Enhanced solubility of piperine using hydrophilic carrier-based potent solid dispersion systems.

CONTEXT: Piperine alkaloid, an important constituent of black pepper, exhibits numerous therapeutic properties, whereas its usage as a drug is limited due to its poor solubility in aqueous medium, which leads to poor bioavailability. OBJECTIVE: Herein, a new method has been developed to improve the solubility of this drug based on the development of solid dispersions with improved dissolution rate using hydrophilic carriers such as sorbitol (Sor), polyethylene glycol (PEG) and polyvinyl pyrrolidone K30 (PVP) by solvent method. Physical mixtures of piperine and carriers were also prepared for comparison. METHODS: The physicochemical properties of the prepared solid dispersions were examined using SEM, TEM, DSC, XRD and FT-IR. In vitro dissolution profile of the solid dispersions was recorded and compared with that of the pure piperine and physical mixtures. The effect of these carriers on the aqueous solubility of piperine has been investigated. RESULTS: The solid dispersions of piperine with Sor, PEG and PVP exhibited superior performance for the dissolution of piperine with a drug release of 70%, 76% and 89%, respectively after 2 h compared to physical mixtures and pure piperine, which could be due to its transformation from crystalline to amorphous form as well as the attachment of hydrophilic carriers to the surface of poorly water-soluble piperine. CONCLUSION: Results suggest that the piperine solid dispersions prepared with improved in vitro release exhibit potential advantage in delivering poorly water-soluble piperine as an oral supplement.

Engineering a horseradish peroxidase C stable to radical attacks by mutating multiple radical coupling sites.

Peroxidases have great potential as industrial biocatalysts. In particular, the oxidative polymerization of phenolic compounds catalyzed by peroxidases has been extensively examined because of the advantage of this method over other conventional chemical methods. However, the industrial application of peroxidases is often limited because of their rapid inactivation by phenoxyl radicals during oxidative polymerization. In this work, we report a novel protein engineering approach to improve the radical stability of horseradish peroxidase isozyme C (HRPC). Phenylalanine residues that are vulnerable to modification by the phenoxyl radicals were identified using mass spectrometry analysis. UV-Vis and CD spectra showed that radical coupling did not change the secondary structure or the active site of HRPC. Four phenylalanine (Phe) residues (F68, F142, F143, and F179) were each mutated to alanine residues to generate single mutants to examine the role of these sites in radical coupling. Despite marginal improvement of radical stability, each single mutant still exhibited rapid radical inactivation. To further reduce inactivation by radical coupling, the four substitution mutations were combined in F68A/F142A/F143A/F179A. This mutant demonstrated dramatic enhancement of radical stability by retaining 41% of its initial activity compared to the wild-type, which was completely inactivated. Structure and sequence alignment revealed that radical-vulnerable Phe residues of HPRC are conserved in homologous peroxidases, which showed the same rapid inactivation tendency as HRPC. Based on our site-directed mutagenesis and biochemical characterization, we have shown that engineering radical-vulnerable residues to eliminate multiple radical coupling can be a good strategy to improve the stability of peroxidases against radical attack.

Overview on the biotechnological production of L-DOPA.

L-DOPA (3,4-dihydroxyphenyl-L-alanine) has been widely used as a drug for Parkinson's disease caused by deficiency of the neurotransmitter dopamine. Since Monsanto developed the commercial process for L-DOPA synthesis for the first time, most of currently supplied L-DOPA has been produced by the asymmetric method, especially asymmetric hydrogenation. However, the asymmetric synthesis shows critical limitations such as a poor conversion rate and a low enantioselectivity. Accordingly, alternative biotechnological approaches have been researched for overcoming the shortcomings: microbial fermentation using microorganisms with tyrosinase, tyrosine phenol-lyase, or p-hydroxyphenylacetate 3-hydroxylase activity and enzymatic conversion by immobilized tyrosinase. Actually, Ajinomoto Co. Ltd commercialized Erwinia herbicola fermentation to produce L-DOPA from catechol. In addition, the electroenzymatic conversion system was recently introduced as a newly emerging scheme. In this review, we aim to not only overview the biotechnological L-DOPA production methods, but also to briefly compare and analyze their advantages and drawbacks. Furthermore, we suggest the future potential of biotechnological L-DOPA production as an industrial process.

