利用微波-热效应进行限制性内切核酸酶反应的可行性验证

限制性内切酶催化的酶解反应是分子生物学实验中的常用技术.鉴于酶切反应时间长,有人提出采用微波释放的热效应催化限制性内切酶解反应,以期缩短酶解时间.但微波是否能代替传统酶切反应还需验证.本研究设计了一系列的酶切 试验,验证微波酶切是否有效.结果表明：短时间（2 min）微波炉高火处理未导致DNA降解及内切酶失活.对于质粒的单酶切和部分双酶切,短时间（2 min）微波炉处理可替代水浴加热完成酶解反应.对于部分双酶切体系,微波炉处理酶切不完全,不能替代温水浴.使用微波酶解需严格控制时间,切勿时间过长.     

Online microwave D-cleavage LC-ESI-MS/MS of intact proteins: site-specific cleavages at aspartic acid residues and disulfide bonds

An online nonenzymatic digestion method utilizing a microwave-heated flow cell and mild acid hydrolysis at aspartic acid (D) for rapid protein identification is described. This methodology, here termed microwave D-cleavage, was tested with proteins ranging in size from 5 kDa (insulin) to 67 kDa (bovine serum albumin) and a bacterial cell lysate ( Escherichia coli). A microwave flow cell consisting of a 5 microL total volume reaction loop connected to a sealed reaction vessel was introduced into a research grade microwave oven. With this dynamic arrangement, the injected sample was subjected to microwave radiation as it flowed through the reaction loop and was digested in less than 5 min. Different digestion times can be achieved by varying the sample flow rate and/or length of the loop inside the microwave flow cell. The microwave flow cell can be operated individually with the output being collected for matrix assisted laser ionization/desorption (MALDI) mass spectrometry (MS) or connected online for liquid chromatography (LC) electrospray ionization (ESI)-MS. In the latter configuration, the microwave flow cell eluates containing digestion products were transferred online to a reversed phase liquid chromatography column for direct ESI-MS and ESI-MS/MS analyses (specifically, Collision Induced Dissociation, CID). Concurrently with the microwave D-cleavage step, disulfide bond reduction/cleavage was achieved by the coinjection of dithiothreitol (DTT) with the sample prior to online microwave heating and online LC-MS analysis and so eliminating the need for alkylation of the reduced protein. All protein standards, protein mixtures, and proteins in a bacterial cell lysate analyzed by this new online methodology were successfully identified via a SEQUEST database search of fragment ion mass spectra. Overall, online protein digestion and identification was achieved in less than 40 min total analysis time, including the chromatographic step.     

Controlled Microwave Heating Accelerates Rolling Circle Amplification

Rolling circle amplification (RCA) generates single-stranded DNAs or RNA, and the diverse applications of this isothermal technique range from the sensitive detection of nucleic acids to analysis of single nucleotide polymorphisms. Microwave chemistry is widely applied to increase reaction rate as well as product yield and purity. The objectives of the present research were to apply microwave heating to RCA and indicate factors that contribute to the microwave selective heating effect. The microwave reaction temperature was strictly controlled using a microwave applicator optimized for enzymatic-scale reactions. Here, we showed that microwave-assisted RCA reactions catalyzed by either of the four thermostable DNA polymerases were accelerated over 4-folds compared with conventional RCA. Furthermore, the temperatures of the individual buffer components were specifically influenced by microwave heating. We concluded that microwave heating accelerated isothermal RCA of DNA because of the differential heating mechanisms of microwaves on the temperatures of reaction components, although the overall reaction temperatures were the same.     

Microwave-accelerated energy-efficient esterification of free fatty acid with a heterogeneous catalyst

This paper shows energy-efficiency of microwave-accelerated esterification of free fatty acid with a heterogeneous catalyst by net microwave power measurement. In the reaction condition of 5 wt% sulfated zirconia and 1:20 M ratio of oil to methanol at 60°C and atmospheric pressure, more than 90% conversion of the esterification was achieved in 20 min by microwave heating, while it took about 130 min by conventional heating. Electric energy consumption for the microwave heating in this accelerated esterification was only 67% of estimated minimum heat energy demand because of significantly reduced reaction time.     

