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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.158, #1)

DNA Interaction with PtCl₂(LL) (LL = Chelating Diamine Ligand: N,N-Dimethyltrimethylendiamine) Complex by Nahid Shahabadi; Soheila Kashanian; Kani Shalmashi; Hamideh Roshanfekr (pp. 1-10).

The [PtCl₂(LL)] complex, as a cisplatin derivative, which LL is diamine chelate ligand (N,N-dimethyltrimethylendiamine), was synthesized and characterized by elemental analysis (CHN) mass, ¹H, and ¹³C nuclear magnetic resonance techniques. Then the binding of this complex to calf thymus DNA was investigated by various physicochemical methods such as spectrophotometric, circular dichroism, spectrofluorometric, melting temperature, and viscosimetric techniques. Upon addition of the complex, important changes were observed in the characteristic UV–Vis bands (hypochromism) of calf thymus DNA, increase in melting temperature and some changes in specific viscosity. Also, the fluorescence spectral characteristics showed an increase in the fluorescence intensity of methylene blue–DNA solutions in the presence of increasing amounts of metal complex, indicating PtCl₂(LL) is able to displace the methylene blue bound to DNA but not as complete as intercalative molecules. The experimental results showed that the platinum complex is bound to DNA non-intercalatively, and an outside binding is the preferred mode of interaction.

Keywords: Pt (II) complex; CT-DNA; Outside binding; DNA melting

Separation and purification of aloe polysaccharides by a combination of membrane ultrafiltration and aqueous two-phase extraction by Jian-min Xing; Fen-fang Li (pp. 11-19).

A two-step process was developed for the purification of polysaccharides from the pulp of Aloe varavia using aqueous two-phase system (ATPS) extraction and a novel copolymer ultrafiltration membrane. The first step was ATPS under optimal separations conditions using a total composition of 18% PEG2000, 25% ammonium sulfate, pH 3.0, and 0.3 M NaCl. To form the copolymer membrane, poly(acrylonitrile-acrylamide-styrene) was prepared by solution polycondensation using azoisobutyronitrile as initiator. Then, membranes were formed from the dissolved copolymer by the phase inversion method. Copolymer structure was investigated by infrared spectrum and thermogravimetric analysis (TGA). The copolymer membrane surface and cross section were observed by scanning electron microscopy. The water flux of this membrane was 26.33 mL/(cm² h), and retention was 96% for bovine serum albumin and 34% for dextran T40000. The separation and purification of aloe polysaccharide were carried using this copolymer membrane following ATPS. The TGA of aloe polysaccharide demonstrated a high purity of the polysaccharide. By gas chromatographic analysis, it was shown that mannose is the main monosaccharide in the aloe polysaccharide, and only a few glucose residues are present.

Keywords: Ultrafiltration membrane; Water flux; Aqueous two phase; Aloe polysaccharide; Purification

Optimization of the Production Medium for Biosynthesis of Antifungal Antibiotic Ak-111-81 by Phosphate-Deregulated Mutant of Streptomyces hygroscopicus by Victoria Gesheva (pp. 20-24).

Experimental mathematical designs were applied for optimization of a nutrient medium for biosynthesis of the antifungal antibiotic AK-111-81 by phosphate-deregulated mutant of Streptomyces hygroscopicus 111-81. Antifungal antibiotic AK-111-81 possesses well-expressed activity against Fusarium graminearum and other phytopathogenic fungi. The level of the production of the antibiotic AK-111-81 on this medium is more than three times higher than on the initial medium. The optimized quantitative composition of the nutrient culture media is (g/l): glucose, 20; soy meal, 18; ammonium succinate, 3; CaCO₃, 1.

Keywords: Streptomyces hygroscopicus ; Phosphate-deregulated mutant; Optimal production medium; Optimization; Antifungal antibiotic AK-111-81

Constitutive Expression and Optimization of Nutrients for Streptokinase Production by Pichia pastoris Using Statistical Methods by Ravi Nagaraj Vellanki; Ravichandra Potumarthi; Lakshmi Narasu Mangamoori (pp. 25-40).