Microgravity Separation of Alginate Empty Capsules from Encapsulated Pancreatic Islets Using a Microfluidic System.

Although microencapsulated pancreatic islets have merits, such as ease of transplantation, viability and functionality improvement, and immune protection in vivo, the co-production of alginate empty capsules during the encapsulation of islets with alginate makes them unusable for biomedical application. In previous research, the removal of empty alginate capsules with high yield was achieved using density-gradient centrifugation. Here, we report advanced microgravity-based separation techniques in a microfluidic format for alginate empty capsules. The optimal separation conditions were mathematically evaluated using Stokes' law and the separation of the encapsulation product was accomplished. A microfluidic chip was designed with two inlets and two outlets at different elevations to mimic the vertical percoll gradient in density-gradient centrifugation. The separation of alginate empty capsules using microgravitational force resulted in effective separation of encapsulated islets from alginate empty capsules with more than 70% efficiency. Moreover, no loss of encapsulated islets was expected because the process is a one-pot separation, unlike the previous method. This type of microgravitational particle separation could be used both for the fractionization of heterogeneous encapsulated cells and to remove empty capsules.

Enhancing the activity of Bacillus circulans xylanase by modulating the flexibility of the hinge region.

Enzymes undergo multiple conformational changes in solution, and these dynamics are considered to play a critical role in enzyme activity. Hinge-bending motions, resulting from reciprocal movements of dynamical quasi-rigid bodies, are thought to be related to turnover rate and are affected by the physical properties of the hinge regions. In this study, hinge identification and flexibility modification of the regions by mutagenesis were conducted to explore the relationship between hinge flexibility and catalytic activity. Bacillus circulans xylanase was selected for the identification and mutation of the hinge regions. As a result, turnover rate (V(max)) was improved approximately twofold in mutants that have more rigid hinge structure, despite the decrease in K(m) and V(max)/K(m). This result indicates that the rigidly mutated hinge has positive effects on B. circulans xylanase activity.

High-rate partial nitritation using porous poly(vinyl alcohol) sponge.

Poly(vinyl alcohol) (PVA) has been utilized as a support material for the immobilization of nitrifying bacteria without the comprehensive survey of partial nitritation. In the present study, the activities of nitrifiers and the maximum nitrogen conversion rate of partial nitritation with PVA sponge-cubes were specified according to different conditions. The selective enrichment of ammonia-oxidizing bacteria (AOB) on PVA sponge-cubes was achieved by the competition between AOB and nitrite-oxidizing bacteria for dissolved oxygen. The efficiency of ammonia oxidation was proportional to the concentration of HCO3 (-) with the molar ratio of HCO3 (-)-C/NH4 (+)-N = 1.91 and a half of the ratio was applied to the further experiments to ensure stable partial nitritation. The maximum nitrogen conversion rate of partial nitritation was dependent on the volume, not the size of sponge-cubes. The partial nitritation showed the superior rate performance of 3.09 kg N/m(3) day with the packing ratio of 32 % of 5 × 5 × 5 mm(3) PVA sponge-cubes.

Statistical Analysis of the Role of Cavity Flexibility in Thermostability of Proteins.

Conventional statistical investigations have primarily focused on the comparison of the simple one-dimensional characteristics of protein cavities, such as number, surface area, and volume. These studies have failed to discern the crucial distinctions in cavity properties between thermophilic and mesophilic proteins that contribute to protein thermostability. In this study, the significance of cavity properties, i.e., flexibility and location, in protein thermostability was investigated by comparing structural differences between homologous thermophilic and mesophilic proteins. Three dimensions of protein structure were categorized into three regions (core, boundary, and surface) and a comparative analysis of cavity properties using this structural index was conducted. The statistical analysis revealed that cavity flexibility is closely related to protein thermostability. The core cavities of thermophilic proteins were less flexible than those of mesophilic proteins (averaged B' factor values, -0.6484 and -0.5111), which might be less deleterious to protein thermostability. Thermophilic proteins exhibited fewer cavities in the boundary and surface regions. Notably, cavities in mesophilic proteins, across all regions, exhibited greater flexibility than those in thermophilic proteins (>95% probability). The increased flexibility of cavities in the boundary and surface regions of mesophilic proteins, as opposed to thermophilic proteins, may compromise stability. Recent protein engineering investigations involving mesophilic xylanase and protease showed results consistent with the findings of this study, suggesting that the manipulation of flexible cavities in the surface region can enhance thermostability. Consequently, our findings suggest that a rational or computational approach to the design of flexible cavities in surface or boundary regions could serve as an effective strategy to enhance the thermostability of mesophilic proteins.

