Hyaluronic acid-graphene quantum dot nanocomposite: Potential target drug delivery and cancer cell imaging.

Nowadays, the use of nanoparticle-based drug delivery systems has received much more attention. In this regard, here, graphene quantum dots (GQD) were used as drug carriers as well as imaging agents for cancer cells. In order to optimize the dose of the drug and reduce its side effects for healthy cells, hyaluronic acid was decorated on the surface of GQD to target cancer cells. The morphology and size of the synthesized nanoparticles alone and conjugated with hyaluronic acid were investigated using scanning electron microscopy (SEM) and transmission electron microscopy (TEM); TEM images revealed a particles size of ∼5.67 and ∼8.69 nm, respectively. In the presence of 1-ethyl-3-[3(dimethylamino)propyl]carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS), hyaluronic acid was bounded to dopamine hydrochloride and was prepared to react with GQD. After synthesis of graphene quantum dot-hyaluronic acid nanocomposite, curcumin (CUR) as a drug model was loaded on the synthesized nanocarriers, and its loading percentage was measured. The results showed that 98.02% of the drug was loaded on the nanocarriers. Also, the conjugation of each agent on the nanocarrier was approved by photoluminescence spectroscopy, Fourier transform infrared spectroscopy (FTIR), and UV-visible absorption techniques, and the results showed that the reactions were performed correctly. The effect of GQD, graphene quantum dot-hyaluronic acid, CUR, graphene quantum dot-hyaluronic acid-CUR on the viability of HeLa and L929 cells was evaluated by the MTT test. The results showed that the synthesized nanocarrier is completely biocompatible, and the drug nanocarriers reduce HeLa cell viability significantly due to the mediation of hyaluronic acid-CD44 for drug cell uptake. Simultaneously with drug delivery, the other goal of these nanocarriers is to image cancer cells by emitting fluorescent light. Fluorescent microscopy showed that these nanocarriers were adsorbed on HeLa cells, unlike L929 cells.

Enhanced survival of Lacticaseibacillus rhamnosus in simulated gastrointestinal conditions using layer-by-layer encapsulation.

OBJECTIVE: The release behavior of Lacticaseibacillus rhamnosus from single bilayer microcapsules of alginate-chitosan (AC) and its double bilayer (ACAC) was investigated in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Methods Multilayer polyelectrolyte AC microcapsules were fabricated using the layer-by-layer (LbL) self-assembly technique through electrostatic interactions. Results AC and ACAC microcapsules kept their integrity and mechanical stability in simulated gastric conditions. Bacterial cells remained inside microcapsules in SGF and dissolution of microcapsules was observed in SIF. To improve the bacterial survivability, L. rhamnosus was co-encapsulated in a double bilayer of AC hydrogels with calcium carbonate as an antacid agent. Conclusions The LbL self-assembly technology provides stable and target release for ACAC microcapsules. Therefore, the double bilayer polyelectrolyte microcapsules have a remarkable potential for successful application in the targeted and controlled delivery of different probiotics and drugs.

Enhanced hyaluronic acid production in Streptococcus zooepidemicus by an optimized culture medium containing hyaluronidase inhibitor.

This study describes the hyaluronic acid (HA) production by S. zooepidemicus ATCC 43079, and the effect of the hyaluronidase enzyme on HA levels. The hyaluronidase production, glucose consumption, and lactate formation were recorded during fermentation. The HA production, and productivity at different amounts of glucose, yeast extract and pH were evaluated by response surface statistical approach in presence of 6-O-palmytoil-l-ascorbic acid as a chemical inhibitor for biocatalyst hyaluronidase. Under optimum conditions, HA production was increased two-fold from 190 ± 17 mg L-1 in basal medium to 384.6 ± 7.5 mg L-1 in the optimized medium containing enzyme inhibitor. Furthermore, the results indicated that the chemical inhibitor could suppress the biocatalyst activity and prevent the HA loss at the end of the exponential phase of S. zooepidemicus ATCC 43079.

