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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.163, #7)

A Novel Magneto-fluorescent Nano-bioprobe for Cancer Cell Targeting, Imaging and Collection by Yicheng Wu; Maoquan Chu; Bizhi Shi; Zonghai Li (pp. 813-825).

Silica-coated magnetic polystyrene nanospheres (MPN) containing CdTe/CdS quantum dots (QDs) and Fe₃O₄ nanoparticles were prepared, and novel anti-EGFR antibodies were conjugated onto these magneto-fluorescent nanocomposites (MPN–QDs–SiO₂) for cancer cell targeting, imaging and collection. Transmission electron microscopy (TEM), scanning electron microscopy (SEM) images and energy-dispersive x-ray spectrometry (EDS) data showed that the MPN had been successfully coated with QDs and a silica shell, and the nanocomposites obtained with negative charged surfaces were well dispersed. The bioconjugates could be used for specifically labeling and separating cancer cells (MDA-MB-435S, SMMC-7721), but did not recognize and separate the K562 cells because the human epidermal growth factor receptor (EGFR) was not expressed on the surface. Because the anti-EGFR antibody, which we have developed, could specifically recognize certain cancer cells that highly expressed EGFR on their surface, these nanoscale bioconjugates, synchronously exhibiting fluorescence and magnetism, may be used in novel bioprobes for labeling and collecting rare cancer cells, which may be beneficial for early cancer diagnosis.

Keywords: Quantum dots; Magnetic nanospheres; Silica shell; Antibody; Cancer cell

The Slope Method: A Tool for Analyzing Semi-Continuous Data by Aaron Douglas Smith; Mark T. Holtzapple (pp. 826-835).

The MixAlco process is a biorefinery that converts lignocellulose into useful chemicals and hydrocarbon fuels via mixed-acid fermentation. For a semi-continuous-staged fermentation train, during each transfer, discrete amounts of material are moved between fermentors and data are tabulated. Because of natural day-to-day variations, the data are inherently noisy. To calculate performance parameters (e.g., yield, conversion, selectivity, productivity), the average flowrate of each stream component must be determined. To minimize error associated with noise, three data analysis methods were compared: Average, Accumulation, and Slope. The Average method determines the flowrate by averaging the amounts moved each transfer. The Accumulation method stores the solids and liquids that exit the fermentation train in separate vessels. After an extended time period, the mass in each storage vessel is measured so the average flowrate can be calculated. The Slope method calculates the flowrate of material in each stream from the slope of the moving cumulative sum with respect to time. For all three methods, the measured rates were virtually identical; thus accuracy was not affected by the method. However, for the examples presented, the Average method had >40% error and the Slope method <4% error; thus, precision was significantly affected by the method. The Accumulation method calculated the flowrate with a single data point so it is not possible to determine the statistical error.

Keywords: Slope method; Error reduction; Semi-continuous; Average method; Accumulation method; Data analysis; Staged fermentations; Analytical methods; MixAlco process

Extracellular Production and Characterization of Two Streptomyces l-Asparaginases by Tadashi Hatanaka; Hirokazu Usuki; Jiro Arima; Yoshiko Uesugi; Yukihiro Yamamoto; Yuya Kumagai; Akihiro Yamasato; Takafumi Mukaihara (pp. 836-844).

l-Asparaginase (ASNase) has proved its use in medical and food industries. Sequence-based screening showed the thermophilic Streptomyces strain Streptomyces thermoluteus subsp. fuscus NBRC 14270 (14270 ASNase) to positive against predicted ASNase primary sequences. The 14270 ASNase gene and four l-asparaginase genes from Streptomyces coelicolor, Streptomyces avermitilis, and Streptomyces griseus (SGR ASNase) were expressed in Streptomyces lividans using a hyperexpression vector: pTONA5a. Among those genes, only 14270 ASNase and SGR ASNase were successful for overexpression and detected in culture supernatants without an artificial signal peptide. Comparison of the two Streptomyces enzymes described above demonstrated that 14270 ASNase was superior to SGR ASNase in terms of optimum temperature, thermal stability, and pH stability.

