Drug-polymer microparticles produced by supercritical assisted atomization

The supercritical assisted atomization (SAA) was proposed as a new technique to produce composite microparticles for drug controlled release. Ampicillin trihydrate and chitosan were selected as model drug and carrier, respectively, and 1% v/v acetic acid aqueous solution was used as solvent. The effect of the polymer/drug ratio on particle morphology and drug release rate was evaluated. SEM analysis indicated that non-coalescing spherical microparticles formed by chitosan/ampicillin were produced by SAA. All coprecipitates produced have a sharp particle distribution, with diameters ranging between about 0.1 and 6 microm. SAA composite microparticles were characterized by X-ray, DSC, EDX and UV-vis analysis. A solid solution of the chitosan and ampicillin was produced and a stabilizing effect of the polymer on the drug has resulted that protects ampicillin from thermal degradation. A prolonged release from SAA coprecipitates with respect to raw drug and physical mixtures of chitosan and ampicillin was obtained; moreover, the polymer/drug ratio has revealed to be a controlling parameter for drug release. Drug release mechanisms characteristic of swelling-controlled systems were observed, with ampicillin release depending on both relaxation and diffusive mechanisms. An empirical binomial equation was used to describe experimental data, showing a fair good agreement with ampicillin release data if both the relaxational and the diffusional parameters are function of the polymer/drug ratio.     

Supercritical assisted atomization: A novel technology for microparticles preparation of an asthma-controlling drug

The objective of this study was to produce microparticles of a new asthma-controlling drug by supercritical assisted atomization (SAA), proposed as an alternative to conventional jet-milling process. SAA is based on the solubilization of supercritical carbon dioxide in a liquid solution containing the drug; the ternary mixture is then sprayed through a nozzle, and microparticles are formed as a consequence of the enhanced atomization. SAA process parameters studied were precipitator temperature, nozzle diameter, and drug concentration in the liquid solution. Their influence was evaluated on morphology and size of precipitated particles. Spherical particles with mean particle size ranging from 1 to 3 μm of the new anti-asthma drug were produced by SAA. The mass median aerodynamic diameter (MMAD) of the SAA micronized particles and of the conventional jet-milled drug was used to compare, the results obtainable using the 2 techniques. Particularly, MMADs from 1.6 to 4.0 μm were obtained by SAA at the optimum operating conditions and by varying the concentration of the solution injected. MMAD of 6.0 μm was calculated for the jet-milled drug. SAA samples also exhibited narrower particle size distribution (PSD). A good control of particle size and distribution together with no drug degradation was obtained by SAA process. Published: October 22, 2005     

Cyclodextrins micrometric powders obtained by supercritical fluid processing

Supercritical fluid technology offers the possibility to produce dry powder formulations of biocompatible materials, overcoming the drawbacks of classical micronization processes. In this work, Supercritical Assisted Atomization (SAA) has been used to micronize alpha-cyclodextrin (alpha-CD) and hydroxypropyl-beta-cyclodextrin (HP-beta-CD). Some process parameters, such as precipitation temperature and solute concentration in the liquid solution, have been studied to evaluate their influence on morphology and size of precipitated particles. Cyclodextrins (CDs) micronization has been successful: well-defined spherical microparticles of alpha-CD and HP-beta-CD have been produced. Particle size analysis revealed that sharp distributions have been obtained: 95% of particles have diameters ranging between 0.1 and 5 microm for both CDs. X-ray and DSC analyses have been also performed to investigate CDs modifications induced by SAA processing: amorphous particles have been obtained in both cases, whereas raw alpha-CD was crystalline and raw HP-beta-CD was amorphous.     

海参i-型溶菌酶在毕赤酵母基因工程菌中优化表达及纯化

将实验室已构建的毕赤酵母基因工程茵(pPIC9K-SjLys/GS115)作为海参i-型溶茵酶生产菌株,本研究分别从甲醇浓度、培养基pH、温度和诱导时间对其产酶发酵条件进行优化.实验得出甲醇诱导浓度为1.0％,发酵培养基初始pH 6.0,温度30℃,培养96 h为最佳目的蛋白表达条件,其发酵液中海参i-型溶菌酶含量达10.63 mg/L.将发酵液经离心和超滤浓缩后得到上清液,再经离子交换和凝胶过滤层析纯化获得海参i-型溶菌酶产品,其酶活力达826.44 U/mg.经测定该酶对革兰氏阳性菌溶壁微球菌和革兰氏阴性菌副溶血弧菌均具有明显的抑菌作用.     

