Covalent immobilization of recombinant Citrobacter koseri transaminase onto epoxy resins for consecutive asymmetric synthesis of L-phosphinothricin.

Transaminase responsible for alienating prochiral ketone compound is applicable to asymmetric synthesis of herbicide L-phosphinothricin (L-PPT). In this work, the covalent immobilization of recombinant transaminase from Citrobacter koseri (CkTA) was investigated on different epoxy resins. Using optimum ES-105 support, a higher immobilized activity was obtained via optimizing immobilization process in terms of enzyme loading, coupling time and initial PLP concentration. Crucially, due to blocking unreacted epoxy groups on support surface with amino acids, the reaction temperature of blocked immobilized biocatalyst was enhanced from 37 to 57 °C. Its thermostability at 57 °C was also found to be superior to that of free CkTA. The Km value was shifted from 36.75 mM of free CkTA to 39.87 mM of blocked immobilized biocatalyst, demonstrating that the affinity of enzyme to the substrate has not been apparently altered. Accordingly, the biocatalyst performed the consecutive synthesis of L-PPT for 11 cycles (yields>91%) with retaining more than 91.13% of the initial activity. The seemingly the highest reusability demonstrates this biocatalyst has prospective for reducing the costs of consecutive synthesis of L-PPT with high conversion.

Improving the catalytic activity of ß-glucosidase from Coniophora puteana via semi-rational design for efficient biomass cellulose degradation.

In order to improve the degradation activity of ß-glucosidase (CpBgl) from Coniophora puteana, the structural modification was conducted. The enzyme activity of mutants CpBgl-Q20C and CpBgl-A240S was increased by 65.75% and 58.58%, respectively. These mutants exhibited maximum activity under the same conditions as wild-type CpBgl (65 â„ƒ and pH 5.0), slightly improved stabilities compared that of the wild-type, and remarkably enhanced activities in the presence of Mn2+ or Fe2+. The Vmax of CpBgl-Q20C and CpBgl-A240S was increased to 138.18 and 125.14 µmol/mg/min, respectively, from 81.34 µmol/mg/min of the wild-type, and the catalysis efficiency (kcat/Km) of CpBgl-Q20C (335.79 min-1/mM) and CpBgl-A240S (281.51 min-1/mM) was significantly improved compared with that of the wild-type (149.12 min-1/mM). When the mutant CpBgl-Q20C were used in the practical degradation of different biomasses, the glucose yields of filter paper, corncob residue, and fungi mycelia residue were increased by 17.68%, 25.10%, and 20.37%, respectively. The spatial locations of the mutation residues in the architecture of CpBgl and their unique roles in the enzyme-substrate binding and catalytic efficiency were probed in this work. These results laid a foundation for evolution of other glycoside hydrolases and the industrial bio-degradation of cellulosic biomass in nature.

Formation of tungstate-containing cluster ions by polyoxotungstate anions under matrix-assisted laser desorption/ionization conditions in the gas phase.

The gas-phase studies of transition-metal oxides continue to attract interest as such oxides are being used as catalysts in various oxidation processes. In this paper, singly negatively charged heteropolyoxotungstate and isopolyoxotungstate ion clusters were produced from Keggin-type polyoxotungstates by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR MS). It was found that the ion series [(PO(3))(WO(3))(n)](-), [(WO(3))(n)](-) and [(OH)(WO(3))(n)](-) were the main fragment ions in the mass spectra and the matrix greatly influenced the resulting cluster ion abundances. [(PO(3))(WO(3))(3)](-), [(WO(3))(3)](-) and [(OH)(WO(3))(4)](-) were the most intense ions in each series when 2-(4-hydroxyphenylazo)benzoic acid was the matrix, whereas [(PO(3))(WO(3))(4)](-), [(WO(3))(6)](-) and [(OH)(WO(3))(4)](-) were the most intense when dithranol (DIT) was the matrix. In addition, a new kind of hybrid ion [W(2)C(14)H(7)O(8)](-) was produced through the reaction of DIT and [(OH)(WO(3))](-) in the plume of the gas phase. These results highlight the utility of the MALDI-FT method for obtaining novel ion clusters and also show the stability of these clusters.

Efficient high-solids enzymatic hydrolysis of corncobs by an acidic pretreatment and a fed-batch feeding mode.

Corncob is an abundant and renewable resource that could be enzymatically hydrolyzed to fermentable sugar. A major impediment in corncob utilization is the low hydrolysis efficiency at high-solids content. This study attempted different pretreatment methods and fed-batch modes to achieve a 25% solids content hydrolysis with high yields. Natural corncobs were compared with acid-treated and acid-alkali-treated corncobs in terms of kinetics parameters, conversion rate and glucose titer. By feeding in batches, a "low amount and high frequency" mode (10%-3%-3%-3%-3%-3%, every 5 h) was confirmed to be optimal for a 25% high-solids hydrolysis system with a cellulase loading of 12 mg/g (7.3 FPU/g), resulted with an 84.4% glucose yield at 96 h. Our results demonstrated that combination of both optimized pretreatment method and fed-batch mode were a favored process model for high-solids hydrolysis of lignocellulose, boosting cellulose hydrolysis efficiency and sugar yields on an industrial scale.