The Pichia pastoris clone producing streptokinase (SK) was optimized for its nutritional requirements to improve intracellular expression using statistical experimental designs and response surface methodology. The skc gene was ligated downstream of the native glyceraldehyde 3-phosphate dehydrogenase promoter and cloned in P. pastoris. Toxicity to the host was not observed by SK expression using YPD medium. The transformant producing SK at level of 1,120 IU/ml was selected, and the medium composition was investigated with the aim of achieving high expression levels. The effect of various carbon and nitrogen sources on SK production was tested by using Plackett–Burman statistical design and it was found that dextrose and peptone are the effective carbon and nitrogen sources among all the tested. The optimum conditions of selected production medium parameters were predicted using response surface methodology and the maximum predicted SK production of 2,136.23 IU/ml could be achieved with the production medium conditions of dextrose (x1), 2.90%; peptone (x2), 2.49%; pH, 7.2 (x3), and temperature, 30.4 (x4). Validation studies showed a 95% increase in SK production as compared to that before optimization at 2,089 IU/ml. SK produced by constitutive expression was found to be functionally active by plasminogen activation assay and fibrin clot lysis assay. The current recombinant expression system and medium composition may enable maximum production of recombinant streptokinase at bioreactor level.

Keywords: Pichia pastoris ; Streptokinase; Constitutive expression; Placket–Burman design; Response surface methodology (RSM); Optimization

Production of γ-Decalactone by a Psychrophilic and a Mesophilic Strain of the Yeast Rhodotorula aurantiaca by Mohamed Alchihab; Jacqueline Destain; Mario Aguedo; Lamia Majad; Hakim Ghalfi; Jean-Paul Wathelet; Philippe Thonart (pp. 41-50).

Among 18 psychrophilic strains isolated near the Antarctic Station, the psychrophilic strain Rhodotorula aurantiaca A19 was selected for its ability of growth and γ-decalactone production at low temperatures. The effects of temperature, initial pH, and castor oil concentration on the growth and γ-decalactone production by a psychrophilic and a mesophilic strain of R. aurantiaca were investigated. The highest γ-decalactone production in flasks (5.8 g/l) was obtained with the strain A19 at 14 °C and initial pH 7.0 in medium containing 20 g/l castor oil. On the other hand, these factors did not affect the production of γ-decalactone by the mesophilic strain. In fermentor, a γ-decalactone concentration of 6.6 g/l was reached with the strain A19, whereas a maximum of 0.1 g/l was obtained with the mesophilic strain. Our results suggest that the ability to synthesize γ-decalactone is a particularity of the strain A19, since the mesophilic strain (no. 30645) produced small amounts, and the other (no. 31354) did not exhibit this property. It is, to our knowledge, the first report of γ-decalactone production by R. aurantiaca and furthermore by a psychrophilic yeast strain. Moreover, the amount of γ-decalactone obtained in fermentor with the strain 19 was on the order of concentrations usually described in patents.

Keywords: Rhodotorula aurantiaca ; Psychrophilic; Mesophilic; Castor oil; γ-Decalactone

Can Hg(II) be Determined via Quenching of the Emission of Green Fluorescent Protein from Anemonia sulcata var. smaragdina? by Serap Seyhan Bozkurt; Levent Cavas (pp. 51-58).

Anemonia sulcata var. smaragdina is a widely distributed Cnidarian species along Turkish coastlines. It is also a well-known example of a facultative symbiotic life form in sea ecosystems. Green fluorescent proteins (GFPs) in Anemonia sulcata var. smaragdina have vital roles in this symbiotic form. The fluorescence quenching by Hg(II) in the supernatants obtained from A. sulcata var. smaragdina was shown in this study. According to results, there was a statistical significant relationship (R ² = 0.9913) between increased Hg(II) concentration and decreased fluorescence intensity of GFP supernatants obtained from A. sulcata var. smaragdina. Mn(II), Fe(II), and Al(II) showed no interference effect and did not change the fluorescence intensity of GFP supernatants obtained from A. sulcata var. smaragdina. In conclusion, the fluorescence quenching of GFPs by Hg(II) can be a novel method to determine the Hg(II) levels in aqueous solution. Therefore, further researches are strongly warranted because of the possible potential applications of the fluorescence quenching of GFPs by Hg(II).

Keywords: Anemonia sulcata var. smaragdina ; Hg(II); Fluorescence; Quenching; Heavy metals

Expression Analysis and Characteristics of Profilin Gene from Silkworm, Bombyx mori by Zuoming Nie; Jiangtao Xu; Jian Chen; Zhengbing Lv; Dan Wang; Qing Sheng; Yi Wu; Xuedong Wang; Xiangfu Wu; Yaozhou Zhang (pp. 59-71).

