微柱高效液相色谱法测定苦荞中芦丁的研究

研究了用微柱高效液相色谱法测定苦荞中芦丁的方法。苦荞中的芦丁用 80 %甲醇加热回流提取 ,以WatersXterraTMRP1 8( 1.0× 5 0mm ,2 .5 μm)色谱柱为固定相 ,1%的醋酸和甲醇为流动相 ,在该色谱条件下 ,苦荞中主要的芦丁在 1.0min内可达到基线分离 ,用紫外二极管矩阵检测器检测 ,方法可用于苦荞中的芦丁的测定 ,结果满意。     

微波法从槐花米中提取芦丁的实验研究

以槐花米为原料,芦丁产率为评价指标,进行微波辐射下提取芦丁的工艺研究,考察了微波功率、提取温度、时间和固液比等对芦丁产率的影响。结果表明,优化工艺条件为:槐花米2 g,澄清石灰水50 mL,温度120℃,功率500 W,提取时间为20 min。优化工艺条件下,粗产品的产率为3.5%,纯度为83.6%。微波法提取芦丁具有提取率高、提取时间短、产品纯度高等优点,为综合开发该资源提供了有益的参考。     

A Novel Cyclodextrin Glycosyltransferase from Alkaliphilic Amphibacillus sp. NPST-10: Purification and Properties

Screening for cyclodextrin glycosyltransferase (CGTase)-producing alkaliphilic bacteria from samples collected from hyper saline soda lakes (Wadi Natrun Valley, Egypt), resulted in isolation of potent CGTase producing alkaliphilic bacterium, termed NPST-10. 16S rDNA sequence analysis identified the isolate as Amphibacillus sp. CGTase was purified to homogeneity up to 22.1 fold by starch adsorption and anion exchange chromatography with a yield of 44.7%. The purified enzyme was a monomeric protein with an estimated molecular weight of 92 kDa using SDS-PAGE. Catalytic activities of the enzyme were found to be 88.8 U mg(-1) protein, 20.0 U mg(-1) protein and 11.0 U mg(-1) protein for cyclization, coupling and hydrolytic activities, respectively. The enzyme was stable over a wide pH range from pH 5.0 to 11.0, with a maximal activity at pH 8.0. CGTase exhibited activity over a wide temperature range from 45 °C to 70 °C, with maximal activity at 50 °C and was stable at 30 °C to 55 °C for at least 1 h. Thermal stability of the purified enzyme could be significantly improved in the presence of CaCl(2). K(m) and V(max) values were estimated using soluble starch as a substrate to be 1.7 ± 0.15 mg/mL and 100 ± 2.0 μmol/min, respectively. CGTase was significantly inhibited in the presence of Co(2+), Zn(2+), Cu(2+), Hg(2+), Ba(2+), Cd(2+), and 2-mercaptoethanol. To the best of our knowledge, this is the first report of CGTase production by Amphibacillus sp. The achieved high conversion of insoluble raw corn starch into cyclodextrins (67.2%) with production of mainly β-CD (86.4%), makes Amphibacillus sp. NPST-10 desirable for the cyclodextrin production industry.     

One Step Large-Scale Separation and Purification of Rutin from Boenninghausenia sessilicarpa by Countercurrent Chromatography

In this study, a preparative countercurrent chromatography (CCC) method for isolation and purification of the bioactive component rutin directly from the ethanol extract of Boenninghausenia sessilicarpa was successfully established by using n-butanol-ethyl acetate-water as the two-phase solvent system. The upper phase of n-butanol-ethyl acetate-water (4:1:5, v/v) was used as the stationary phase of CCC. Under the optimum conditions, 112 mg of rutin at 98.6% purity was obtained from 2.0 g of the crude extract in a single CCC separation. The peak fraction of CCC was identified by negative ESI, ¹H NMR, and ¹³C NMR.     

Separation and Purification of Rosmarinic Acid and Rutin from Glechoma hederacea L. using High-Speed Counter-Current Chromatography

Rosmarinic acid and rutin were successfully separated from Glechoma hederaceaL. using high-speed counter-current chromatography for the first time. Eleven milligrams of rosmarinic acid (chromatographic purity 97.2 %) and 10 mg of rutin (chromatographic purity 98.1 %) were obtained from 100 mg ethyl acetate extract and 100 mg n -butanol extract of Glechoma hederacea L., respectively, with the separation procedure less than 2 h. Their structures were characterized by UV, MS, and NMR. The established methods were simple, fast, and convenient, which can be applied to the preparation of reference substances of rosmarinic acid and rutin.     

