Overcoming oral insulin delivery barriers: application of cell penetrating peptide and silica-based nanoporous composites

Oral insulin delivery has received the most attention in insulin formulations due to its high patient compliance and, more importantly, to its potential to mimic the physiologic insulin secretion seen in non-diabetic individuals. However, oral insulin delivery has two major limitations: the enzymatic barrier that leads to rapid insulin degradation, and the mucosal barrier that limits insulin’s bioavailability. Several approaches have been actively pursued to circumvent the enzyme barrier, with some of them receiving promising results. Yet, thus far there has been no major success in overcoming the mucosal barrier, which is the main cause in undercutting insulin’s oral bioavailability. In this review of our group’s research, an innovative silica-based, mucoadhesive oral insulin formulation with encapsulated-insulin/cell penetrating peptide (CPP) to overcome both enzyme and mucosal barriers is discussed, and the preliminary and convincing results to confirm the plausibility of this oral insulin delivery system are reviewed. In vitro studies demonstrated that the CPP-insulin conjugates could facilitate cellular uptake of insulin while keeping insulin’s biologic functions intact. It was also confirmed that low molecular weight protamine (LMWP) behaves like a CPP peptide, with a cell translocation potency equivalent to that of the widely studied TAT. The mucoadhesive properties of the produced silica-chitosan composites could be controlled by varying both the pH and composition; the composite consisting of chitosan (25 wt-%) and silica (75 wt-%) exhibited the greatest mucoadhesion at gastric pH. Furthermore, drug release from the composite network could also be regulated by altering the chitosan content. Overall, the universal applicability of those technologies could lead to development of a generic platform for oral delivery of many other bioactive compounds, especially for peptide or protein drugs which inevitably encounter the poor bioavailability issues.     

Effect of a double‐structured microporous polymer support on the catalytic activity,stability and aggregation behavior of immobilized enzymes

A double‐structured microporous polymer composite support consisting of a microporous skeleton for protection of immobilized enzymes and a gel‐like polymer bearing different functionalities has been synthesized for immobilization of various enzymes. Among three approaches based on two types of interactions (physical and chemical), we found that under our experimental conditions the covalent bonding between amine moieties of enzymes and epoxide groups of the support was the best, with enzyme leaching being reduced to as low as zero for nine enzymes and catalytic activities increased by factors of 1–4 for two lipases. The improvement in catalytic activity could be attributed to the amphiphilic soft gel which might create a kind of lipid–water interface or microenvironment in aqueous solution, favoring the interfacial activation of certain lipases. Furthermore, the thermal and operational stability, and reusability of all the enzymes could be enhanced to varying degrees. We also found that enzyme aggregates of different sizes could be formed on the spherical support surface at quite low enzyme concentrations, which might also influence the final catalytic activities of the immobilized enzymes. © 2015 Society of Chemical Industry     

Crystallographic and mutational analyses of an extremely acidophilic and acid-stable xylanase: biased distribution of acidic residues and importance of Asp37 for catalysis at low pH

Xylanase C from Aspergillus kawachii has an optimum pH of 2.0 and is stable at pH 1.0. The crystal structure of xylanase C was determined at 2.0 A resolution (R-factor = 19.4%). The overall structure was similar to those of other family 11 xylanases. Asp37 and an acid-base catalyst, Glu170, are located at a hydrogen-bonding distance (2.8 A), as in other xylanases with low pH optima. Asp37 of xylanase C was replaced with asparagine and other residues by site-directed mutagenesis. Analyses of the wild-type and mutant enzymes showed that Asp37 is important for high enzyme activity at low pH. In the case of the asparagine mutant, the optimum pH shifted to 5.0 and the maximum specific activity decreased to about 15% of that of the wild-type enzyme. On structural comparison with xylanases with higher pH optima, another striking feature of the xylanase C structure was found; the enzyme has numerous acidic residues concentrated on the surface (so-called 'Ser/Thr surface' in most family 11 xylanases). The relationship of the stability against extreme pH conditions and high salt concentrations with the spacially biased distribution of charged residues on the proteins is discussed.      

