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 mono-layered 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.

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The three-dimensional (3-D) scaffold serves as a structural substrate and as a niche for cell proliferation to ensure tissue regeneration. Ideal scaffolds should have porous structures with high pore interconnectivity to allow cell adherence, differentiation and proliferation besides suitable mechanical strength and biodegradability without inflicting any immune response. Cross-linker is one of major factors that affect to mechanical and biological properties of scaffolds. In this study, different chemical cross-linker effects on scaffold architecture were examined. Porous 3D scaffolds based on carrageenan and alginate (CA) were successfully fabricated by freeze-drying technique and using various cross-linkers like glutaraldehyde (GA), genipin, ethyl (dimethylamino propyl) carbodiimide/ N-Hydroxysuccinimide (EDC/NHS). The chemical cross-linker effects on CA scaffold was characterized by scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and thermogravimetry (TG). Human fibroblast cell line (L929) was seeded into the fabricated scaffold and the cell proliferation was assessed by MTT and live/dead assay. Overall results suggested the potential cross-linkers for ideal CA biomaterial could be EDC/NHS among other agents tested as the scaffold CA_EN was found to be porous, interconnected, physically and mechanically stable. Comparing to matrices with other cross-linkers higher cell attachment, better cellular response, higher metabolic activity could be observed in scaffold synthesized using EDC/NHS as cross-linker.

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The present study is aimed at developing an economical medium for the production of α-amylase from Bacillus subtilis S8-18, a marine sediment isolate from Palk Bay, with various agricultural by-products which are cheap and rich in starch. These products included wheat bran, wheat husk, rice bran, rice husk and potato peel and used to replace soluble starch present in the LB broth (synthetic medium). The rice husk was found to be the best to influence enzyme production significantly (61186 IU mL^-1) when compared to the yield of 30026 IU mL^-1 obtained by commercial starch. Hence, LB broth containing rice husk was termed as economical medium. Besides, the effect of various nutritional and physiological factors on enzyme production was also investigated. Furthermore, the desizing efficiency of α-amylases produced by synthetic and economical medium was evaluated through various assays like reducing sugar estimation, weight loss assay, drop absorbency assay, SEM and FTIR analyses. In addition, a commercial α-amylase from B. subtilis was also used in desizing analyses for comparative purpose. It revealed that the α-amylase from economical medium was highly effective in desizing the cotton fabrics as that of the commercial enzyme and much superior to the enzyme produced through synthetic medium.

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In the current study, controlled unidirectional freeze casting method was employed to fabricate highly porous gelatin scaffolds. Different gelatin concentration, 1, 3, and 5 wt.% were dissolved in distilled water and the constant value of glutaraldehyde cross-linking agent (0.5 wt.%) was added to the solution. Then, the solutions freeze casted at different cooling rates of 1, 3 and 6 °C/min and freeze dried. Finally, pore morphology, mechanical properties and water adsorption characteristics of scaffolds were assessed. Results showed that by increasing in gelatin concentration, the pore shapes change from oblate and polygon to almost round but the cooling rate had no obvious effect on pore morphology. Compressive strengths of the scaffolds improved as a function of increasing in cooling rate and gelatin concentration from 20 to 1150 kPa. The value of water adsorption was decreased with augmentation in gelatin concentration and cooling rate in the range of 2000 to 500%. Therefore, this study suggests that the use of controllable freeze casting method and cooling rate can tailor the pore morphology, mechanical properties and water adsorption of gelatin scaffolds and could be a novel approach to avoid the use of toxic dose of cross-linking agent like glutaraldehyde.

Calcium hydroxide cements (CHCs), as a direct pulp-capping material, have been widely used for several decades. In spite of the well-known advantages of CHCs they have some disadvantages as well, such as high solubility, no inherent adhesive qualities and low mechanical strength. Earlier studies have been undertaken to improve these deficiencies by implementing changes to modify the conventional CHCs. The aim of the present research is to demonstrate the potential rectification of the aforementioned deficiencies of the commercially available CHCs by adding hydroxyapatite (HA) nanoparticles without deteriorating their advantages. Here, the synthesized HA nanoparticles were added into the CHCs in two different portions of 3 and 7 wt.%. Scanning electron microscope (SEM) was used to observe and analyze the microstructure, and X-ray energy dispersive analysis (EDX) was used to analyze the elemental composition of the new CHCs. In addition, setting time, mechanical strength, pH, calcium (Ca) release and antibacterial behavior were measured to assess how HA nanoparticles influence the characteristics of CHCs. The results showed that adding 3 wt.% HA nanoparticles can optimally improve the mechanical strength of the cement and increase Ca release rate as a mineralization promoter without deteriorating the antibacterial behavior.