微波/超声波抽提褐煤黄腐酸类物质的比较研究

采用正交试验分别对微波和超声波方法抽提褐煤黄腐酸类物质的工艺进行了研究和对比。结果显示,微波抽提较优的条件为压力0.6MPa、抽提温度150℃、固液比1：10（g/mL）、抽提时间8min;超声提取较优的条件为超声频率50KHz、加热温度60℃、固液比（g/mL）1：20、超声时间30min。并采用元素分析及官能团测定...     

腐植酸作为天然有机物(NOM)的主要类型对环糊精聚氨酯去除水中异味的影响

目前,水处理机构普遍认为,天然有机物会妨碍活性炭的吸附位点与异味化合物结合,进而导致对甲萘烷醇和2-甲基异茨醇的清除效率下降。腐植酸也被认为具有相同的作用。对于城市水处理系统来说,饮用水中异味化合物如甲萘烷醇和2-甲基异茨醇的清除至关重要。已证实,环糊精聚氨酯可清除水中有机污染物,但不会显著降低天然有机物的含量。我们希望确定在有天然有机物存在的条件下,该聚合物是否能够有选择地去除甲萘烷醇和2-甲基异茨醇。本研究以腐植酸为天然有机物代表,因腐植酸约占天然有机物的70%。研究表明,不同浓度腐植酸的存在不会影响甲萘烷醇和2-甲基异茨醇的清除,使用环糊精聚合物,90%的异味化合物被去除。同时,紫外分析表明只有少量的腐植酸(3%～20%)被去除。     

Improvement of cotton cloth soil removal by inclusion of alkaline cellulase from Bacillus sp. KSM-635 in detergents

Alkaline cellulase from Bacillus sp. KSM-635 (EC 3.2.1.4) had reproducible detergent effects on cotton cloth that was artificially or naturally soiled with oily and/or particulate matter, under European washing conditions. The detergent effects of the cellulase, in combination with surfactants, apparently were the result of enzymatic action on amorphous regions of cotton fibers in which soil was trapped. The contribution of cellulase to soil removal increased as (i) the amount of soil in the amorphous regions of fibers in test fabrics was increased and (ii) the inhibition, by soil that adhered to the fibers' surfaces, of the action of the cellulase on fibers was reduced. Alkaline cellulase had the potential to replace, in part, both surfactants and zeolite in detergents, and it reduced washing time and allowed washing at lower temperatures under European washing conditions. The marked detergent effect of cellulase on naturally soiled cotton fabric was visually apparent, and it inhibited the accumulation of ash, calcium, and other inorganic components on cotton fibers during wash-and-wear cycles. These contributions of cellulase to the cleanliness of cotton fabrics were clearly increased by repeated wash-and-wear cycles. Cotton fabrics were not degraded by washing with the cellulase because effective hydrolysis by the cellulase occurred only in the amorphous regions of cotton fibers.     

Characterization of a Bioflocculant (MBF-UFH) Produced by Bacillus sp. AEMREG7

A bioflocculant named MBF-UFH produced by a Bacillus species isolated from sediment samples of Algoa Bay of the Eastern Cape Province of South Africa was characterized. The bacterial identification was through 16S rDNA sequencing; nucleotide sequences were deposited in GenBank as Bacillus sp. AEMREG7 with Accession Number {"type":"entrez-nucleotide","attrs":{"text":"KP659187","term_id":"768700521","term_text":"KP659187"}}KP659187. The production of the bioflocculant was observed to be closely associated with cell growth. The bioflocculant had the highest flocculating activity of 83.2% after 72 h of cultivation, and approximately 1.6 g of purified MBF-UFH was recovered from 1 L of fermentation broth. Its chemical analyses indicated that it is a glycoprotein composed of polysaccharide (76%) and protein (14%). Fourier transform infrared spectroscopy (FTIR) revealed that it consisted of hydroxyl, amide, carboxyl and methoxyl as the functional moieties. Scanning electron microscopy (SEM) revealed the amorphous structure of MBF-UFH and flocculated kaolin clay particles. The maximum flocculating activity of 92.6% against kaolin clay suspension was achieved at 0.3 mg/mL over pH ranges of 3–11 with the peak flocculating rate at pH 8 in the presence of MgCl₂. The bioflocculant retained high flocculating activity of 90% after heating at 100 °C for 1 h. MBF-UFH appears to have immense potential as an alternative to conventional chemical flocculants.     

