D-allulose-3-epimerase (DPEase) is the key enzyme for isomerization of D-fructose to D-allulose. In order to improve its thermal stability, short amphiphilic peptides (SAP) were fused to the N-terminal of DPEase. SDS-PAGE analysis showed that the heterologously expressed DPEase folded correctly in Bacillus subtilis, and the protein size was 33 kDa. After incubation at 40 °C for 48 h, the residual enzyme activity of SAP1-DSDPEase was 58%. To make the recombinant B. subtilis strain reusable, cells were immobilized with a composite carrier of sodium alginate (SA) and titanium dioxide (TiO2). The results showed that 2% SA, 2% CaCl2, 0.03% glutaraldehyde solution and a ratio of TiO2 to SA of 1:4 were optimal for immobilization. Under these conditions, up to 82% of the activity of immobilized cells could be retained. Compared with free cells, the optimal reaction temperature of immobilized cells remained unchanged at 80 °C but the thermal stability improved. After 10 consecutive cycles, the mechanical strength remained unchanged, while 58% of the enzyme activity could be retained, with a conversion rate of 28.8% achieved. This study demonstrated a simple approach for using SAPs to improve the thermal stability of recombinant enzymes. Moreover, addition of TiO2 into SA during immobilization was demonstrated to increase the mechanical strength and reduce cell leakage.
Bacillus subtilis/metabolism* ; Carbohydrate Epimerases/genetics* ; Enzyme Stability ; Enzymes, Immobilized/metabolism* ; Fructose ; Hydrogen-Ion Concentration ; Racemases and Epimerases ; Temperature

Enzymatic conversion is very important to produce functional rare sugars, but the conversion rate of single enzymes is generally low. To increase the conversion rate, a dual-enzyme coupled reaction system was developed. Dual-enzyme coupled reaction system was constructed using D-psicose-3-epimerase (DPE) and L-rhamnose isomerase (L-RhI), and used to convert D-fructose to D-psicose and D-allose. The ratio of DPE and L-RhI was 1:10 (W/W), and the concentration of DPE was 0.05 mg/mL. The optimum temperature was 60 degrees C and pH was 9.0. When the concentration of D-fructose was 2%, the reaction reached its equilibrium after 10 h, and the yield of D-psicose and D-allose was 5.12 and 2.04 g/L, respectively. Using the dual-enzymes coupled system developed in the current study, we could obtain sugar syrup containing functional rare sugar from fructose-rich raw material, such as high fructose corn syrup.
Aldose-Ketose Isomerases ; metabolism ; Carbohydrate Epimerases ; metabolism ; Fructose ; chemistry ; Glucose ; chemistry ; Hydrogen-Ion Concentration ; Temperature

Rare sugar is a kind of important low-energy monosaccharide that is rarely found in nature and difficult to synthesize chemically. D-allose, a six-carbon aldose, is an important rare sugar with unique physiological functions. It is radical scavenging active and can inhibit cancer cell proliferation. To obtain D-allose, the microorganisms deriving D-psicose 3-epimerase (DPE) and L-rhamnose isomerase (L-RhI) have drawn intense attention. In this paper, DPE from Clostridium cellulolyticum H10 was cloned and expressed in Bacillus subtilis, and L-RhI from Bacillus subtilis 168 was cloned and expressed in Escherichia coli BL21 (DE3). The obtained crude DPE and L-RhI were then purified through a HisTrap HP affinity chromatography column and an anion-exchange chromatography column. The purified DPE and L-RhI were employed for the production of rare sugars at last, in which DPE catalyzed D-fructose into D-psicose while L-RhI converted D-psicose into D-allose. The conversion of D-fructose into D-psicose by DPE was 27.34%, and the conversion of D-psicose into D-allose was 34.64%.
Aldose-Ketose Isomerases ; metabolism ; Bacillus subtilis ; enzymology ; Carbohydrate Epimerases ; metabolism ; Clostridium cellulolyticum ; enzymology ; Escherichia coli ; metabolism ; Fructose ; metabolism ; Glucose ; metabolism

OBJECTIVETo purify a novel galactose mutarotase (TTE1925) from Thermoanaerobacter tengcongensis for crystallization and X-ray diffraction.

METHODSThe 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).

RESULTThe 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.

CONCLUSIONSWe 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.

Bacterial Proteins ; biosynthesis ; chemistry ; isolation & purification ; Carbohydrate Epimerases ; biosynthesis ; chemistry ; isolation & purification ; Cloning, Molecular ; Crystallization ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Thermoanaerobacter ; enzymology ; genetics ; Transformation, Bacterial

Heparin and heparan sulfate are a class of glycosaminoglycans for clinical anticoagulation. Heparosan N-sulfate-glucuronate 5-epimerase (C5, EC 5.1.3.17) is a critical modifying enzyme in the synthesis of heparin and heparan sulfate, and catalyzes the inversion of carboxyl group at position 5 on D-glucuronic acid (D-GlcA) of N-sulfoheparosan to form L-iduronic acid (L-IdoA). In this study, the heparin C5 epimerase gene Glce from zebrafish was expressed and molecularly modified in Escherichia coli. After comparing three expression vectors of pET-20b (+), pET-28a (+) and pCold Ⅲ, C5 activity reached the highest ((1 873.61±5.42) U/L) with the vector pCold Ⅲ. Then we fused the solution-promoting label SET2 at the N-terminal for increasing the soluble expression of C5. As a result, the soluble protein expression was increased by 50% compared with the control, and the enzyme activity reached (2 409±6.43) U/L. Based on this, site-directed mutations near the substrate binding pocket were performed through rational design, the optimal mutant (V153R) enzyme activity and specific enzyme activity were (5 804±5.63) U/L and (145.1±2.33) U/mg, respectively 2.41-fold and 2.28-fold of the original enzyme. Modification and expression optimization of heparin C5 epimerase has laid the foundation for heparin enzymatic catalytic biosynthesis.
Animals ; Carbohydrate Epimerases ; biosynthesis ; chemistry ; genetics ; Escherichia coli ; Gene Expression ; Heparin ; metabolism ; Heparitin Sulfate ; metabolism ; Iduronic Acid ; metabolism ; Zebrafish Proteins ; biosynthesis ; chemistry ; genetics

