Enzyme-catalyzed direct three-component aza-Diels–Alder reaction using lipase from Candida sp.99–125

The direct three-component aza-Diels–Alder reaction was conducted with lipase as a catalyst for the first time. Under the optimized conditions, the aza-Diels–Alder reaction catalyzed by lipase from Candida sp.99–125 provided the products in moderate to excellent yields. Meanwhile, the endo/exo ratio reached up to 88:12.     

无溶剂体系中固定化脂肪酶Candida sp. 99-125催化合成油酸低碳醇酯

采用固定化脂肪酶Candida sp.99-125在无溶剂体系中催化合成油酸低碳醇酯,考察了加酶量、温度、酸醇摩尔比和醇结构对油酸酯化率的影响。结果表明,加酶量为底物质量的3%,最适温度为20℃,酸与醇摩尔比为1∶1,甲醇对该酶有一定的毒性,由于空间位阻效应,该酶对伯醇具有高选择性,对仲醇、叔醇的选择性低,且对长链脂肪酸催化活性高,对带支链的多元酸、多元醇活性低。该酶重复使用5次,酶活性基本没有降低。与传统的化学法相比,用该酶催化合成酯类化合物的色泽更浅。     

Recombinant Expression and Characterization of an Organic-Solvent-Tolerant α-Amylase from Exiguobacterium sp. DAU5

The enzyme from halophilic microorganisms often has unique properties such as organic-solvent-tolerance. In this study, a novel organic-solvent-tolerant α-amylase gene was cloned from the mild halophile Exiguobacterium sp. DAU5. The open reading frame (ORF) of the enzyme consisted of 1,545 bp and encoded 514 amino acids, the primary sequence revealed that it belongs to the glycoside hydrolase (GH) family 13. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed an AmyH monomer of 57 kDa. The enzyme exhibited maximal activity at 40 °C in pH?8.5 glycine–NaOH buffer, and the activity was strongly inhibited by Zn²⁺, Cu²⁺, and Fe²⁺. The α-amylase AmyH exhibited high hydrolysis activity toward soluble starch, and the major hydrolysis products were maltose, maltotriose, and maltopentaose; the AmyH could not efficiently hydrolyze oligosaccharides smaller than maltoheptaose, nor could it act on the β-1,4 or α-1,6 glucosidic bonds in xylan or pullulan, respectively. In addition, the α-amylase exhibited better tolerance to organic solvents, as it was stable in the presence of dimethylsulfoxide (DMSO), methanol, ethanol, and acetone. Base on all of these results, the enzyme could be useful for practical application in the bakery industry and in biotechnological processes that occur in the presence of organic solvents.     

Identification and Functional Characterization of an α-Amylase with Broad Temperature and pH Stability from Paenibacillus sp.

Amylases are important industrial enzymes that have been applied widely in the food, detergent, and pulp industries and fermentation processes. In the present study, a gene encoding an alpha-amylase from the genomic DNA library of Paenibacillus sp. was identified and characterized. The amylase gene designated amy1 was shown to consist of 1,980 bp and shared sequence identity towards α-amylase genes from other Bacillus sp. The deduced amino acid sequence for Amy1 indicated 80 % sequence identity with other Bacillus strains. Heterologous expression of recombinant Amy1 in Escherichia coli BL21(DE3) facilitated the recovery of this protein in soluble form. Enzyme kinetic data revealed Amy1 to have a K _m of 23.83 mg/mL and K _cat of 48.74 min^?1 and K _cat /K _m of 2 min^?1 mg^?1 mL^?1 for starch. The activity of this protein was found to be enhanced by Mn²⁺, and furthermore, Amy1 remained active at a broad pH range (4–10) and temperature (30–90 °C). The ability of Amy1 to act on food waste under broad temperature and pH conditions, together with its ability to produce simple sugars, shows many advantages for further application in the food industry.     

Synthesis and Characterization of Silver–PVA Nanocomposite for Sensor and Antibacterial Applications

Silver–polyvinyl alcohol (PVA) nanocomposite is synthesized by a simple chemical reduction method in aqueous media and characterized by various techniques such as UV–Visible spectroscopy, transmission electron microscopy, X-ray diffraction and infrared spectroscopy. The morphology of the obtained silver nanoparticles is spherical in shape with an average diameter of 24 nm. The prepared silver–PVA nanocomposite is demonstrated to detect the concentration of heavy metal contaminant cadmium in water based on linear change in surface plasmon resonance absorption strength. The antibacterial tests show the inhibiting action of this nanocomposite against pathogenic bacteria Escherichia coli and Pseudomonas aeruginosa (gram negative), Bacillus cereus and Staphylococcus aureus (gram positive), which are found commonly in water. These observed optical and antibacterial properties, suggest the possible utilization of prepared silver–PVA nanocomposite in the water purification application.     