This study aimed to investigate release of cefuroxime (CF) and calcium from polycaprolactone (PCL)/Calcium sulfate (CaS) implants (PCL:CaS-2:1–10% CF; PCL:CaS-2:1–20% CF; PCL:CaS-1:1–10% CF) for treating infectious bone diseases. Bioactivity, crystallinity and strength and release profiles under standard and pressurized release conditions were studied. PCL:CaS-2:1–20% CF had slower release than 10% loading. These groups had no significant change in CF and Ca release in response to pressure. PCL:CaS-1:1 group had slowest release despite having higher CaS probably due to more compaction of discs. In contrary, pressure caused significant differentiation of CF and Ca release. Presence of CaS enhanced mechanical properties and bioactivity of discs. SEM and XPS results showed Calcium-phosphate containing accumulations on surfaces upon SBF incubations. CF loaded implants were applied in rabbit osteomyelitis model. In vivo CF release was enhanced with increased CaS proportion, suggesting that in vivo release conditions are more close to pressurized in vitro conditions. In the control group, there was still some inflammation in the bone and no complete coverage with bone was achieved in defect site. Discs provided suitable surface for regeneration of bone. However, bone formation in PCL:CaS 1:1 disc implanted group was more complete and regular than 2:1 group.

In this paper, stability analysis and controller design problem in microbial continuous culture with discrete time delay is studied. The dissimilation process of glycerol to 1,3-propanediol cannot avoid the disturbances caused by time delay. Time delay can limit and degrade the achievable performance of controlled systems, and even induce instability. Based on the biodynamical model, some properties of its solutions are discussed. And we investigate how the time delay affects the stability of the system. LMIs method is applied to find a feedback controller to assure the stability of the closed-loop system. The simulation results indicate that the controller might be feasible for the continuous bioprocess controlling.

Candida albicans is a common fungal pathogen that causes systemic and superficial infections in most immunocompromised patients. Fluconazole, a synthetic triazole antifungal agent is the most prescribed drug used in treating this pathogen. But due to its poor solubilization in water and the emergence of resistant strains against this antimycotic drug, we aimed at devising a unique microemulsion drug delivery system for fluconazole against candidiasis. A clear oil-in-water microemulsion system, consisting of clove oil as oil phase, tween 20 as surfactant and water as aqueous phase was developed using ternary phase diagram. Physico-chemical characterization was done to understand their internal physico-chemical state. The bulk drug, fluconazole that measured several microns in length was reduced to 10–65 nm range with no means of high-energy methods as confirmed by transmission electron microscopy. The very small and uniform spherical structure of the drug loaded microemulsion system could be of high impact to the biological system as the efficacy of fluconazole is greatly improved when compared to its conventional bulk form. The optimized microemulsion exhibited significantly higher anti-fungal activity at a minimum concentration (8μg/ml) of fluconazole as examined by fluorescence and scanning electron microscopy. Thus, our report discloses an excellent oral drug delivery system.

2,3-butanediol (2,3-BD) is a major byproduct of 1,3-propanediol (1,3-PDO) fermentation by Klebsiella pneumoniae ZG25. It not only consumes large amounts of carbon source and NADH to diminish synthesis of 1,3-PDO, but also serves as an obstacle for high purity 1,3-PDO in downstream processes. To decrease the formation of 2,3-BD and make an intrinsic improvement of 1,3-PDO production, the budC gene in K. pneumoniae, coding 2,3-BD dehydrogenase, which is a key gene of 2,3-BD pathway, was successfully knocked out using Red recombination system in this paper. The results of the mutant fed-batch fermentation showed that the 1,3-PDO concentration, productivity per cell dry weight and conversion rate were up to 880 mmol l⁻¹, 22.0 mmol l⁻¹ h⁻¹, and 0.700 mol mol⁻¹, respectively, and increased by 10%, 15% and 11% compared with the parent strain. Meanwhile, 2,3-BD was still found in fermentation broth with 2,3-BD metabolic pathway blocked, which implied K. pneumoniae possesses a pathway of 2,3-BD cycle as a replenishment pathway.

Amylin has been implicated in type II diabetes due to its inherent property to misfold into toxic aggregates. Although it has been shown that amylin interacts with cell membranes, no study to date has monitored the association process using a direct approach. The present study uses confocal microscopy to identify the localization of carboxyfluorescein-labeled amylin in RIN-5F cells. In addition, the size of the aggregates that forms were evaluated using nanoparticle tracking analysis (NTA). In support of previous findings, amylin was observed to interact with and remain associated to the cell membrane. The cell membrane-associated aggregates spanned a size range of 130–800 nm.

