Catalyst Compartmentalization in Dynamic Kinetic Resolutions

Catalytic nanoreactors, prepared by encapsulation of Zeolite H-Beta using a Layer-by-Layer (LbL) assembly of polyelectrolytes, have been examined in the Dynamic Kinetic Resolution (DKR) of secondary alcohols. The encapsulation of the acidic zeolite racemisation catalyst protects the pH-sensitive enzyme. This approach represents a convenient method of catalyst protection in multi-catalyst systems.     

A Catalytic Nanoreactor Based on in Vivo Encapsulation of Multiple Enzymes in an Engineered Protein Nanocompartment

Bacterial protein compartments concentrate and sequester enzymes, thereby regulating biochemical reactions. Here, we generated a new functional nanocompartment in Escherichia coli by engineering the MS2 phage capsid protein to encapsulate multiple cargo proteins. Sequestration of multiple proteins in MS2‐based capsids was achieved by SpyTag/SpyCatcher protein fusions that covalently crosslinked with the interior surface of the capsid. Further, the functional two‐enzyme indigo biosynthetic pathway could be targeted to the engineered capsids, leading to a 60 % increase in indigo production in vivo. The enzyme‐loaded particles could be purified in their active form and showed enhanced long‐term stability in vitro (about 95 % activity after seven days) compared with free enzymes (about 5 % activity after seven days). In summary, this engineered in vivo encapsulation system provides a simple and versatile way for generating highly stable multi‐enzyme nanoreactors for in vivo and in vitro applications.     

Immobilization of glucose oxidase in alginate-chitosan microcapsules

In order to improve its stability and catalytic rate in flour, the immobilization of glucose oxidase (GOX) was investigated in this work. The enzyme was encapsulated in calcium alginate-chitosan microspheres (CACM) using an emulsification-internal gelation-GOX adsorption-chitosan coating method. The interaction between alginate and chitosan was confirmed by infrared spectroscopy (IR). The resultant CACM in wet state, whose morphology was investigated by scanning electron microscopy (SEM), was spherical with a mean diameter of about 26 μm. The GOX load, encapsulation efficiency and activity of the CACM-GOX were influenced by concentration of chitosan, encapsulation time and encapsulation pH. The highest total enzymatic activity and encapsulation efficiency was achieved when the pH of the adsorption medium was near the isoelectric point (pI) of GOX, approximately pH 4.0. In addition, the molecular weight of chitosan also evidently influenced the encapsulation efficiency. Storage stabilities of GOX samples were investigated continuously over two months and the retained activity of CACM-GOX was 70.4%, markedly higher than the 7.5% of free enzyme. The results reveal the great potential of CACM-GOX as a flour improver.     

Kinetic behaviour and stability of glucose oxidase entrapped in liposomes

Glucose oxidase (EC 1.1.3.4) was encapsulated in liposomes (prepared from phosphatidyl choline and cholesterol) by the dehydration–rehydration method. The enzymatic activities of native and liposomal glucose oxidase were followed by the amount of H₂O₂ obtained in the enzymatic β‐D ‐glucose oxidation. Some characteristics of the liposomal and free glucose oxidase were compared. The enzyme encapsulated in liposomes showed an apparent inhibition by glucose at concentrations higher than 0.28 mol dm^?3 with a substrate inhibition constant of 0.95 ± 0.12 mol dm^?3. The enzyme entrapped showed an apparent K_m value higher than that of the free enzyme. The apparent V_max of liposomal enzyme decreased by a factor of 0.35 with respect of that of the native enzyme. The optimum temperature of the free and entrapped enzymes remained similar but the liposomal enzyme showed maximal activity at a more acid pH (5.2). The thermal and proteolytic stabilities were enhanced by encapsulation in liposomes. The stabilization factors (relationship between half‐lives of entrapped form and free enzyme) at 45, 50 and 55 °C for liposomal glucose oxidase were 2.6, 1.6 and 1.6, respectively. Copyright © 2003 Society of Chemical Industry     

Preparation of glucose oxidase immobilized in different carriers using radiation polymerization