Biochemical Characterization of Thermostable and Detergent-Tolerant β-Agarase,PdAgaC, from Persicobacter sp. CCB-QB2

Applied Biochemistry and Biotechnology - Persicobacter sp. CCB-QB2 belonging to the family Flammeovirga is an agarolytic bacterium and exhibits a diauxic growth in the presence of tryptone and...     

Synthesis and Characterization of Novel Layered Lithium Sodium Silicate （Silinaite） and the Mesoporous Materials from Silinaite

Novel layered silinaite has been synthesized using an aqueous mixture of water glass, LiOH and NaOH under hydrothermal crystallization. Subsequently transformation of silinaite into mesoporous materials (SDM) was achieved at mild condition using cetyltrimethylammonium bromide as structure-directing agents. The resulting samples were characterized by XRD, SEM, FTIR nitrogen adsorption-desorption isotherms and catalytic performance in bulky molecular involved reaction. The results revealed that synthesized mesoporous materials derived from the silinaite exhibited an ordered hexagonal crystal structure with average pore diameter 2.7 nm and BET surface area 817m2/g. The SDM-supported ZnCl2 catalyst, prepared by impregnation-evaporation method, retained the mesoporous structure and showed high selectivity in alkylation of benzene with benzyl chloride.     

Study on the Synthesis and Characterization of Novel Immobilized β-Cyclodextrin Polymer (Ⅰ)

A series of novel β-cyclodextrin polymers was synthesized by immobilization of β-cyclodextrin on the chloromethylated crosslinked polystyrene carriers. The synthetic conditions such as reaction time, temperature, molar feeding ratio of reactants, the degree of crosslinking of polystyrene and the catalysts used were studied in detail and the chemical and physical structures of the formed β-CDpolymers were characterized. Results show that the preparation method is simple and the amount of β-CD immobilized is high. As biomedical adsorbents, they were tested for removal of various endogenous and exogenous toxins such as phenols, aromatic amins, barbitals and unconjugated bilirubin. Results indicate that the adsorptión capacity for those toxins can be enhanced by the inclusion interaction among the β-CD, the substrate molecules and the β-CD polymers.     

Iodo–sesquiterpene hydroquinone and brominated indole alkaloids from the Thai sponge Smenospongia sp.

6′-Iodoaureol, an iodo–sesquiterpene hydroquinone, 6′-aureoxyaureol, a bissesquiterpene hydroquinone, and four brominated indole alkaloids 3–6 were isolated from the Thai sponge Smenospongia sp. Additionally, four known sesquiterpene hydroquinones 12–15, ten known brominated indole alkaloids 7–11, 17–18, and 20–22, ergosterol, and furospinosulin-1 were also isolated. Compound 1 is the first reported iodo–sesquiterpene hydroquinone and compounds 7–11 were isolated for the first time from natural sources. The structures were characterized by spectroscopic analyses as well as comparison with literature data. Several of these metabolites were evaluated for cytotoxic activity against seven tumor cell lines.     

Immobilized β-cyclodextrin on surface-modified carbon-coated cobalt nanomagnets: reversible organic contaminant adsorption and enrichment from water

Surface-modified magnetic nanoparticles can be used in extraction processes as they readily disperse in common solvents and combine high saturation magnetization with excellent accessibility. Reversible and recyclable adsorption and desorption through solvent changes and magnetic separation provide technically attractive alternatives to classical solvent extraction. Thin polymer layered carbon-coated cobalt nanoparticles were tagged with β-cyclodextrin. The resulting material reversibly adsorbed organic contaminants in water within minutes. Isolation of the immobilized inclusion complex was easily carried out within seconds by magnetic separation due to the strong magnetization of the nanomagnets (metal core instead of hitherto used iron oxide). The trapped molecules were fully and rapidly recovered by filling the cyclodextrin cavity with a microbiologically well accepted substitute, e.g., benzyl alcohol. Phenolphthalein was used as a model compound for organic contaminants such as polychlorinated dibenzodioxins (PCDDs) or bisphenol A (BPA). Fast regeneration of nanomagnets (compared to similar cyclodextrin-based systems) under mild conditions resulted in 16 repetitive cycles (adsorption/desorption) at full efficiency. The high removal and regeneration efficiency was examined by UV-vis measurements at chemical equilibrium conditions and under rapid cycling (5 min). Experiments at ultralow concentrations (160 ppb) underline the high potential of cyclodextrin modified nanomagnets as a fast, recyclable extraction method for organic contaminants in large water streams or as an enrichment tool for analytics.     

Purification of Pseudomonas sp. Lipase by Continuous Elution Electrophoresis Based on Pb^2＋ Precipitation Method

A Pb^2 precipitation method was designed to get rid of the impure proteins in a lipase. The results show that it was a simple way in the primary treatment of the crude samples and about 20% impure proteins were removed in the precipitation step. Further, continuous elution electrophoresis was also applied as a preparative technique for attaining the highly pure lipase. During the continuous elution electrophoresis, the enzyme was eluted as a single peak and 5.7-fold purification was achieved in a yield of 54.3%. The two steps finally yielded an electrophoretically homogeneous enzyme.     