Newly isolated and partially purified trimeric l-methionine-N-carbamoylase from Brevibacillus reuszeri HSN1 was immobilized by covalent coupling to a well-known support material, Eupergit® C. Approximately 80% enzyme activity yield was achieved with ≈61% binding of a soluble protein from a solution containing 5 mg/mL protein. The immobilized preparation was found to be quite unstable due to a poor multisubunit covalent interaction of trimeric enzyme. Additional cross-linking with polyaldehyde–dextran was done to sustain the biotechnological application of immobilized enzyme. The temperature and pH optima of immobilized enzyme were increased by 10°C and 0.5 unit, respectively. The enzyme was significantly stabilized and retained ≈93% enzyme activity when incubated at 60°C for 60 Min, whereas free enzyme lost ≈50% activity. It was recycled nine times with ≈100% conversion efficiency when batch experiments were carried out at 35°C, pH 7.5, for the 180 Min cycle, using 5% N-carbamoyl-l-methionine as the substrate. The half-life of the immobilized preparation was determined as 23 cycles and is significant. Approximately 50% of enzyme activity was retained even after 5 months of storage at 4°C, whereas free enzyme lost complete enzyme activity. Hence, we could enhance the stability of l-methionine-N-carbamoylase to make it a potential biocatalyst for biotechnological production of α-amino acids.

Single base mismatched oligonucleotides related to the rpoB gene of Mycobacterium tuberculosis, the mutations of which cause drug resistance of the infectious agent, were detected and discriminated using a surface plasmon resonance biosensor system. Thiol-modified oligonucleotides of the selected sequence (the probe) and 1-mercapto-6-hexanol were immobilized on a gold sensor surface. Hybridization between immobilized probe P2 and perfectly matched target T2 as well as a single base mismatched target TN was investigated in buffer solutions of various stringencies. Discrimination of perfectly matched and single base mismatched targets is achieved due to a difference in the level of their hybridization with the immobilized probe depending on stringency of the buffer solution. In 0.5×SSC buffer solution (7.5 mM sodium citrate, pH 7, containing 75 mM NaCl), sensor response at T2 injection into the measuring sensor cell was 16 times that at TN injection. The experimental results on surface hybridization between the studied oligonucleotides demonstrated a good correlation with theoretical calculations of thermodynamic parameters of these interactions in the solution. The described approach could be proposed as a basis for creating a biosensor for real-time label-free diagnostics of drug-resistant tuberculosis.

Chinese hamster ovary (CHO) cells producing β-galactosidase (β-gal) were successfully cultured on silicone-based porous microcarriers (ImmobaSil FS) in a 1 L stirred-tank perfusion bioreactor. We studied the growth, metabolism, and productivity of free and immobilized cells to understand cellular activity in immobilized conditions. CHO cells attached to ImmobaSil FS significantly better than to other microcarriers. Scanning electron microscope images showed that the CHO cells thoroughly colonized the porous surfaces of the ImmobaSil FS, exhibiting a spherical morphology with microvilli that extended to anchorage cells on the silicone surface. In perfusion culture, the concentration of the attached cells reached 8 × 10⁸ cells/mL of carrier, whereas those that remained freely suspended reached 2 × 10⁷ cells/mL medium. The β-gal concentration reached more than 5 unit/mL in perfusion culture, more than fivefold that of batch culture. The maximum concentration per microcarrier was proportional to the initial cell density. The specific growth rate, the specific β-gal production rate, the percentage of S phase, and the oxygen uptake rate were all relatively lower for immobilized cells than freely suspended cells in the same bioreactor, indicating that not only do cells survive and grow to a greater extent in a free suspension state, but they are also metabolically more active than viable cells inside the pores of the microcarriers.

The application of Fe₃O₄ nanoparticles to the separation of desulfurizing bacterial cells and their influence on the desulfurization activity and reusability of the two bacterial strains Rhodococcus erythropolis FMF and R. erythropolis IGTS8 were investigated. Magnetite nanoparticles were synthesized via the reverse coprecipitation method. Transmission electron microscopy (TEM) images showed that the magnetite nanoparticles had sizes of 5.35 ± 1.13 (F1 nanoparticles) and 8.74 ± 1.18 nm (F2 nanoparticles) when glycine was added during the synthesis of nanoparticles and when it was absent from the reaction mixture, respectively. Glycine was added after the synthesis of both F1 and F2 nanoparticles to stabilize the nanoparticle dispersion. TEM images of cells treated with magnetite nanoparticles indicated that F1 nanoparticles were immobilized on the surface of bacterial cells more evenly than the F2 nanoparticles. Desulfurization activities of the F1 magnetite nanoparticle–coated R. erythropolis FMF and R. erythropolis IGTS8 cells (with sulfur-removal percentage values of 70 ± 4 and 73 ± 3, respectively), as examined with the spectrophotometric Gibbs assay (based on dibenzothiophene degradation and sulfur-removal percentage), were not significantly different from those for the free bacterial cells (67 ± 3 and 69 ± 4, respectively). These results indicate that magnetite nanoparticles cannot affect the desulfurization activity of cells examined in this work. Isolation of bacterial cells from the suspension using a magnet and evaluation of desulfurization activity of separated cells showed that Fe₃O₄ nanoparticles can provide a high-efficiency recovery of bacterial cells from a suspension, with the reused magnetite nanoparticle–coated bacterial cells being able to maintain their desulfurization activity efficiently.