Irreversible lysozyme inactivation and aggregation induced by stirring: kinetic study and aggregates characterisation

Stirring strongly enhanced irreversible inactivation and aggregation of lysozyme being studied as a model enzyme. From 0 to 740 rpm (equivalent to impeller tip speeds from 0 to 0.77 m s^–1), the inactivation kinetic constant was proportional to the power imparted by the impeller. Collisions between inactive and native molecules induced inactivation of the latter and led to lysozyme aggregation. These fractal aggregates of lysozyme were made of monomers, dimers and trimers.     

Effects of supercritical CO2 exposure and depressurization on immobilized lipase activity

Lipozyme IM20 from Novo Nordisk (Denmark) was examined after various treatments. Conditions were chosen to reflect those that would be considered in the design of an industrial process. A two-level factorial design was employed to assess the effects of pressurization/depressurization cycles, rate of depressurization and exposure length. A significant three-factor interaction was observed. Lowest residual activity was observed for runs in which the depressurization rate was 86–89 bar min^–1. Incubation for 12 h also yielded low residual activity but only when exposing the immobilized enzyme to one cycle. The highest residual activity was obtained for immobilized enzymes repeatedly exposed for periods of 12 h (5 times) with a depressurization rate of 4.3 to 4.45 bar min^–1. This effect may be due to the extraction of an inhibiting compound. Tuning process parameters can lead to a seven-fold change in residual activity.     

Continuous supercritical emulsions extraction: a new technology for biopolymer microparticles production

Supercritical emulsion extraction (SEE) was recently proposed for the production of biopolymer microparticles starting from oil‐in‐water emulsions. This technology can improve the product quality because of the fast and selective extraction of the dispersed oily phase by using supercritical carbon dioxide (SC‐CO₂). However, until now, SEE was proposed in batch configuration, sharing with the traditional processes an intrinsically discontinuous operation and problems of batches reproducibility and process yield. In this study, by using a countercurrent packed column, the SEE process was proposed in a continuous operating mode (SEE‐CM) for the production of poly‐lactic‐co‐glycolic acid (PLGA) microparticles. The new process design takes advantage of the large contact area between the SC‐CO₂ and emulsion allowing the production of PLGA microparticles with controlled and narrow size distributions in only few minutes. SEE‐CM operating parameters such as pressure, temperature, and flow rate ratios were analyzed and the process efficiency in terms of recovered material and its size distribution compared with SEE (batch mode operation) and conventional evaporation technology. PLGA microparticles showed a mean particle size between 1–3 µm (depending on the droplet sizes) with a SD that was always smaller than that associated with particles produced by discontinuous processes. Single and double emulsions were successfully treated and the microparticles physico‐chemical properties showed no morphological and structural differences between the SEE‐CM‐produced microparticles and the ones obtained by conventional evaporation technology. Biotechnol. Bioeng. 2011; 108:676–686. © 2010 Wiley Periodicals, Inc.     

Expression of functional recombinant human lysozyme in transgenic rice cell culture

Using particle bombardment-mediated transformation, a codon-optimized synthetic gene for human lysozyme was introduced into the calli of rice (Oryza sativa) cultivar Taipei 309. The expression levels of recombinant human lysozyme in the transformed rice suspension cell culture approached approximately 4% of total soluble protein. Recombinant human lysozyme was purified to greater than 95% homogeneity using a two-step chromatography process. Amino acid sequencing verified that the N-terminus of the mature recombinant human lysozyme was identical to native human lysozyme. This indicates that the rice RAmy3D signal peptide was correctly cleaved off from the human lysozyme preprotein by endogenous rice signal peptidase. Recombinant human lysozyme was found to have the same molecular mass, isoelectric point and specific activity as native human lysozyme. The bactericidal activity of recombinant human lysozyme was determined by turbidimetric assay using Micrococcus lysodeikticus in 96-well microtiter plates. The bactericidal activity of lysozyme on Gram-negative bacteria was examined by adding purified lysozyme to mid-log phase cultures of E. coli strain JM109. In this study, significant bactericidal activity was observed after E.coli cells were exposed to recombinant human lysozyme for 60min. Both native and recombinant human lysozyme displayed the same thermostability and resistance to degradation by low pH. The potential for using rice-derived lysozyme as an antimicrobial food supplement, particularly for infant formula and baby foods, is discussed.     

