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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.168, #6)

Use of Styrene as Sole Carbon Source by the Fungus Exophiala oligosperma: Optimization and Modeling of Biodegradation, Pathway Elucidation, and Cell Membrane Composition by Eldon R. Rene; Przemysław Bernat; Jerzy Długoński; Maria C. Veiga; Christian Kennes (pp. 1351-1371).

Biodegradation of styrene by Exophiala sp. was tested at different initial concentrations (19.3–170.6 mg l⁻¹), pH (2.8–8.7), and temperatures (19.8–45.1 °C), for 120 h according to a 2³ full-factorial central composite design. The specific growth rate (SGR, per hour) and specific styrene utilization rate (SUR, milligrams of styrene per milligram of biomass per hour) values were used as the response variables for optimization purposes. The interactions between concentration and temperature (P = 0.022), and pH and temperature (P = 0.010) for SGR, and interactions between concentration and temperature (P = 0.012) for SUR were found to be statistically significant. The optimal values for achieving high SGR (0.15 h⁻¹) and SUR (0.3622 mg styrene mg⁻¹ biomass h⁻¹) were calculated from the regression model equation. Those values are C o = 89.1 mg l⁻¹, pH = 5.4, and T = 31.5 °C for SGR and C o = 69.2 mg l⁻¹, pH = 5.5, and T = 32.4 °C for SUR. It was also observed that the Exophiala strain degrades styrene via phenylacetic acid, involving initial oxidation of the vinyl side chain. Besides, in the presence of styrene, changes in the fatty acids profile were also observed. It is hypothesized that an increasing amount of linoleic acid (18:2) may be involved in the protection of the fungus against toxic substrate.

Keywords: Biodegradation; Filamentous fungi; Microbial growth; Modeling; Styrene degradation pathway; Membrane fatty acids

Stabilization of the Cellulase Enzyme Complex as Enzyme Nanoparticle by Imre Hegedüs; Jenő Hancsók; Endre Nagy (pp. 1372-1383).

The native Celluclast BG cellulase enzyme complex consists of different enzymes which can also degrade great substrate molecules as native celluloses. This enzyme complex has been covered by a very thin, a few nanometers thick, polymer layer, in order to improve its stability. It has been proved that the polymer layer around the enzyme molecules does not hinder the digestion as great substrates as crystalline cellulose polymer. The stability of the prepared enzyme nanoparticles (PE) could significantly be increased comparing to that of the native one what was proved by results of the total cellulose activity measured. The pretreated enzyme complex holds its activity often a few magnitudes of orders longer in time than that of the native enzyme complex (enzyme without pretreatment). It retains its activity at least ten times longer than that of the native one, at a temperature range between 20 and 37 °C. The pretreated enzyme complex can have about 50 % of its original activity during 12 h of incubation at even 80 °C, while the native cellulase one totally lost it during 6 h incubation time. The activity of PE has not been significantly reduced even at extreme pH values, namely in the pH range of 1.5 to 12.

Keywords: Enzyme activity; Enzyme stabilization; Single enzyme nanoparticle; Enzyme complex; Cellulase

Cre-lox66/lox71-Based Elimination of Phosphotransacetylase or Acetaldehyde Dehydrogenase Shifted Carbon Flux in Acetogen Rendering Selective Overproduction of Ethanol or Acetate by Vel Berzin; Michael Kiriukhin; Michael Tyurin (pp. 1384-1393).

Acetogen strain Clostridium sp. MT1121 produced 300 mM acetate (p < 0.005) and 321 mM ethanol (p < 0.005) from synthesis gas (syngas) blend 60 % CO and 40 % H₂. Clostridium sp. MT1121 was metabolically engineered to eliminate production of either acetate or acetaldehyde during syngas fermentation. We used Cre-lox66/lox71-based gene removal system to eliminate either phosphotransacetylase (pta), or acetaldehyde dehydrogenase (aldh). The resulted biocatalyst with eliminated pta increased ethanol yield to 610 mM (p < 0.005). Inactivation of pta rendered only 502 mM of ethanol (p < 0.005). The acetogen biocatalyst with eliminated aldh produced 450 mM acetate (p < 0.005). The role of cell energy pool preservation for re-directed carbon flux is discussed. This is the first report on time- and cost-efficient gene elimination in acetogens using lox66/lox71 gene elimination system.

Keywords: Acetogens; Gene elimination; pta ; aldh ; Syngas fermentation; Electrotransformation

Evaluation of Metal Ions and Surfactants Effect on Cell Growth and Exopolysaccharide Production in Two-Stage Submerged Culture of Cordyceps militaris by Jian-Dong Cui; Ya-Nan Zhang (pp. 1394-1404).

During the two-stage submerged fermentation of medicinal mushroom Cordyceps militaris, it was found that K⁺, Ca²⁺, Mg²⁺, and Mn²⁺ were favorable to the mycelial growth. The EPS production reached the highest levels in the media containing Mg²⁺ and Mn²⁺. However, Ca²⁺ and K⁺ almost failed to increase significantly exopolysaccharides (EPS) production. Sodium dodecyl sulfate (SDS) significantly enhanced EPS production compared with that of without adding SDS when SDS was added on static culture stage of two-stage cultivation process. The presence of Tween 80 in the medium not only simulated mycelial growth but also increased EPS production. By response surface methods (RSM), EPS production reached its peak value of 3.28 g/L under optimal combination of 27.6 mM Mg²⁺, 11.1 mM Mn²⁺, and 0.05 mM SDS, which was 3.76-fold compared with that of without metal ion and surfactant. The results obtained were useful in better understanding the regulation for efficient production of EPS of C. militaris in the two-stage submerged culture.