A recombinant Bombyx mori profilin protein (rBmPFN) was overexpressed in Escherichia coli BL21. Purified rBmPFN was used to generate anti-BmPFN polyclonal antibody, which were used to determine the subcellular localization of BmPFN. Immunostaining indicated that profilin can be found in both the nucleus and cytoplasm but is primarily located in the cytoplasm. Real-time RT-PCR and Western blot analyses indicated that, during the larvae stage, profilin expression levels are highest in the silk gland, followed by the gonad, and are lowest in the fatty body. Additionally, BmPFN expression begins during the egg stage, increases during the larvae stage, reaches a peak during the pupa stage, and decreases significantly in the moth. Therefore, we propose that BmPFN may play an important role during larva stage development, especially in the silk gland.

Keywords: Profilin; Expression analysis; Subcellular localization; Real-time PCR; Bombyx mori

Improvement on Citric Acid Production in Solid-state Fermentation by Aspergillus niger LPB BC Mutant Using Citric Pulp by Cristine Rodrigues; Luciana Porto de Souza Vandenberghe; Juliana Teodoro; Ashok Pandey; Carlos Ricardo Soccol (pp. 72-87).

Citric acid (CA) production has been conducted through a careful strain selection, physical–chemical optimization and mutation. The aim of this work was to optimize the physical–chemical conditions of CA production by solid-state fermentation (SSF) using the Aspergillus niger LPB BC strain, which was isolated in our laboratory. The parental and mutant strain showed a good production of CA using citric pulp (CP) as a substrate. The physical–chemical parameters were optimized and the best production was reached at 65% moisture, 30 °C and pH 5.5. The influence of the addition of commercial and alternative sugars, nitrogen sources, salts, and alcohols was also studied. The best results (445.4 g of CA/kg of CP) were obtained with sugarcane molasses and 4% methanol (v/w). The mutagenesis induction of LPB BC was performed with UV irradiation. Eleven mutant strains were tested in SSF where two mutants showed a higher CA production when compared to the parental strain. A. niger LPB B3 produced 537.6 g of CA/kg of CP on the sixth day of fermentation, while A. niger LPB B6 produced 616.5 g of CA/kg of CP on the fourth day of fermentation, representing a 19.5% and 37% gain, respectively.

Keywords: Citric acid; CP; Aspergillus niger ; Solid-state fermentation; Mutation; UV

Influence of Silica-Derived Nano-Supporters on Cellobiase After Immobilization by Peng Wang; Xiaoke Hu; Sean Cook; Huey-Min Hwang (pp. 88-96).

Core shell magnetite nanoparticle (CSMN) was successfully synthesized with diameter around 125 nm according to the determination with scanning electronic microscopy. SBA-15 with diameter around 31 nm was synthesized in our previous work as another supporter for immobilized degradation enzymes. The aim of this study was to investigate the influence of silica-derived nano-supporters on cellobiase after immobilization. With covalent method, glutaraldehyde was introduced to immobilize cellobiase. The immobilized enzyme efficiency, specific activity, and its characterization, including optimum pH, pH stability, optimum temperature for enzyme reaction, and enzyme thermal stability were investigated. Results show that the method of enzyme immobilization on both nano-supporters could improve cellobiase stability under low pH and high temperature conditions compared with the free enzyme. In the aspect of immobilization efficiency, SBA had higher amount of bounded protein than that of CSMN, but had lower specific enzyme activity than CSMN, assumably due to the change in silica surface properties caused by process of supporter synthesis.

Keywords: Cellobiase; Immobilization; Core shell magnetite nanoparticle; SBA

Activity of some Mucolytics Against Bacterial Adherence to Mammalian Cells by Mohamed M. Hafez; Mohammad M. Aboulwafa; Mahmoud A. Yassien; Nadia A. Hassouna (pp. 97-112).