Effect of polyols on the conformational stability and biological activity of a model protein lysozyme

The purpose of this study was to investigate the stabilizing action of polyols against various protein degradation mechanisms (eg, aggregation, deamidation, oxidation), using a model protein lysozyme. Differential scanning calorimeter (DSC) was used to measure the thermodynamic parameters, mid point transition temperature and calorimetric enthalpy, in order to evaluate conformational stability. Enzyme activity assay was used to corroborate the DSC results. Mannitol, sucrose, lactose, glycerol, and propylene glycol were used as polyols to stabilize lysozyme against aggregation, deamidation, and oxidation. Mannitol was found to stabilize lysozyme against aggregation, sucrose against deamidation both at neutral pH and at acidic pH, and lactose against oxidation. Stabilizers that provided greater conformational stability of lysozyme against various degradation mechanisms also protected specific enzyme activity to a greater extent. It was concluded that DSC and bioassay could be valuable tools for screening stabilizers in protein formulations.     

Preparation of budesonide and budesonide-PLA microparticles using supercritical fluid precipitation technology

The objective of this study was to prepare and characterize microparticles of budesonide alone and budesonide and polylactic acid (PLA) using supercritical fluid (SCF) technology. A precipitation with a compressed antisolvent (PCA) technique employing supercritical CO₂ and a nozzle with 100-μm internal diameter was used to prepare microparticles of budesonide and budesonide-PLA. The effect of various operating variables (temperature and pressure of CO₂ and flow rates of drug-polymer solution and/or CO₂) and formulation variables (0.25%, 0.5%, and 1% budesonide in methylene chloride) on the morphology and size distribution of the microparticles was determined using scanning electron microscopy. In addition, budesonide-PLA particles were characterized for their surface charge and drug-polymer interactions using a zeta meter and differential scanning calorimetry (DSC), respectively. Furthermore, in vitro budesonide release from budesonide-PLA microparticles was determined at 37°C. Using the PCA process, budesonide and budesonide-PLA microparticles with mean diameters of 1 to 2 μm were prepared. An increase in budesonide concentration (0.25%–1% wt/vol) resulted in budesonide microparticles that were fairly spherical and less aggiomerated. In addition, the size of the microparticles increased with an increase in the drug-polymer solution flow rate (1.4–4.7 mL/min) or with a decrease in the CO₂ flow rate (50–10 mL/min). Budesonide-PLA microparticles had a drug loading of 7.94%, equivalent to ∼80% encapsulation efficiency. Budesonide-PLA microparticles had a zeta potential of— 37±4 mV, and DSC studies indicated that SCF processing of budesonide-PLA microparticles resulted in the loss of budesonide crystallinity. Finally, in vitro drug release studies at 37°C indicated 50% budesonide release from the budesonide-PLA microparticles at the end of 28 days. Thus, the PCA process was successful in producing budesonide and budesonide-PLA microparticles. In addition, budesonide-PLA microparticles sustained budesonide release for 4 weeks.     

Solvent effects on the structure,dynamics and activity of lysozyme

Kuntz and Kauzmann have argued that dehydrating a protein results in conformational changes. In contrast, Rupleyet al. have developed a hydration model which involves no significant change in conformation; the onset of enzyme activity in hen egg-white lysozyme at hydration values of about 0.2 g water/g protein they ascribe rather to a solvation effect. Using a direct difference infra-red technique we can follow specific hydration events as water is added to a dry protein. Conformational studies of lysozyme using laser Raman spectroscopy indicate changes in conformation with hydration that are complete just before measurable activity is found. Parallel nuclear magnetic resonance measurements of exchangeability of the main chain amide hydrogens, as a function of hydration from near dryness, suggest a hydration-related increase in conformational flexibility which occurs before-and is probably necessary for-the Raman-detected conformational changes. Very recent inelastic neutron scattering measurements provides direct evidence of a flexibility change induced by hydration, which is apparently necessary before the enzyme can achieve adequate flexibility for it to begin to function.     