Attachment of alginate microcapsules onto plasma-treated PDMS sheet for retrieval after transplantation.

Although transplantation of microencapsulated islets has been proposed as a therapy for the treatment of diabetes mellitus, limited retrievability of the cells has impeded its medical usage. To achieve retrieval of microencapsulated islets, capsules were attached to polydimethylsiloxane (PDMS) with a biocompatible adhesive. Because the hydrophobic nature of the PDMS surface prevents attachment, surface modification is essential. Alginate microcapsules were attached to modified PDMS sheets, and the mechanical stability of the resulting constructs was determined. Acrylic acid (AA) and acrylamide (AM) mixtures were grafted on the surfaces of PDMS sheets using a two-step oxygen plasma treatment (TSPT). TSPT-PDMS was characterized according to water contact angle and zeta-potential measurements. The contact angle was altered by changing the ratio of AM to AA to generate hydrophilic surface. Evaluation of the surface charge at pH 2, 7, and 12 confirmed the presence of polar groups on the modified surface. Microcapsules were attached to TSPT-PDMS using Histoacryl® and shown to be in a monolayered and half-exposed state. The shear stress resistance of alginate capsules attached to the PDMS sheet indicates the possibility of transplantation of encapsulated cells without scattering in vivo. This method is applicable to retrieve microencapsulated porcine islets when required.

In vitro evolution of glutathione S-transferase using a plasmid display system based on the GAL4 DNA-binding domain.

Enzyme characteristics, such as thermal stability and catalytic activity, can be improved in a targeted manner. However, the screening of target mutants is time-consuming and requires highly experimental downstream efforts. Here, we describe a simple strategy based on plasmid display and limited proteolysis for the rapid and easy screening of highly stable and active mutants from a library. When glutathione S-transferase was used as a model enzyme, the resulting mutants obtained in the first round of screening were approximately two- to sevenfold more thermostable than the wild-type enzyme at 50 °C, with similar enzyme activity. This methodology is therefore powerful for the in vitro enrichment and screening of thermostable and active mutants. It can reduce downstream experimental effort and can create a high-quality library using relatively simple steps.

Separation of empty microcapsules after microencapsulation of porcine neonatal islets.

Pancreatic islet transplantation is used to treat diabetes mellitus that has minimal complications and avoids hypoglycemic shock. Conformal microencapsulation of pancreatic islets improves their function by blocking immunogenic molecules while protecting fragile islets. However, production of empty alginate capsules during microencapsulation causes enlargement of the transplantation volume of the encapsulated islets and interferes with efficient transfer of nutrients and insulin. In this study, empty alginate capsules were separated after microencapsulation of neonatal porcine islet-like cell clusters (NPCC) using density-gradient centrifugation. Densities of NPCC and alginate capsules were determined using Percoll. Encapsulation products following alginate removal were 97 % of products, with less than 10 % of the capsules remaining empty. The viability of this process compared with manually-selected encapsulated islets indicates the separation process does not harm islets.

Thermostabilization of Bacillus subtilis lipase A by minimizing the structural deformation caused by packing enhancement.

Enzyme thermostabilization is a critical research topic due to potential industrial benefits. Among the various reasons to increase enzyme thermostability, enhancement of residual packing at the core of the enzyme structure has been commonly accepted as a successful strategy. However, structural changes that occur with residual packing enhancement may decrease enzyme activity. In this study, a strategy to minimize structural deformation by calculating the overlapping packing volume of a single-point mutation followed by applying a double-point mutation was suggested. Four double mutants, A38V_K23A, A75V_T83A, G80A_N106A, and G172A_V100A, were selected for the in vitro experiment; three of the four showed enhancements in both thermostability and catalytic activity. In particular, G80A_N106A showed 2.78 times higher catalytic activity compared with wild type.

Development of thermostable Candida antarctica lipase B through novel in silico design of disulfide bridge.