Effects of bioactive peptides derived from feather keratin on plasma cholesterol level, lipid oxidation of meat, and performance of broiler chicks.

In this research, the effect of mixed feather bioactive peptides (MFBPs) added in water, on intestinal health, meat quality, and plasma cholesterol level of broiler chickens, was evaluated. A total of 80 day-old male broiler chicks (Ross 308) were randomly divided into two treatments with four replication pens. The dietary treatments were the drinking water with no additives (control) and drinking water containing 50 mg/L of MFBPs. Live weight and feed intake were measured at the end of starter (1-10 days), grower (11-24 days), and finisher (25-36 days) periods by calculating the average daily gain and feed conversion ratio. The results indicate that body weight gain was greater (P < 0.05) in birds that received MFBPs in the final period. At 24 days of age, the villus height and muscle layer thickness in different parts of the intestine were higher in birds that received bioactive peptides but epithelial thickness was lower than that in control birds (P < 0.05). In addition, the administration of MFBPs decreased (P < 0.01) serum total cholesterol, triglyceride, and low-density lipoprotein in broilers. Supplementation with MFBPs significantly reduced (P < 0.01) the malondialdehyde (MDA) amount in the thigh muscle. In conclusion, using the MFBPs in the diet of broilers could improve meat quality, cholesterol concentration in serum, and gut health.

Stability and cytotoxicity of DPPH inhibitory peptides derived from biodegradation of chicken feather.

The antioxidant activity and cell viability of feather hydrolysates obtained with the Bacillus licheniformis were evaluated using an in-vitro model. The results indicate that feathers-derived peptides under 3 kDa have antioxidant activity with IC50 values of 5.03 ± 0.215 mg/mL by using DPPH antioxidant assay. Although the antioxidant activity of the peptides under 3 kDa preserved after applying diverse heating (from 20 to 100 °C), they lost their activity under strongly acidic or alkaline conditions. Antioxidant activity of the mixed feather bioactive peptides (MFBPs) obtained with partial purification of peptides under 3 kDa was with IC50 amount of 0.169 mg/mL ± 0.004 using DPPH radical scavenging assay. Also, MFBPs within an amount range of from 0.0048 to 5.0 mg/mL, illustrated no cytotoxicity to gingival fibroblast blood cell lines. In light of our results, the obtained value-added peptides could be useful in different food products as a future functional ingredient with antioxidant potency.

Preparation, purification, and characterization of low-molecular-weight hyaluronic acid.

OBJECTIVE: The use and commercial value of hyaluronic acid (HA) as an important element in the pharmaceutical, biomedical, and cosmetics industry is because of its purity. Four recombinant strains of Corynebacterium glutamicum containing different genes were used to produce HA. RESULTS: The production parameters were measured and strain 183.2, with the highest amount of HA (2.15 mg/ml), was selected for further experiments. HA was precipitated by different ratios of ethanol-isopropanol at 4 °C and - 20 °C. Active charcoal (1%) was added to the solvent precipitation mixture at pH 5 and 10. Finally, to achieve more purity and separation, gel filtration chromatography was used. The best result was obtained using an ethanol-isopropanol ratio of 1:1 of at - 20 °C, followed by active charcoal treatment at the acidic pH, and three fractions of the chromatography with molecular weights of 27, 27-110, and < 27 KDa were more analyzed with electrophoresis and FTIR. CONCLUSIONS: The present study described a simple, economical, and reproducible method resulting in a high yield for low-MW HA from C. glutamicum.

The effect of lactobacillus cell size on its probiotic characteristics.