Glucose oxidase (EC 1.1.3.4) was immobilized on different polymeric materials using different immobilization techniques (entrapping by γ‐irradiation, and covalent binding using epichlorohydrin). Studies were carried out to increase the thermal stability of glucose oxidase (GOD) for different applications. The activity and stability of the resulting biopolymers have been compared with those of free GOD. The effect of different polyvinyl alcohol/polyacrylamide (PVA/PAAm) compositions of the copolymer carrier on the enzymatic activity of the immobilized GOD was studied. The maximum enzymatic activity was obtained with the composition ratio of PVA/PAAm of 60:40. The behaviour of the free and immobilized enzyme was analysed as a function of pH. A broadening in the pH profile (5.5–8) was observed for immobilized preparations. The activity and stability of the resulting biopolymers produced by immobilization of GOD onto different carriers have been compared, in both aqueous and organic media, with those of the free GOD. The enzyme's tolerance toward both heat and organic solvent was enhanced by immobilization onto polymers. The addition of different concentrations of organic solvents (10–50%, v/v) to the enzyme at higher temperature (60 °C) was found to stabilize the enzyme molecule. The strongest stabilizing effect on the enzymatic activity was achieved at a concentration of 10%. Copyright © 2005 Society of Chemical Industry     

Enhancing the stability of immobilized catalase on activated carbon with gelatin encapsulation

Gelatin (Gel) encapsulation onto activated carbon (AC) with catalase (CAT) was developed as an alternative method for CAT immobilization in this study. The immobilized CAT with AC encapsulated by Gel as the supporter accounted for 65.69% of the native enzyme activity. Furthermore, the properties of the immobilized CATs were characterized by Fourier transform infrared spectroscopy and scanning electron microscopy. Among free CAT and the two immobilized CATs, the immobilized CAT with AC encapsulated by Gel as the supporter showed the highest relative enzymatic activity and a high stability in a broad range of pH values and temperatures, and its residual activity was 80% after 15 uses, whereas the immobilized CAT with AC as the supporter was retained at a level of only 50% under the same conditions. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 130: 1498–1502, 2013     

药物肠吸收机制在胰岛素口服制剂设计中的应用

提高口服胰岛素的生物利用度一直是药学工作者研究的热点内容。介绍了药物在肠内经细胞间和跨细胞转运吸收的一般机制,详述了胰岛素在胃肠道中所面临的物理和化学屏障、酶屏障以及对此采取的相应措施,建议：在胰岛素制剂加工过程中,采用温和的pH值和温度环境并尽量避免使用有机溶剂,以最大限度保留药物的生物活性;对胰岛素采用微囊包衣以降低胃肠道的化学屏障或酶屏障对其稳定性的影响;开发广谱酶抑制剂并用于胰岛素口服制剂,以对抗肠道内的蛋白酶对其降解。     

Modulation of serratiopeptidase transdermal patch by lipid-based transfersomes

Serratiopeptidase is a proteolytic enzyme obtained from serratia marcescens strain E-15 and used as anti-inflammatory and analgesic drug. Serratiopeptidase undergoes first pass metabolism, causes the gastrointestinal disturbance and systemic toxicity after oral administration. To overcome the limitations of serratiopeptidase, transdermal drug delivery system is an alternative method. So, the aim of present work was to modulate serratiopeptidase transdermal patch by lipid-based transfersomes. Particle size of drug was the major concern to cross stratum corneum which acts as a barrier. This difficulty was surmounted by modulating the vesicles such as transfersomes which carries the drug into the skin by passing the barrier of stratum corneum. Serratiopeptidase was encapsulated in transfersomes in different ratios of lecithin and cholesterol. Particle size of transfersomes, folding endurance, thickness, tensile strength, adhesion test, encapsulation efficiency, in vitro and in vivo release, Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimeter (DSC) studies of patch were used as characterization parameters. Serratiopeptidase transfersomes size was found to be 50 µm with smooth surface. The promising entrapment efficiencies of transfersomes and formulation were found to be 96.76 and 98.7%, respectively. In vitro and in vivo release studies showed controlled and steady release of serratiopeptidase for 24 h. FTIR and DSC confirmed the encapsulation of drug in patch without interaction. It is concluded that transfersomes are interesting carriers for enzymatic drugs for topical application.     