Keywords: Asparaginase; Streptomyces ; Extracellular expression; Secretion; Asparagine

Applications of Loop-Mediated Isothermal DNA Amplification by Shijun Fu; Guanggang Qu; Shijin Guo; Lin Ma; Na Zhang; Songlin Zhang; Sanyang Gao; Zhiqiang Shen (pp. 845-850).

During the last 10 years, with the development of loop-mediated isothermal amplification (LAMP) method, it has been widely applied in nucleic acid analysis because of its simplicity, rapidity, high efficiency, and outstanding specificity. This method employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. Expensive equipment are not necessary to acquire a high level of precision, and there are fewer preparation steps compared to conventional PCR and real-time PCR assays. This paper briefly summarized the applications of LAMP method in pathogenic microorganisms, genetically modified ingredients, tumor detection, and embryo sex identification.

Keywords: Loop-mediated isothermal amplification; Pathogenic microorganisms; Detection; Application

Preconditioning of Axillary Buds in Thidiazuron-Supplemented Liquid Media Improves In Vitro Shoot Multiplication in Nyctanthes arbor-tristis L. by Anushi A. Jahan; M. Anis; Ibrahim M. Aref (pp. 851-859).

An efficient tissue culture technology has been designed for mass multiplication of Nyctanthes arbor-tristis L. by preculturing nodal explants in thidiazuron (TDZ)-supplemented liquid Murashige and Skoog (MS) media. Direct inoculation of nodal segments on semi-solid MS medium augmented with various concentrations of TDZ (0.1 to 0.9 μM) produced shoots but with low regeneration response and few shoots per explant. Hence, nodal explants were pretreated with greater concentrations of TDZ (5 to 100 μM) in liquid MS media for different durations (4, 8, 12, and 16 days) with the aim of improving shoot regeneration response from cultured explants. After pretreatment, explants were transferred to agar-solidified hormone-free MS medium. Best response in terms of percent regeneration (94%), number of shoots per explant (20.00 ± 1.15), and greatest shoot length (7.23 ± 0.83 cm) were obtained with nodal segments pretreated in75 μM TDZ for 8 days. Similarly, root induction was obtained from pulse-treated microshoots for 24 h with 200 μM indole-3-butyric acid (IBA) followed by their transfer to 1/2 MS medium which produced an average of 5.50 ± 0.92 roots per microshoot. The rooted plantlets were transplanted to soil with 80% success rate.

Keywords: Medicinal plant; Harsinghar; Nodal explants; Pretreatment

Plantlet Regeneration from Callus Cultures of Selected Genotype of Aloe vera L.—An Ancient Plant for Modern Herbal Industries by Mangal S. Rathore; J. Chikara; N. S. Shekhawat (pp. 860-868).

Aloe vera L., a member of Liliaceae, is a medicinal plant and has a number of curative properties. We describe here the development of tissue culture method for high-frequency plantlet regeneration from inflorescence axis-derived callus cultures of sweet aloe genotype. Competent callus cultures were established on 0.8% agar-gelled Murashige and Skoog’s (MS) basal medium supplemented with 6.0 mg l⁻¹ of 2,4-dichlorophenoxyacetic acid (2,4-D) and 100.0 mg l⁻¹ of activated charcoal and additives (100 mg l⁻¹ of ascorbic acid, 50.0 mg l⁻¹ each of citric acid and polyvinylpyrrolidone, and 25.0 mg l⁻¹ each of l-arginine and adenine sulfate). The callus cultures were cultured on MS medium containing 1.5 mg l⁻¹ of 2,4-D, 0.25 mg l⁻¹ of Kinetin (Kin), and additives with 4% carbohydrate source for multiplication and long-term maintenance of regenerative callus cultures. Callus cultures organized, differentiated, and produced globular embryogenic structures on MS medium with 1.0 mg l⁻¹ of 2,4-D, 0.25 mg l⁻¹ of Kin, and additives (50.0 mg l⁻¹ of ascorbic acid and 25.0 mg l⁻¹ each of citric acid, l-arginine, and adenine sulfate). These globular structures subsequently produced shoot buds and then complete plantlets on MS medium containing 1.0 mg l⁻¹ of 6-benzylaminopurine and additives. A hundred percent regenerated plantlets were hardened in the greenhouse and stored under an agro-net house/nursery. The regeneration system defined could be a useful tool not only for mass-scale propagation of selected genotype of A. vera, but also for genetic improvement of plant species through genetic transformation.