Production of acrylic acid from Acrylonitrile by immobilization of Arthrobacter nitroguajacolicus ZJUTB06-99.

Immobilized cells of Arthrobacter nitroguajacolicus ZJUTB06-99 capable of producing nitrilase was used for biotransformation of acrylonitrile to acrylic acid. Six different entrapment matrixes were chosen to search for a suitable support in terms of nitrilase activity. Ca-alginate proved to be more advantageous over other counterparts in improvement of the biocatalyst activity and bead mechanical strength. The effects of sodium alginate concentration, CaCl2 concentration, bead diameter, and ratio by weight of cells to alginate, on biosynthesis of acrylic acid by immobilized cells were investigated. Maximum activity was obtained under the conditions of 1.5% sodium alginate concentration, 3.0% CaCl2 concentration, and 2-mm bead size. The beads coated with 0.10% polyethylenimine (PEI) and 0.75% glutaraldehyde (GA) could tolerate more phosphate and decrease leakage amounts of cells from the gel. The beads treated with PEI/ GA could be reused up to 20 batches without obvious decrease in activities, which increased about 100% compared with the untreated beads with a longevity of 11 batches.

[The protective effects to the function of kidney and long by clearing of cytokines in patients with open-heart surgery].

OBJECTIVE: To observe the effect of cytokines absorption on renal and respiratory function in patients with open-heart surgery. METHODS: 30 patients undergoing valve replacement with cardiopulmonary bypass (CPB) were randomly divided into two groups. A sulfonated polyacrylonitrile hemofilter (AN69) that has been used to absorb cytokines was connected into the efferent limb of CPB in Group A (n = 15), and a cellulose triacetate hemofilter (CT 190G) instead of AN69 was used as controls (Group B, n = 15). The levels of plasma pro-inflammatory (TNF-alpha, IL-6, IL-8) anti-inflammatory cytokines (IL-10, IL-1ra), C-reactive protein (CRP) levels, and post-operation renal and respiratory function were compared between the two groups. Blood samples were analysed for TNF-alpha and IL-6 and IL-8 and C-reactive protein (CRP). The changes in renal, respiratory function were also observed. RESULTS: (1) At the end of CPB, TNF-alpha 10 ng/L +/- 3 ng/L and IL-6 115 ng/L +/- 22 ng/L levels in Group A were significantly lower than that in Group B 13 ng/L +/- 3 ng/L, 134 ng/L +/- 29 ng/L) respectively (P < 0.05 in all). There is no statistical differences in plasma IL-10 and IL-1ra levels between the two groups. (2) After 24 hours of CPB, the magnitude of increased body temperature, heart rate, white blood cell and plasma CRP in Group A [1.6 degrees C +/- 0.2 degrees C, 15/min +/- 4/min, (17 +/- 3) x 10(9)/L, 56 mg/L +/- 13 mg/L], were significantly lower than that in Group B [2.1 degrees C +/- 0.2 degrees C, 23/min +/- 6/min, (22 +/- 3) x 10(9)/L, 69 mg/L +/- 15 mg/L] respectively (P < 0.05 in all). (3) After 24 hours of CPB, the levels of 24h urinary protein excretion and urinary N-acetyl-beta-D-glucosaminidase (NAG) were significantly lower in Group A when compared to that in Controls (0.20 g/d +/- 0.08 g/d vs 0.30 g/d +/- 0.14 g/d, 28 U/L +/- 11 U/L vs 38 U/L +/- 13 U/L respectively), P < 0.05 in all. The level of creatinine clearance (Ccr) in Group A (68 +/- 7) ml.min(-1).1.73 m(-2) was significantly elevated than that in Group B (57 +/- 11) ml.min(-1).1.73 m(-2) (P < 0.05). (4) One hour after the end of CPB, the magnitude of increased plateau airway pressure (P(Plateau)) and peak airway pressure (P(Peak)) in Group A were significantly lower than that in Controls (P < 0.01 in all). The duration that need mechanical ventilation after operation in Group A (4.9 h +/- 0.6 h) was much shorter than that in Group B (5.8 h +/- 0.8 h, P < 0.05). CONCLUSIONS: Lowering the plasma levels of cytokines by extracorporeal absorption may attenuate systemic inflammatory response and protect lung and kidney function in patients with open-heart surgery.

Biosynthesis of iminodiacetic acid from iminodiacetonitrile by immobilized recombinant Escherichia coli harboring nitrilase.

Iminodiacetic acid (IDA) is widely used as an intermediate in the manufacture of chelating agents, glyphosate herbicides and surfactants. In the current work, the fragment with the length of 1,110 bp encoding the Acidovorax facilis nitrilase was obtained. The recombinant nitrilase expressed in Escherichia coli BL21 (DE3) was successfully used in the production of IDA from iminodiacetonitrile. To improve the stability of operation, the recombinant cells were entrapped in polyvinyl alcohol (PVA) and sodium alginate (SA) copolymer. The maximum relative nitrilase activity with 98.1% was further observed at 1.0% SA, 8.0% PVA, 1.0% CaCl(2), and 5.0% wet cells, under conditions of 1.0% iminodiacetonitrile in distilled water and a temperature of 40°C, respectively. The entrapped cells facilitated easy separation and good recycling compared with free cells. Moreover, the immobilized cells showed good operation and storage stability. This report is the first to describe IDA preparation using immobilized recombinant E. coli harboring nitrilase.

