Preservation of <em>Bacillus firmus</em> Strain 37 and Optimization of Cyclodextrin Biosynthesis by Cells Immobilized on Loofa Sponge

The preservation of Bacillus firmus strain 37 cells by lyophilization was evaluated and response surface methodology (RSM) was used to optimize the β-cyclodextrin (β-CD) production by cells immobilized on loofa sponge. Interactions were studied with the variables temperature, pH and dextrin concentration using a central composite design (CCD). Immobilization time influence on β-CD production was also investigated. B. firmus strain 37 cells remained viable after one year of storage, showing that the lyophilization is a suitable method for preservation of the microorganism. From the three-dimensional diagrams and contour plots, the best conditions for β-CD production were determined: temperature 60 °C, pH 8, and 18% dextrin. Considering that the amount of dextrin was high, a new assay was carried out, in which dextrin concentrations of 10, 15, and 18% were tested and the temperature of 60 °C and pH 8 were maintained. The results achieved showed very small differences and therefore, for economic reasons, the use of 10% dextrin is suggested. Increasing the immobilization time of cells immobilized on synthetic sponge the β-CD production decreased and did not change for cells immobilized on loofa sponge. The results of this research are important for microorganism preservation and essential in the optimization of the biosynthesis of CD.     

THERMAL ACTIVATION OF IMMOBILIZED PAPAIN

Papain (Papainase, EC 3.4.22.2) was immobilized on porous silica beadsby cross linking with glutaraldehyde. The thermal activation of this immobilized papainin aqueous system was found at a temperature range from 50 to 90℃. The higher thetemperature, the more active the immobilized papain will possess. At the same time,the durability of the immobilized papain on heating was greatly improved. The effect ofadditives and salts on the activity of the immobilized papain were also studied. The resultsshowed that the additives and some of the salts studied could markedly enhance the activityof the immobilized papain at elevated temperature.     

固定化过氧化氢酶的制备及其抗氧化作用

以烟用醋酸纤维的生物化学改性为目标,研究了以壳聚糖为载体时,吸附交联固定化过氧化氢酶的条件,并考察了固定化酶的性质。结果表明,固定化的最佳条件为:加酶量(酶活2×104C IU/m l)6m l,3%壳聚糖20m l,乙二醛浓度6%(w/v),交联剂用量100m l,吸附时间0.5 h,交联时间2.5h,酶活收率可达42.9%。过氧化氢酶固定化后,动学参数Km值为61.7mmol/L;对活性氧具有较好清除作用。     

Calculation of the dynamic temperature characteristics of a heated graphite tube used in electrothermal atomic absorption measurements

A method and computer program were developed for calculating the temperature of any part of a graphite furnace tube at any instant of time after onset of the heating cycle. Results for tubes of the Massman design show large differences in spatial temperature distributions during and at the end of the heating cycle when equilibrium is reached. When constant current is applied to contoured tubes, greater heating rates at the tube centres are obtained than with standard tubes.     

Properties of Immobilized Laccase on Mesostructured Cellular Foam Silica and its Use in Dye Decolorization

Laccase was immobilized on mesostructured cellular foam (MCF), a kind of mesoporous silica with large pore size by adsorption–cross linking method. The effects of immobilization time, temperature, pH, amount of enzyme and content of glutaraldehyde on the immobilization were optimized. The activities and stabilities towards pH and temperature of the immobilized enzyme were studied, and significantly improved enzymatic properties and good operational stability were obtained for the immobilized laccase. Dye decolorization tests showed that the immobilized enzyme could decolorize Alizarin Red and Indigo Blue solution fast and efficiently in the presence of ABTS.     

有机相中蚕丝固定化脂肪酶催化酯化反应性能研究

研究了有机相中蚕丝固定化脂肪酶催化酶化反应的催化活性。考究了有机溶剂,底物、反应温度,ＰＨ值和体系含水量等因素对固定化脂肪酶催化活性的影响。结果表明,以异辛烷为有机溶剂,在反应温度为５０℃ＰＨ值为７．４时,酶催化活性最好。     