Keywords: Edible; In vitro ; Sweet aloe; Hardening and soilrite

Characterization of a Defined Cellulolytic and Xylanolytic Bacterial Consortium for Bioprocessing of Cellulose and Hemicelluloses by Benedict C. Okeke; Jue Lu (pp. 869-881).

Diminishing fossil fuel reserve and increasing cost of fossil hydrocarbon products have rekindled worldwide effort on conversion of lignocellloloses (plant biomass) to renewable fuel. Inedible plant materials such as grass, agricultural, and logging residues are abundant renewable natural resources that can be converted to biofuel. In an effort to mimic natural cellulolytic–xylanolytic microbial community in bioprocessing of lignocelluloses, we enriched cellulolytic–xylanolytic microorganisms, purified 19 monocultures and evaluated their cellulolytic–xylanolytic potential. Five selected isolates (DB1, DB2, DB7, DB8, and DB13) were used to compose a defined consortium and characterized by 16S ribosomal RNA gene sequence analysis. Nucleotide sequence blast analysis revealed that DB1, DB2, DB7, DB8, and DB13 were respectively similar to Pseudoxanthomonas byssovorax (99%), Microbacterium oxydans (99%), Bacillus sp. (99%), Ochrobactrum anthropi (98%), and Klebsiella trevisanii (99%). The isolates produced an array of cellulolytic–xylanolytic enzymes (filter paper cellulase, β-glucosidase, xylanase, and β-xylosidase), and significant activities were recorded in 30 min. Isolates DB1 and DB2 displayed the highest filter paper cellulase: 27.83 and 31.22 U mg⁻¹, respectively. The highest β-glucosidase activity (18.07 U mg⁻¹) was detected in the culture of isolate DB1. Isolate DB2 produced the highest xylanase activity (103.05 U mg⁻¹), while the highest β-xylosidase activity (7.72 U mg⁻¹) was observed with DB13. Use of microbial consortium in bioprocessing of lignocelluloses could reduce problems such as incomplete synergistic enzymes, end-product inhibition, adsorption, and requirement for high amounts of enzymes in direct use of enzymes.

Keywords: Bacterial consortium; rRNA gene sequence analysis; Cellulolytic–xylanolytic enzymes; Cellulose; Hemicelluloses; Bioprocessing

Characterization of Biosorption Process of Acid Orange 7 on Waste Brewery’s Yeast by YunHai Wu; Yue Hu; ZhengWei Xie; ShiXun Feng; Bin Li; XianMiao Mi (pp. 882-894).

The use of cheap, high-efficiency, and ecofriendly adsorbent has been studied as an alternative way for the removal of dyes from wastewater. This paper investigated the use of waste brewery’s yeast for the removal of acid orange 7 from aqueous solution. The optimum removal of acid orange 7 was found to be 3.561 mg/g at pH 2.0, 10 mg/L initial concentration and 303 K. The kinetic studies indicated that the biosorption process of acid orange 7 agreed well with the pseudo-second-order model. The external diffusion is the rate-controlling step of the initial fast adsorption (<20 min) and then the intraparticle diffusion dominated the mass transfer process. Langmuir, Freundlich, and Dubinin–Radushkevich models were applied to describe the biosorption isotherm of acid orange 7 by waste brewery’s yeast. Langmuir isotherm model fits the equilibrium data, at all the studied temperatures, better than the other isotherm models which indicates monolayer dye biosorption process. The highest monolayer biosorption capacity was found to be 2.27 × 10⁻³ mol/g at 303 K. The calculated thermodynamic parameters (ΔG, ΔS, ΔH) showed the biosorption process to be spontaneous and exothermic in nature. Amine or amino, amide, carboxyl, phosphate groups are responsible for the dyes biosorption based on the result of Fourier transform infrared analysis.