Boron–Ligand Cooperation: The Concept and Applications

The term boron–ligand cooperation was introduced to describe a specific mode of action by which certain metal-free systems activate chemical bonds. The main characteristic of this mode of action is that one covalently bound substituent at the boron is actively involved in the bond activation process and changes to a datively bound ligand in the course of the bond activation. Within this review, how the term boron–ligand cooperation evolved is reflected on and examples of bond activation by boron–ligand cooperation are discussed. It is furthermore shown that systems that operate via boron–ligand cooperation can complement the reactivity of classic intramolecular frustrated Lewis pairs and applications of this new concept for metal-free catalysis are summarized.     

Efficient Immobilization of Porcine Pancreatic α-Amylase on Amino-Functionalized Magnetite Nanoparticles: Characterization and Stability Evaluation of the Immobilized Enzyme

The potential of the modified magnetic nanoparticles for covalent immobilization of porcine pancreatic α-amylase has been investigated. The synthesis and immobilization processes were simple and fast. The co-precipitation method was used for synthesis of magnetic iron oxide (Fe₃O₄) nanoparticles (NPs) which were subsequently coated with silica through sol–gel reaction. The amino-functionalized NPs were prepared by treating silica-coated NPs with 3-aminopropyltriethoxysilane followed by covalent immobilization of α-amylase by glutaraldehyde. The optimum enzyme concentration and incubation time for immobilization reaction were 150 mg and 4 h, respectively. Upon this immobilization, the α-amylase retained more than 50 % of its initial specific activity. The optimum pH for maximal catalytic activity of the immobilized enzyme was 6.5 at 45 °C. The kinetic studies on the immobilized enzyme and its free counterpart revealed an acceptable change of K_m and V_max. The Km values were found as 4 and 2.5 mM for free and immobilized enzymes, respectively. The V_max values for the free and immobilized enzymes were calculated as 1.75 and 1.03 μmol mg^?1 min^?1, in order, when starch was used as the substrate. A quick separation of immobilized amylase from reaction mixture was achieved when a magnetically active support was applied. In comparison to the free enzyme, the immobilized enzyme was thermally stable and was reusable for 9 cycles while retaining 68 % of its initial activity.     

Production and Characterization of Extracellular α-Amylase Produced by Wickerhamia sp. X-Fep

A yeast isolate able to produce high levels of extracellular ??-amylase was selected from a collection of 385 yeasts and identified as Wickerhamia sp. by the sequence of the D1/D2 domain of the 26?S rDNA gene. Part of the nucleotide sequence of the amy1-W gene was cloned, and a sequence of 191 amino acids deduced from this gene was analyzed. The peptide contains three characteristic well-conserved regions in the active sites of ??-amylases (EC 3.2.1.1). The enzyme was purified and in situ activity showed only one band with amylolytic activity. The molecular mass of the ??-amylase was estimated at 54?kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Enzymatic activity on soluble starch as substrate was optimal at pH 5?C6 and 50 °C. This thermostable enzyme was inhibited by EDTA?CNa₂ and 1,10-phenanthroline; the activity of the dialyzed enzyme was reactivated with Ca²⁺ and Mg²⁺ cations, which indicates that the ??-amylase is a metalloenzyme. ??-Amylase production was induced by starch and maltose and repressed by glucose. The high yield and productivity found in this work makes this Wickerhamia sp. strain a promising candidate for the biotechnological production of ??-amylase.     

Effect of Sol–Gel Encapsulation on Lipase Structure and Function: A Small Angle Neutron Scattering Study

The application of small angle neutron scattering (SANS) to the characterisation of sol–gel hosts containing biomolecules offers the opportunity to explore the relationship between gel structure and catalyst. A model system involving the immobilisation of Candida antarctica lipase B (CALB) was investigated.Gels were produced by fluoride-catalysed hydrolysis of fixed ratios of tetramethylorthosilicate (TMOS) and methyltrimethoxysilane (MTMS). Phase separation between the enzyme and the evolving sol–gel matrix was minimised by incorporating glycerol into the sol–gel precursor solution. The potential stabilising effect of the NaF catalyst upon the enzyme was also investigated. Scattering studies were conducted on both immobilised lipase, and lipase in free solution. Scattering studies on free enzyme provided evidence of multiple populations of enzyme aggregates and showed that choice of solvent affected the degree of aggregation. Both NaF and glycerol affected neutron scattering, indicating changes in lipase conformation. Increasing glycerol concentration increased the degree of aggregation and produced differences in solvent packing on the surface of protein molecules. Initial evidence from SANS data indicated that the presence of the enzyme during gel formation conferred structural changes on the gel matrix. Modelling the effect of sol–gel encapsulation on lipase requires comparison of data from free enzyme to the immobilised form. Removal of the enzyme from the sol–gel structure, post gelation, is necessary to better characterise the modified matrix. This methodological problem will be the subject of future investigations.