Screening for probiotic characteristics is usually associated with a series of assays and a large number of isolates to be tested, which can be sometimes costly and frustrating. For this reason, finding some indicators to predict the probiotic potential would be of great significance. In this study, 10 Lactobacillus strains including L. sakei, L. reuteri, L. delbrueckii subsp. lactis, L. delbrueckii subsp. delbrueckii, L. plantarum, L. acidophilus, L. rhamnosus, L. paracasei, L. salivarius, and L. gasseri were characterized by cell morphology and growth properties. The strains were then examined in terms of some probiotic characteristics including resistance to acid and bile conditions, ability to adhesion to intestinal epithelial cells, antioxidant activity, aggregation characteristics, antibacterial activity, hemolytic activity, and resistance to different antibiotics. Correlations between different quantitative features were analyzed using Pearson's coefficient (r). Results of this study provided first-time evidence for the effects of cell length on probiotic features. Based on statistical analysis, long Lactobacillus strains had often higher antioxidant and aggregation activities. Moreover, these long strains were usually more sensitive to acid and bile conditions and resulted in a lower CFU yield compared to short strains. By conducting morphological tests at the first step of screening, some strains would gain higher priority because of predicting a high performance in some of the desired characteristics. Therefore, the cost and time required for the subsequent tests would be significantly reduced.

Evaluation of microbial-induced calcite precipitation performance for soil surface improvement and toxicity assessment of the biostabilizer.

Microbial-induced calcite precipitation (MICP) is an environmentally friendly process that can be used to enhance soil surface stability against wind erosion. In this study, the performance of the MICP process on soil surface improvement was investigated using Staphylococcus warneri IR-103 bacteria. The biostabilizer, containing S. warneri suspension and a cementation solution consisting of 0.5 mM CaCl2 and 1.5 mM urea, was sprayed on fine-grain soil to induce a surface MICP reaction. Soil surface strength was measured using a penetrometer test, and wind tunnel tests were conducted to evaluate the soil surface's resistance to wind erosion. Scanning electron microscopy (SEM) analysis of the treated soils was conducted to visualize carbonate crystal formations within and on the soil particles. Additionally, X-ray diffraction (XRD) was used to confirm the presence and identify the crystal structures. The ecotoxicological assessment of the biostabilizer was carried out by performing phytotoxicity and oral/dermal/ocular in vivo acute toxicity experiments due to a few case reports of S. warneri's harmfulness and virulence of coagulase-negative staphylococci, highlighting the need for safety measures for workers and end-users. Mixing cementation solution with bacterial suspension in yeast-ammonium chloride medium increased soil strength and durability. The biostabilizer did not harm the seed germination of Agropyron desertorum, and the soil surface remained resistant to wind erosion. Rat oral/dermal acute toxicity tests revealed no adverse effects during the 14-day observation period. The LD50 (median lethal dose) cut-off value of the biostabilizer in oral and dermal administrations was 5000 and 1000 mg/kg body weight, respectively. Ocular administration of a 0.1 mL drop did not induce eye irritation in rabbits. In conclusion, the use of the biostabilizer for wind erosion control appears to be technically and environmentally feasible and justifiable.

Multi-Objective Optimization of Copper Bioleaching: Comparative Study of Pure and Co-Cultured Cultivation.

Background: Bioleaching is a practical method to recover metals from low-grade mineral sulfides. The most frequent bacteria involved in the bioleaching of metals from ores are Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans. Experimental design is a method through which the optimum activity condition will be obtained, avoiding numerous trials and errors. Objectives: This study aimed to optimize the bioleaching condition of two indigenous iron- and sulfur-oxidizing bacteria from the Meydouk mine, Iran, and evaluate their function in a semi-pilot operation in pure and mixed cultures. Material and Methods: After treatment with sulfuric acid, the bacterial DNA was extracted, and further 16S rRNA was sequenced to characterize the bacterial species. The cultivation condition of these bacteria was optimized using Design-expert (6.1.1 version) software. The copper recovery rate and the differentiation in the ORP rate in the percolation columns were also investigated. These strains were isolated from the Meydouk mine for the first time. Results: 16S rRNA analysis revealed that both bacteria belong to the Acidithiobacillus genus. The factors with the most significant impact on Acidithiobacillus ferrooxidans with their optimum level were temperature=35 °C, pH=2.5, and initial FeSO4 concentration=25 g.L-1. Also, initial sulfur concentration had the most significant impact on Acidithiobacillus thiooxidans with the optimum level of 35 g.L-1. Moreover, the mixed culture determined higher bioleaching efficiency compared with the case of employing the pure cultures. Conclusions: Utilizing a mixture of both bacteria, Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans elevated the Cu recovery rate due to the synergetic function of the strains. Also, introducing an initial dosage of sulfur and pre-acidification could elevate metal recovery efficiency.