An Imprinted Cross-Linked Enzyme Aggregate (iCLEA) of Sucrose Phosphorylase: Combining Improved Stability with Altered Specificity

The industrial use of sucrose phosphorylase (SP), an interesting biocatalyst for the selective transfer of α-glucosyl residues to various acceptor molecules, has been hampered by a lack of long-term stability and low activity towards alternative substrates. We have recently shown that the stability of the SP from Bifidobacterium adolescentis can be significantly improved by the formation of a cross-linked enzyme aggregate (CLEA). In this work, it is shown that the transglucosylation activity of such a CLEA can also be improved by molecular imprinting with a suitable substrate. To obtain proof of concept, SP was imprinted with α-glucosyl glycerol and subsequently cross-linked with glutaraldehyde. As a consequence, the enzyme’s specific activity towards glycerol as acceptor substrate was increased two-fold while simultaneously providing an exceptional stability at 60 °C. This procedure can be performed in an aqueous environment and gives rise to a new enzyme formulation called iCLEA.     

基于纳米氢氧化铝颗粒的Pickering乳液制备

以异丙醇铝为原料,采用醇盐水解?水热法制备勃姆石型纳米氢氧化铝颗粒,优化制备条件;以所制颗粒为稳定剂、角鲨烯为油相,通过超声破碎法制备Pickering乳液,考察了颗粒浓度、水相成分、超声时间及功率对Pickering乳液粒径及稳定性的影响。结果表明,水热温度200℃、水热时间2 h条件下,可制得结晶度高且均一的勃姆石型纳米氢氧化铝颗粒,平均粒径为55.70?9.20 nm,多分散性指数(PDI)为0.187?0.011;所制Pickering乳液平均粒径为1870?55 nm,PDI=0.120?0.010,可在室温下稳定储存120 d以上,且生物相容性良好,有望应用于生物医药领域。     

用于质子交换膜燃料电池的高温无机质子传导材料研究进展

质子交换膜是质子交换膜燃料电池（PEMFC）的核心部件，其主要作用是传导质子。无机质子传导材料作为一种新型的质子传导介质，近年来逐渐引起了人们的关注。本文主要介绍了小分子磷酸、无机沸石材料、固体酸和无机氧化物陶瓷材料等几种高温无机质子传导材料，并对它们的性能和特点进行了评述。主要结论如下：小分子磷酸质子传导率高，但是容易泄露；无机沸石材料化学稳定性好，但质子传导率尚有提高的空间；无机氧化物陶瓷材料力学性能和化学温度性能均很好，但质子传导率相对较低；固体酸质子传导率优异，高温稳定性也好，是最有希望在PEMFC中获得推广应用的材料。     

Enhancement of enzyme activity through three-phase partitioning: crystal structure of a modified serine proteinase at 1.5 A resolution

Three-phase partitioning is fast developing as a novel bioseparationstrategy with a wide range of applications including enzymestability and enhancement of its catalytic activity. Despiteall this, the enzyme behaviour in this process still remainsunknown. A serine proteinase, proteinase K, was subjected tothree-phase partitioning (TPP). A 3 ml volume of proteinaseK solution (3 mg/ml in 0.05 M acetate buffer, pH 6.0) was broughtto 30% (w/v) ammonium sulphate saturation by addition of saturatedammonium sulphate. tert-Butanol (6 ml) was added to this solutionand the mixture was incubated at 25°C for 1 h. The precipitatedprotein in the mid-layer was dissolved in 3 ml of 0.05 M acetatebuffer, pH 6.0. The specific activity of the processed enzymewas estimated and was found to be 210% of the original enzymeactivity. In order to understand the basis of this remarkableenhancement of the enzyme activity, the structure of the TPP-treatedenzyme was determined by X-ray diffraction at 1.5 Å resolution.The overall structure of the TPP-treated enzyme is similar tothe original structure in an aqueous environment. The hydrogenbonding system of the catalytic triad is intact. However, thewater structure in the substrate binding site has undergonea rearrangement as some of the water molecules are either displacedor completely absent. Two acetate ions were identified in thestructure. One is located in the active site and seems to mimicthe role of water in the enzyme activity and stability. Theother is located at the surface of the molecule and is involvedin stabilizing the local structure of the enzyme. The most strikingobservation in respect of the present structure pertains toa relatively higher overall temperature factor (B = 19.7 Ų)than the value of 9.3 Ų in the original enzyme. As aresult of a higher B-factor, a number of residues, particularlytheir side chains, were found to adopt more than one conformation.It appears that the protein exists in an excited state whichmight be helping the enzyme to function more rapidly than theoriginal enzyme in aqueous media. Summarily, the basis of increasedenzymatic activity could be attributed to (i) the presence ofan acetate ion at the active site and (ii) its excited stateas reflected by an overall higher B-factor.     