In this article, some mucolytic agents were tested for their activity to prevent bacterial adherence to mammalian cells. Preliminary screening for antiadherent activity showed that ambroxol, bromhexine, ammonium chloride, and ammonium acetate but neither guaiphenesin nor carbocysteine significantly reduced the adherence of the tested clinical isolates to cultured mammalian cells. The antiadherent effect of such agents was observed when mammalian cells were treated with these agents either before or after bacterial adherence, and this effect was exhibited in a dose-dependent manner. The minimum concentrations of ambroxol, bromhexine, ammonium chloride, and ammonium acetate required for mammalian cells treatment to prevent bacterial adherence were 2.5, 5, 50, and 20 ng/ml, respectively, whereas significantly higher mucolytic concentrations were required to eradicate bacteria that adhered to mammalian cells. Upon treatment of mammalian cells with mucolytics, the maximum reduction in adherence of the tested isolates attained by ambroxol, bromhexine, ammonium chloride, ammonium acetate were 99%, 98%, 75%, and 54% to that of control, respectively. Insignificant difference existed between the antiadherent activities of ambroxol and bromhexine, while both agents had significantly higher effect than ammonium chloride and ammonium acetate. Pretreatment of the immobilized mucin with ambroxol, bromhexine, ammonium chloride, or ammonium acetate reduced the adherence of Pseudomonas aeruginosa, Escherichia coli, and staphylococcal isolates to this receptor analogue. A strong correlation was observed for the antiadherent activity of the tested mucolytics in case of mammalian cells and immobilized mucin. Moreover, pretreatment of the immobilized receptor analogues chondroitin sulfate-B and heparin with the abovementioned agents significantly reduced the adherence of Staphylococcus aureus, P. aeruginosa, and E. coli isolates to such immobilized glycoproteins.

Keywords: Bacterial adherence; Mammalian cells; Mucolytics

Overproduction of Glucoamylase by a Deregulated Mutant of a Thermophilic Mould Thermomucor indicae-seudaticae by Pardeep Kumar; T. Satyanarayana (pp. 113-125).

Thermomucor indicae-seudaticae, a glucoamylase-producing thermophilic mould, was mutagenised using nitrous acid and gamma (⁶⁰Co) irradiation in a sequential manner to isolate deregulated mutants for enhanced production of glucoamylase. The mutants were isolated on Emerson YpSs agar containing a non-metabolisable glucose analogue 2-deoxy-d-glucose (2-DG) for selection. The preliminary screening for glucoamylase production using starch–iodine plate assay followed by quantitative confirmation in submerged fermentation permitted the isolation of several variants showing varying levels of derepression and glucoamylase secretion. The mutant strain T. indicae-seudaticae CR19 was able to grow in the presence of 0.5 g l⁻¹ 2-DG and produced 1.8-fold higher glucoamylase. As with the parent strain, glucoamylase production by T. indicae-seudaticae CR19 in 250-ml Erlenmeyer flasks attained a peak in 48 h of fermentation, showing higher glucoamylase productivity (0.67 U ml⁻¹ h⁻¹) than the former (0.375 U ml⁻¹ h⁻¹). A large-scale cultivation in 5-l laboratory bioreactor confirmed similar fermentation profiles, though the glucoamylase production peak was attained within 36 h attributable to the better control of process parameters. Although the mutant grew slightly slow in the presence of 2-DG and exhibited less sporulation, it showed faster growth on normal Emerson medium with a higher specific growth rate (0.138 h⁻¹) compared to the parent strain (0.123 h⁻¹). The glucoamylase produced by both strains was optimally active at 60 °C and pH 7.0 and displayed broad substrate specificity by cleaving α-1,4- and α-1,6-glycosidic linkages in starch, amylopectin, amylose and pullulan. Improved productivity and higher specific growth rate make T. indicae-seudaticae CR19 a useful strain for glucoamylase production.

Keywords: 2-Deoxy-d-glucose; Mutagenesis; Deregulated mutant; Glucoamylase; γ-Irradiation; Thermomucor indicae-seudaticae

Fifty-gigahertz Microwave Exposure Effect of Radiations on Rat Brain by Kavindra Kumar Kesari; J. Behari (pp. 126-139).