树枝状大分子聚酰胺-胺用于氨基酰化酶的固定化研究

以树枝状大分子修饰的硅胶为载体,戊二醛为交联剂,对氨基酰化酶进行固定化研究。考察了树枝状大分子的代数、戊二醛浓度、反应温度与时间对氨基酰化酶固定化效果的影响,并且考察了该固定化酶的最佳酶解条件。结果表明,随着树枝状大分子代数的增加,固定化的酶量随之增大,同时,固定后的酶仍然保持较高的活性。     

Radiation synthesis of a water-soluble temperature sensitive polymer, activated copolymer and applications in immobilization of proteins

In this work the radiation polymerization of N-isopropylacrylamide (NIPAAM) in aqueous solutions has been carried out and a water-soluble, temperature sensitive polymer and copolymer were obtained by using γ-rays from Co-60 source at room temperature. We have gained the optimum dose and dose—rate of radiation synthesis of linear polyNIPAAM through determining conversion yield and viscosity. In order to immobilize protein (BSA) and enzyme (HRP) into this water-soluble polymer, we prepared an activated copolymer, poly ( N-isopropylacrylamide-co-N-acryloxysuccinimide). The BSA and HRP has been immobilized onto the activated copolymer. The BSA (HRP) / copolymer conjugates still kept the original thermally sensitive properties of the linear polyNIPAAM. The conjugation yield of BSA to the activated copolymer decreased with increasing of dose. The thermal stability of the immobilized HRP was stable at 0 °C for a long time and has, at least, 4 days stability at room temperature. Immobilized HRP activity was lowered when the temperature was raised above its LCST. This phenomenon was reversible and the immobilized HRP regained activity below its LCST. The optimum pH of the immobilized HRP shifted from ca.5 upward to ca.7.     

以HPD-750大孔树脂为载体材料固定化果胶酶

HPD-750树脂是中极性大孔吸附树脂,生物相容性好,机械性能稳定,具有较大的比表面积,可用于固定化酶载体材料。本文以HPD-750大孔树脂为载体固定化果胶酶,研究各因素对固定化酶的影响,并采用正交试验对固定化条件进行优化。结果表明,当pH为4.0、固定化温度为45℃、固定化时间为4h、加酶量为0.16g/mL时,固定化酶活力可达5146U/mg。以HPD-750大孔树脂为载体材料制备的固定化酶相较于游离酶具有更好的酸碱稳定性和热稳定性。在循环使用10次后,酶活力依然保留80%以上;4℃储藏25d之后,其酶活力仍保留60%以上。与D311大孔树脂、聚丙烯酰胺和海藻酸钠微球制备的固定化酶相比,HPD-750大孔树脂固定化酶的活性、操作稳定性、机械稳定性和储存稳定性都较好。该结果说明,HPD-750大孔树脂可作为固定化酶较好的载体材料。     

Experimental Results and Analysis for Adsorption Ice-Making System with Consolidated Adsorbent

An adsorption ice-making machine has been built with a single consolidated adsorber and activated carbon-methanol pair. A consolidated adsorbent block made of activated carbon mixed with a binder with good heat transfer properties has been developed and implemented in the adsorber. The design is focused on the adsorber consisting of copper finned tubes and carbon blocks. Experimental tests have been performed suitable for ice making. This paper describes the experimental results of such an ice-maker operating with an intermittent cycle and a cycle time of 35 minutes. The thermal conditions used to test the cycle are: 115°C heat source, 22°C heat sink, the evaporator temperature corresponding to the chilled ethylene glycol temperature is –7°C. At this evaporating pressure, the mass transfer resistance controls the adsorption process. Test results show that the COP reaches 0.07 whereas the SCP (specific cooling power) is 11 W kg^–1 activated carbon. A two-bed adsorptive prototype ice-making machine operating with a heat and mass recovery cycle has also been made for onboard adsorption refrigeration in fishing boats. Good performances have been achieved due to improved mass transfer and the new ice maker can produce 18–20 kg h^–1 of flake ice at mean temperature of –7°C.     

The preparation of analytically stable,immobilized stationary phase coatings on colums packing materials

Summary A coating of silicone SE-54 on Chromosorb W has been converted to an analytically stable, immobilized stationary phase layer by cross-linking with an organic peroxide. Up to 10% by weight has been applied in this manner. Excellent high temperature qualities are exhibited, such as low bleeding and good base line stability, during typical analyses.Poly (2,6-dimethyl-p-phenylene oxide) has also been crosslinked as a surface coating on Chromosorb W with an organic peroxide. From 3 to 4% by weight has been immobilized in this way. The resultant packing material, following high temperature conditioning, has been examined for use as an adsorbent of trace substances, similar to the applications to which such adsorbents as Tenax GC have commonly been applied, e.g. trace analysis of air. It appears to exhibit satisfactory properties for this analytical purpose.     