Complementary DNAs encoding human stefins A (HSA) and B (HSB) were synthesized using Pichia-preferred codons by overlap extension PCR. The full-length genes were ligated downstream of the glyceraldehyde-3-phosphate dehydrogenase promoter in the Pichia expression vector pGAPZαC and successfully expressed in Pichia pastoris strain X-33. Functional recombinant HSA and HSB proteins were purified from culture medium at yields of 121.3 ± 13.5 (n = 3) and 95.4 ± 4.1 (n = 3) mg/L, respectively. Using this expression strategy, we demonstrated that high levels of bioactive recombinant HSA and HSB can be produced by fermentation in P. pastoris.

The endoglucanase Cel5A from Thermobifida fusca was cloned and expressed in Escherichia coli BL21(DE3). The carboxymethyl cellulase (CMCase) activity in shake flasks and 3-L fermentation scale reached 46.8 and 656.6 IU/mL, respectively. The CMCase activity in 3-L fermentation scale represented the highest yield of T. fusca Cel5A reported so far. Recombinant Cel5A was purified and characterized in detail. The optimum temperature of recombinant enzyme was 80 °C, and the half-life of the enzyme was 132 H at 50 °C and 65 H at 60 °C. The activity of recombinant Cel5A was retained more than 90% over the range of pH 5.0–10.0 with maximal activity at pH 5.5. Using carboxymethyl cellulose as the substrate, the K_m and V_max values were 5.1 mg/mL and 48.7 IU/mg, respectively. The enzyme showed superstability in surfactants and was retained above 90% activity after treatment with sodium dodecyl sulfate, linear alkyl benzene sulfonate, fatty alcohol polyoxyethylene (9) ether, and polyoxyethylene (10) nonyl phenyl ether at 25 °C for 1 H, indicating that the enzyme could be a valuable component in detergents. The potential mechanism of this stability was investigated by analysis of the electrostatic potential of the surface of the enzyme.

Three heavy-chain and three kappa (κ)-chain transcripts were amplified from hybridoma cells secreting a monoclonal antibody (mAb) against transferrin receptor. Sequence analysis via IMGT/V-QUEST yielded the functional/aberrant prediction. Two functional κ-chain transcripts, Vκ2 and Vκ3, and one functional VH1 were revealed. Comprehensive bioinformatics analyses including sequence alignment, phylogenetic tree, somatic hypermutation prediction, and three-dimensional-molecular structure modeling were used to predict the origin of the two κ-chain transcripts. The results of bioinformatics analysis suggest that Vκ3 is derived from the myeloma partner of the hybridoma; Vκ2 is derived from B-cell. Functional transcripts VH1 and Vκ2 and Vκ3 were then used to construct two chimeric antibodies chi-C2 (Vκ2–VH1) and chi-C3 (Vκ3–VH1), respectively. Antigen-binding experiments showed that only chi-C2 remained the same affinity as its parental mAb. Possible explanations for the coexistence of two functional κ-chain transcripts and the different affinity of the two chimeric antibodies are discussed.

The marine diatom Phaeodactylum tricornutum, a widely used forage species, has a storage lipid content of up to 30% dry cell weight. To explore the mechanism behind the high storage lipid accumulation in this diatom, acetyl-CoA carboxylase (ACCase), which catalyzes the first committed step of the fatty acid biosynthetic pathway, was characterized in this study. A homogeneous type of ACCase (PtACC) was identified from P. tricornutum by homology searches. The first exon of the ACCase gene (PtACC-1) was cloned. PtACC-1 was fused with a Myc epitope tag and cloned into plasmid pMD18 driven by the LacZ promoter and expressed in Escherichia coli. The expression of the PtACC-1-Myc protein was verified by Western blot. The neutral lipid content in transformed E. coli increased substantially by twofold as determined by Nile red fluorescent dye staining. Concomitantly, ACCase activity increased by 1.72-fold. The fatty acid composition, analyzed by GC-MS, demonstrated a significant difference in the ratio of saturated fatty acids and monounsaturated fatty acids (MUFAs). MUFAs of PtACC-1 expressing cells increased by 13%. This study represents the first characterization of the key domains of ACCase from a diatom and demonstrates high neutral lipid accumulation in E. coli expressing PtACC-1, providing an additional genetic resource with the potential for biodiesel development.