Keywords: Cordyceps militaris ; The two-stage submerged culture; Exopolysaccharides; Metal ion; Surfactant

Lipase-Catalyzed Synthesis of Cocoa Butter Equivalent from Palm Olein and Saturated Fatty Acid Distillate from Palm Oil Physical Refinery by Ibrahim O. Mohamed (pp. 1405-1415).

Cocoa butter equivalent was prepared by enzymatic acidolysis reaction of substrate consisting of refined palm olein oil and palmitic–stearic fatty acid mixture. The reactions were performed in a batch reactor at a temperature of 60 °C in an orbital shaker operated at 160 RPM. Different mass ratios of substrates were explored and the compositions of the five major triacylglycerol (TAG) of the structured lipids were identified and quantified using cocoa butter-certified reference material IRMM-801. The reaction resulted in production of cococa butter equivent with TAG compostion (POP 26.6 %, POS 42.1, POO 7.5, SOS 18.0 %, and SOO 5.8 %) and melting temperature between 34.7 and 39.6 °C which is close to that of the cocoa butter. The result of this research demonstrated the potential use of saturated fatty acid distillate (palmitic and stearic fatty acids) obtained from palm oil physical refining process into a value-added product.

Keywords: Cocoa butter equivalent; Lipase; Triacylglycerol; Acidolysis; Interesterification; Palm oil; Structured lipids

Synthesis and Preliminary In Vitro Biological Evaluation of 5-Chloro-2-(Substituted Phenyl)Benzo[d]Thiazole Derivatives Designed As Novel Antimelanogenesis Agents by Young Mi Ha; Yohei Uehara; Daeui Park; Hyoung Oh Jeong; Ji Young Park; Yun Jung Park; Ji Yeon Lee; Hye Jin Lee; Yu Min Song; Hyung Ryong Moon; Hae Young Chung (pp. 1416-1433).

We describe the design, synthesis, and biological activities of 5-chloro-2-(substituted phenyl)benzo[d]thiazole derivatives as novel tyrosinase inhibitors. Among them, 4-(5-chloro-2,3-dihydrobenzo[d]thiazol-2-yl)-2,6-dimethoxyphenol (MHY884) and 2-bromo-4-(5-chloro-benzo[d]thiazol-2-yl)phenol (MHY966) showed inhibitory activity higher than or similar to kojic acid, against mushroom tyrosinase. Therefore, we carried out kinetic studies on the two compounds with potent tyrosinase inhibitory effects. Kinetic analysis of tyrosinase inhibition revealed that all of these compounds are competitive inhibitors. MHY884 and MHY966 effectively inhibited tyrosinase activity and reduced melanin levels in B16 cells treated with α-melanocyte stimulating hormone (α-MSH). These data strongly suggest that the newly synthesized compounds MHY884 and MHY966 could suppress production of melanin via inhibition of tyrosinase activity.

Keywords: 5-Chloro-2-(substituted phenyl)benzo[d]thiazole derivatives; Tyrosinase inhibitor; Docking simulation; Melanin contents

Synthesis and Characterization of Doxorubicin-Loaded Poly(Lactide-co-glycolide) Nanoparticles as a Sustained-Release Anticancer Drug Delivery System by I. Amjadi; M. Rabiee; M. S. Hosseini; M. Mozafari (pp. 1434-1447).

The objective of the present study was to prepare a polymeric drug delivery system with no burst effect. To attain this goal, doxorubicin (Dox) as an effective anticancer drug was loaded into poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) to improve the drug performance and also maximize the release period. After the synthesis process, the freshly made PLGA NPs with two different lactide-to-glycolide ratios (75:25 and 50:50) were evaluated physically and chemically. To determine the encapsulation efficiency, a centrifugation method was applied. Also, the drug loading effect on particle size, polydispersity index, and zeta potential was examined. The results indicated that the NPs had nearly the same diameters around 360 nm, and the entrapment efficiencies for 75:25 PLGA and 50:50 PLGA were reported around 39 and 48 %, respectively. A slight increase in all parameters was observed due to the increase of the drug loading content. The primary release was 7.91 % (w/w) and 14.70 % (w/w) for 75:25 and 50:50 drug-loaded NPs, respectively; no burst effect was observed. After 20 days, the drug release was around 70.98 and 62.22 % of the total entrapped drug for 75:25 and 50:50 drug-loaded NPs, respectively. Finally, it was found that Dox was an appropriate anticancer agent with good capability to be encapsulated in polymeric NPs and could be released from the carriers with no burst effect and favor rate.

Keywords: Doxorubicin; PLGA; Drug release; Nanoparticles; Encapsulation efficiency; In vitro

Chicken Egg Yolk Antibody (IgY) Controls Solobacterium moorei Under In Vitro and In Vivo Conditions by Xiaoyu Li; He Liu; Yongping Xu; Fanxing Xu; Linhui Wang; Jiansong You; Shuying Li; Liji Jin (pp. 1448-1458).