Efficacy of hyaluronic acid, absorbable collagen sponge, and their combination in minimizing bisphosphonate-related osteonecrosis of the jaws (BRONJ) after dental extraction: a preliminary animal histomorphometric study.

INTRODUCTION: There is no study on the effectiveness of hyaluronic acid (HA) placement either with or without absorbable collagen sponge (ACS) in reducing or preventing bisphosphonate-related osteonecrosis of the jaws (BRONJ). This preliminary animal study examined the efficacy of this clinically important treatment. METHODS: For simulating BRONJ, zoledronic acid was administered to 40 rats for 5 weeks. Two weeks later, a right first molar was extracted from each rat. The rats were randomized into four groups of socket treatments: control (empty extraction socket) or with sockets filled with ACS, HA, or HA+ACS (n=4×10). After 2 weeks, 5 rats in each group were sacrificed and subjected to histopathologic and histomorphometric evaluation. Eight weeks post-surgically, the rest of rats were euthanized and histologically examined. The Kruskal-Wallis test was used to compare the four treatments at each time point (α=0.05). RESULTS: Six rats were lost overall. In the second week, vascularization was higher in ACS group (P<0.05); osteoclast activity was not different between groups (P>0.05); empty lacunae were the most and fewest in control and HA+ACS groups, respectively (P<0.05); eosinophil infiltration was maximum in HA group (P<0.05); lymphocyte counts were maximum and minimum in the HA+ACS and ACS groups, respectively (P<0.05); the highest and lowest neutrophil counts were seen in ACS and control groups, respectively (P<0.05); and the extent of live bone did not differ between groups (P>0.05). In the eighth week, vascularization was not different in groups (P>0.05); the highest and lowest osteoclast activities were seen in the control and HA+ACS groups, respectively (P<0.05); empty lacunae were the most and fewest in control and HA+ACS, respectively (P<0.05); maximum and minimum numbers of eosinophils were in control and HA+ACS groups, respectively (P<0.05); HA and control groups exhibited the highest and lowest lymphocyte counts, respectively (P<0.05); the lowest and highest neutrophil counts were observed in HA+ACs and control groups, respectively (P<0.05); and the highest and lowest extents of the live bone were observed in HA+ACS and control groups, respectively (P<0.05). CONCLUSIONS: Within the limitations of this preliminary animal study, HA and especially HA+ACS seem a proper method for preventing or treating BRONJ.

Production of a recombinant alkane hydroxylase (AlkB2) from Alcanivorax borkumensis.

Alcanivorax borkumensis is an oil-degrading marine bacterium. Its genome contains genes coding for three cytochrome P450s and two integral membrane alkane hydroxylases (AlkB1 & AlkB2), all assumed to perform hydroxylation of different linear or branched alkanes. Although, the sequence of alkB2 has been determined, the molecular characterization and the substrate specificity of AlkB2 require more precise investigation. In this study, AlkB2 from A. borkumensis SK2 was expressed in Escherichia coli to examine the functionality of AlkB2 as a hydroxylating enzyme. Furthermore, the activity of the enzyme in the presence of the accessory proteins, rubredoxin (RubA) and rubredoxin reductase (RubB), produced in E. coli BL21(DE3)plysS cells, was determined. Recombinant AlkB2 is produced in an active form and rubredoxin is the intermediate electron donor to AlkB2 and can replace AlkG function, when NADH is the prime electron donor.