Covalent immobilization of cytochrome P450 BM3 (R966D/W1046S) on glutaraldehyde activated SPIONs

Selective hydroxylation of the C‐H bond of saturated hydrocarbon chains at room temperature is the signature of an invaluable biocatalyst, cytochrome P450 BM3 from Bacillus megaterium. Despite this remarkable ability, because of the enzyme's inherent low stability and dependence on electron supply by expensive NADPH, developing stable and economic BM3 systems is a challenging subject. To improve BM3 stability, facilitate its reuse, and reduce the process cost, this study suggests covalent immobilization of R966D/W1046S P450 BM3 on glutaraldehyde pre‐activated super paramagnetic iron oxide nanoparticles (SPIONs). This double mutant consumes less expensive cofactors like NADH and BNAH and its immobilization on magnetic support facilitates its separation and reuse. Free and immobilized enzyme performances were evaluated by 10‐pNCA hydroxylation and BM3 selectivity (hydroxylation at ω (1–3) positions of a fatty acid) was confirmed in a reaction involving myristic acid. The enzyme activity recovery was up to 60 % with 100 % enzyme binding efficiency. BM3‐SPIONs were easily separated from the reaction medium by applying a magnet, and recycled for 5 times, after which they could still present half of their initial activity. The enzyme storage stability was significantly improved: after one month of storage at 4 °C, the immobilized enzyme showed 80 % residual activity toward NADH while the soluble enzyme was inactive after a week. Binding an enzyme to fabricated SPIONs is a promising technique to increase enzyme stability and prevent downstream contamination in biocatalytic processes. In this context, BM3‐SPIONs can be a practical model system in cost‐effective large‐scale applications of such enzymes.

     

Stabilization of the Reductase Domain in the Catalytically Self‐Sufficient Cytochrome P450BM3 by Consensus‐Guided Mutagenesis

The multidomain, catalytically self‐sufficient cytochrome P450 BM‐3 from Bacillus megaterium (P450_BM3) constitutes a versatile enzyme for the oxyfunctionalization of organic molecules and natural products. However, the limited stability of the diflavin reductase domain limits the utility of this enzyme for synthetic applications. In this work, a consensus‐guided mutagenesis approach was applied to enhance the thermal stability of the reductase domain of P450_BM3. Upon phylogenetic analysis of a set of distantly related P450s (>38 % identity), a total of 14 amino acid substitutions were identified and evaluated in terms of their stabilizing effects relative to the wild‐type reductase domain. Recombination of the six most stabilizing mutations generated two thermostable variants featuring up to tenfold longer half‐lives at 50 °C and increased catalytic performance at elevated temperatures. Further characterization of the engineered P450_BM3 variants indicated that the introduced mutations increased the thermal stability of the FAD‐binding domain and that the optimal temperature (T_opt) of the enzyme had shifted from 25 to 40 °C. This work demonstrates the effectiveness of consensus mutagenesis for enhancing the stability of the reductase component of a multidomain P450. The stabilized P450_BM3 variants developed here could potentially provide more robust scaffolds for the engineering of oxidation biocatalysts.     