Solobacterium moorei is a causative agent in diseases such as oral halitosis, bacteremia, and necrobacillosis-associated thrombophlebitis. The objective of this study was to determine the effectiveness of chicken egg yolk antibody (IgY) in controlling S. moorei. Intact S. moorei cells were used as an immunogen to immunize four White Leghorn laying hens. IgY, extracted from egg yolks obtained from these immunized hens, was purified using water dilution, two-step salt precipitation, and ultrafiltration. The purity of the IgY obtained was approximately 87.3 %. The antibody titer of the IgY was determined by enzyme-linked immunosorbent assay. The antibody titer peaked at 10,000 following the third immunization. In order to evaluate the inhibitory effects of the specific IgY, the growth of S. moorei in liquid media was measured every 12 h using a microplate reader at 600 nm. Biofilm formation of S. moorei was quantified by staining with crystal violet. The specific binding ability of IgY was further confirmed by the use of immunofluorescence and immunoelectron microscopy. Growth and biofilm formation of S. moorei were significantly (P < 0.05) inhibited by 20 and 40 mg/ml specific IgY compared with the control. The specific IgY also decreased the bacterial level in the oral cavity of mice after infection with S. moorei. This study demonstrates that the growth and biofilm formation of S. moorei can be effectively inhibited by specific IgY. As a result, IgY technology may have application in the control of diseases caused by S. moorei.

Keywords: Solobacterium moorei ; Egg yolk antibody; Inhibition; Biofilm; Immunofluorescence and immunoelectron microscopy

In Vitro Biomineralization and Bulk Characterization of Chitosan/Hydroxyapatite Composite Microparticles Prepared by Emulsification Cross-Linking Method: Orthopedic Use by Hamida Maachou; Kheireddine Bal; Youcef Bal; Alexandre Chagnes; Gerard Cote; Djamel Aliouche (pp. 1459-1475).

Chitosan/hydroxyapatite composite microparticles were prepared by a solid-in-water-in-oil emulsification cross-linking method. The characteristics and activity in presence of simulated body fluid for 14 and 21 days were investigated. The size distribution, surface morphology, and microstructure of these biomaterials were evaluated. The scanning electron microscopy revealed an aggregate of microparticles with a particle size, ranged from 4 to 10 μm. The deposited calcium phosphate was studied using X-ray diffraction analysis, Fourier transform infrared spectroscopy, and inductively coupled plasma/atomic emission spectroscopy analysis of phosphorus. These results show that the mineral, formed on microparticles, was a mixture of carbonated hydroxyapatite and calcite. Scanning electron microscopy revealed that calcium phosphate crystals growth was in form of rods organized as concentric triangular packets interconnected to each other by junctions. Interaction between chitosan and growing carbonated hydroxyapatite and calcite crystals are responsible for a composite growth into triangular and spherical shapes. The results demonstrated that these microparticles were potential materials for bone repair.

Keywords: Hydroxyapatite; Chitosan; Microparticle; Composite; Emulsification; Biomineralization

Polyamines Induced by Osmotic Stress Protect Synechocystis sp. PCC 6803 Cells and Arginine Decarboxylase Transcripts Against UV-B Radiation by Apiradee Pothipongsa; Saowarath Jantaro; Aran Incharoensakdi (pp. 1476-1488).

The effect of UV-B radiation on growth and polyamines content of Synechocystis sp. PCC 6803 subjected to either NaCl or sorbitol stress was investigated. Cells could not grow in the presence of 350 mM NaCl or 500 mM sorbitol under normal white light. However, cells grown in BG11 under osmotic stress imposed by NaCl or sorbitol followed by ultraviolet-B (UV-B) irradiation for 2 h showed higher cell density than those under the same condition but no osmotic stress. The chlorophyll fluorescence parameter (F _v/F _m) also showed an apparent decrease upon UV-B irradiation. Intracellular polyamines increased by about 2- and 4-fold in NaCl- and sorbitol-stressed cells, respectively. When these cells were irradiated with UV-B for 1 h, a further 3-fold increase in polyamines content was detected in NaCl-stressed but not sorbitol-stressed cells. Synechocystis cells contained adc1 and adc2 genes encoding arginine decarboxylase (ADC) with only adc1 showing upregulation by NaCl or sorbitol stress. NaCl- or sorbitol-stressed cells contained about 5-fold higher level of adc1 transcript than did the unstressed cells after 1-h irradiation with UV-B, suggesting the protection of adc1 transcript by accumulated polyamines, due to NaCl or sorbitol stress, against UV-B radiation damage. ADC levels as analyzed by Western blot showed upregulation by UV-B in NaCl-stressed but not sorbitol-stressed cells.

Keywords: Arginine decarboxylase; Osmotic stress; Polyamines; Synechocystis sp. PCC 6803; UV-B radiation

A Low-Temperature-Active Alkaline Pectate Lyase from Xanthomonas campestris ACCC 10048 with High Activity over a Wide pH Range by Peng Yuan; Kun Meng; Yaru Wang; Huiying Luo; Pengjun Shi; Huoqing Huang; Tao Tu; Peilong Yang; Bin Yao (pp. 1489-1500).

Alkaline pectate lyases are favorable for the textile industry. Here, we report the gene cloning and expression of a low-temperature-active alkaline pectate lyase (PL D) from Xanthomonas campestris ACCC 10048. Deduced PL D consists of a putative 27-residue signal peptide and a catalytic domain of 320 residues belonging to family PF09492. Recombinant PL D (r-PL D) produced in Escherichia coli was purified to electrophoretic homogeneity with a single step of Ni²⁺–NTA affinity chromatography and showed an apparent molecular weight of ~38 kDa. The pH and temperature optima of r-PL D were found to be 9.0 °C and 30 °C, respectively. Compared with its microbial counterparts, r-PL D had higher activity over a wide pH range (>45 % of the maximum activity at pH 3.0–12.0) and at lower temperatures (>35 % of activity even at 0 °C). The K _m and V _max values of r-PL D for polygalacturonic acid were 4.9 g l⁻¹ and 30.1 μmol min⁻¹ mg⁻¹, respectively. Compared with the commercial compound pectinase from Novozymes, r-PL D showed similar efficacy in reducing the intrinsic viscosity of polygalacturonic acid (35.1 % vs. 36.5 %) and in bioscouring of jute (10.25 % vs. 10.82 %). Thus, r-PL D is a valuable additive candidate for the textile industry.