Modified β-casein (mβ-CN) was investigated as an efficient additive for thermal reversibility of human carbonic anhydrase II (HCA II) at pH 7.75. The mβ-CN was obtained via modification of β-casein (β-CN) acidic residues using Woodward's reagent K. The effects of mβ-CN on the reversibility and stability of HCA II were determined by differential scanning calorimetry, UV–vis, and 1-anilinonaphthalene-8-sulfonic acid fluorescence spectroscopic methods. The mβ-CN, as an additive, enhanced thermal reversibility of HCA II by 33%. Together, our results indicated that mβ-CN is very efficient in decreasing thermal aggregation and enhancing the stability of HCA II. Using theoretical studies, we propose that the mechanism for thermal reversibility is mediated through formation of a salt bridge between the Woodward part of mβ-CN and the Zn ion of HCA II.

Porous scaffolds consisting of β-tricalcium phosphate (β-TCP) were successfully fabricated via selective laser sintering. The scaffolds had a controlled microstructure and totally interconnected porous structure. The microstructure and mechanical properties were studied. The bioactivity and degradability of scaffolds were evaluated through the simulated body fluid (SBF) cultivation experiment. The formation of a biologically active carbonate apatite layer on the surface after immersion in SBF was demonstrated using scanning electron microscope, energy dispersive X-ray, and Fourier transform infrared spectroscopy. Fast nucleation and growth of the carbonate apatite crystals were observed to occur all through the specimen surfaces. The phenomenon was explained in terms of the distribution and dispersion of inorganic phases in the scaffolds and the ionic activity products of the apatite in the SBF. The calculation results of weight loss and Ca/P molar ratio also suggest the good bioactivity and degradability of the scaffolds. These indicate that the β-TCP porous ceramic scaffold is a potential candidate scaffold for bone tissue engineering.

The pfah2 gene coding for a novel hydrophobin PfaH2 from the ascomycete Paecilomyces farinosus was identified during sequencing of random clones from a cDNA library. The corresponding protein sequence of PfaH2 deduced from the cDNA comprised 134 amino acids (aa). A 16 aa signal sequence preceded the N-terminus of the mature protein. PfaH2 belonged to the class Ia hydrophobins. The protein was isolated using trifluoroacetic acid extraction and purified via SDS-PAGE and high-performance liquid chromatography. The surface activity of the recently described PfaH1 and of PfaH2 was compared by the determination of contact angles (CAs) on glass slides and Teflon tape, and the CA of distilled water droplets was measured on glass slides coated with hydrophobin PfaH1 or PfaH2. Surprisingly, both hydrophobins adsorbed to hydrophilic surfaces and changed their physicochemical properties to a similar quantitative extent, although little aa sequence homology was found.

The novel protease from Exiguobacterium profundum BK-P23 was partially purified by ammonium sulfate precipitation and further purified Mono Q 5/50 and Superdex 200 10/300 column chromatography. The enzyme was purified 10.23-fold with a yield of 14%. The molecular weight was estimated to be 52 kDa by SDS-PAGE. The enzyme was most active at a pH of 8.0 and temperature of 40°C and the enzyme was stable between a pH of 7 and 10 and up to a temperature of 50°C. The enzyme activity was enhanced by CaCl₂ but was slightly inhibited by CoCl₂, MgSO₄, and AgNO₃. In addition, this enzyme was completely inhibited by phenylmethylsulfonyl fluoride, indicating that this enzyme was a serine protease. Furthermore, the alkaline protease was more stable in the presence of surfactants such as Triton X-100, which was followed by Tween 80 and SDS. Moreover, the enzyme was highly stable in the presence of 1% oxidizing agent (H₂O₂). The enzyme also has significant stability (70%–80%) in a few organic solvents. Thus, the increased stability of the enzyme in the presence of oxidizing agent, surfactants, and organic solvents may find potential applications in the detergent industry and peptide synthesis in nonaqueous media.