Posterolateral spinal fusion with nano-hydroxyapatite-collagen/PLA composite and autologous adipose-derived mesenchymal stem cells in a rabbit model.

Spinal fusion is routinely performed to treat low back pain caused by degeneration of intervertebral discs. An autologous bone graft derived from the iliac crest is the standard procedure used for spinal fusion. However, several shortcomings, including pseudarthrosis, pain and the need for blood transfusion are known to be associated with the procedure. Our study analysed the effectiveness of a new mineralized collagen matrix, nano-hydroxyapatite-collagen-polylactic acid (nHAC-PLA), combined with autologous adipose-derived mesenchymal stem cells (ADMSCs) as a graft material for posterolateral spinal fusion in a rabbit model. Forty rabbits were randomly divided into four groups: autologous iliac crest bone group (ACB), nHAC-PLA composite group (nHAC-PLA), autologous iliac crest bone mixed with nHAC-PLA composite group (ACB + nHAC-PLA), and nHAC-PLA composite combined with ADMSCs (ADMSCs + nHAC-PLA). The viability and the proliferation of the ADMSCs seeded on the scaffolds were evaluated by live/dead kit and MTT assay in vitro, respectively. Lumbar posterolateral fusions were assessed by manual palpation, radiographical and histological procedures, mechanical strength and scanning electronic microscopy (SEM) in 10 weeks of observation. The results showed that the rate of fusion was significantly higher in the ACB and ADMSCs + nHAC-PLA groups than that in the nHAC-PLA and ACB + nHAC-PLA groups. It was not significantly higher in the ACB group than in the ADMSCs + nHAC-PLA group. From microstructural analysis of the samples using histological staining methods, there was more new bone-like tissue formation in the ACB and ADMSCs + nHAC-PLA groups than that in the other two groups at the 10th postoperative week. Our study demonstrated the effective impact of nHAC-PLA combined with ADMSCs in rabbit posterolateral spinal fusion.

Functional improvement of infarcted heart by co-injection of embryonic stem cells with temperature-responsive chitosan hydrogel.

Transplantation of embryonic stem cells (ESCs) can improve cardiac function in treatment of myocardial infarction. The low rate of cell retention and survival within the ischemic tissues makes the application of cell transplantation techniques difficult. In this study, we used a temperature-responsive chitosan hydrogel (as scaffold) combined with ESCs to maintain viable cells in the infarcted tissue. Temperature-responsive chitosan hydrogel was prepared and injected into the infarcted heart wall of rat infarction models alone or together with mouse ESCs. The result showed that the 24-h cell retention and 4 week graft size of both groups was significantly greater than with a phosphate buffered saline control. After 4 weeks of implantation, heart function, wall thickness, and microvessel densities within the infarct area improved in the chitosan + ESC, chitosan, and ESC group more than the PBS control. Of the three groups, the chitosan + ESC performed best. Results of this study indicate that temperature-responsive chitosan hydrogel is an injectable scaffold that can be used to deliver stem cells to infarcted myocardium. It can also increase cell retention and graft size. Cardiac function is well preserved, too.

One-dimensional copper(II) coordination polymers bridged both by mu-trans-oxamidates and phenyldicarboxylates: Synthesis, crystal structure and DNA binding studies.

Two new one-dimensional copper(II) polymers with formulae of [Cu(2)(H(2)O)(2)(dmapox)(ipa)(2)](n) (1) and [Cu(2)(H(2)O)(2)(dmapox)(tpa)(2)](n) (2), where dmapox, ipa and tpa stand for the dianion of N,N'-bis[3-(dimethylamino)propyl]oxamide, isophthalic acid and terephthalic acid respectively, have been synthesized and characterized by elemental analysis, conductivity measurement, IR and electronic spectral studies. The crystal structures of the two complexes have been determined by X-ray single-crystal diffraction. The structures of 1 and 2 consist of one-dimensional copper(II) coordination polymeric chain constructed both by the bis-tridentate trans-dmapox and bis-monodentate phenyldicarboxylate bridging ligands. In the two complexes, the environment around the copper(II) atoms can be described as distorted square-pyramid and the Cu...Cu separations through mu-trans-dmapox and phenyldicarboxylato bridging ligands are 5.245(5) A and 8.212(3) A for 1, 5.237(8) A and 11.171(1) A for 2, respectively. The binding properties of the two copper(II) polymers with herring sperm DNA (HS-DNA) have been investigated by using absorption and emission spectral and electrochemical techniques and viscometry. The results show that the two copper(II) complexes interact with the HS-DNA in the mode of intercalation with the intrinsic binding constants of 1.22(+/-0.2)x10(4)M(-1) and 1.45(+/-0.3)x10(4)M(-1) for 1 and 2, respectively.