Microwave-enhanced reductive amination via Schiff's base formation for block copolymer synthesis

Reductive amination via Schiff's base formation is a widely used reaction for laboratory and industrial applications ranging from protein immobilization to nanoparticle synthesis. One major limitation of this reaction is the slow kinetics and hence, it can take several days for the reaction to reach completion. Here we demonstrate that electromagnetic microwave can be used to accelerate the rate of reduction amination. To demonstrate proof of concept, we utilized the reductive amination between reducing end of dextran and primary amine from N-Boc-ethylenediamine as a model reaction. The reaction was conducted at room temperature to demonstrate that the enhancement was mainly due to electromagnetic effects of the microwave rather than thermal effects. We show that reductive amination reaction time was reduced from 72 h to 4 h using microwave irradiation. These results indicate non-thermal microwave effects to expedite reductive amination for synthesizing copolymers. The efficient conjugation of dextran using reductive amination provides an important tool for developing biocompatible copolymers using carbohydrates.     

Accelerated esterification of free fatty acid using pulsed microwaves

It was demonstrated that pulsed microwave irradiation is a more effective method to accelerate the esterification of free fatty acid with a heterogeneous catalyst than continuous microwave irradiation. A square-pulsed microwave with a 400 Hz repetition rate and a 10-20% duty cycle with the same energy as the continuous microwave were used in this study. The pulsed microwaves improved the esterification conversion from 39.9% to 66.1% after 15 min in comparison with the continuous microwave under the same reaction conditions. These results indicated that pulsed microwaves with repetitive strong power could enhance the efficiency of biodiesel production relative to the use of continuous microwave with mild power.     

Effects of microwave irradiation on triglyceride transesterification: Experimental and theoretical studies

An improved reaction rate of biodiesel fuel formation was observed under microwave irradiation. Such irradiation is effective for not only heating during the reaction but also preheating. The advantages observed in this study suggest that a continuous process, that is, the introduction of reactant preheated by microwave irradiation into reactor will be feasible. The reasons for the improved reaction rate are explained through the use of optimized structures, dipole moments, IR spectra and activation energies obtained using molecular orbital calculations. Planar triolein formed by microwave irradiation has higher reactivity, a lower dipole moment, a lower activation energy and stronger vibration around the carboxyl carbon, and is more reactive than triolein with a higher dipole moment.     

一种快速高效获取丝状真菌PCR反应模板的方法

建立一种快速高效获取丝状真菌PCR反应模板的方法,提高丝状真菌PCR鉴定效率。通过单因素法对机械破壁联合微波法进行条件优化,利用优化后的方法获取13株不同种属丝状真菌的PCR反应模板,同时与Chelex-100法、机械破壁法作对比,以试剂盒抽提法作为阳性对照,进行ITS序列扩增,琼脂糖凝胶电泳检测扩增结果。机械破壁联合微波法获取丝状真菌PCR反应模板的最佳条件为40 Hz机械破壁1 min、微波700 W高温裂解3 min,采取该法与试剂盒抽提法获得的模板均成功扩增13株不同种属丝状真菌ITS序列,且PCR鉴定结果一致;Chelex-100法获得的模板成功扩增6株丝状真菌ITS序列;机械破壁法获得的模板虽成功扩增9株丝状真菌ITS序列,但扩增效果欠佳。机械破壁联合微波法能够有效获取丝状真菌PCR反应模板,与试剂盒抽提法相比具有操作简便、快速高效的优点,提高丝状真菌PCR鉴定效率。     

Applications of microwave dielectric heating in environment-related heterogeneous gas-phase catalytic systems

The application of microwave dielectric heating in a range of environment-related heterogeneous catalytic reaction systems has been reviewed. The reactions investigated include the decomposition of hydrogen sulfide, the reduction of sulfur dioxide with methane, the reformation of methane by carbon dioxide, the hydrodesulfurization of thiophene, and the oxidative coupling of methane. The interaction of microwave irradiation with heterogeneous catalytic systems and its consequence for the microwave heating behaviour of catalysts have been examined. The effect/mechanism of microwave dielectric heating on heterogeneous catalytic reaction systems has also been discussed.     