Polymethyl methacrylate (PMMA) nanofiber membrane (NFM) was synthesized by an electrospinning technique. These membranes were utilized as a support for immobilization of xylanase enzyme to study its pH stability, thermal stability, and reusability. The morphology of aligned NFM was studied by optical microscopy and scanning electron microscopy. The PMMA NFM was functionalized with phenylenediamine and activated with glutaraldehyde to yield an aldehyde group on its surface for covalent immobilization of xylanase. The Fourier transform infrared analysis of the covalently immobilized xylanase confirmed that the enzyme was immobilized on PMMA NFM via amide linkages. The immobilization efficiency of covalently bound xylanase was found experimentally to be 90%. A forward shift in pH optima from 6.0–7.0 (soluble enzyme) to 7.0–9.0 (immobilized enzyme) was observed after xylanase immobilization. The pH and temperature stability of xylanase were enhanced upon its covalent immobilization. The immobilized enzyme was active on repeated use and retained ∼80% of its initial activity after 11 reaction cycles. The improved thermal and operational stability of the covalently immobilized enzyme on PMMA NFM might be advantageous for industrial applications.

Ten protein sequences, each of caffeic acid-O-methyltransferase (COMT) and caffeoyl-coenzyme A-O-methyltransferase (CCoAOMT), catalyzing methylation of precursors of monolignol from selected dicots and monocots have been analyzed and compared on the basis of their amino acid sequence, motifs/domains, three-dimensional (3D) structure, and substrate binding. The isoelectric points of all the COMT and CCoAOMT sequences analyzed were found to vary in the pH range of 5 to 6. Molecular weight analyses suggested CCoAOMT to be smaller monomeric proteins (27–29 kDa) as compared with those of COMTs (39–40 kDa), which were dimeric. On the basis of phylogenetic analysis, COMT and CCoAOMT were clustered into two major groups, each of which could be further divided into two subgroups of monocots and dicots. Modeling and superimposition of COMT and CCoAOMT sequences of alfalfa (Medicago sativa) revealed that both were quite different at the 3D levels, although they had similarity in the core region. Molecular docking of 16 putative substrates (intermediates of monolignol biosynthesis pathway) revealed that both enzymes interact with all 16 substrates in a similar manner, with thiol esters being the most potent and binding of these putative substrates to CCoAOMT being more efficient.

Although megaprimer PCR mutagenesis has been used routinely in protein directed evolution, users sometimes encounter technical hurdles, particularly inefficiency during amplification when large fragments are used or the template is difficult to be amplified. Instead of methodology development, here we simply overcome the limitation by optimizing megaprimer PCR conditions via orthogonal array design of the four PCR components in three levels of each: template, primer, Mg²⁺, and dNTPs. For this, only nine PCRs need to be performed. The strategy (termed as OptiMega) was not only successfully applied for the construction of one multiple-site saturation mutagenesis library of halohydrin dehalogenase HheC, which failed to be constructed previously using the standard QuikChange™ protocol, but also expanded the construction of two high-quality random mutagenesis libraries of HheA and HheC. Most importantly, OptiMega offers a quick and simple way of constructing random mutagenesis libraries by eliminating the ligation step. Our results demonstrated that the OptiMega strategy could greatly strengthen the potential of megaprimer PCR mutagenesis for library construction.

Pectinases are among the most widely distributed enzymes in bacteria, fungi, and plants. Almost all the commercial preparations of pectinases are produced from fungal sources. Mucor circinelloides ITCC-6025 produced polygalacturonase when grown in Riviere's medium containing pectin (methyl ester) as the sole source of carbon. Immobilization of purified polygalacturonase was done on silica gel with 86% efficiency. The enzyme took 60 Min to bind maximally on the support. The immobilized enzyme showed maximum activity at a temperature of 45°C (4.57 U/mg) and pH 5.4. The immobilized enzyme was reused for four cycles as it retained almost 55% of its activity. The immobilized enzyme treatment increased the formation of higher alcohols and phenolics during the course of wine formation from apple and plum juices, whereas there was a decrease in the amount of carbohydrates. The enzyme treatment also resulted in clarification of wine; there was an increase in transmittance at 650 nm (201.78% in the case of apple wine and 223.4% in the case of plum wine) as compared to the control (untreated wine).

Waste money bills (WMB) that are no longer legal tender are nonrecyclable and are generally useless. In this work, we used this cellulose-rich material for ethanol fermentation for the first time. Torrefaction of this nonlignocellulosic waste material was attempted to examine whether such material could benefit from this process as a conventional lignocellulosic material does. Effects of two important parameters, that is, residence times (20, 40, and 60 Min) and temperatures (140, 160, 180, 200, and 220°C), on the torrefaction yield were studied under an inert atmosphere. Glucose and ethanol yields were compared using a factorial experimental design. The highest glucose yield (81.59 mg/mL) was obtained with a torrefaction treatment consisting of 40 min at 180 °C, and it was increased 44.89% compared to untreated WMB. Based on ethanol feasibility studies conducted on WMB, this estimated quantity of glucose could be produced for subsequent fermentation to ethanol (38.92 mg/mL) and it was increased 47.92% compared to the untreated sample. The fermentation rate was also enhanced by adding 0.4 mM benzoic acid under anaerobic conditions. It is concluded that production of ethanol from WMB would reduce waste management costs and thus would be profitable.