Design, construction, and expression of recombinant human interferon beta gene in CHO-s cell line using EBV-based expression system.

BACKGROUND AND PURPOSE: Codon optimization has been considered as a powerful strategy to increase the expression level of protein therapeutics in mammalian cells. As an empirical approach to study the effects of the codon usage and GC content on heterologous gene expression in suspension adapted Chinese hamster ovary (CHO-s) cells, we redesigned the recombinant human interferon beta (rhIFN- ß) gene based on the codon preference of the CHO cell in a way to increase the GC content in the third position of each codon. EXPERIMENTAL APPROACH: The nucleotide sequence of the codon-optimized rhIFN-ß was synthesized in parallel with the wild-type and expressed transiently in CHO-s cells using Epstein-Bar virus (EBV)-based expression system. The protein expression of the rhIFN-ß by codon-optimized and wild-type genes were quantified using ELISA test. FINDINGS / RESULTS: The results indicated a 2.8-fold increase in the expression level of the biologically active form of the rhIFN-ß by codon-optimized sequence. CONCLUSION AND IMPLICATIONS: These results shed light on the capability of codon optimization to create a stable CHO cell for scaling up the production of recombinant therapeutics such as rhIFN-ß.

Regenerative medicine as a novel strategy for AMD treatment: a review.

Age-related macular degeneration (AMD) is known as a major cause of irreversible blindness in elderly adults. The segment of the retina responsible for central vision damages in the disease process. Degeneration of retinal pigmented epithelium (RPE) cells, photoreceptors, and choriocapillaris associated with aging participate for visual loss. In 2010, AMD involved 6.6% of all blindness cases around the world. Some of the researches have evaluated the replacing of damaged RPE in AMD patients by using the cells from various sources. Today, the advancement of RPE differentiation or generation from stem cells has been gained, and currently, clinical trials are testing the efficiency and safety of replacing degenerated RPE with healthy RPE. However, the therapeutic success of RPE transplantation may be restricted unless the transplanted cells can be adhered, distributed and survive for long-term in the transplanted site without any infections. In recent years a variety of scaffold types were used as a carrier for RPE transplantation and AMD treatment. In this review, we have discussed types of scaffolds; natural or synthetic, solid or hydrogel and their results in RPE replacement. Eventually, our aim is highlighting the novel and best scaffold carriers that may have potentially promoting the efficacy of RPE transplantation.

Hyaluronic acid production enhancement via genetically modification and culture medium optimization in Lactobacillus acidophilus.

Hyaluronic acid (HA) is a natural polymer with various molecular weights that specify multiple biological roles. Traditionally, HA is obtained from animal waste and conventional pathogenic streptococci. However, there are challenges in these processes such as the presence of exotoxins, hyaluronidase, and viral contamination. In order to reduce these problems, this study was conducted to produce HA using recombinant bacterium that is generally recognized as safe (GRAS), and thereafter increase production through experimental design. At first, some lactic acid bacteria were screened and evaluated for HA production. Accordingly, among the selected bacteria, Lactobacillus acidophilus PTCC1643 produced about 0.25 g HA/L in the 48th hour of cultivation, and was thus selected as an alternative host for heterologous HA production. An expression vector containing HA synthase genes was transformed into L. acidophilus by electroporation. Consequently, HA production increased to 0.4 g/L. Eventually, response surface method (RSM) was used, which increased HA production to 1.7 g/L. This is approximately 7-fold higher than that produced at first. The resulting HA was characterized by FTIR spectroscopy and its molecular weight was estimated using agarose gel electrophoresis. In conclusion, L. acidophilus could be a safe, effective, and novel HA producer with industrial potential and commercial prospects.

A proposed feeding strategy for the overproduction of recombinant proteins in Escherichia coli.