表面活性剂对海洋微生物溶菌酶活性的影响

以海洋微生物溶菌酶（ⅧL）为研究对象,分别检验几种表面剂对MBL活性的影响,着重研究烷基多苷（APG）对其活性的影响。结果表明,APG与阳离子烷基多苷（矾PG）分别提高MBL相对酶活性为21％,15％,SDS降低该酶活性约为15％,Tween20和Tween80对MBL活性的影响不明显。MBL含量大于5．0mg／mL时,对大肠杆菌、金黄色葡萄、白色念珠球菌有抑菌作用。0．5％～1．5％的APG无明显抑菌作用。将5．0mg／mMBL与1．0mg／mLAPG复配后（简称CEP）,发现APG能明显增强MBL抑菌作用,CEP具有较好地杀菌作用;CEP在54℃培养箱中放置14d后,其杀菌率保持不变,说明CEP的杀菌性能的稳定性良好。     

Co-segregation of alleles at a microsatellite locus within the macrophage expressed lysozyme gene and levels of serum lysozyme activity in two half-sib families of Polish Black and White Lowland cattle

Alleles at a microsatellite locus within the macrophage expressed lysozyme gene were shown to co-segregate with lysozyme activity in two half-sib families of Polish Black and White Lowland cattle. The bimodal distribution of lysozyme activities in both progeny groups is concordant with the occurrence of the alternative paternal alleles. The microsatellite is linked to a locus for high lysozyme activity that accounts for 70–95% of the phenotypic variation of both offspring groups considering the lysozyme activities of animals being older than 1 month.     

人源溶菌酶结构与功能的研究进展

人溶菌酶是一类人体内天然存在的能够溶解细菌细胞壁的碱性蛋白的总称。其作用特征是能够裂解肽聚糖中的N-乙酰氨基葡萄糖与N-乙酰氨基甲酸之间的β-(1,4)-糖苷键。人溶菌酶具有抗菌、抗炎、抗病毒和增强免疫力等多种特性,因此在国内外市场上应用广泛。本文就人溶菌酶的结构特点、表达部位、功能表达以及应用情况进行综述。     

Enhanced antimicrobial activity of a peptide derived from human lysozyme by arylation of its tryptophan residues

Antimicrobial peptides are valuable agents to fight antibiotic resistance. These amphipatic species display positively charged and hydrophobic amino acids. Here, we enhance the local hydrophobicity of a model peptide derived from human lysozyme (107RKWVWWRNR115) by arylation of its tryptophan (Trp) residues, which renders a positive effect on Staphylococcus aureus and Staphylococcus epidermidis growth inhibition. This site‐selective modification was accessed by solid‐phase peptide synthesis using the non‐proteinogenic amino acid 2‐aryltryptophan, generated by direct C‐H activation from protected Trp. The modification brought about a relevant increase in growth inhibition: S. aureus was fully inhibited by arylation of Trp 112 and by only 10% by arylation of Trp 109 or 111, respect to the non‐arylated peptide. On the other hand, S. epidermidis was fully inhibited by the three arylated peptides and the parent peptide. The minimum inhibitory concentration was significantly reduced for S. aureus depending on the arylation site. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Improved antimicrobial activity of h‐lysozyme (107–115) by rational Ala substitution

The most challenging target in the design of new antimicrobial agents is the development of antibiotic resistance. Antimicrobial peptides are good candidates as lead compounds for the development of novel anti‐infective drugs. Here we propose the sequential substitution of each Ala residue present in a lead peptide with known antimicrobial activity by specific amino acids, rationally chosen, that could enhance the activity of the resultant peptide. Taking the fragment 107–115 of the human lysozyme as lead, two‐round screening by sequentially replacing both Ala residues (108 and 111) by distinct amino acids resulted in a novel peptide with 4‐ and 20‐fold increased antimicrobial activity against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213, respectively. These results reinforce the strategy proposed, which, in combination with simple and easy screening tools, will contribute to the rapid development of new therapeutic peptides required by the market. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