Enzymatic synthesis of propyl gallate in an organic solvent was studied using cell-associated tannase (E.C. 3.1.1.20) of Bacillus massiliensis. Lyophilized biomass showing tannase activity was used as a biocatalyst. The influence of buffer pH and strength, water activity, temperature, biocatalyst loading, gallic acid concentration, and 1-propanol concentration was studied by the one-factor-at-a-time method. Subsequently, response surface methodology was applied based on a central composite design to determine the effects of three independent variables (biocatalyst loading, gallic acid concentration, and 1-propanol concentration) and their mutual interactions. A total of 20 experiments were conducted, and a statistical model was developed, which predicted the maximum propyl gallate yield of 20.28 μg/mL in the reaction mixture comprising 40.4 mg biocatalyst, 0.4 mM gallic acid, and 6.52 % (v/v) 1-propanol in 9.5 mL benzene at 30°C. The subsequent verification experiments established the validity of the model. Under optimal conditions, 25% conversion of gallic acid to propyl gallate was achieved on a molar basis. The absence of the need for enzyme purification and subsequent immobilization steps and good conversion efficiency makes this enzyme system an interesting one. Reports on the applications of bacterial whole cell systems for synthetic reactions in organic solvents are scarce, and perhaps this is the first report on bacterial cell-associated tannase-mediated esterification in a nonaqueous medium.

l-N-carbamoylase was isolated from Brevibacillus reuszeri HSN1 and purified to homogeneity in three steps, which is a reasonably short protocol for native l-N-carbamoylase. The enzyme purification protocol resulted in ≈60-fold purification of l-N-carbamoylase with specific activity of 145 µmol/Min/mg. The subunit and native molecular mass were found to be 44.3 and 132 kDa, respectively. Temperature and pH optima were determined as 50°C and 8.5, respectively. The enzyme had retained ≈86% activity at 50°C when incubated for 60 Min and the half-life was determined as 180 Min at 50°C. N-carbamoyl-l-methionine (l-N-CMet) was found to be a preferred substrate with K_m and V_max values of ≈13.5 mM and ≈103 µmol/Min/mg, respectively. The broad substrate specificity with derivatives of N-carbamoyl amino acids is advantageous to be a better biocatalyst for production of corresponding l-α-amino acids. The enzyme activity was enhanced by 73% in the presence of 0.8 mM Mn²⁺ ion during the biotransformation. In the batch experiment, ≈97% conversion of 5.0% l-N-CMet into enantiomerically pure l-methionine was achieved in 10 H when carried out at pH 8.0, 45°C, and 15% wet (w/v) cell loading, under controlled conditions. The overall merits of this enzyme show promise as a potential biocatalyst for l-α-amino acid production.

The properties of cellulase that was attached to the surface of the chitosan carrier in aqueous–ionic liquid (IL; 1,3-dimeth-ylimidazolium dimethylphosphate) mixture were studied. The optimal temperature for immobilized cellulase in aqueous–IL mixed solutions was 60 °C. The immobilized cellulase acquired the highest relative activity at a ratio of 1:4 (IL to water, v/v), compared to activity levels of 79% and 7%, when the ratio of IL to water (v/v) was 0:1 and 1:0, respectively. At 80 °C, the immobilized cellulase in the aqueous–IL mixture conserved 46.3% activity after 120 Min. The immobilized cellulase can be effectively reused three times. After 4 weeks, the activity of immobilized cellulase maintained 83.5%. The Michaelis constant (K_m) and maximum reaction velocity (V_m) values for the immobilized cellulase were 4.8 mg/L and 0.156 mg/(mL Min), respectively. To the best of our knowledge, this is the first report on the properties of chitosan-immobilized cellulase in aqueous–IL.

Bioconversion of resveratrol is mainly achieved using plant cells and genetically modified microorganisms. We proposed a reaction system for resveratrol production using resting cells of a non–genetically modified strain, Alternaria sp. MG1, a resveratrol-producing endophytic fungus isolated from the grape. Effects of phenylalanine concentration, inoculum size, resting time, bioconversion medium, cell age, and pH on resveratrol production in the bioconversion process were investigated and their levels were optimized. The resulting optimal bioconversion medium was 0.2 mM phosphate buffer (pH 6.5), 0.1 g/L MgSO₄, 0.2 g/L CaSO₄, and 4.66 mM phenylalanine. Resting cells obtained from cultures of liquid potato-glucose medium after 4 days proved to be at the most suitable cell age for the bioconversion process with high resveratrol production and nonobvious cell growth. Highest resveratrol production (1.376 µg/L) was observed under the obtained optimal conditions of inoculum size, 12.16% (wet cell weight in 100 mL medium), and resting time, 21.3 H. The study provides a new way to produce resveratrol and establishes an essential reaction system for further study of the biosynthesis pathway of resveratrol in microorganisms, especially fungi.