Hybrid Nanoreactors: Coupling Enzymes and Small-Molecule Catalysts within Virus-Like Particles

Virus-like particles (VLPs) provide unique scaffolds for the construction of coupled catalytic systems by attachment and encapsulation of catalysts within their hollow interiors. The interior of VLPs provides an environment where catalysts of biological or synthetic origins can be confined, protected, and colocalized in close proximity with catalysts of different types. Herein, we utilize the P22VLP as a scaffold to construct a synthetic hybrid catalyst by attachment of a small organometallic catalyst to the interior colocalized with an encapsulated enzyme. This produces a complex and active coupled biomimetic catalyst system. By combining both enzymatic and synthetic catalysts together, new biological synthetic hybrid materials can be produced that incorporate the best of both catalytic systems.     

Nano‐reactors for controlling the selectivity of the free radical grafting of maleic anhydride onto polypropylene in the melt

This paper reports on a successful application of the concept of nanoreactors to effectively controlling the selectivity of the free radical grafting of maleic anhydride (MAH) onto polypropylene (PP) in the melt, an industrially relevant process. More specifically, a free radical initiator of type ROOR was first confined into (or encapsulated by) the galleries of an organically modified montmorillonite (o‐MMT) whose interdistance was 2.4 nm. Primary free radicals (RO ·) formed inside the o‐MMT galleries had to diffuse out before they could react with the PP backbone. The controlled release of the primary free radicals significantly increased the grafting degree of MAH onto PP and greatly reduced the level of the chain scission of the latter. Those results were better understood by electron spin resonance studies on model systems and by Monte Carlo simulations. POLYM. ENG. SCI. 46:1443–1454, 2006. © 2006 Society of Plastics Engineers.     

Improvement of catalytic efficiency of chloroperoxidase by its covalent immobilization on SBA-15 for azo dye oxidation

Chloroperoxidase from the fungus Caldariomyces fumago was covalently immobilized on SBA-15 mesoporous material and assayed for the enzymatic oxidation of four azo dyes. All dyes were oxidized by the free and the immobilized enzyme to different extent. Acid Blue 120 and Direct Blue 85 dyes were decolorized almost completely. The catalytic efficiency, k _cat/K _M, of the immobilized enzyme was 27, 2.9, 137 and 28 times higher than the free enzyme for Basic Blue 41, Disperse Blue 85, Acid Blue 120 and Direct Black 22 oxidation, respectively. The immobilized enzyme displayed improved thermostability and a similar pH profile compared to the free enzyme. The immobilized chloroperoxidase showed excellent storage stability and maintained 100 % catalytic activity after 90 days at both 4 and 25 °C.     

重组耐热β-葡萄糖苷酶的固定化及其对黄酮糖苷的定向转化

为提高黄酮糖苷类化合物酶法转化的效率,降低酶使用成本,对重组耐热β-葡萄糖苷酶的固定化条件进行了研究,比较了固定化酶和游离酶的酶学性质差异,同时研究了固定化酶转化3种黄酮糖苷的时效曲线及转化效率。结果表明：重组耐热β-葡萄糖苷酶的固定化最优条件为在pH值6.0,室温条件下,酶活力2 U/mL,海藻酸钠质量分数1.5%,氯化钙质量分数2.5%,戊二醛质量分数1.25%,颗粒硬化时间2.5 h,此条件下固定化酶颗粒的酶活力为0.64 U/mg,酶固定化率可达62.5%。固定化酶较游离酶具有更优的酸碱稳定性和热稳定性,且重复使用20次后仍具有55%以上的残余酶活力。固定化酶能够高效定向转化异槲皮素、大豆苷以及淫羊藿次苷I等3种黄酮糖苷,生成活性更强的黄酮苷元槲皮素、大豆苷元和淫羊藿素,其摩尔转化率分别为99.37%、97.21%和97.93%,产率分别为0.776、1.091和0.714 g/（L·h）。     

SBA-15/酶纳米反应器的扩散和动力学

将猪胰脂肪酶固定于不同孔径SBA-15载体的孔道内,将已固定酶的尺寸分布均一的一维孔道看作纳米反应器,研究了酶催化水解反应的内扩散和动力学特征。蒂勒模数和有效扩散系数随孔径尺寸的变化以及动力学参数和内扩散之间的关系说明:当孔径小于9.4nm时,内扩散限制明显;孔径大于10.9nm时,内扩散对酶催化水解过程的影响减小。     