A theoretical DFT investigation of the lysozyme mechanism: computational evidence for a covalent intermediate pathway

To test the occurrence of local particularities during the unfolding of Ca2+-loaded goat alpha-lactalbumin (GLA) we replaced Trp60 and -118, either one or both, by Phe. In contrast with alternative studies, our recombinant alpha-lactalbumins are expressed in Pichia pastoris and do not contain the extra N-terminal methionine. The substitution of Trp60 leads to a reduction of the global stability. The effect of the Trp118Phe substitution on the conformation and stability of the mutant, however, is negligible. Comparison of the fluorescence spectra of these mutants makes clear that Trp60 and -118 are strongly quenched in the native state. They both contribute to the quenching of Trp26 and -104 emission. By the interplay of these quenching effects, the fluorescence intensity changes upon thermal unfolding of the mutants behave very differently. This is the reason for a discrepancy of the apparent transition temperatures derived from the shift of the emission maxima (Tm,Fl lambda) and those derived from DSC (Tm,DSC). However, the transition temperatures derived from fluorescence intensity (Tm,Fl int) and from DSC (Tm,DSC), respectively, are quite similar, and thus, no local rearrangements are observed upon heat-induced unfolding. At room temperature, the occurrence of specific local rearrangements upon GdnHCl-induced denaturation of the different mutants is deduced from the apparent free energies of their transition state obtained from stopped-flow fluorescence measurements. By phi-value analysis it appears that, while the surroundings of Trp118 are exposed in the kinetic transition state, the surroundings of Trp60 remain native.     

X-ray structure of Glu 53 human lysozyme.

The three-dimensional structure of a modified human lysozyme (HL), Glu 53 HL, in which Asp 53 was replaced by Glu, has been determined at 1.77 A resolution by X-ray analysis. The backbone structure of Glu 53 HL is essentially the same as the structure of wild-type HL. The root mean square difference for the superposition of equivalent C alpha atoms is 0.141 A. Except for the Glu 53 residue, the structure of the active site region is largely conserved between Glu 53 HL and wild-type HL. However, the hydrogen bond network differs because of the small shift or rotation of side chain groups. The carboxyl group of Glu 53 points to the carboxyl group of Glu 35 with a distance of 4.7 A between the nearest carboxyl oxygen atoms. A water molecule links these carboxyl groups by a hydrogen bond bridge. The active site structure explains well the fact that the binding ability for substrates does not significantly differ between Glu 53 HL and wild-type HL. On the other hand, the positional and orientational change of the carboxyl group of the residue 53 caused by the mutation is considered to be responsible for the low catalytic activity (ca. 1%) of Glu 53 HL. The requirement of precise positioning for the carboxyl group suggests the possibility that the Glu 53 residue contributes more than a simple electrostatic stabilization of the intermediate in the catalysis reaction.     

Antimicrobial peptides derived from goose egg white lysozyme

Peptide fragments possessing antimicrobial activity were obtained by protease digestion of goose egg white lysozyme. Digested peptide purified from RP-HPLC which showed no lysozyme activity exhibited bactericidal activity toward Gram-negative and Gram-positive bacteria. LC/MS–MS and automated Edman degradation revealed the amino acid sequence to be Thr-Ala-Lys-Pro-Glu-Gly-Leu-Ser-Tyr. This sequence corresponds to amino acid positions 20–28, located at the N-terminal outer part of goose lysozyme. The peptide acted on bacterial membrane as shown by scanning electron microscopy. The mechanism of action could be explained from a helical structure that may be formed by the centered Pro residue and the terminal Lys residue after the peptide attaches to a cell membrane. This is the first study to report that a peptide derived from the protease digests of G-type lysozyme possesses antimicrobial activity with broad spectrum activity. Our result is comparative to the previous reports of Chicken lysozyme and T4 phage lysozyme, which showed antimicrobial activity after digestion with protease. These results might contribute to the usage of antimicrobial peptides engineered by genetic or chemical synthesis.