Lipase B from Candida antarctica (CalB) is a versatile biocatalyst for various bioconversions. In this study, the thermostability of CalB was improved through the introduction of a new disulfide bridge. Analysis of the B-factors of residue pairs in CalB wild type (CalB-WT) followed by simple flexibility analysis of residues in CalB-WT and its designated mutants using FIRST server were newly proposed to enhance the selective power of two computational tools (MODIP and DbD v1.20) to predict the possible disulfide bonds in proteins for the enhancement of thermostability. Five residue pairs (A162-K308, N169-F304, Q156(-) L163, S50-A273, and S239C-D252C) were chosen and the respective amino acid residues were mutated to cysteine. In the results, CalB A162C-K308C showed greatly improved thermostability while maintaining its catalytic efficiency compared to that of CalB-WT. Remarkably, the temperature at which 50% of its activity remained after 60-min incubation (T6°50) of CalB A162C_K308C was increased by 8.5°C compared to that of CalB-WT (55 and 46.5°C, respectively). Additionally, the half-life at 50°C of CalB A162C-K308C was 4.5-fold higher than that of CalB-WT (220 and 49 min, respectively). The improvement of thermostability of CalB A162C-K308C was elucidated at the molecular level by molecular dynamics (MD) simulation.

A hydrophilic and hydrophobic organic solvent mixture enhances enzyme stability in organic media.

Binary mixtures of hydrophilic and hydrophobic solvents were assessed for their ability to balance enzyme activity with the conservation of enzyme stability in organic media. Acetone, dioxane and dodecane were chosen as model organic solvents, and subtilisin Carlsberg and horseradish peroxidase (HRP) were chosen as model enzymes. Residual enzyme activities were measured to monitor enzyme stability, and the fluorescence intensity of HRP was monitored to investigate structural changes due to the presence of an organic solvent. Enzyme stability increased with the increasing hydrophobicity of the solvent mixture used, and a solvent mixture with a high log P value (~ >4) was capable of conserving enzyme stability. Enzyme stability in organic media can be conserved therefore with a mixture of hydrophilic and hydrophobic solvents: this approach might be used as a general and practical strategy for optimizing enzyme activity and stability for industrial applications.

Continuous biodiesel production using in situ glycerol separation by membrane bioreactor system.

Biodiesel is one of the most promising renewable fuel sources. Candida antarctica lipase B (CalB) has been used for biodiesel production because of its high activity and stability. However, CalB can only be utilized in industrial biodiesel production if the enzyme deactivation by methanol and the negative effects of glycerol can be mitigated. Methanol inhibition can be avoided by utilizing a stepwise addition of methanol, but there is no suitable method to reduce the glycerol effect. This study aims to use a membrane bioreactor system to remove glycerol during biodiesel production. In addition, methanol inhibition can be reduced by continuously feeding methanol through the membrane system. This continuous membrane bioreactor system can be used for efficient biodiesel production.

Enzymatic analysis of the effect of naturally occurring Leu138Pro mutation identified in SHV ß-lactamase on hydrolysis of penicillin and ampicillin.

BACKGROUND: The aim of this study was to analyze the significance of leucine to proline substitution at position 138(Leu138Pro) on the hydrolysis of penicillin and ampicillin that we identified in the blaSHV gene of clinical Escherichia coli swine isolate. RESULTS: Kinetic analysis of the mutant proteins showed that K(m) value of the purified L138P mutant was comparatively higher than SHV-1, SHV-33 and SHV-33(L138P) enzyme for penicillin and ampicillin. Docking simulation of the SHV-1 and SHV-(L138P) enzymes also confirmed that ß-lactamases preferred penicillin to ampicillin and the SHV-1 had a higher binding affinity for antibiotics compared to the SHV-(L138P) and other mutants. CONCLUSIONS: Our result demonstrated that L138P has a reduced role in penicillin and ampicillin hydrolyzing properties of SHV ß-lactamases. These naturally occurring mutations rendering reduced function of the existing protein could trigger the emergence or acquisition of more effective alternative mechanisms for ß-lactam hydrolysis.

Partial nitrification using an electrolytic aerating bioreactor with ammonia-oxidizing bacteria-dominant activated sludge.