酶固定化技术用载体材料的研究进展

酶的固定化是生物技术中最为活跃的研究领域之一.作为固定化酶技术的重要组成部分,载体的结构及性能在很大程度上直接影响着所得固定化酶的催化活性及操作稳定性.综合性能优良的载体材料的设计与制备是固定化酶技术领域的一个非常重要的研究内容.通过对传统材料的改性和新型载体材料的研究开发,必将促进固定化酶在各个领域的广泛应用.本文综述了近年来国内外有关固定化酶载体材料的研究现状和发展趋势.     

聚ε-己内酯的微米硅球固定化猪胰脂肪酶促合成

本文采用微米硅球固定化猪胰脂肪酶为催化剂合成聚ε-己内酯, 以期获得具有较高分子量、 良好生物相容性和使用安全性的生物可降解医用高分子材料.     

Genetic transformation of immobilized competent cells

This study describes the investigation of the possibility of genetic transformation of already immobilized competent cells by plasmids. The preliminary prepared competent cells were entrapped into granules of an insoluble carrier, a cryogel of poly(vinyl alcohol). The specific activity of organophosphate hydrolase and ampicillin resistance conferred by pOPf1 plasmid were used as markers of successful transformation of the immobilized competent cells. The effect of main experimental conditions of transformation usually used for free cells, i.e., time of incubation of cells with DNA solution, temperature, and time of heat shock, on the transformation efficiency of the immobilized competent cells has been studied. A num ber of important factors of preparation of immobilized transformed cells, i.e., the concentration of im mobilized competent cells inside the granules, the concentration of DNA solution used for transformation, have been shown to affect the OPH-activity of the final immobilized transformants. The possibility of transformation of the immobilized competent cells by both single- and double-stranded plasmid DNA molecules has been demonstrated.     

Improving off-line accelerated tryptic digestion. Towards fast-lane proteolysis of complex biological samples

Off-line digestion of proteins using immobilized trypsin beads is studied with respect to the format of the digestion reactor, the digestion conditions, the comparison with in-solution digestion and its use in complex biological samples. The use of the filter vial as the most appropriate digestion reactor enables simple, efficient and easy-to-handle off-line digestion of the proteins on trypsin beads. It was shown that complex proteins like bovine serum albumin (BSA) need much longer time (89 min) and elevated temperature (37 degrees C) to be digested to an acceptable level compared to smaller proteins like cytochrome c (5 min, room temperature). Comparing the BSA digestion using immobilized trypsin beads with conventional in-solution digestion (overnight at 37 degrees C), it was shown that comparable results were obtained with respect to sequence coverage (>90%) and amount of missed cleavages (in both cases around 20 peptides with 1 or 2 missed cleavages were detected). However, the digestion using immobilized trypsin beads was considerable less time consuming. Good reproducibility and signal intensities were obtained for the digestion products of BSA in a complex urine sample. In addition to this, peptide products of proteins typically present in urine were identified.     

Rapid PCR in a continuous flow device

Continuous flow polymerase chain reaction (CFPCR) devices are compact reactors suitable for microfabrication and the rapid amplification of target DNAs. For a given reactor design, the amplification time can be reduced simply by increasing the flow velocity through the isothermal zones of the device; for flow velocities near the design value, the PCR cocktail reaches thermal equilibrium at each zone quickly, so that near ideal temperature profiles can be obtained. However, at high flow velocities there are penalties of an increased pressure drop and a reduced residence time in each temperature zone for the DNA/reagent mixture, that potentially affect amplification efficiency. This study was carried out to evaluate the thermal and biochemical effects of high flow velocities in a spiral, 20 cycle CFPCR device. Finite element analysis (FEA) was used to determine the steady-state temperature distribution along the micro-channel and the temperature of the DNA/reagent mixture in each temperature zone as a function of linear velocity. The critical transition was between the denaturation (95 degrees C) and renaturation (55 degrees C-68 degrees C) zones; above 6 mm s(-1) the fluid in a passively-cooled channel could not be reduced to the desired temperature and the duration of the temperature transition between zones increased with increased velocity. The amplification performance of the CFPCR as a function of linear velocity was assessed using 500 and 997 base pair (bp) fragments from lambda-DNA. Amplifications at velocities ranging from 1 mm s(-1) to 20 mm s(-1) were investigated. The 500 bp fragment could be observed in a total reaction time of 1.7 min (5.2 s cycle(-1)) and the 997 bp fragment could be detected in 3.2 min (9.7 s cycle(-1)). The longer amplification time required for detection of the 997 bp fragment was due to the device being operated at its enzyme kinetic limit (i.e., Taq polymerase deoxynucleotide incorporation rate).     