Solvent-free synthesis of chiral Schiff-base ligands based on ferrocene under microwave irradiation and application to enantioselective nitroaldol (Henry) reaction

Chiral Schiff-bases 3a-f based on ferrocene were designed and synthesized using solvent-free methods by mixing ferrocene carbaldehyde 1 with amino alcohols and amines 2a-f under microwave irradiation and classical method for the enantioselective nitroaldol (Henry) reaction. The Schiff-bases were obtained in shorter reaction times and improved yield under microwave irradiation method over classical method. The highest enantioselectivity was observed in ligand 3e (95% ee) when CH(2)Cl(2) was used as solvent.     

Solid acid and microwave-assisted hydrolysis of cellulose in ionic liquid

Solid acid-catalyzed hydrolysis of cellulose in ionic liquid was greatly promoted by microwave heating. H-form zeolites with a lower Si/Al molar ratio and a larger surface area showed a relatively higher catalytic activity. These solid catalysts exhibited better performance than the sulfated ion-exchanging resin NKC-9. Compared with conventional oil bath heating mode, microwave irradiation at an appropriate power significantly reduced the reaction time and increased the yields of reducing sugars. A typical hydrolysis reaction with Avicel cellulose produced glucose in around 37% yield within 8 min.     

Salt-assisted acid hydrolysis of chitosan to oligomers under microwave irradiation

The effect of inorganic salts such as sodium chloride on the hydrolysis of chitosan in a microwave field was investigated. While it is known that microwave heating is a convenient way to obtain a wide range of products of different molecular weights only by changing the reaction time and/or the radiation power, the addition of some inorganic salts was shown to effectively accelerate the degradation of chitosan under microwave irradiation. The molecular weight of the degraded chitosan obtained by microwave irradiation was considerably lower than that obtained by traditional heating. Moreover, the molecular weight of degraded chitosan obtained by microwave irradiation assisted under the conditions of added salt was considerably lower than that obtained by microwave irradiation without added salt. Furthermore, the effect of ionic strength of the added salts was not linked with the change of molecular weight. FTIR spectral analyses demonstrated that a significantly shorter time was required to obtain a satisfactory molecular weight by the microwave irradiation-assisted inorganic salt method than by microwave irradiation without inorganic salts and conventional technology.     

Rapid solid-phase peptide synthesis using thermal and controlled microwave irradiation.

A rapid and efficient microwave-assisted solid-phase synthesis method is described for the preparation of the nonapeptide WDTVRISFK, using conventional Fmoc/Bu(t) orthogonal protection strategy. The synthesis protocol is based on the use of cycles of pulsed microwave irradiation with intermittent cooling of the reaction during the removal of the Fmoc protecting group and during the coupling. The desired nonapeptide was obtained in highest yield and purity by employing MicroKan technology. The chemical reactions were carried out in a single-mode microwave reactor, equipped with a fiber-optic probe to monitor the reaction temperature continuously.     

Inactivation of Lactobacillus Bacteriophage PL-1 by Microwave Irradiation

The effect of microwave irradiation on the survival of bacteriophage PL-1, which is specific for Lactobacillus casei, was studied using a commercial 2,450 MHz microwave oven. The phages were inactivated by microwave irradiation according to almost first-order reaction kinetics. The rate of phage inactivation was not affected by the difference in the continuous or intermittent irradiation, nor by the concentrations of phages used, but was affected by the volume of phage suspensions, which prevented the loss of generated heat. Microwave irradiation of phage suspensions produced a number of ghost phages with empty heads, but fragmentation of the tail was hardly noticed. The breakage of phage genome DNA was primarily caused by the heat generated by microwave irradiation, whereas the phage DNA was not affected by the same temperature achieved by heat from outside. Thus we concluded that the phage-inactivating effect of microwave irradiation was mainly attributed to a thermal microwave effect, which was much stronger than a simple thermal exposure.     