Carbohydrate Epimerases/metabolism* ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Esophageal Neoplasms/genetics* ; Gene Expression Regulation, Neoplastic ; Humans ; Ketone Oxidoreductases/metabolism* ; MAP Kinase Signaling System ; Matrix Metalloproteinase 2/genetics* ; Matrix Metalloproteinase 9 ; Neoplasm Invasiveness/genetics*

Despite recent economic prosperity, Korea still has high prevalence of tuberculosis. Molecular biologic characterization of Korean Mycobacterium tuberculosis strains might provide a deeper understanding of the forces contributing to the spread of tuberculosis in Korea. Therefore, we analyzed the cell lysate proteome of a representative Korean Mycobacterium tuberculosis isolate (K01) in comparison with laboratory reference strains H37Rv and H37Ra. Seven spots were strongly expressed only in K01 strain compared with M. tuberculosis H37Rv and H37Ra. Through continuous MALDI-MS analysis, these spots were identified as hypothetical protein Rv3849, secreted immunogenic protein Mpt64, Acetyl/propionyl-CoA Carbpxylase (AccD1), alkyl hydroperoxide reductase C (AhpC), N-acetylmuramyl-L-alanine amidase, a putative UDP glucose epimerase, and a transposase. A deeper study of these proteins may provide a clue in the development of effective new anti-tuberculosis vaccines against Korean M. tuberculosis isolates.
Korea ; Mycobacterium tuberculosis* ; Mycobacterium* ; Peroxiredoxins ; Prevalence ; Proteome* ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Transposases ; Tuberculosis ; UDPglucose 4-Epimerase ; Vaccines

OBJECTIVEA comparative study on the role of Campylobacter jejuni (CJ) HB9313 and galE mutant in inducing experimental sciatic nerve damage was conducted in guinea pigs in order to explore whether CJ lipo-oligosaccharide (LOS) is critical component associated with peripheral nerve lesions and find experimental evidence for the presumption of molecular mimicry on the pathogenesis of Guillain-Barre syndromes (GBS) with CJ antecedent infection.

METHODSA total of 32 guinea pigs were randomly divided into four groups: parental strain group (n = 10), galE mutant group (n = 10), control group (n = 6) and PBS group (n = 6), and immunized with the whole cell antigens of CJ HB9313 with Freund's adjuvant (FA), the whole cell antigens of galE mutant (without ganglioside-like structure) with FA, PBS with FA, and PBS alone, respectively. Enzyme-linked immunosorbent assay (ELISA) was employed to detect anti-LOS and anti-ganglioside GM1 antibodies in sera of these animals, and comparative morphologic studies of pathologic changes were carried out on the sciatic nerves, including examination of teasing fibers, examination of semithin sections made from epon-embedded tissue blocks under light microscope and transmission electron microscope.

RESULTSELISA results indicated that after immunization, the levels of anti-LOS IgG antibody were significantly elevated in animals from parental strain group and galE mutant group as compared with those before immunization (P < 0.01). No statistically significant difference was found between the two groups. However, the mean optical densities (ODs) of IgG antibody against GM1 at 14 and 28 day after immunization, in parental strain group, were 0.661 +/- 0.290 and 0.984 +/- 0.025, respectively, significantly higher than those of galE mutant group, which were 0.193 +/- 0.078 and 0.180 +/- 0.063 (P < 0.01). The results of morphologic examination on sciatic nerves showed that for teased-fiber study, incidence of pathologic abnormalities of teased fibers from animals of galE mutant group was 4.9% (98/2000), significantly lower than that from parental strain group, which was 16% (320/2000), characterized by predominantly axonal degeneration. The difference between them was highly significant statistically (P < 0.01). Examination of semithin sections of sciatic nerves also revealed that obvious pathological changes occurred in the animals from parental strain group, while only minimal abnormalities could be seen from galE mutant group, there was a significant differences between them (P < 0.01). In parental strains group, the predominant pathologicanl change was axonal degeneration with considerable variation in severity. These morphologic changes were confirmed by electron microscopy.

CONCLUSIONCompared with parental strain, galE mutant without ganglioside-like structure no longer could induce anti-GM1 antibodies, nor induce obvious immune damage of peripheral nerves in experimental guinea pigs. The results of this study provide a strong support to the hypothesis of molecular mimicry as a pathogenesis in patients with GBS following CJ antecedent infection.

Animals ; Antibodies, Bacterial ; blood ; Campylobacter jejuni ; genetics ; immunology ; pathogenicity ; G(M1) Ganglioside ; immunology ; Guillain-Barre Syndrome ; etiology ; Guinea Pigs ; Immunization ; Lipopolysaccharides ; immunology ; UDPglucose 4-Epimerase ; physiology