Keywords: Biosorption; Waste brewer’s yeast; Acid orange 7; Thermodynamic

Cloning and Expression of Che a 1, the Major Allergen of Chenopodium album in Escherichia coli by Fatemeh Vahedi; Mojtaba Sankian; Malihe Moghadam; Maryam Mohaddesfar; Sirous Ghobadi; Abdol Reza Varasteh (pp. 895-905).

Chenopodium album is a weedy annual plant in the genus Chenopodium. C. album pollen represents a predominant allergen source in Iran. The main C. album pollen allergens have been described as Che a 1, Che a 2, and Che a 3. The aim of this work was to clone the Che a 1 in Escherichia coli to establish a system for overproduction of the recombinant Che a 1 (rChe a 1). In order to clone this allergen, the pollens were subjected to RNA extraction. A full-length fragment encoding Che a 1 was prepared by polymerase chain reaction amplification of the first-strand cDNA synthesized from extracted RNA. Cloning was carried out by inserting the cDNA into the pET21b (+) vector, thereafter the construct was transformed into E. coli Top10 cells and expression of the protein was induced by IPTG. The rChe a 1 was purified using histidine tag in recombinant protein by means of Ni–NTA affinity chromatography. IgE immunoblotting, ELISA, and inhibition ELISA were done to evaluate IgE binding of the purified protein. In conclusion, the cDNA for the major allergen of the C. album pollen, Che a 1, was successfully cloned and rChe a 1 was purified. Inhibition assays demonstrated allergic subjects sera reacted with rChe a 1 similar to natural Che a 1 in crude extract of C. album pollen. This study is the first report of using the E. coli as a prokaryotic system for Che a 1 cloning and production of rChe a 1.

Keywords: Allergy; Recombinant allergen; Chenopodium album ; Che a 1; Cloning

Bioconversion of d-glucose into d-glucosone by Glucose 2-oxidase from Coriolus versicolor at Moderate Pressures by Amin Karmali; José Coelho (pp. 906-917).

Glucose 2-oxidase (pyranose oxidase, pyranose:oxygen-2-oxidoreductase, EC 1.1.3.10) from Coriolus versicolor catalyses the oxidation of d-glucose at carbon 2 in the presence of molecular O₂ producing d-glucosone (2-keto-glucose and d-arabino-2-hexosulose) and H₂O₂. It was used to convert d-glucose into d-glucosone at moderate pressures (i.e. up to 150 bar) with compressed air in a modified commercial batch reactor. Several parameters affecting biocatalysis at moderate pressures were investigated as follows: pressure, [enzyme], [glucose], pH, temperature, nature of fluid and the presence of catalase. Glucose 2-oxidase was purified by immobilized metal affinity chromatography on epoxy-activated Sepharose 6B-IDA-Cu(II) column at pH 6.0. The rate of bioconversion of d-glucose increased with the pressure since an increase in the pressure with compressed air resulted in higher rates of conversion. On the other hand, the presence of catalase increased the rate of reaction which strongly suggests that H₂O₂ acted as inhibitor for this reaction. The rate of bioconversion of d-glucose by glucose 2-oxidase in the presence of either nitrogen or supercritical CO₂ at 110 bar was very low compared with the use of compressed air at the same pressure. The optimum temperature (55°C) and pH (5.0) of d-glucose bioconversion as well as kinetic parameters for this enzyme were determined under moderate pressure. The activation energy (E _a) was 32.08 kJ mol⁻¹ and kinetic parameters (V _max, K _m, K _cat and K _cat/K _m) for this bioconversion were 8.8 U mg⁻¹ protein, 2.95 mM, 30.81 s⁻¹ and 10,444.06 s⁻¹ M⁻¹, respectively. The biomass of C. versicolor as well as the cell-free extract containing glucose 2-oxidase activity were also useful for bioconversion of d-glucose at moderate pressures. The enzyme was apparently stable at moderate pressures since such pressures did not affect significantly the enzyme activity.