Preparation and nanoencapsulation of l-asparaginase II in chitosan-tripolyphosphate nanoparticles and in vitro release study

This paper describes the production, purification, and immobilization of l-asparaginase II (ASNase II) in chitosan nanoparticles (CSNPs). ASNase II is an effective antineoplastic agent, used in the acute lymphoblastic leukemia chemotherapy. Cloned ASNase II gene (ansB) in pAED4 plasmid was transformed into Escherichia coli BL21pLysS (DE3) competent cells and expressed under optimal conditions. The lyophilized enzyme was loaded into CSNPs by ionotropic gelation method. In order to get optimal entrapment efficiency, CSNP preparation, chitosan/tripolyphosphate (CS/TPP) ratio, and protein loading were investigated. ASNase II loading into CSNPs was confirmed by Fourier transform infrared (FTIR) spectroscopy, and morphological observation was carried out by transmission electron microscopy. Three absolute CS/TPP ratios were studied. Entrapment efficiency and loading capacity increased with increasing CS and TPP concentration. The best ratio was applied for obtaining optimal ASNase II-loaded CSNPs with the highest entrapment efficiency. Size, zeta potential, entrapment efficiency, and loading capacity of the optimal ASNase II-CSNPs were 340 ± 12 nm, 21.2 ± 3 mV, 76.2% and 47.6%, respectively. The immobilized enzyme showed an increased in vitro half-life in comparison with the free enzyme. The pH and thermostability of the immobilized enzyme was comparable with the free enzyme. This study leads to a better understanding of how to prepare CSNPs, how to achieve high encapsulation efficiency for a high molecular weight protein, and how to prolong the release of protein from CSNPs. A conceptual understanding of biological responses to ASNase II-loaded CSNPs is needed for the development of novel methods of drug delivery.     

Strategies for lipase‐catalyzed production and the purification of structured phospholipids

This work provides different strategies for the enzymatic modification of the fatty acid composition in soybean phosphatidylcholine (PC) and the subsequent purification. Enzymatic transesterification reactions with caprylic acid as acyl donor were carried out in continuous enzyme bed reactors with a commercial immobilized lipase (Lipozyme RM IM) as catalyst. Operative stability of the immobilized lipase was examined under solvent and solvent‐free conditions. The long reaction time required to have a high incorporation, combined with rapid deactivation of the enzyme, makes the solvent‐free transesterification reaction unfavorable. Performing the reaction in the presence of solvent (hexane) makes it possible to have high incorporation into PC and deactivation of the lipase is less pronounced as compared to solvent‐free operations. For solvent‐free operation, it is suggested to recycle the reaction mixture through the packed bed reactor, as this would increase incorporation of the desired fatty acids, due to increased contact time between substrate and enzyme in the column. Removal of free fatty acids from the reaction mixture can be done by ultrafiltration; however, parameters need to be selected with care in order to have a feasible process. No changes are observed in the phospholipid (PL) distribution during ultrafiltration, and other techniques as column chromatography may be required if high purity of individual PL species is desired. LC/MS analysis of transesterified PC revealed the presence of 8:0/8:0‐PC, showing that acyl migration takes place during the acidolysis reaction.     

Enzyme immobilization: Part 2: Immobilization of alkaline phosphatase on Na-bentonite and modified bentonite

Alkaline phosphatase from calf intestinal mucous membrane was immobilized on unmodified and modified bentonites. The activity of the immobilized enzyme was examined under varying experimental conditions. The effects of various factors such as concentration of enzyme solution, pH and temperature, stirring and various thermodynamic parameters were evaluated. The highest enzyme activity, for free and immobilized enzyme, was obtained in 0.1 M phosphate buffer at pH 7. Optimal enzyme activity of both free and adsorbed enzyme was reached at pH = 10. The best time period of magnetic stirring was 2 h at 4 °C. The adsorption isotherm was modeled by Langmuir equation. The effect of substrate concentration on enzymatic activity of the free and immobilized enzymes showed a good fit to the Michaelis–Menten plots. The immobilized enzyme exhibited activity comparable to the soluble enzyme after storage at 30 °C. Thermal stability and resistance against proteolytic attack were also evaluated.     