Keywords: Xanthomonas campestris ACCC 10048; Alkaline pectate lyase; Bioscouring

Effect of Acetic Acid on Saccharomyces Carlsbergensis ATCC 6269 Batch Ethanol Production Monitored by Flow Cytometry by Cláudia Freitas; Elisabete Neves; Alberto Reis; Paula C. Passarinho; Teresa Lopes da Silva (pp. 1501-1515).

Bioethanol produced from lignocellulosic materials has been considered a sustainable alternative fuel. Such type of raw materials have a huge potential, but their hydrolysis into mono-sugars releases toxic compounds such as weak acids, which affect the microorganisms' physiology, inhibiting the growth and ethanol production. Acetic acid (HAc) is the most abundant weak acid in the lignocellulosic materials hydrolysates. In order to understand the physiological changes of Saccharomyces carlsbergensis when fermenting in the presence of different acetic acid (HAc) concentrations, the yeast growth was monitored by multi-parameter flow cytometry at same time that the ethanol production was assessed. The membrane potential stain DiOC₆(3) fluorescence intensity decreased as the HAc concentration increased, which was attributed to the plasmic membrane potential reduction as a result of the toxic effect of the HAc undissociated form. Nevertheless, the proportion of cells with permeabilized membrane did not increase with the HAc concentration increase. Fermentations ending at lower external pH and higher ethanol concentrations depicted the highest proportions of permeabilized cells and cells with increased reactive oxygen species levels. Flow cytometry allowed monitoring, near real time (at-line), the physiological states of the yeast during the fermentations. The information obtained can be used to optimize culture conditions to improve bioethanol production.

Keywords: Lignocellulosic materials, bioethanol; Saccharomyces carlsbergensis ; Acetic acid; Flow cytometry

Development of a Defined Medium for Arachidonic Acid Production by Mortierella alpina Using a Visualization Method by Xin Liu; Xiaojun Ji; Hongman Zhang; Ninghua Fu; Liexiang Yan; Zhongtao Deng; He Huang (pp. 1516-1527).

Defined medium for arachidonic acid (ARA) production by Mortierella alpina was optimized for its metabolomics study. For this purpose, a visualization method (VM) was applied for the first time. Experiments were designed according to the uniform design with four factors (concentrations of glucose, NaNO₃, KH₂PO₄ and MgSO₄·7H₂O) for each at nine levels. Dry cell weight (DCW), ARA yield in DCW [percent (w/w)] and ARA content in total fatty acids [percent (w/w)] were considered as the three objectives. Optimization of single-objective function and multi-objective function of two objectives and three objectives was attempted. Optimal DCW, ARA yield and ARA content were predicted to occur in a medium that contained (grams per litre): glucose 35, NaNO₃ 1, KH₂PO₄ 7.5 and MgSO₄·7H₂O 2.6. Upon verification, the average tested DCW (12.95 g/l), ARA yield (18.89 %) and ARA content (42.36 %) were fairly close to the predicted values (12.88 g/l, 9.68 % and 35.57 %, respectively). Moreover, DCW, ARA yield and ARA content from the optimum medium increased by 35.68, 47.23 and 30.90 % compared with control, respectively, indicating that VM had succeeded in exploiting the biomass growth and ARA production by M. alpina.

Keywords: Arachidonic acid; Defined medium; Mortierella alpina ; Optimization; Visualization method

Metal-Chelating Nanopolymers for Antibody Purification from Human Plasma by Deniz Aktaş Uygun; R. Hilal Şenay; Ceren Türkcan; Sinan Akgöl; Adil Denizli (pp. 1528-1539).

The purification of immunoglobulin G (IgG) from human plasma was performed by using a novel metal-chelated adsorbent with nano size. The non-porous nanoparticles were produced by surfactant free emulsion polymerization of ethylene glycol dimethacrylate (EDMA) and 2-methacryloylamidohistidine (MAH). Then, Cu(II) ions were chelated on the nanoparticles. The nano-poly(EDMA-MAH) nanoparticles were characterized by Fourier transform infrared, scanning electron microscope, atomic force microscope and elemental analysis. The non-porous nanoparticles were spherical form and have 100–250 nm size distribution. The maximum IgG adsorption capacity of the Cu(II) chelated nanoparticles was found to be 463 mg/g polymer at pH 7.0 in HEPES buffer. Desorption of IgG was performed by 1.0 M NaCl and desorption rate was found to be 97 %. IgG was obtained from human plasma with purity of 94 % (up to 578 mg/g polymer). The non-porous nanoparticles allowed one-step purification of IgG from human plasma.

Keywords: IgG; Nanoparticle; IMAC; Purification

In Vitro Inhibition Effect of Some Dihydroxy Coumarin Compounds on Purified Human Serum Paraoxonase 1 (PON1) by Mahmut Erzengin; Ismet Basaran; Umit Cakir; Aynur Aybey; Selma Sinan (pp. 1540-1548).