大尺寸SiO2大孔材料固定化漆酶

以表面固定Cu2+的改性大尺寸SiO2大孔材料作为载体,考察了时间、pH和给酶量对漆酶固定化效果的影响,并对固定化漆酶的活性和稳定性进行了研究。结果表明:5 h时吸附达到平衡,pH为4.5、漆酶与载体比例为5 mg·g-1时固定化效果最好,酶活回收率可达到100.4%;固定化漆酶的最适pH和最适温度较游离漆酶的均有升高且范围变宽,固定化后,漆酶的pH稳定性和热稳定性都得到显著提高;固定化漆酶的K m值略高于游离漆酶的;固定化漆酶具有良好的操作稳定性,与底物反应反复操作10批次后剩余酶活为72.7%。     

Immobilization of Candida antarctica Lipase B by Adsorption to Green Coconut Fiber

An agroindustrial residue, green coconut fiber, was evaluated as support for immobilization of Candida antarctica type B (CALB) lipase by physical adsorption. The influence of several parameters, such as contact time, amount of enzyme offered to immobilization, and pH of lipase solution was analyzed to select a suitable immobilization protocol. Kinetic constants of soluble and immobilized lipases were assayed. Thermal and operational stability of the immobilized enzyme, obtained after 2 h of contact between coconut fiber and enzyme solution, containing 40 U/ml in 25 mM sodium phosphate buffer pH 7, were determined. CALB immobilization by adsorption on coconut fiber promoted an increase in thermal stability at 50 and 60 °C, as half-lives (t _1/2) of the immobilized enzyme were, respectively, 2- and 92-fold higher than the ones for soluble enzyme. Furthermore, operational stabilities of methyl butyrate hydrolysis and butyl butyrate synthesis were evaluated. After the third cycle of methyl butyrate hydrolysis, it retained less than 50% of the initial activity, while Novozyme 435 retained more than 70% after the tenth cycle. However, in the synthesis of butyl butyrate, CALB immobilized on coconut fiber showed a good operational stability when compared to Novozyme 435, retaining 80% of its initial activity after the sixth cycle of reaction.     

青霉素酰化酶在介孔分子筛MCM-41上的固定化研究

通过改变酶固定化时的pH值、温度、时间以及酶用量,研究了固定化条件对MCM-41载体上固定化青霉素酰化酶的酶活及其稳定性的影响,明确了酶活及其稳定性随固定化条件的变化规律,初步分析了青霉素酰化酶在介孔分子筛MCM-41上的固定化过程。     

Physical immobilization of Rhizopus oryzae lipase onto cellulose substrate: activity and stability studies

Rhizopus oryzae lipase (ROL) was immobilized by adsorption onto oxidized cellulose fibers and regenerated films. The maximum adsorption level increases with the raise in the amount of carboxylic groups on cellulose surface confirming that adsorption is being governed mainly by electrostatic interaction between the enzyme and the substrate. This hypothesis was further confirmed by zeta-potential measurements showing a decrease in the zeta-potential of the fibers after enzyme adsorption. XPS analysis showed an intensification of the N 1s peak attesting the presence of the enzyme on the surface. The effect of temperature, pH and solvent polarity on the immobilized enzyme activity and stability was investigated. The catalytic esterification of oleic acid with n-butanol has been carried on using hexane as an organic solvent. A high conversion yield was obtained (about 80%) at 37 degrees C with a molar ratio of oleic acid to butanol 1:1 and 150IU immobilized lipase. The adsorption achieved two successive cycles with the same efficiency, and started to lose its activity during the third cycle.