Immobilized cunner fish trypsin was used to inactivate pectin methylesterase (PME). The effects of different reaction conditions (e.g., incubation time, PME concentration, and temperature) on PME inactivation and kinetics of inactivation were investigated. Temperature, incubation time, and PME concentration significantly affected the extent of PME inactivation. Generally, higher temperature, longer incubation time, and low PME concentration caused more PME inactivation. The immobilized fish trypsin had higher capacity to inactivate PME than immobilized bovine trypsin. The inactivation efficiency of the immobilized fish trypsin was about 20% higher than that of its bovine counterpart. However, PME inactivated by both trypsins regained partial activity during storage at 4°C, with immobilized fish trypsin–treated PME regaining more of its original activity than the immobilized bovine trypsin–treated PME. Heat-denatured PME was hydrolyzed more extensively by immobilized fish trypsin than by its bovine counterpart. The rate constants increased, whereas the D-values decreased with temperature for both immobilized fish and bovine trypsins. The inactivation rate constants of immobilized fish trypsin at all the temperatures investigated (i.e., 15–35°C) were higher than those of immobilized bovine trypsin. Furthermore, the activation energy (E_a) of PME inactivation by immobilized fish trypsin was lower than that of immobilized bovine trypsin.

Development of techniques utilizing waste without any additional energy or rare catalysts is a starting point for becoming sustainable. In the present work, the complex utilization of greenhouse residues was studied on a commercial scale. Only the energy produced by the process (8%) was used to run the technology, thanks to multilevel heat recuperation and high methane yields (over 340 m³ volatile solid t⁻¹). Manifestations of labile carbon in relation to available nitrogen, methane yields, and the formation of inhibitors were investigated in detail. The results sweep away many false beliefs about the ratios of carbon to nitrogen and highlight the role of the availability of carbon in phytomass utilization.

In recent years, the development of nanotechnology opens up new prospects for biomedical applications of unmodified and chemically modified diamond nanoparticles (DNPs). The problem of biocompatibility of DNPs is thus of primary importance. The first step in the modification of DNPs is usually the introduction of ‒OH groups, which can bind other functional groups. One of the basic methods to introduce ‒OH groups onto DNPs is the Fenton reaction. The aim of this study was to compare the effect of unmodified DNPs and nanoparticles modified by the Fenton reaction on human endothelial cells. Ultradisperse diamond (UDD) was modified by the Fenton reaction introducing surface ‒OH groups. Immortalized human umbilical cord endothelial cells (HUVEC-ST) were incubated with 2–100 µg/mL nanopowders in the opti-MEM medium. For comparison, graphite powder (GRAF and GRAF+OH) was also employed. UDD and GRAF augmented generation of reactive oxygen species in the cells after 24 H incubation, estimated by oxidation of 2′,7′-dichlorofluorescin diacetate (H2DCF-DA). Cellular production of nitric oxide, estimated with DAF-FM-DA (3-amino-4-aminomethyl 2′,7′-dichlorofluorescein diacetate), was also affected by UDD and GRAF after 24 H. Fenton-modified OH, in contrast to unmodified diamond, decreased NO production. Detonation nanoparticles also affected the cellular content of glutathione and activities of main antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione S-transferase).

This article was published online on 5 February 2013. Errors in the byline and affiliation line were subsequently identified. This notice is included in the online and print versions to indicate that both have been corrected 18 April 2013.

Porous scaffolds consisting of β-tricalcium phosphate (β-TCP) were successfully fabricated via selective laser sintering. The scaffolds had a controlled microstructure and totally interconnected porous structure. The microstructure and mechanical properties were studied. The bioactivity and degradability of scaffolds were evaluated through the simulated body fluid (SBF) cultivation experiment. The formation of a biologically active carbonate apatite layer on the surface after immersion in SBF was demonstrated using scanning electron microscope, energy dispersive X-ray, and Fourier transform infrared spectroscopy. Fast nucleation and growth of the carbonate apatite crystals were observed to occur all through the specimen surfaces. The phenomenon was explained in terms of the distribution and dispersion of inorganic phases in the scaffolds and the ionic activity products of the apatite in the SBF. The calculation results of weight loss and Ca/P molar ratio also suggest the good bioactivity and degradability of the scaffolds. These indicate that the β-TCP porous ceramic scaffold is a potential candidate scaffold for bone tissue engineering.