiose 2-epimerase (CE), found mainly in anaerobes, reversibly converts D-glucose residues at the reducing end of ß-1,4-linked oligosaccharides to D-mannose residues. In this study, we characterized CE-like proteins from various aerobes (Flavobacterium johnsoniae NBRC 14942, Pedobacter heparinus NBRC 12017, Dyadobacter fermentans ATCC 700827, Herpetosiphon aurantiacus ATCC 23779, Saccharophagus degradans ATCC 43961, Spirosoma linguale ATCC 33905, and Teredinibacter turnerae ATCC 39867), because aerobes, more easily cultured on a large scale than anaerobes, are applicable in industrial processes. The recombinant CE-like proteins produced in Escherichia coli catalyzed epimerization at the C2 position of cellobiose, lactose, epilactose, and ß-1,4-mannobiose, whereas N-acetyl-D-glucosamine, N-acetyl-D-mannosamine, D-glucose, and D-mannose were inert as substrates. All the CEs, except for P. heparinus CE, the optimum pH of which was 6.3, showed highest activity at weakly alkaline pH. CEs from D. fermentans, H. aurantiacus, and S. linguale showed higher optimum temperatures and thermostability than the other enzymes analyzed. The enzymes from D. fermentans, S. linguale, and T. turnerae showed significantly high k(cat) and K(m) values towards cellobiose and lactose. Especially, T. turnerae CE showed a very high k(cat) value towards lactose, an attractive property for the industrial production of epilactose, which is carried out at high substrate concentrations.
Subject(s)
Bacteria, Aerobic/enzymology , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Carbohydrate Epimerases/isolation & purification , Carbohydrate Epimerases/metabolism , Cellobiose/metabolism , Aerobiosis , Bacteria, Aerobic/chemistry , Bacterial Proteins/classification , Carbohydrate Epimerases/classification , Enzyme Assays , Enzyme Stability , Escherichia coli/genetics , Glucose/metabolism , Hydrogen-Ion Concentration , Isoenzymes/classification , Isoenzymes/isolation & purification , Isoenzymes/metabolism , Kinetics , Lactose/metabolism , Mannose/metabolism , Phylogeny , Recombinant Proteins/classification , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Species Specificity , Stereoisomerism , Substrate Specificity , TemperatureABSTRACT
Overexpression of recombinant N-acetyl-D-glucosamine 2-epimerase, one of the key enzymes for the synthesis of N-acetylneuraminic acid, in E. coli led to the formation of protein inclusion bodies. In this study we report the recovery of active epimerase from inclusion bodies by direct solubilization with Tris buffer. At pH 7.0, 25% of the inclusion bodies were solubilized with Tris buffer. The specific activity of the solubilized proteins, 2.08±0.02 U/mg, was similar to that of the native protein, 2.13±0.01 U/mg. The result of circular dichroism spectroscopy analysis indicated that the structure of the solubilized epimerase obtained with pH 7.0 Tris buffer was similar to that of the native epimerase purified from the clarified cell lysate. As expected, the extent of deviation in CD spectra increased with buffer pH. The total enzyme activity recovered by solubilization from inclusion bodies, 170.41±10.06 U/l, was more than 2.5 times higher than that from the clarified cell lysate, 67.32±5.53 U/l. The results reported in this study confirm the hypothesis that the aggregation of proteins into inclusion bodies is reversible and suggest that direct solubilization with non-denaturing buffers is a promising approach for the recovery of active proteins from inclusion bodies, especially for aggregation-prone multisubunit proteins.
Subject(s)
Carbohydrate Epimerases/isolation & purification , Carrier Proteins/isolation & purification , Animals , Bioengineering , Buffers , Carbohydrate Epimerases/genetics , Carbohydrate Epimerases/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Circular Dichroism , Escherichia coli/enzymology , Escherichia coli/genetics , Inclusion Bodies/enzymology , Protein Multimerization , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Solubility , SwineABSTRACT
N-Acetyl-D: -neuraminic acid (Neu5Ac) can be produced from N-acetyl-D: -glucosamine (GlcNAc) and pyruvate by a chemoenzymatic process in which an alkaline-catalyzed epimerization transforms GlcNAc to N-acetyl-D: -manosamine (ManNAc). ManNAc is then condensed biocatalytically with pyruvate in the presence of N-acetyl-D: -neuraminic acid lyase (NAL) or by a two-step, fully enzymatic process involving bioconversions of GlcNAc to ManNAc and ManNAc to Neu5Ac using N-acetyl-D: -glucosamine 2-epimerase (AGE) and NAL. There are some drawbacks to this technique, such as lengthy reaction time, and the low conversion rate when the soluble forms of the enzymes are used in the two-step enzymatic process. In this study, the Escherichia coli-expressed AGE and NAL in the supernatant were purified by FP-based affinity chromatography and then immobilized on Amberzyme oxirane resin. These two immobilized enzymes, with a specific activity of 78.18 U/g for AGE and 69.30 U/g for NAL, were coupled to convert GlcNAc to Neu5Ac directly in one reactor. The conversion rate of the two-step reactions from GlcNAc to Neu5Ac was approximately 73% within 24 h. Furthermore, the immobilized AGE and NAL could both be used up to five reaction cycles without loss of activity or significant decrease of the conversion rate.