3D Structure Elucidation of Thermostable L2 Lipase from Thermophilic Bacillus sp. L2

The crystallization of proteins makes it possible to determine their structure by X-ray crystallography, and is therefore important for the analysis of protein structure-function relationships. L2 lipase was crystallized by using the J-tube counter diffusion method. A crystallization consisting of 20% PEG 6000, 50 mM MES pH 6.5 and 50 mM NaCl was found to be the best condition to produce crystals with good shape and size (0.5 × 0.1 × 0.2 mm). The protein concentration used for the crystallization was 3 mg/mL. L2 lipase crystal has two crystal forms, Shape 1 and Shape 2. Shape 2 L2 lipase crystal was diffracted at 1.5 Å and the crystal belongs to the orthorhombic space group P2₁2₁2₁, with unit-cell parameters a = 72.0, b = 81.8, c = 83.4 Å, α = β = γ = 90°. There is one molecule per asymmetric unit and the solvent content of the crystals is 56.9%, with a Matthew’s coefficient of 2.85 Å Da⁻¹. The 3D structure of L2 lipase revealed topological organization of α/β-hydrolase fold consisting of 11 β-strands and 13 α-helices. Ser-113, His-358 and Asp-317 were assigned as catalytic triad residues. One Ca²⁺ and one Zn²⁺ were found in the L2 lipase molecule.     

Cloning, Expression, and Characterization of Thermotolerant Manganese Superoxide Dismutase from Bacillus sp. MHS47

A superoxide dismutase gene from thermotolerant Bacillus sp. MHS47 (MnSOD47) was cloned, sequenced, and expressed. The gene has an open reading frame of 612 bp, corresponding to 203 deduced amino acids, with high homology to the amino acid sequences of B. thuringiensis (accession no. EEN01322), B. anthracis (accession no. NP_846724), B. cereus (accession no. ZP_04187911), B. weihenstephanensis (accession no. YP_001646918), and B. pseudomycoides. The conserved manganese-binding sites (H28, H83, D165, and H169) show that MnSOD47 has the specific characteristics of the manganese superoxide dismutase (MnSOD) enzymes. MnSOD47 expressed an enzyme with a molecular weight of approximately 22.65 kDa and a specific activity of 3537.75 U/mg. The enzyme is active in the pH range 7-8.5, with an optimum pH of 7.5, and at temperatures in the range 30-45 °C, with an optimum temperature of 37 °C. Tests of inhibitors and metal ions indicated that the enzyme activity is inhibited by sodium azide, but not by hydrogen peroxide or potassium cyanide. These data should benefit future studies of MnSODs in other microorganisms and the biotechnological production of MnSOD47, and could also be used to develop a biosensor for the detection of antioxidants and free radical activity. In the future, this basic knowledge could be applicable to the detection of cancer risks in humans and therapeutic treatments.     

Purification and Characterization of Organic Solvent and Detergent Tolerant Lipase from Thermotolerant Bacillus sp. RN2

The aim of this study was to characterize the organic solvent and detergent tolerant properties of recombinant lipase isolated from thermotolerant Bacillus sp. RN2 (Lip-SBRN2). The isolation of the lipase-coding gene was achieved by the use of inverse and direct PCR. The complete DNA sequencing of the gene revealed that the lip-SBRN2 gene contains 576 nucleotides which corresponded to 192 deduced amino acids. The purified enzyme was homogeneous with the estimated molecular mass of 19 kDa as determined by SDS-PAGE and gel filtration. The Lip-SBRN2 was stable in a pH range of 9-11 and temperature range of 45-60 °C. The enzyme was a non metallo-monomeric protein and was active against pNP-caprylate (C8) and pNP-laurate (C12) and coconut oil. The Lip-SBRN2 exhibited a high level of activity in the presence of 108% benzene, 102.4% diethylether and 112% SDS. It is anticipated that the organic solvent and detergent tolerant enzyme secreted by Bacillus sp. RN2 will be applicable as catalysts for reaction in the presence of organic solvents and detergents.     

Removal of Cadmium Ions from Aqueous Samples by Synechocystis sp.