Human serum paraoxonase 1 (PON1; EC 3.1.8.1) is a high-density lipoprotein associated, calcium-dependent enzyme that hydrolyses aromatic esters, organophosphates and lactones and can protect the low-density lipoprotein against oxidation. In this study, in vitro inhibition effect of some dihydroxy coumarin compounds namely 6,7-dihydroxy-3-(2-methylphenyl)-2H-chromen-2-one (A), 6,7-dihydroxy-3-(3-methylphenyl)-2H-chromen-2-one (B) and 6,7-dihydroxy-3-(4-methylphenyl)-2H-chromen-2-one (C) on purified PON1 were investigated by using paraoxon as a substrate. PON1 was purified using two-step procedures, namely ammonium sulphate precipitation and Sepharose-4B-l-tyrosine-1-naphthylamine hydrophobic interaction chromatography. The purified enzyme had a specific activity of 11.76 U/mg. The dihydroxy coumarin derivatives of A and B compounds inhibited PON1 enzyme activity in a noncompetitive inhibition manner with K _i of 0.0080 ± 0.256 and 0.0003 ± 0.018 mM values, respectively. C compound exerted an uncompetitive inhibition of PON1 enzyme activity with K _i of 0.0010 ± 0.173 mM. Moreover, dihydroxy coumarin derivatives of A, B and C compounds were effective inhibitors on purified human serum PON1 activity with IC₅₀ of 0.012, 0.022 and 0.003 mM values, respectively. IC₅₀ value of unsubstituted 6,7 dihydroxy coumarin was found as 0.178 mM. The present study has demonstrated that PON1 activity is very highly sensitive to studied coumarin derivatives.

Keywords: Paraoxonase 1; In vitro inhibition; Coumarin derivatives

A Green Approach for Synthesis of Gold and Silver Nanoparticles by Leishmania sp. by Fatemeh Ramezani; Ali Jebali; Bahram Kazemi (pp. 1549-1555).

The application of microorganisms for the synthesis of metal nanoparticles as an eco-friendly and promising approach is ongoing. In this paper, an attempt has been made to investigate the capability of Leishmania sp. for synthesis of metal nanoparticles from aqueous silver and gold ions. The samples were analyzed by a UV–Vis spectroscopy and the results showed the absorbance peak values at 420 and 540 nm, respectively, for the surface plasmon resonance of silver and gold nanoparticles. The surface morphology of the nanoparticles in solution was visualized by atomic force microscope and scanning electron microscope images, which showed the production of metallic nanoparticles by this protozoan. Fourier transform infrared spectroscopy analyses confirmed the presence of different bands of protein as capping and stabilizing agent on the nanoparticles surfaces. The synthesized silver and gold nanoparticles were with dimensions ranging between 10 and 100 nm for silver and 50–100 nm for gold. These results of the present study have demonstrated the efficiency of this protozoan for synthesis of nanoparticles, by offering the merits of environmentally friendly, amenability, and time saving for large-scale production.

Keywords: Biosynthesis; Gold; Leishmania ; Nanoparticles; Silver

Effects of SPORL and Dilute Acid Pretreatment on Substrate Morphology, Cell Physical and Chemical Wall Structures, and Subsequent Enzymatic Hydrolysis of Lodgepole Pine by Xinping Li; Xiaolin Luo; Kecheng Li; J. Y. Zhu; J. Dennis Fougere; Kimberley Clarke (pp. 1556-1567).

The effects of pretreatment by dilute acid and sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) on substrate morphology, cell wall physical and chemical structures, along with the subsequent enzymatic hydrolysis of lodgepole pine substrate were investigated. FE-SEM and TEM images of substrate structural morphological changes showed that SPORL pretreatment resulted in fiber separation, where SPORL high pH (4.2) pretreatment exhibited better fiber separation than SPORL low pH (1.9) pretreatment. Dilute acid pretreatment produced very poor fiber separation, consisting mostly of fiber bundles. The removal of almost all hemicelluloses in the dilute acid pretreated substrate did not overcome recalcitrance to achieve a high cellulose conversion when lignin removal was limited. SPORL high pH pretreatment removed more lignin but less hemicellulose, while SPORL low pH pretreatment removed about the same amount of lignin and hemicelluloses in lodgepole pine substrates when compared with dilute acid pretreatment. Substrates pretreated with either SPORL process had a much higher cellulose conversion than those produced with dilute acid pretreatment. Lignin removal in addition to removal of hemicellulose in SPORL pretreatment plays an important role in improving the cellulose hydrolysis of the substrate.

Keywords: Enzymatic hydrolysis; SPORL; Morphology; Wall structures; Sulfonation of lignin

Detoxification and Fermentation of Pyrolytic Sugar for Ethanol Production by Hui Wang; Darrell Livingston; Radhakrishnan Srinivasan; Qi Li; Philip Steele; Fei Yu (pp. 1568-1583).

The sugars present in bio-oil produced by fast pyrolysis can potentially be fermented by microbial organisms to produce cellulosic ethanol. This study shows the potential for microbial digestion of the aqueous fraction of bio-oil in an enrichment medium to consume glucose and produce ethanol. In addition to glucose, inhibitors such as furans and phenols are present in the bio-oil. A pure glucose enrichment medium of 20 g/l was used as a standard to compare with glucose and aqueous fraction mixtures for digestion. Thirty percent by volume of aqueous fraction in media was the maximum additive amount that could be consumed and converted to ethanol. Inhibitors were removed by extraction, activated carbon, air stripping, and microbial methods. After economic analysis, the cost of ethanol using an inexpensive fermentation medium in a large scale plant is approximately $14 per gallon.