An electrolytic aerating bioreactor was used to partially nitrify ammonia from wastewater. Activated sludge was cultured for 8 months to increase the population of ammonia-oxidizing bacteria (AOB) and then used in the bioreactor. The maximum ammonia removal rate was 0.64 mM NH(3)/l h in a 50 ml reactor using 5.4 g mixed liquor suspended solids per litre of AOB-dominant activated sludge.

Improving the organic solvent resistance of lipase a from Bacillus subtilis in water-ethanol solvent through rational surface engineering.

Given that lipase is an enzyme applicable in various industrial fields and water-miscible organic solvents are important reaction media for developing industrial-scale biocatalysis, a structure-based strategy was explored to stabilize lipase A from Bacillus subtilis in a water-ethanol cosolvent. Site-directed mutagenesis of ethanol-interacting sites resulted in 4 mutants, i.e., Ser16Gly, Ala38Gly, Ala38Thr, and Leu108Asn, which were stable in 50% ethanol and had up to 1.8-fold higher stability than the wild-type. In addition, Leu108Asn was more thermostable at 45 °C than the wild type. The results discussed in this study not only provide insights into strategies for enzyme engineering to improve organic solvent resistance but also suggest perspectives on pioneering routes for constructing enzyme-based biorefineries to produce value-added fuels and chemicals.

Improving the catalytic performance of xylanase from Bacillus circulans through structure-based rational design.

Endo-1,4-ß-xylanase is one of the most important enzymes employed in biorefineries for obtaining fermentable sugars from hemicellulosic components. Herein, we aimed to improve the catalytic performance of Bacillus circulans xylanase (Bcx) using a structure-guided rational design. A systematic analysis of flexible motions revealed that the R49 component of Bcx (i) constrains the global conformational changes essential for substrate binding and (ii) is involved in modulating flexible motion. Site-saturated mutagenesis of the R49 residue led to the engineering of the active mutants with the trade-off between flexibility and rigidity. The most active mutant R49N improved the catalytic performance, including its catalytic efficiency (7.51-fold), conformational stability (0.7 °C improvement), and production of xylose oligomers (2.18-fold higher xylobiose and 1.72-fold higher xylotriose). The results discussed herein can be applied to enhance the catalytic performance of industrially important enzymes by controlling flexibility.

Biological nitrate removal in industrial wastewater treatment: which electron donor we can choose.

Biological denitrification was reviewed regarding its potential application to treating nitrate in industrial wastewater. Although heterotrophic denitrification is an efficient and well-developed process, some carbon content in wastewater is essential to maintain bacterial activity. Because of the high operating cost of heterotrophic denitrification caused by the required addition of a carbon source and potential "carbon breakthrough", the study of autotrophic denitrification has attracted the interest of numerous researchers. Many advances in autotrophic processes have been made in the application of novel concepts and reaction schemes. While the main advantage of autotrophic bacteria rests on the reduction of operating costs by the replacement of an external carbon source with a cheaper electron donor, further decrease in cost requires additional refinement of these processes, including further improvement of reactor structure and optimization of reaction conditions. In the long term, new concepts are required for a compact wastewater treatment process. This review addresses the state of the art of each electron donor candidate for its potential application to the treatment of industrial wastewater containing nitrate.

Strain development of Streptomyces sp. for tacrolimus production using sequential adaptation.

An immunosuppressant tacrolimus-producing strain of Streptomyces sp. TST8 was isolated and developed by the TS corporation in Korea using the sequential adaptation of media containing tacrolimus (600-1600 mg/l). The aim of the tacrolimus sequential adaptation protocol was to select those cells with tacrolimus resistance and to reduce product inhibition of the tacrolimus-producing strain. The developed strains produced more tacrolimus than the original strain. In particular, the TST10 strain adapted in the medium containing 900 mg/l of tacrolimus produced 972 mg/l of tacrolimus in the final titer after 7 days of cultivation in a 5-l jar fermenter. This is the largest final titer of tacrolimus produced by a specific strain to date. Because the sequential adaptation protocol is limited by the solubility of tacrolimus in water, the final tacrolimus titer of TST11 adapted in the medium containing 1600 mg/l of tacrolimus was lower than that of TST10. The developed strains and the development method using sequential adaptation can facilitate the efficient and economical production of tacrolimus.