Abstract

The biosorption of cadmium ions from aqueous solution by dried, immobilized dried and immobilized live Synechocystis sp. was investigated. Sorption of plain Ca-alginate beads, which were used as substrate for immobilization, was also studied for comparison. Removal efficiency of biosorbents was studied as a function of pH (2-8 Volesky , B. ( 1990 ) Biosorption of Heavy Metals ; CRC Press : Boca Raton , FL . Wase , J. , Forster , C.F. (1997) Biosorbents for Metal Ions ; Taylor & Francis : London. Malkoç , E. ; Nuho?lu , Y. ( 2005 ) Investigations of nickel (II) removal from aqueous solutions using tea factory waste . J. Hazard. Mater. , 127 : 120 – 128 . Lodeiro , P. ; Cordero , B. ; Barriada , J.L. ; Herrero , R. ; Sastre de Vicente , M.E. ( 2005 ) Biosorption of cadmium by biomass of brown marine macroalgae . Bioresource Technol. , 96 : 1796 – 1803 . Shen , J. ; Duvnjak , Z. ( 2005 ) Adsorption isotherms for cupric and cadmium ions on corncob particles . Sep. Sci. Technol. , 40 : 1461 – 1481 . Kat?rc?o?lu , H. ; Asl?m , B. ; Türker , A.R. ; At?c? , T. ; Beyatl? , Y. ( 2008 ) Removal of cadmium(II) ion from aqueous system by dry biomass, immobilized live and heat-dried Oscillatoria sp. H1 isolated from freshwater (Mogan Lake) . Bioresource Technol. , 99 ( 10 ): 4185 – 4191 . Pavasant , P. ; Apiratikul , R. ; Sungkhum , V. ; Suthiparinyanont , P. ; Wattanachira , S. ; Marhaba , T.F. ( 2006 ) Biosorption of Cu²⁺, Cd²⁺, Pb²⁺, and Zn²⁺ using dried marine green macroalga Caulerpa lentillifera. Bioresource Technol. , 97 : 2321 – 2329 . ), temperature (20–40°C), initial cadmium ion concentration (50–300 mg/L), and contact time (0–120 min). The maximum biosorption capacities of the dried, immobilized dried, and immobilized live Synechocystis sp. and plain Ca-alginate beads were found as 75.7, 4.9, 4.3, and 3.9 mg/g, respectively at optimum conditions. Biosorption equilibrium was established in about 15 min. Dried biomass of Synechocystis sp. was found to be more suitable and an efficient biosorbent for the removal of cadmium ion from aqueous solution. Both of the isotherm models (Langmuir and Freundlich) were suitable for describing the biosorption of cadmium by the dried biomass of Synechocystis sp. All the tested cyanobacterial forms could be recovered more than 90% and reused in five biosorption–desorption cycles without any considerable loss in the biosorption capacity.     

Production of polyhydroxy fatty acids from linoleic acid by Clavibacter sp. ALA2

Hydroxy fatty acids are important industrial materials. We isolated a microbial culture, Clavibacter sp. ALA2, which converts linoleic acid to many polyhydroxy fatty acids. Structures of the products were determined as: 12,13,17-trihydroxy-9(Z)-octadecenoic (THOA, main product), 12-[5-ethyl-2-tetrahydrofuranyl]-7,12-dihydroxy-9(Z)-dodecenoic (ETDDA), and 12-[5-ethyl-2-tetrahydrofuranyl]-12-hydroxy-9(Z)-dodecenoic (ETHDA) acid. The yield of THOA was 25% and the relative amount of the products were THOA/ETDDA/ETHDA =9:1.3:1. The structures of the hydroxy unsaturated fatty acids resemble those of plant self-defense substances.     

Biosynthetic pathway of diepoxy bicyclic FA from linoleic acid by Clavibacter sp. ALA2

The biosynthetic pathway of two bicyclic FA, 12∶17, 13∶17-diepoxy-9(Z)-octadecenoic acid (DEOA) and 7-hydroxy-12∶17, 13∶17-diepoxy-9(Z)-octadecenoic acid (hDEOA), by Clavibacter sp. ALA2 was investigated. When cultivated with linoleic acid as a substrate, the strain produced 12,13,17-trihydroxy-9(Z)-octadecenoic acid (THOA), DEOA, and hDEOA as well as other FA. To clarify the synthetic route to these bicyclic FA, the strain was cultivated with purified THOA as a starting substrate. THOA was consumed almost completely by the strain with sequential generation of DEOA and hDEOA. Moreover, the strain produced hDEOA when cultivated with purified DEOA. Therefore, it was confirmed that THOA was a precursor of these bicyclic FA and that hDEOA was generated from DEOA. Based on our previously reported result that linoleic acid is first converted to 12,13-dihydroxy-9(Z)-octadecenoic acid (DHOA) and the present results, the overall biosynthetic pathway for the diepoxy bicyclic FA from linoleic acid was postulated as: linoleic acid→DHOA→THOA→DEOA→hDEOA.     