1,2-Mannobioside mimic: synthesis, DC-SIGN interaction by NMR and docking, and antiviral activity

The design and preparation of carbohydrate ligands for DC-SIGN is a topic of high interest because of the role played by this C-type lectin in immunity and infection processes. The low chemical stability of carbohydrates against enzymatic hydrolysis by glycosylases has stimulated the search for new alternatives more stable in vivo. Herein, we present a good alternative for a DC-SIGN ligand based on a mannobioside mimic with a higher enzymatic stability than the corresponding disaccharide. NMR and docking studies have been performed to study the interaction of this mimic with DC-SIGN in solution demonstrating that this pseudomannobioside is a good ligand for this lectin. In vitro studies using an infection model with Ebola pseudotyped virus demonstrates that this compound presents an antiviral activity even better than the corresponding disaccharide and could be an interesting ligand to prepare multivalent systems with higher affinities for DC-SIGN with potential biomedical applications.     

Cellular Biocatalysts Using Synthetic Genetic Circuits for Prolonged and Durable Enzymatic Activity

Cellular biocatalysts hold great promise for the synthesis of difficult to achieve compounds, such as complex active molecules. Whole-cell biocatalysts can be programmed through genetic circuits to be more efficient, but they suffer from low stability. The catalytic activity of whole cells decays under stressful conditions, such as prolonged incubation times or high temperatures. In nature, microbial communities cope with these conditions by forming biofilm structures. In this study, it is shown that the use of biofilm structures can enhance the stability of whole-cell biocatalysts. We employed two different strategies to increase the stability of whole-cell catalysts and decrease their susceptibility to high temperature. In the first approach, the formation of a biofilm structure is induced by controlling the expression of one of the curli component, CsgA. The alkaline phosphatase (ALP) enzyme was used to monitor the catalytic activity of cells in the biofilm structure. In the second approach, the ALP enzyme was fused to the CsgA curli fiber subunit to utilize the protective properties of the biofilm on enzyme biofilms. Furthermore, an AND logic gate is introduced between the expression of CsgA and ALP by toehold RNA switches and recombinases to enable logical programming of the whole-cell catalyst for biofilm formation and catalytic action with different tools. The study presents viable approaches to engineer a platform for biocatalysis processes.     

Mechanism Study on the Severe Foaming of Rhamnolipid in Fermentation

Although the biosurfactant rhamnolipid has been previously characterized as having low foam ability, its fermentation is largely impeded by severe foaming. Hence, the investigation of this paradox is critically important for improving the mass production of rhamnolipid. Unexpectedly, the hydrophobic cell, instead of rhamnolipid, has been claimed to explain such severe foaming in rhamnolipid fermentation. This study tried to systematically investigate the severe foaming in fermentation, aiming to propose an effective strategy for foam control. The overflowing foam sustained a super high stability in terms of half‐time for over 30 min. The major product of rhamnolipid largely contributed to the severe foaming in the fermentation process whereas other products like cells elicited much more limited effects. Furthermore, the foam stability of the fermentation broth increased with rhamnolipid concentration and noticeably increased with agitation speed. In the classic Bikerman foam test system without stirring, rhamnolipid showed foam stability as low as Tween 20 which is well known for its poor foam stability. However, in a stirring Bikerman system, rhamnolipid exhibited a foam stability almost as high as sodium dodecyl sulfate (SDS) at 10 g/L and even surpassed SDS at a higher concentration of 20 g/L. Hence, the extraordinarily increased foam stability of rhamnolipid with both agitation and concentration could explain the severe foaming at its late‐stage fermentation when rhamnolipid‐rich solution is mechanically agitated.     

配位剂对低浓度硫酸铬镀铬的影响

环保、成本较低的低浓度硫酸盐三价铬电镀装饰性铬受到广泛关注.在低浓度硫酸铬体系中,研究了配位剂对镀液和镀层性能的影响.实验结果表明,以甲酸钠和苹果酸作为复合配位剂的镀液性能较好.该镀液在pH =3.0,θ=40℃时,所得镀层光亮,电流密度范围较宽,镀液稳定性达10 Ah/L,沉积速率大于0.2 μm/min.     