Subject(s)
Acetylglucosamine/metabolism , Carbohydrate Epimerases/metabolism , Carrier Proteins/metabolism , Enzymes, Immobilized/metabolism , N-Acetylneuraminic Acid/metabolism , Oxo-Acid-Lyases/metabolism , Bioreactors , Carbohydrate Epimerases/genetics , Carbohydrate Epimerases/isolation & purification , Carrier Proteins/genetics , Carrier Proteins/isolation & purification , Catalysis , Cloning, Molecular , DNA, Bacterial/genetics , Enzymes, Immobilized/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Hexosamines/metabolism , Hydrogen-Ion Concentration , Kinetics , Oxo-Acid-Lyases/genetics , Oxo-Acid-Lyases/isolation & purification , Pyruvic Acid/metabolism , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolismABSTRACT
UDP-alpha-D-galacturonic acid (UDP-GalA) is a key precursor for the synthesis of various bacterial and plant polysaccharides. UDP-glucuronic acid 4-epimerase (UGlcAE) catalyses the reversible conversion of UDP-alpha-D-glucuronic acid to UDP-GalA. UGlcAEs isolated from bacterial species have different biochemical properties when compared with the isoenzymes from the plant dicot species, Arabidopsis. However, little is known about the specificity of UGlcAE in Poaceae species. Therefore, we cloned and expressed in Escherichia coli several maize and rice UGlcAE genes, and compared their enzymatic properties with dicot homologs from Arabidopsis. Our data show that UGlcAE isoforms in different plant species have different enzymatic properties. For example, the Poaceae UGlcAE enzymes from rice and maize have significantly lower K(i) for UDP-xylose when compared with the Arabidopsis enzymes. The epimerases from different plant species are very specific and unlike their bacterial homolog in Klebsiella pneumoniae, can only use UDP-GlcA or UDP-GalA as their substrate. This study demonstrates that although members of the plant UGlcAE isoforms are highly conserved, the in vitro enzymatic activity of specific Poaceae isoform(s) may be regulated differently by specific nucleotide or nucleotide sugar.
Subject(s)
Carbohydrate Epimerases/chemistry , Carbohydrate Epimerases/metabolism , Oryza/enzymology , Zea mays/enzymology , Amino Acid Sequence , Arabidopsis/enzymology , Carbohydrate Epimerases/isolation & purification , Cloning, Molecular , Isoenzymes/chemistry , Isoenzymes/isolation & purification , Isoenzymes/metabolism , Molecular Sequence Data , Species Specificity , Time FactorsABSTRACT
Cellobiose 2-epimerase (CE, EC 5.1.3.11) catalyzes the reversible epimerization of cellobiose to 4-O-beta-D-glucopyranosyl-D-mannose. In this study, we found a CE gene in the genome sequence of non-cellulolytic Bacteroides fragilis NCTC 9343. The recombinant enzyme, expressed in Escherichia coli cells, catalyzed a hydroxyl stereoisomerism at the C-2 positions of the reducing terminal glucose and at the mannose moiety of cello-oligosaccharides, lactose, beta-mannobiose (4-O-beta-D-mannopyranosyl-D-mannose), and globotriose [O-alpha-D-galactopyranosyl-(1-->4)-O-beta-D-galactopyranosyl-(1-->4)-D-glucose]. The CE from B. fragilis showed less than 40% identity to reported functional CEs. It exhibited 44-63% identities to N-acyl-D-glucosamine 2-epimerase-like hypothetical proteins of unknown function in bacterial genome sequences of the phyla Firmicutes, Bacteroidetes, Proteobacteria, Chloroflexi, and Verrucomicrobia. On the other hand, it showed less than 26% identity to functional N-acyl-D-glucosamine 2-epimerases. Based on the amino acid homology and phylogenetic positions of the functional epimerases, we emphasize that many genes for putative N-acyl-D-glucosamine 2-epimerases and related hypothetical proteins of unknown function reported to date in the bacterial genomes should be annotated as CE-like proteins or putative CEs.
Subject(s)
Bacteroides fragilis/enzymology , Bacteroides fragilis/genetics , Carbohydrate Epimerases/genetics , Carbohydrate Epimerases/metabolism , Cellobiose/metabolism , Genes, Bacterial , Amino Acid Sequence , Bacteroides fragilis/cytology , Biocatalysis , Carbohydrate Epimerases/chemistry , Carbohydrate Epimerases/isolation & purification , Escherichia coli/genetics , Gene Expression , Hydrogen-Ion Concentration , Kinetics , Molecular Sequence Data , Phylogeny , Sequence Alignment , Substrate Specificity , TemperatureABSTRACT
OBJECTIVE: To purify a novel galactose mutarotase (TTE1925) from Thermoanaerobacter tengcongensis for crystallization and X-ray diffraction. METHODS: The tte 1925 gene was subcloned into the prokaryotic expression vector pGEX-6P-1 and overexpression was obtained in the E.coli BL21 (DE3) through transformation of the right recombinant plasmid that had been verified by colony PCR and sequencing. Soluble fusion protein with glutathione S-transferase tag expressed highly by the induction of isopropyl beta-D-thiogalactoside and was purified in a three-step procedure, which included Glutathione Sepharose 4B affinity, ion chromatography (Resource Q 6 mL), and gel filtration chromatography (10/300 superdex 200). RESULT: The purity of the purified protein was over 99% and a large amount of claval crystals whose X-ray diffraction reached 1.9 A were obtained. CONCLUSIONS: We successfully prepared TTE1925 with high purity and obtained crystals for X-ray diffraction. These work paved the way for the further research on the 3-D structure of TTE1925 and its biological mechanism.