Keywords: Ethanol; Bio-oil; Fast pyrolysis; Fermentation; Glucose; Inhibitor

Cunninghamella as a Microbiological Model for Metabolism of Histamine H₃ Receptor Antagonist 1-[3-(4-tert-Butylphenoxy)propyl]piperidine by Elżbieta Pękala; Paulina Kubowicz; Dorota Łażewska (pp. 1584-1593).

The aim of the study was to analyze the ability of the microorganism Cunninghamella to carry out the biotransformation of 1-[3-(4-tert-butylphenoxy)propyl]piperidine (DL76) and to compare the obtained results with in silico models. Biotransformation was carried out by three strains of filamentous fungus: Cunninghamella echinulata, Cunninghamella blakesleeana, and Cunninghamella elegans. Most probable direction of DL76 metabolic transition was the oxidation of the methyl group in the tert-butyl moiety leading to the formation of the metabolite with I° alcohol properties. This kind of reaction was conducted by all three strains tested. However, only in the case of C. blakesleeana that biotransformation product had a structure of carboxylic acid. CYP2C19 was identified by Metasite software to be the isoform of major importance in the oxidation process in the tert-butyl moiety of DL76. In silico data coincide with the results of experiments conducted in vitro. It was confirmed that Cunninghamella fungi are a very good model to study the metabolism of xenobiotics. The computational methods and microbial models of metabolism can be used as useful tools in early ADME-Tox assays in the process of developing new drug candidates.

Keywords: Cunninghamella; Biotransformation; Microbiological model; In vitro metabolism; In silico metabolism

Development and Characterization of Microsatellite Markers (SSR) in Sesamum (Sesamum indicum L.) Species by B. Spandana; V. Prathap Reddy; G. John Prasanna; G. Anuradha; S. Sivaramakrishnan (pp. 1594-1607).

Microsatellites, also known as simple sequence repeats (SSRs), are the class of repetitive DNA sequences present throughout the genome of many plant and animal species. Recent advances in molecular genetics had been the introduction of microsatellite markers to investigate the genetic structuring of natural plant populations. We have employed an enrichment strategy for microsatellite isolation by using multi-enzymes digestion, microsatellite oligoprobes, and streptavidin magnetic beads in Sesamum (Sesamum indicum L.). More than 200 SSR motifs were detected (SSR motifs ≥2 repeat units or 6 bp); 80 % of the clones contained SSR motifs. When regarding SSRs with four or more repeat units and a minimum length of 10 bp, 132 of them showed repeats. Eighteen SSR markers were initially characterized for optimum annealing temperature using a gradient PCR technique. Among the 18 SSR markers characterized, five were found to be polymorphic and used to analyze 60 Sesamum germplasm accessions. The maximum number of alleles detected was four with a single primer and the least number of two alleles with three primers with an average PIC value of 0.77. SSRs are a valuable tool for estimating genetic diversity and analyzing the evolutionary and historical development of cultivars at the genomic level in sesame breeding programs.

Keywords: Sesame (Sesamum indicum L.); SSR; Microsatellite loci; Genetic variability

Use of Transgenic Oryzacystatin-I-Expressing Plants Enhances Recombinant Protein Production by P. Pillay; T. Kibido; M. du Plessis; C. van der Vyver; G. Beyene; B. J. Vorster; K. J. Kunert; U. Schlüter (pp. 1608-1620).

Plants are an effective and inexpensive host for the production of commercially interesting heterologous recombinant proteins. The Escherichia coli-derived glutathione reductase was transiently expressed as a recombinant model protein in the cytosol of tobacco plants using the technique of leaf agro-infiltration. Proteolytic cysteine protease activity progressively increased over time when glutathione reductase accumulated in leaves. Application of cysteine protease promoter–GUS fusions in transgenic tobacco identified a cysteine protease NtCP2 expressed in mature leaves and being stress responsive to be expressed as a consequence of agro-infiltration. Transgenic tobacco plants constitutively expressing the rice cysteine protease inhibitor oryzacystatin-I had significantly lower cysteine protease activity when compared to non-transgenic tobacco plants. Lower cysteine protease activity in transgenic plants was directly related to higher glutathione reductase activity and also higher glutathione reductase amounts in transgenic plants. Overall, our work has demonstrated as a novel aspect that transgenic tobacco plants constitutively expressing an exogenous cysteine protease inhibitor have the potential for producing more recombinant protein which is very likely due to the reduced activity of endogenous cysteine protease.

Keywords: Cysteine protease; Cysteine protease inhibitor; Transient expression; Tobacco agro-infiltration; Glutathione reductase

Flocculating Property of Extracellular Polymeric Substances Produced by a Biofilm-Forming Bacterium Acinetobacter junii BB1A by Krishna K. Yadav; Amit K. Mandal; Ipsita K. Sen; Soumyananda Chakraborti; Syed S. Islam; Ranadhir Chakraborty (pp. 1621-1634).

Extracellular polymeric substances (EPS) produced by a biofilm-producing bacterium Acinetobacter junii BB1A were characterized. Purified EPS on analysis revealed neutral sugar (73.21 %), uronic acid (10.12 %), amino sugars (0.23 %), α- amino acids (11.13 %), and aromatic amino acids (1.23 %). Infrared spectrometry revealed the presence of hydroxyl, carboxyl, and amide groups. The average molecular weight of the polysaccharide (PS) fraction of EPS was ~2 × 10⁵. Gas liquid chromatography analysis of PS revealed the presence of three main sugar residues, namely, mannose, galactose, and arabinose (molar ratio of 3:1:1). Cation-independent flocculation above 90 % was observed in the pH range of 4–5 with EPS dosage of 30 mg l⁻¹ at 20 °C. The emulsifying activity of EPS was 66.6 % with toluene, 60 % with n-hexadecane, 53.3 % with olive oil, and least activity of 13.3 % with kerosene. Proteinase K- and trichloroacetic acid-treated EPS showed reduction in flocculation and emulsification, suggesting the significant role of protein component. Energy dispersive X-ray spectroscopy was used to provide direct evidence of biosorption of Co(II), Cd(II), and Hg(II) by EPS.