Microwave assisted alkali-catalyzed transesterification of Pongamia pinnata seed oil for biodiesel production

In this study, microwave assisted transesterification of Pongamia pinnata seed oil was carried out for the production of biodiesel. The experiments were carried out using methanol and two alkali catalysts i.e., sodium hydroxide (NaOH) and potassium hydroxide (KOH). The experiments were carried out at 6:1 alcohol/oil molar ratio and 60 °C reaction temperature. The effect of catalyst concentration and reaction time on the yield and quality of biodiesel was studied. The result of the study suggested that 0.5% sodium hydroxide and 1.0% potassium hydroxide catalyst concentration were optimum for biodiesel production from P. pinnata oil under microwave heating. There was a significant reduction in reaction time for microwave induced transesterification as compared to conventional heating.     

Preparation of carboxymethyl chitosan in aqueous solution under microwave irradiation

Carboxymethyl chitosan was prepared by reacting chitosan with chloroacetic acid in water under microwave irradiation. The effect of the reaction conditions was investigated and optimal conditions were identified. The influence of mass ratio of chloroacetic acid to chitosan, microwave power and pH on the degree of substitution or intrinsic viscosity were further studied. The degree of substitution of the carboxymethyl chitosan synthesized exceeded 0.85.     

Limiting racemization and aspartimide formation in microwave-enhanced Fmoc solid phase peptide synthesis.

Microwave energy represents an efficient manner to accelerate both the deprotection and coupling reactions in 9-fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis (SPPS). Typical SPPS side reactions including racemization and aspartimide formation can occur with microwave energy but can easily be controlled by routine use of optimized methods. Cysteine, histidine, and aspartic acid were susceptible to racemization during microwave SPPS of a model 20mer peptide containing all 20 natural amino acids. Lowering the microwave coupling temperature from 80 degrees C to 50 degrees C limited racemization of histidine and cysteine. Additionally, coupling of both histidine and cysteine can be performed conventionally while the rest of the peptide is synthesized using microwave without any deleterious effect, as racemization during the coupling reaction was limited to the activated ester state of the amino acids up to 80 degrees C. Use of the hindered amine, collidine, in the coupling reaction also minimized formation of D-cysteine. Aspartimide formation and subsequent racemization of aspartic acid was reduced by the addition of HOBt to the deprotection solution and/or use of piperazine in place of piperidine.     

Enzymatic synthesis of cetyl oleate in a solvent-free medium using microwave irradiation and physicochemical evaluation

Abstract

A cosmetic ester, cetyl oleate was synthesized using microwave irradiated system. The esterification reaction was carried using Candida antarctica lipase B in a solvent-free media. The influence of various reaction parameters was studied, and the efficiency of Fermase CALB^TM10000 was compared with other enzymes. Equilibrium conversion of 97.5% was obtained within 20?min at 60?°C temperature, 1:2 oleic acid to cetyl alcohol molar ratio and 4% w/w dose of lipase. A comparative study showed that microwave irradiation is a much more efficient method than ultrasound irradiation and conventional heating. Fermase CALB^TM10000 was reusable over 6 enzymatic cycles as its stability improved under microwave system. Physicochemical parameters of cetyl oleate were tested in order to analyze its suitability for further cosmetic use.     

微波辐射法合成苄基-2-萘基醚的研究

在微波辐射条件下,以2-萘酚和氯化苄为原料,用氢氧化钠作碱剂,碘化钾作催化剂,以水和N,N-二甲基甲酰胺(DMF)为溶剂合成了苄基-2-萘基醚;采用单因素实验法,考察了反应物的摩尔比、催化剂用量、微波功率、辐射时间等因素对苄基-2-萘基醚产率的影响。实验结果表明:在n(2-萘酚):n(氢氧化钠):n(氯化苄):n(碘化钾)=1:1.1:1:0.018,水7 ml,DMF 25 ml,微波功率320 W和辐射时间75 s的优化条件下,苄基-2-萘基醚的产率可达88.03%。