Different feeding strategies for the production of human interferon-gamma using an isopropyl beta-D-thiogalactoside-inducible expression system in recombinant Escherichia coli BL21(DE3) (plasmid pET3a-ifngamma) were studied. Four fed-batch modes were designed to compare the effect of mu (specific growth rate) on recombinant-protein production, substrate consumption, by-product formation and plasmid stability during pre- and post-chemical induction in high-cell-density cultures of E. coli. It was found that Y(p/s), the product/substrate yield of interferon-gamma was significantly affected by mu throughout the process, but product/biomass yield (Y(p/x)) was influenced by mu at the pre-induction stage. By applying an efficient feeding strategy, in which the mu was maintained at the maximum attainable level, recombinant protein was accumulated up to a level of 60% of the total cell protein and its productivity was increased significantly. In this case, the overall productivities of biomass and recombinant protein were 6.36 g l(-1) h(-1) and 2.1 g l(-1) h(-1) respectively, in comparison with 1.91 g l(-1) h(-1) and 0.16 g l(-1) h(-1) during exponential feeding, in which the specific growth rate was kept constant throughout the entire process.

Equilibrium Isotherm, Kinetic Modeling, Optimization, and Characterization Studies of Cadmium Adsorption by Surface-Engineered Escherichia coli

Background: Amongst the methods that remove heavy metals from environment, biosorption approaches have received increased attention because of their environmentally friendly and cost-effective feature, as well as their superior performances. Methods: In the present study, we investigated the ability of a surface-engineered Escherichia coli, carrying the cyanobacterial metallothionein on the cell surface, in the removal of Ca (II) from solution under different experimental conditions. The biosorption process was optimized using central composite design. In parallel, the kinetics of metal biosorption was studied, and the rate constants of different kinetic models were calculated. Results: Cadmium biosorption is followed by the second-order kinetics. Freundlich and Langmuir equations were used to analyze sorption data; characteristic parameters were determined for each adsorption isotherm. The biosorption process was optimized using the central composite design. The optimal cadmium sorption capacity (284.69 nmol/mg biomass) was obtained at 40°C (pH 8) and a biomass dosage of 10 mg. The influence of two elutants, EDTA and CaCl2, was also assessed on metal recovery. Approximately, 68.58% and 56.54% of the adsorbed cadmium were removed by EDTA and CaCl2 during desorption, respectively. The Fourier transform infrared spectrophotometer (FTIR) analysis indicated that carboxyl, amino, phosphoryl, thiol, and hydroxyl are the main chemical groups involved in the cadmium bioadsorption process. Conclusion: Results from this study implied that chemical adsorption on the heterogeneous surface of E. coli E and optimization of adsorption parameters provides a highly efficient bioadsorbent.

Controlled surface morphology and hydrophilicity of polycaprolactone toward human retinal pigment epithelium cells.

Applying scaffolds as a bed to enhance cell proliferation and even differentiation is one of the treatment of retina diseases such as age-related macular degeneration (AMD) which deteriorating photoreceptors and finally happening blindness. In this study, aligned polycaprolactone (PCL) nanofibers were electrospun and at different conditions and their characteristics were measured by scanning electron microscope (SEM) and contact angle. Response surface methodology (RSM) was used to optimize the diameter of fabricated nanofibers. Two factors as solution concentration and voltage value were considered as independent variables and their effects on nanofibers' diameters were evaluated by central composite design and the optimum conditions were obtained as 0.12g/mL and 20kV, respectively. In order to decrease the hydrophobicity of PCL, the surface of the fabricated scaffolds was modified by alkaline hydrolysis method. Contact time of the scaffolds and alkaline solution and concentration of alkaline solution were optimized using Box Behnken design and (120min and 5M were the optimal, respectively). Contact angle measurement showed the high hydrophilicity of treated scaffolds (with contact angle 7.48°). Plasma surface treatment was applied to compare the effect of using two kinds of surface modification methods simultaneously on hydrolyzed scaffolds. The RPE cells grown on scaffolds were examined by immunocytochemistry (ICC), MTT and continuous inspection of cellular morphology. Interestingly, Human RPE cells revealed their characteristic morphology on hydrolyzed scaffold well. As a result, we introduced a culture substrate with low diameter (185.8nm), high porosity (82%) and suitable hydrophilicity (with contact angle 7.48 degree) which can be promising for hRPE cell transplantation.