Biochemical Characterization of An Arginine-Specific Alkaline Trypsin from Bacillus licheniformis

In the present study, we isolated a trypsin-producing strain DMN6 from the leather waste and identified it as Bacillus licheniformis through a two-step screening strategy. The trypsin activity was increased up to 140 from 20 U/mL through culture optimization. The enzyme was purified to electrophoretic homogeneity with a molecular mass of 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the specific activity of purified enzyme is 350 U/mg with Nα-Benzoyl-l-arginine ethylester as the substrate. The optimum temperature and pH for the trypsin are 65 °C and pH 9.0, respectively. Also, the enzyme can be significantly activated by Ba²⁺. This enzyme is relatively stable in alkaline environment and displays excellent activity at low temperatures. It could retain over 95% of enzyme activity after 180 min of incubation at 45 °C. The distinguished activity under low temperature and prominent stability enhance its catalytic potential. In the current work, the open reading frame was obtained with a length of 1371 nucleotides that encoded a protein of 456 amino acids. These data would warrant the B. licheniformis trypsin as a promising candidate for catalytic application in collagen preparation and leather bating through further protein engineering.     

Stability and proteolytic activity of papain in reverse micellar and aqueous media: A kinetic and spectroscopic study

The stability and proteolytic activity of papain were studied in reverse micellar systems, and in aqueous media. In reverse micelles the maximum activity obtained was 80% of the enzyme activity in aqueous solution. Higher papain stability was found in reverse micellar systems compared with that in aqueous solution with half-lives of 24 and 10 days respectively. Electron spin resonance (ESR) spectroscopy studies of aqueous and reverse micellar systems were performed in an attempt to explain the observed enzyme stability and activity profiles. For this purpose a spin label—TEMPOacetamide—was covalently linked to the Cys-25 residue of the papain active center. ESR spectra of labeled papain indicated that catalytic activity of papain could be related to the conformational rigidity near the reaction center. The lower activities obtained in reverse micelles could be a result of the greater degree of mobility and polarity observed in these systems, which can be attributed to papain unfolding. The greater stability found for papain in reverse micelles could be the result of the limited extent of this denaturing process owing to the organized surfactant molecules around the enzyme.     

CRISPR-Mediated Enzyme Fragment Complementation Assay for Quantification of the Stability of Splice Isoforms

Small-molecule splicing modulators exemplified by an FDA-approved drug, risdiplam, are a new pharmacological modality for regulating the expression and stability of splice isoforms. We report a CRISPR-mediated enzyme fragment complementation (EFC) assay to quantify the splice isoform stability. The EFC assay harnessed a 42 amino acid split of a β-galactosidase (designate α-tag), which could be fused at the termini of the target genes using CRISPR/cas9. The α-tagged splice isoform would be quantified by measuring the enzymatic activity upon complementation with the rest of β-galactosidase. This EFC assay retained all the sequences of introns and exons of the target gene in the native genomic environment that recapitulates the cell biology of the diseases of interest. For a proof-of-concept, we developed a CRISPR-mediated EFC assay targeting the exon 7 of the survival of motor neuron 2 (SMN2) gene. The EFC assay is compatible with 384-well plates and robustly quantified the splicing modulation activity of small molecules. In this study, we also discovered that a coumarin derivative, compound 4 , potently modulated SMN2 exon 7 splicing at as low as 1.1 nM.     

