Construction and immobilization of recombinant Bacillus subtilis with D-allulose 3-epimerase / 生物工程学报

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.

Expression optimization and molecular modification of heparin C5 epimerase / 生物工程学报

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.

Production of sugar syrup containing rare sugar using dual-enzyme coupled reaction system / 生物工程学报

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.

Bioconversion of D-fructose to D-allose by novel isomerases / 生物工程学报

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%.

Expression, purification, and crystallization of a novel galactose mutarotase from Thermoanaerobacter tengcongensis / 中国医学科学院学报

<p><b>OBJECTIVE</b>To purify a novel galactose mutarotase (TTE1925) from Thermoanaerobacter tengcongensis for crystallization and X-ray diffraction.</p><p><b>METHODS</b>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).</p><p><b>RESULT</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

Channeling of the intermediates and catalytic facilitation to Rubisco in a multienzyme complex of Calvin cycle enzymes.

Calvin cycle multienzyme complex, consisting of phosphoriboisomerase, phosphoribulokinase and ribulose-1,5-bisphosphate carboxylase (Rubisco), shows ribose-5-phosphate + ATP dependent CO2 fixation activity with a small but discernible lag. Transient time analysis showed that the lag at pH 7 was independent of multienzyme concentration and was significantly lower than the expected transient time calculated from Km and Vmax of the individual enzymes, indicative of channeling of the intermediates in the enzyme complex. Channeling of ribulose-1,5-bisphosphate was found to offer a catalytic advantage to Rubisco. Rubisco shows a decrease in activity during catalysis in ribulose-1,5-bisphosphate dependent CO2 fixation reaction, due to the formation of the catalytic inhibitor. Such a decrease of Rubisco activity was not observed in ribose-5-phosphate + ATP dependent CO2 fixation reaction and the catalytic inhibitor was also not detected. These results suggested that the intermediates are channeled in the complex and channeling offers a catalytic facilitation to Rubisco.

Photoprotection of ultraviolet light irradiated E. Coli. B/r cells.

Ultraviolet irradiated E. Coli. B/r cells recover from UV damage when the cells are kept in dark due to dark repair mechanism. Photoprotection by illumination of the cells in near UV light prior to the exposure to UV light increases the capacity of the cells to induce L-arabinose isomerase synthesis in response to inducer, L-arabinose. The survival of the cells is dependent on the UV dose. The increased synthesis of L-arabinose isomerase after photoprotection is due to the amount of cyclic AMP in the cells.

Produccion de glucosa-isomerasa por Streptomyces phaeochromogenes. / Production of glucose-isomerase from Streptomyces phaeochromogenes

Se estudio la produccion de glucosa-isomerasa utilizando una cepa de Streptomyces phaeochromogenes NRRL B-3559. Se considero la influencia de la composicion del medio cultivo y de las condiciones de aeracion.De tres tipos de colonias bien diferenciadas que el microorganismo es capaz de desarrollar, las grises fueron de la mayor produccion enzimatica. Con el objeto de evitar la variabilidad de la poblacion microbiana y obtener resultados sobre la composicion del medio, se vio que la concentracion de cobalto es critica en cuanto a los niveles de enzima alcanzados.Empleando una concentracion de Cl2Co.6H2 de 0,18 g/l se obtuvieron 1600 UE/l. Los estudios de aeracion realizados en fermentadores con agitacion mecanica del tipo convencional, mostraron que es posible obtener valores de actividad enzimatica similares a los producidos en erlenmeyers agitados, operando a 550 rpm y un flujo de aire de 1 1/1.min. Se demostro que el cultivo estaba adecuadamente aerado en razon de que los valores de consumo y demanda celular de oxigeno fueron semilares