The object of this study is to investigate the effects of 50-GHz microwave radiation on the brain of Wistar rats. Male rats of the Wistar strain were used in the study. Animals of 60-day age were divided into two groups—group 1, sham-exposed, and group 2, experimental (microwave-exposed). The rats were housed in a temperature-controlled room (25 °C) with constant humidity (40–50%) and received food and water ad libitum. During exposure, rats were placed in Plexiglas cages with drilled ventilation holes and kept in an anechoic chamber. The animals were exposed for 2 h a day for 45 days continuously at a power level of 0.86 μW/cm² with nominal specific absorption rate 8.0 × 10⁻⁴ w/kg. After the exposure period, the rats were killed and homogenized, and protein kinase C (PKC), DNA double-strand break, and antioxidant enzyme activity [superoxides dismutase (SOD), catalase, and glutathione peroxidase (GPx)] were estimated in the whole brain. Result shows that the chronic exposure to these radiations causes DNA double-strand break (head and tail length, intensity and tail migration) and a significant decrease in GPx and SOD activity (p = <0.05) in brain cells, whereas catalase activity shows significant increase in the exposed group of brain samples as compared with control (p = <0.001). In addition to these, PKC decreased significantly in whole brain and hippocampus (p < 0.05). All data are expressed as mean ± standard deviation. We conclude that these radiations can have a significant effect on the whole brain.

Keywords: Glutathione peroxidase; Superoxidase; Catalase; Microwave radiation; Protein kinase C

Cloning, High Expression and Purification of Recombinant Human Intereferon-β-1b in Escherichia coli by Dasari Venkata Krishna Rao; Chatadi Tulasi Ramu; Joginapally Venkateswara Rao; Mangamoori Lakshmi Narasu; Adibhatla Kali Satya Bhujanga Rao (pp. 140-154).

Sequential evaluation and process control strategy were employed for impurity profile and high recovery with quality of rhIFN-β-1b expressed in Escherichia coli. The high-level expression was achieved by using codon substitution (AT content of 52.6% at N-terminal region) and optimization of culture conditions. The addition of rifampicin at a concentration of 200 μg/ml has increased the specific product yield of 66 mg optical density⁻¹ l⁻¹ (43.5% of total cellular protein). Eighty-three percent of lipopolysaccharides, 32% of host deoxyribonucleic acid (DNA), and 78% of host cell proteins were removed by 0.75% Triton X-100 and 2 M urea wash. Eleven percent of lipopolysaccharides, 39% of host DNA, and 12% of host cell proteins were removed at the solubilization step. Ninety-two percent of protein refolding was achieved by high-pressure diafiltration method. Refolding by high-pressure diafiltration, bed height, and height equivalent to the theoretical plate value in chromatography column were identified as key parameters for high recovery with purity. Finally, the established process yielded 34% of purified protein with greater than 99% purity and is acceptable for preclinical toxicological studies. The purified rhIFN-β-1b obtained in this study is the highest that has been reported so far.

Keywords: High-level expression; Recombinant human IFN-β-1b; Site directed mutagenesis; Specific product yield; High purity

Poly-β-hydroxybutyrate Production by Fast-Growing Rhizobia Cultivated in Sludge and in Industrial Wastewater by Faouzi Ben Rebah; Danielle Prévost; Rajeshwar Dayal Tyagi; Lassaad Belbahri (pp. 155-163).

In our study, the potential of producing polyhydroxybutyrate (PHB) by cultivating fast-growing rhizobia (Sinorhizobium meliloti, Rhizobium leguminosarum bv. viciae, R. leguminosarum bv. phaseoli and R. leguminosarum bv. trifolii) in sludge and in industrial wastewater was evaluated. Results confirmed the possibility of using sludge as media for rhizobial growth. During growth, substantial quantity of PHB was accumulated and yields varied depending on the media and rhizobial species. Growing in sludge, PHB production did not exceed 3.7% w/w for all strains at the end of experiment (after 72 h). During the growth of S. meliloti, PHB yield varied and the maximum value reached 7.27% w/w after 60 h, with 1% Total Suspend Solid (TSS) sludge. Alkaline sludge pre-treatment affects rhizobial growth but did not improve the PHB accumulation. While growing S. meliloti in industrial wastewater, the PHB yields varied and the highest value was obtained with slaughterhouse wastewater (10.7% w/w) after 35 h of growth. Therefore, this work shows the potential of exploiting PHB production by rhizobia growing in wastewater or sludge which could be applied to bioplastic industry, and confirms the potential of these recyclable wastes for high production of rhizobial cells useable for legumes inoculants production. This study provides an environmentally sound way of sludge and wastewater management and use in diverse biotechnological applications.

Keywords: Poly-β-hydroxybutyrate; Fast-growing rhizobia; Sludge; Industrial wastewater

Evaluation and Characterization of Forage Sorghum as Feedstock for Fermentable Sugar Production by D. Y. Corredor; J. M. Salazar; K. L. Hohn; S. Bean; B. Bean; D. Wang (pp. 164-179).