Enzymatic production of biodiesel from microalgal oil using ethyl acetate as an acyl acceptor.

Microalgae have become an important source of biomass for biodiesel production. In enzymatic transesterification reaction, the enzyme activity is decreased in presence of alcohols. The use of different acyl acceptors such as methyl/ethyl acetate is suggested as an alternative and effective way to overcome this problem. In this study, ethyl acetate was used for the first time in the enzymatic production of biodiesel by using microalga, Chlorella vulgaris, as a triglyceride source. Enzymatic conversion of such fatty acids to biodiesel was catalyzed by Novozym 435 as an efficient immobilized lipase which is extensively used in biodiesel production. The best conversion yield of 66.71% was obtained at the ethyl acetate to oil molar ratio of 13:1 and Novozym 435 concentration of 40%, based on the amount of oil, and a time period of 72 h at 40℃. The results showed that ethyl acetate have no adverse effect on lipase activity and the biodiesel amount was not decreased even after seven transesterification cycles, so ethyl acetate has a great potential to be substituted for short-chain alcohols in transesterification reaction.

Fabrication and characterization of core-shell magnetic chitosan nanoparticles as a novel carrier for immobilization of Burkholderia cepacia lipase.

In this study, the chitosan magnetic core-shell nanoparticles (CMNPs) was synthesized and then used as a support for immobilization of lipase. The characteristics of CMNPs, including morphology, topography and spectra type before and after immobilization were determined. The scanning electron micrographs of the CMNPs showed that they were approximately uniform spheres and the distribution chart indicated that the particles have the mean diameter of 100 nm. Kinetic parameters of Km and Vm were calculated as 1.07 mM and 29.43 U/mg for free B. cepacia lipase and 1.29 mM and 25.82 U/mg for immobilized lipase on CMNPs, respectively. The activity of immobilized lipase was 32 U/mg under optimum temperature and pH. CMNP's were used in trasesterification reaction in order to evaluate the activity of the immobilized enzyme compared to the free enzyme. Immobilization of lipase on CMNPs improved stability and total relative activity of the enzyme. It could be concluded that CMNPs be considered as a suitable carrier for enzyme immobilization.

Rational design of hyper-glycosylated interferon beta analogs: a computational strategy for glycoengineering.

Glycoengineering has been successfully used to improve the physicochemical and pharmaceutical properties of therapeutics. One aspect of glycoengineering is to introduce new N-linked glycosylation consensus sequences (Asn, X, Thr/Ser) into desirable positions in the peptide backbone by mutational insertion to generate proteins with increased sialic acid content. In the current work, human interferon beta (huIFN-ß) was used as a model to identify the potential positions for the addition of new N-glycosylation sites. A computational strategy was employed to predict the structural distortions and functional alterations that might be caused by the change in amino acid sequence. Accordingly, three-dimensional (3D) structures of the designed huIFN-ß analogs were generated by comparative modeling. Molecular dynamics (MD) simulation was carried out to assess the molecular stability and flexibility profile of the structures. Subsequently, for the purpose of glycoengineering huIFN-ß, analogs with 3D structures more similar to the wild-type huIFN-ß and exposed Asn residue in the new N-glycosylation site were identified. These modeling procedures indicated that the addition of the new N-glycosylation site in the loop regions had lower constraining effects on the tertiary structure of the protein. This computational strategy can be applied to avoid alterations in the 3D structure of proteins caused by changes in the amino acid sequences, when designing novel hyper-glycosylated therapeutics. This in turn reduces labor-intensive experimental analyses of each analog.