Enzymatic large‐scale production of biodiesel

Enzymatic production of biodiesel has been the subject of intensive research over the last 5‐10 years. When comparing the economy of enzymatic and chemical produced biodiesel important factors include yield, flexibility in feedstock, value of by‐products, recovery costs for alcohol as well as the cost of enzyme, enzyme lifetime, reaction time are important factors. By 2009 large‐scale enzymatic biodiesel was considered to be cost effective [1]. Since then development work has further improved the technology which is now ready for the market. We have developed a full enzymatic two‐step process for converting oils to biodiesel, and a pretreatment unit which can be installed in an existing chemical biodiesel plant to enable it to process high free fatty acid feedstocks.     

Improvement in the Cleaning Performance Towards Protein Soils in Laundry Detergents by Protease Immobilization on the Silica Nanoparticles

This work aimed to understand the effect of protease immobilization on silica nanoparticles and how such immobilization affects protease performance as catalysis for enhancing the removal of protein soils. Detergent products contain many components that may affect the free enzyme activity and stability. Various factors such as temperature, pH and humidity are know to affect enzyme activity and cleaning efficiency. Therefore, the effect of enzyme immobilization on the removal of protein based soil was investigated on cotton fabrics as the model soil. The effect of temperature and humidity on the stability of free and immobilized enzyme was compared. It was found that the immobilized enzyme increased cleaning efficiency toward protein soil removal on cotton fabrics, whereas the free enzyme imposed a small effect on the enzymatic activity towards the same soil substrates. In addition, the stability of the immobilized enzyme against temperature and humidity was much higher than its corresponding value by free enzyme.     

Galacto-oligosaccharide production with immobilized β-galactosidase in a packed-bed reactor vs. free β-galactosidase in a batch reactor

We report here that the usage of immobilized enzyme in a continuous packed bed reactor (PBR) can be a good alternative for GOS production instead of the traditional use of free enzyme in a batch reactor. The carbohydrate composition of the product of the PBR with immobilized enzyme was comparable to that of the batch reactor with free enzyme. The stability of the immobilized enzyme at a lactose concentration of 38% (w/v) and at 50 °C was very high: the half-life time of the immobilized enzyme was approximately 90 days. The enzymatic productivity of GOS production using immobilized enzyme in a PBR can be more than six times higher than that of GOS production with free enzyme in a batch reactor. Besides, when aiming for an equal volumetric productivity to the batch process in designing a PBR, the volume of the PBR can be much smaller than that of the batch reactor, depending on the enzyme dosage and the run time of a single batch.     

Zwitterionic poly(carboxybetaine) microgels for enzyme (chymotrypsin) covalent immobilization with extended stability and activity

There is emerging evidence that biocompatible zwitterionic materials can prevent nonspecific interactions within protein systems and increase protein stability. Here, a zwitterionic microgel was synthesized from poly (carboxybetaine methyl methacrylate) (pCB) using an inverse emulsion, free radical polymerization reaction technique. The microgel was loaded with a model enzyme, α-chymotrypsin (ChT), using a post-fabrication loading technique. A reaction scheme was developed and studied for covalent immobilization of ChT within the microgel. Confocal laser microscopy studies showed that immobilized ChT (i-ChT) was distributed within the hydrogel. The enzyme-immobilized microgels showed excellent reusability (72% of its initial activity after 10 uses) and could undergo several freezing/drying/rehydration cycles while retaining enzymatic activity. The i-ChT activity, half-life, and conformational stability were studied at varying pH and temperatures with results compared to free ChT in buffer. ChT immobilized within pCB hydrogel showed increased enzymatic stability as observed by a 13°C increase in the temperature at which i-ChT loses activity compared to free ChT. Furthermore, enzyme half-life increased up to seven-fold for the pCB immobilized ChT, and the increased stability resulted in higher activity at elevated pH. The i-ChT was most active at pH of 8.5 and was partially active up to the pH of 10.2.