Biosorption of Cadmium by Citrobacter sp. JH 11-2 Isolated from Mining Site Soil

The wide use of cadmium (Cd) in batteries and semiconductors poses human and environmental health hazards when these materials are disposed. Citrobacter sp. JH 11-2, isolated from soil at an abandoned mining site, shows potential for use as a biosorbent for Cd removal from aqueous solution. A minimal inhibitory concentration of 300 mg L^?1 indicated high tolerance of the strain to Cd. The strain effectively removed 47.7% of the Cd from a 100 mg L^?1 solution within 160 h. Cell fractioning revealed that most of the Cd (43.5%) was in the membrane fraction, while 38.5% was in the peptidoglycan layer and 18.0% was in the cytoplasmic fraction. Fourier transform infrared spectroscopy (FTIR) confirmed the presence of functional groups on dried Citrobacter sp. JH 11-2 cells that can adsorb or complex Cd ions. A Langmuir model provided a good fit to Cd removal by the cells, which followed pseudo-second-order kinetics. Results support further development of Citrobacter sp. JH 11-2 as a biosorbent for Cd removal.     

Synthesis of Wax Esters from Crude Fish Fat by Lipase of Burkholderia sp. EQ3 and Commercial Lipases

The lipase from Burkholderia sp. EQ3 was used to synthesize wax esters in comparison with commercial lipases. The supernatant of Burkholderia sp. EQ3 was collected from a liquid basal medium with 1 % fish oil after 12 h cultivation (1.90 U/ml of lipase activity). The crude lipase was prepared by acetone precipitation of the culture supernatant (4.70 U/mg and 9.40 purification folds). The crude fish fat obtained by hexane extraction of waste fat from the wastewater pond of a canned tuna factory and cetyl alcohol were used for wax esters synthesis. Five commercial lipases were screened in comparison with crude lipase from Burkholderia sp. EQ3 in wax esters synthesis. The optimum conditions for wax esters synthesis from crude fish fat using Novozyme 435 were enzyme 1 U, substrate molar ratio of crude fish fat to cetyl alcohol 1:2 (115.30 mg of crude fish fat and 66.67 mg of cetyl alcohol) in hexane at 37 °C and 200 rpm with 90.81 % (TLC–FID peak area) after one h of reaction. The optimum conditions for the synthesis by crude lipase from Burkholderia sp. EQ3 were crude lipase 40 U, substrate molar ratio of crude fish fat and cetyl alcohol 1:2 in isooctane at 30 °C and 200 rpm with 95.07 % (TLC–FID peak area) after 6 h of reaction. The synthesized wax esters were mainly composed of cetyl palmitate and cetyl oleate.     

海藻酸钙包埋枝孢霉对水中Cu2+吸附性能研究

利用海藻酸钙包埋枝孢霉对水中Cu2+吸附性能进行研究。实验结果表明:当海藻酸钙质量分数为3%,CaC l2质量分数为4%,菌体积分数为15%时,包埋制得的固定化小球具有较好的机械性能和较高的吸附量,生物吸附平衡时间3 h。固定化空白小球和活菌的最佳pH值分别为3.5和4.0。在质量浓度为30—500 mg/L时,吸附过程较好地符合Langmu ir吸附模型。在浓度为0.1 mol/L的多种解吸剂中,HNO3解吸效果最好,解吸率达到93.84%。包埋小球重复利用3次,吸附性能没有明显变化。     

响应面法优化微波提取扶芳藤抗氧化物

用响应面法研究了微波法提取扶芳藤抗氧化物质的最佳工艺。考察了乙醇体积分数(X1)、乙醇用量(X2)、提取时间(X3)、提取温度(X4)4个因素的影响。微波提取的优化工艺条件是:乙醇体积分数49.4%、乙醇用量44 mL、提取时间9 m in、提取温度41℃,与模型预测值基本相符。说明微波方法可较好地应用于扶芳藤抗氧化物质的提取,通过响应面法得到一个能较好预测实验结果的模型方程。     

Separation and Purification of Polyhydroxyalkanoates from Newly Isolated Comamonas sp. EB172 by Simple Digestion with Sodium Hydroxide

A simple, mild, and effective process for the recovery of intracellular polyhydroxyalkanoate from a newly isolated gram-negative wild-type bacteria Comamonas sp. EB172 was developed using sodium hydroxide. Various parameters such as sodium hydroxide concentration, digestion time, and temperature were examined for their effect on polyhydroxyalkanoate recovery. The results showed that polyhydroxyalkanoate with 88.6% purity and 96.8% recovery yield were obtained by incubating the dried cells with 0.05 M sodium hydroxide at 4°C for 1 h, followed by purification steps using ethanol and water. Removal of non-polymeric cellular materials from the Comamonas sp. EB172 was increased under alkaline solution as a result of enhanced cell wall permeability. In addition, the presence of glycerol in the polymer suspension proved that saponification of the lipid layer in the bacterial cell wall occurred due to sodium hydroxide reaction.