Sorghum is a tropical grass grown primarily in semiarid and drier parts of the world, especially areas too dry for corn. Sorghum production also leaves about 58 million tons of by-products composed mainly of cellulose, hemicellulose, and lignin. The low lignin content of some forage sorghums such as brown midrib makes them more digestible for ethanol production. Successful use of biomass for biofuel production depends on not only pretreatment methods and efficient processing conditions but also physical and chemical properties of the biomass. In this study, four varieties of forage sorghum (stems and leaves) were characterized and evaluated as feedstock for fermentable sugar production. Fourier transform infrared spectroscopy and X-ray diffraction were used to determine changes in structure and chemical composition of forage sorghum before and after pretreatment and the enzymatic hydrolysis process. Forage sorghums with a low syringyl/guaiacyl ratio in their lignin structure were easy to hydrolyze after pretreatment despite the initial lignin content. Enzymatic hydrolysis was also more effective for forage sorghums with a low crystallinity index and easily transformed crystalline cellulose to amorphous cellulose, despite initial cellulose content. Up to 72% hexose yield and 94% pentose yield were obtained using modified steam explosion with 2% sulfuric acid at 140 °C for 30 min and enzymatic hydrolysis with cellulase (15 filter per unit (FPU)/g cellulose) and β-glucosidase (50 cellobiose units (CBU)/g cellulose).

Keywords: Forage sorghum; FTIR; XRD; Enzymatic hydrolysis; Dilute acid; Pretreatment

Key Factors Regarding Decolorization of Synthetic Anthraquinone and Azo Dyes by A. Boonyakamol; T. Imai; P. Chairattanamanokorn; T. Higuchi; M. Sekine (pp. 180-191).

The factors affecting decolorization of anthraquinone dye represented by Reactive Blue 4 (RB4) and azo dye represented by Methyl Orange (MO) were studied in batch experiments under mesophilic (35 °C) and thermophilic (55 °C) anaerobic conditions. The results indicated differences in decolorization properties of the dyes with different chromophore structures. In abiotic conditions, MO could be decolorized by a physicochemical reaction when it was sterilized at 121 °C together with sludge cells or glucose. RB4 only showed absorption onto the cell mass. The presence of a redox mediator accelerated the decolorizing reaction when supplied together with glucose in the presence of sterilized sludge cells. In biotic conditions, the results indicated that the biological activity of microorganisms was an important factor in decolorization. The main factor involved in decolorization was the conversion of cosubstrate as electron donor, which reacted with dye as an electron acceptor in electron transfer. Redox mediators, anthraquinone-2-sulfonic acid, and anthraquinone could accelerate decolorization even if a small amount (0.2 mM) was applied. On the other hand, a high concentration of redox mediator (1.0 mM) had an inhibitory effect on decolorization especially under thermophilic conditions. In addition, the decolorization of dye was accelerated by increasing treatment temperature, as shown in biotic treatments. Based on these results, increasing the treatment temperature could be used to improve the decolorizing process of textile dye wastewater treatment, especially for recalcitrant dyes such as anthraquinone.

Keywords: Factors of decolorization; Synthetic dye; Abiotic; Biotic; Anaerobic treatment; Redox mediator

Purification and Characterization of a Novel β-Galactosidase with Transglycosylation Activity from Bacillus megaterium 2-37-4-1 by Yumei Li; Hongmei Wang; Lili Lu; Zhengyi Li; Xiaodong Xu; Min Xiao (pp. 192-199).

A novel β-galactosidase of 120 kDa (BgaBM) from Bacillus megaterium 2-37-4-1 was purified, and its gene (bgaBM) was analyzed and expressed. It displayed wide acceptor specificity for transglycosylation with a series of acceptors, including pentose, hexose, hydroxyl, and alkyl alcohol using o-nitrophenyl-β-d-galactoside (ONPG) as a donor. BgaBM preferentially hydrolyzed ONPG in all tested substrates and showed maximum activity at pH 7.5–8.0 and 55 °C. It was stable at pH 6.0–9.0 below 40 °C. The K _m and V _max values for ONPG and lactose were 9.5 mM, 16.6 mM/min and 12.6 mM, 54.4 mM/min, respectively. The nucleotide sequence of the bgaBM gene consists of an ORF of 3,105 bp corresponding to 118 kDa protein, which indicates that BgaBM is a modular enzyme in the glycosyl hydrolase family 2, including conserved sugar-binding domain, acid–base catalyst, and immunoglobulin-like beta-sandwich domain. The possible acid/base and nucleophile sites of BgaBM were estimated to be E481 and E547, respectively. Furthermore, expression of the bgaBM gene in Escherichia coli and purification of the recombinant enzyme were performed. The recombinant enzyme showed similar biochemical characteristics to natural enzyme.