Combining Protein and Strain Engineering for the Production of Glyco-Engineered Horseradish Peroxidase C1A in Pichia pastoris

Horseradish peroxidase (HRP), conjugated to antibodies and lectins, is widely used in medical diagnostics. Since recombinant production of the enzyme is difficult, HRP isolated from plant is used for these applications. Production in the yeast Pichia pastoris (P. pastoris), the most promising recombinant production platform to date, causes hyperglycosylation of HRP, which in turn complicates conjugation to antibodies and lectins. In this study we combined protein and strain engineering to obtain an active and stable HRP variant with reduced surface glycosylation. We combined four mutations, each being beneficial for either catalytic activity or thermal stability, and expressed this enzyme variant as well as the unmutated wildtype enzyme in both a P. pastoris benchmark strain and a strain where the native α-1,6-mannosyltransferase (OCH1) was knocked out. Considering productivity in the bioreactor as well as enzyme activity and thermal stability, the mutated HRP variant produced in the P. pastoris benchmark strain turned out to be interesting for medical diagnostics. This variant shows considerable catalytic activity and thermal stability and is less glycosylated, which might allow more controlled and efficient conjugation to antibodies and lectins.     

Catalysis by Enzymes: The Biological Ammonia Synthesis

Enzymes are nature’s own catalysts and fundamental for life, as they catalyze essentially all biological processes. Compared to inorganic catalysts, however, their structure and function is immensely more complicated. Computational modeling has played an increasing role in elucidating enzyme mechanisms, as it can provide information complementary to experimental techniques. Elucidating enzyme structure and mechanism is not only important to understand the basic functions of life, they can also help to find and develop inorganic catalysts. In this review, an overview of recent computational developments for the enzyme nitrogenase will be given. Nitrogenase catalyzes ammonia synthesis, which is also a very important reaction in inorganic catalysis, and insight on enzyme structure and function could provide understanding on how to design biomimetic catalysts for low temperature ammonia synthesis.     

Cytosolic 5′-Nucleotidase II Silencing in Lung Tumor Cells Regulates Metabolism through Activation of the p53/AMPK Signaling Pathway

Cytosolic 5′-nucleotidase II (cN-II) is an allosteric catabolic enzyme that hydrolyzes IMP, GMP, and AMP. The enzyme can assume at least two different structures, being the more active conformation stabilized by ATP and the less active by inorganic phosphate. Therefore, the variation in ATP concentration can control both structure and activity of cN-II. In this paper, using a capillary electrophoresis technique, we demonstrated that a partial silencing of cN-II in a pulmonary carcinoma cell line (NCI-H292) is accompanied by a decrease in adenylate pool, without affecting the energy charge. We also found that cN-II silencing decreased proliferation and increased oxidative metabolism, as indicated by the decreased production of lactate. These effects, as demonstrated by Western blotting, appear to be mediated by both p53 and AMP-activated protein kinase, as most of them are prevented by pifithrin-α, a known p53 inhibitor. These results are in line with our previous observations of a shift towards a more oxidative and less proliferative phenotype of tumoral cells with a low expression of cN-II, thus supporting the search for specific inhibitors of this enzyme as a therapeutic tool for the treatment of tumors.     

Role of Electrostatic Interactions in Calcitonin Prefibrillar Oligomer-Induced Amyloid Neurotoxicity and Protective Effect of Neuraminidase

Salmon calcitonin is a good model for studying amyloid behavior and neurotoxicity. Its slow aggregation rate allows the purification of low molecular weight prefibrillar oligomers, which are the most toxic species. It has been proposed that these species may cause amyloid pore formation in neuronal membranes through contact with negatively charged sialic acid residues of the ganglioside GM1. In particular, it has been proposed that an electrostatic interaction may be responsible for the initial contact between prefibrillar oligomers and GM1 contained in lipid rafts. Based on this evidence, the aim of our work was to investigate whether the neurotoxic action induced by calcitonin prefibrillar oligomers could be counteracted by treatment with neuraminidase, an enzyme that removes sialic acid residues from gangliosides. Therefore, we studied cell viability in HT22 cell lines and evaluated the effects on synaptic transmission and long-term potentiation by in vitro extracellular recordings in mouse hippocampal slices. Our results showed that treatment with neuraminidase alters the surface charges of lipid rafts, preventing interaction between the calcitonin prefibrillar oligomers and GM1, and suggesting that the enzyme, depending on the concentration used, may have a partial or total protective action in terms of cell survival and modulation of synaptic transmission.