Keywords: Acinetobacter junii BB1A; Flocculating rate; Biofilm; Purification; Adsorption; Bioremediation

Graphene Oxide as a Matrix for the Immobilization of Glucose Oxidase by Liya Zhou; Yanjun Jiang; Jing Gao; Xinqiang Zhao; Li Ma (pp. 1635-1642).

The adsorption of glucose oxidase (GOD) on graphene oxide (GO) nanoparticles without using any cross-linking reagents and/or additional surface modification was studied. Results of Fourier-transform infrared and ultraviolet–visible absorption spectroscopy confirmed that GOD was successfully immobilized on GO surface. The obtained immobilized GOD showed a wide range of pH stability and improved thermal and storage stability. In addition, GO exhibited good biocompatibility, which has potential advantages for biomedical and clinical diagnosis applications.

Keywords: Graphene oxide; Glucose oxidase; Electrostatic attraction; Immobilization; Stability

A Two-Stage Aerobic/Anaerobic Denitrifying Horizontal Bioreactor Designed for Treating Ammonium and H₂S Simultaneously by F. A. Chinalia; L. H. P. Garbossa; J. A. Rodriguez; K. R. Lapa; E. Foresti (pp. 1643-1654).

A two-stage bioreactor was operated for a period of 140 days in order to develop a post-treatment process based on anaerobic bioxidation of sulfite. This process was designed for simultaneously treating the effluent and biogas of a full-scale UASB reactor, containing significant concentrations of NH₄ and H₂S, respectively. The system comprised of two horizontal-flow bed-packed reactors operated with different oxygen concentrations. Ammonium present in the effluent was transformed into nitrates in the first aerobic stage. The second anaerobic stage combined the treatment of nitrates in the liquor with the hydrogen sulfide present in the UASB-reactor biogas. Nitrates were consumed with a significant production of sulfate, resulting in a nitrate removal rate of 0.43 kg N m³ day⁻¹ and ≥92 % efficiency. Such a removal rate is comparable to those achieved by heterotrophic denitrifying systems. Polymeric forms of sulfur were not detected (elementary sulfur); sulfate was the main product of the sulfide-based denitrifying process. S-sulfate was produced at a rate of about 0.35 kg m³ day⁻¹. Sulfur inputs as S–H₂S were estimated at about 0.75 kg m³ day⁻¹ and Chemical Oxygen Demand (COD) removal rates did not vary significantly during the process. DGGE profiling and 16S rRNA identified Halothiobacillus-like species as the key microorganism supporting this process; such a strain has not yet been previously associated with such bioengineered systems.

Keywords: Biogas treatment; Sulfide anaerobic oxidation; Denitrification; Lithotrophic denitrification

Effect of Low Culture Temperature on Urokinase Production in Hollow Fiber Reactor by Shilpa S. Khaparde; Pradip K. Roychoudhury (pp. 1655-1663).

The effect of culture temperature on urokinase production by HT-1080 cell line was studied in batch culture and hollow fiber reactor. Small-scale t-flask experiments revealed that urokinase production could be enhanced and media utilization could be reduced by lowering the culture temperature in production phase. Urokinase production was scaled up using a hollow fiber perfusion reactor system. Temperature of culture was maintained at the physiological 37 °C during growth phase that extended up to 12 days in hollow fiber bioreactor. Subsequently, in the production phase, culture temperature was lowered to 34 °C. Decrease in culture temperature resulted in a significant increase in urokinase production. Proteolytic degradation and inhibition was also minimized. The medium utilization rate was decreased at lower temperature, and hence, a higher economy of production could be obtained.

Keywords: Urokinase; Plasminogen activator; Hollow fiber bioreactor; Physiological shift

Influence of Biofilm Density on Anaerobic Sequencing Batch Biofilm Reactor Treating Mustard Tuber Wastewater by Hongxiang Chai; Wei Kang (pp. 1664-1671).

Considering the characteristics of high salinity, high concentration of organic matter, and high biodegradability, a new and efficient anaerobic sequencing batch biofilm reactor (ASBBR) was chosen as an anaerobic pretreatment unit to treat most organic compounds in mustard tuber wastewater. By changing the biofilm density of the reactor, the test was carried out to find out the influence of biofilm density on effluent COD, the content of the sludge dehydrogenase, and gas production rate. Results showed that under the condition of 30 °C, draining ratio of 1/3, and 2 days of hydraulic retention time, COD removal rate increased from 71.5 to 90.5 % when the biofilm density increased from 15 to 50 %; however, COD removal rate increased from 90.5 to 91.3 % when the biofilm density increased from 50 to 70 %. According to the influence of biofilm density on effluent COD, the content of the sludge dehydrogenase, and gas production rate, ASBBR should take 50 % biofilm density in mustard wastewater treatment. At the same time, these design parameters can be used to guide practical engineering.

Keywords: Anaerobic sequencing batch biofilm reactor (ASBBR); High-salinity wastewater; Mustard tuber wastewater; Biofilm density; COD removal rate

Screened Butanol-Tolerant Enterococcus faecium Capable of Butanol Production by Cindy Ng Wei Ting; Jinchuan Wu; Katsuyuki Takahashi; Ayako Endo; Hua Zhao (pp. 1672-1680).