Keywords: Biocatalysis; Moderate pressures; Glucose 2-oxidase; Coriolus versicolor ; Bioconversion of d-glucose into d-glucosone; Catalase; Kinetic parameters

Combining Enzymatic Esterification with Conventional Alkaline Transesterification in an Integrated Biodiesel Process by Jesper Brask; Marianne Linde Damstrup; Per Munk Nielsen; Hans Christian Holm; Jeroen Maes; Wim De Greyt (pp. 918-927).

An integrated biodiesel process that combines enzymatic esterification and alkaline transesterification is suggested. With focus on the enzymatic step, the paper provides proof of concept and suggestions for further process development. Hence, palm fatty acid distillate (PFAD) has been enzymatically converted to fatty acid methyl esters in a two-step process using the immobilized lipase Novozym 435 in packed-bed columns. With only a small excess of methanol, the first reaction stage could reduce the free fatty acid (FFA) content from 85% to 5%. After removal of water by simple phase separation, it was possible to lower the FFA content to 2.5% in a second reaction stage. Both reaction stages are relatively fast with suggested reaction times of 15 min in column 1 (productivity 10 kg/kg/h) and 30 min in column 2 (productivity 5 kg/kg/h), resulting in 15% FFA after column 1 and 5% FFA after column 2. A lifetime study indicated that approximately 3,500 kg PFAD/kg Novozym 435 can be treated in the first reaction stage before the enzyme has become fully inactivated. With further optimization, the enzymatic process could be a real alternative to today’s sulfuric acid catalyzed process.

Keywords: Fatty acid methyl ester (FAME); Biodiesel; Palm fatty acid distillate (PFAD); Enzyme; Lipase; Novozym 435; Esterification; Packed-bed reactor (PBR)

Ethanol Production from Residual Wood Chips of Cellulose Industry: Acid Pretreatment Investigation, Hemicellulosic Hydrolysate Fermentation, and Remaining Solid Fraction Fermentation by SSF Process by Neumara Luci Conceição Silva; Gabriel Jaime Vargas Betancur; Mariana Peñuela Vasquez; Edelvio de Barros Gomes; Nei Pereira Jr. (pp. 928-936).

Current research indicates the ethanol fuel production from lignocellulosic materials, such as residual wood chips from the cellulose industry, as new emerging technology. This work aimed at evaluating the ethanol production from hemicellulose of eucalyptus chips by diluted acid pretreatment and the subsequent fermentation of the generated hydrolysate by a flocculating strain of Pichia stipitis. The remaining solid fraction generated after pretreatment was subjected to enzymatic hydrolysis, which was carried out simultaneously with glucose fermentation [saccharification and fermentation (SSF) process] using a strain of Saccharomyces cerevisiae. The acid pretreatment was evaluated using a central composite design for sulfuric acid concentration (1.0–4.0 v/v) and solid to liquid ratio (1:2–1:4, grams to milliliter) as independent variables. A maximum xylose concentration of 50 g/L was obtained in the hemicellulosic hydrolysate. The fermentation of hemicellulosic hydrolysate and the SSF process were performed in bioreactors and the final ethanol concentrations of 15.3 g/L and 28.7 g/L were obtained, respectively.

Keywords: Residual wood chips; Acid pretreatment; Hemicellulose; Cellulose; Bioethanol

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.163, #7)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.163, #7)