Due to the complex mechanisms involved in butanol-induced stress response, butanol tolerance phenotype is difficult to engineer even in microorganisms with well-defined genetic backgrounds. We therefore aimed to isolate butanol-tolerant microorganisms from environmental samples as potential alternative hosts for butanol production. Soil samples collected were subjected to butanol stress. A microbial strain capable of 2.5–3 % (w/v) butanol tolerance was isolated and identified as Enterococcus faecium by 16S rDNA analysis. The isolate grew readily under both aerobic and anaerobic conditions and was capable of producing butanol anaerobically. In comparison with the obligate anaerobe Clostridium acetobutylicum, the growth under both aerobic and anaerobic conditions of the isolated strain, together with no detection of butyrate and lack of two-phase fermentation suggests different metabolic networks from the obligate anaerobe C. acetobutylicum. Under anaerobic condition, butanol reached up to 0.4 g l⁻¹ in a batch culture without heterologous introduction of butanol biosynthetic pathway. Besides butanol tolerance, the isolated E. faecium IB1 showed high tolerance to 10 % (w/v) ethanol and 3 % (w/v) isobutanol. With distinct features including high butanol tolerance and natural butanol production, the isolated E. faecium IB1 with minimum metabolic engineering can be explored as a potential host for butanol production.

Keywords: Butanol tolerance; Butanol production; Enterococcus faecium

Characterization of a Glycoside Hydrolase Family 1 β-Galactosidase from Hot Spring Metagenome with Transglycosylation Activity by Richa Gupta; Tanvi Govil; Neena Capalash; Prince Sharma (pp. 1681-1693).

A novel, thermostable, alkalophilic β-d-galactosidase (Mbgl) was isolated from a metagenome of geothermal springs in northern Himalayan region of India. Mbgl was 447 amino acids in size and had conserved catalytic residues E170 and E358, indicating that it belonged to family 1 of glycosyl hydrolases showing maximum homology (89 %) with uncharacterized β-galactosidase of Eubacterium, Meiothermus ruber DSM1279. Temperature and pH optima of Mbgl were 65 °C and 8.0 respectively, and it retained 80 % activity even at pH 10.0. Mbgl was active as a homotetramer, recognized β-(1,4)-d-galactoside as the preferred glycosidic bond, and preferentially hydrolyzed pNPgal with K _m 3.33 mM and k _cat 2,000 s⁻¹. It displayed high transglycosylation activity with wide acceptor specificity including hexoses and pentoses leading to the formation of prebiotic galacto-oligosaccharides whereas its lactose hydrolysis potential was low.

Keywords: β-Galactosidase; Metagenome; Thermostable; Glycosyl hydrolase family 1; Transglycosylation; Galacto-oligosaccharides

Engineering Glucosinolates in Plants: Current Knowledge and Potential Uses by Venkidasamy Baskar; Mayank Anand Gururani; Jae Woong Yu; Se Won Park (pp. 1694-1717).

Glucosinolates (GSL) and their derivatives are well known for the characteristic roles they play in plant defense as signaling molecules and as bioactive compounds for human health. More than 130 GSLs have been reported so far, and most of them belong to the Brassicaceae family. Several enzymes and transcription factors involved in the GSL biosynthesis have been studied in the model plant, Arabidopsis, and in a few other Brassica crop species. Recent studies in GSL research have defined the regulation, distribution, and degradation of GSL biosynthetic pathways; however, the underlying mechanism behind transportation of GSLs in plants is still largely unknown. This review highlights the recent advances in the metabolic engineering of GSLs in plants and discusses their potential applications.

Keywords: Brassicaceae; Glucosinolate; Myrosinase; Transgenic

Saliva Metabolomics Opens Door to Biomarker Discovery, Disease Diagnosis, and Treatment by Aihua Zhang; Hui Sun; Xijun Wang (pp. 1718-1727).

Metabolomics is the systematic study of the unique chemical fingerprints of low molecular weight endogenous metabolites or metabolite profiles in a biological sample. Metabolites that are important indicators of physiological or pathological states can provide information for the identification of early and differential markers for disease and help to understand its occurrence and progression. Analysis of these key biomarkers has become an important role to monitor the state of biological organisms and is a widely used diagnostic tool for disease. Metabolomic analyses are propelling the field of medical diagnostics forward at unprecedented rates because of its ability to reliably identify metabolites that are at the metabolic level in concentration. These advancements have benefited biomarker research to the point where saliva is now recognized as an excellent diagnostic medium for the detection of disease. Saliva contains a large array of metabolites, many of which can be informative for the detection of diseases. Salivary diagnostics offer an easy, inexpensive, safe, and noninvasive approach for disease detection. Discovery of salivary biomarkers that could be used to scrutinize health and disease surveillance has addressed its diagnostic value for clinical applications. Availability of emerging metabolomic techniques gives optimism that saliva can eventually be placed as a biomedium for clinical diagnostics. Comprehensive salivary metabolome will be an important resource for researchers who are studying metabolite chemistry, especially in the fields of salivary diagnostics, and will be helpful for analyzing and hence identifying corresponding disease-related salivary biomarkers. This review presents an overview of the value of saliva as a credible diagnostic tool, the discovery of salivary biomarkers, and the development of salivary diagnostics now and in the future. In particular, proof of principle has been demonstrated for salivary biomarker research.

Keywords: Metabolomics; System biology; Saliva analyses; Metabolites; Biomarkers; Disease diagnostics

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.168, #6)