Keywords: β-Galactosidase; Purification; Bacillus megaterium ; Transglycosylation; Gene

Highly Thermostable Xylanase of the Thermophilic Fungus Talaromyces thermophilus: Purification and Characterization by Ines Maalej; Ines Belhaj; Najla Fourati Masmoudi; Hafedh Belghith (pp. 200-212).

A thermostable xylanase from a newly isolated thermophilic fungus Talaromyces thermophilus was purified and characterized. The enzyme was purified to homogeneity by ammonium sulfate precipitation, diethylaminoethyl cellulose anion exchange chromatography, P-100 gel filtration, and Mono Q chromatography with a 23-fold increase in specific activity and 17.5% recovery. The molecular weight of the xylanase was estimated to be 25kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and gel filtration. The enzyme was highly active over a wide range of pH from 4.0 to 10.0. The relative activities at pH5.0, 9.0, and 10.0 were about 80%, 85.0%, and 60% of that at pH7.5, respectively. The optimum temperature of the purified enzyme was 75°C. The enzyme showed high thermal stability at 50°C (7days) and the half-life of the xylanase at 100°C was 60min. The enzyme was free from cellulase activity. K _m and V _max values at 50°C of the purified enzyme for birchwood xylan were 22.51mg/ml and 1.235μmol min⁻¹ mg⁻¹, respectively. The enzyme was activated by Ag⁺, Co²⁺, and Cu²⁺; on the other hand, Hg²⁺, Ba²⁺, and Mn²⁺ inhibited the enzyme. The present study is among the first works to examine and describe a secreted, cellulase-free, and highly thermostable xylanase from the T. thermophilus fungus whose application as a pre-bleaching aid is of apparent importance for pulp and paper industries.

Keywords: Xylanase; Fungus; Talaromyces thermophilus ; Thermostability

Supplementation of Methionine Enhanced the Ergothioneine Accumulation in the Ganoderma neo-japonicum Mycelia by Wi Young Lee; Eung-Jun Park; Jin Kwon Ahn (pp. 213-221).

The methods for increasing the production of ergothioneine (ERG) were investigated by using the mycelial culture of several mushroom species, primarily Ganoderma neo-japonicum. We first found that ERG was accumulated at the different levels in mycelia and fruiting bodies, respectively, depending on the mushroom species. As a result of adding various amino acids to the mycelial culture medium, methionine (Met) was shown to be the most effective additive. The most preferable condition of the additive was the combination of 4 mM Met and 1 g/l of yeast extract, and the maximum ERG production reached approximately 1.7 mg/l, which corresponds to 2.4 times (0.7 mg/l) that in the basal medium without Met. Although the supplementation of Met enhanced the ERG production, the mycelial growth was significantly inhibited. Furthermore, the analysis of amino acids in the culture medium revealed that the Met additive reduced the consumption rates of most amino acids tested, probably due to the decrease in mycelial growth. Taking these results into consideration, we suggest that the addition of Met to the mycelial culture medium is an efficient way to enhance the ERG production in economically important mushroom species.

Keywords: Ergothioneine; Methionine; Additives; Mycelial culture; Ganoderma neo-japonicum

Effect of Starvation and Shock Loads on the Biodegradation of 4-Chlorophenol in a Discontinuous Moving Bed Biofilm Reactor by Iván Moreno-Andrade; Germán Buitrón; Alejandro Vargas (pp. 222-230).

The influence of starvation (defined as the period without substrate) and shock loads on the performance of a moving bed sequencing batch reactor degrading 4-chlorophenol (4CP) were investigated. The biomass was acclimated to biodegrade 100 mg/L of 4CP, and the colonization of the packing material was followed. Two starvation periods and two shock loads were studied. The degradation capacity of the suspended and the attached biomass present on the moving bed was also evaluated. The experiments showed that, after the starvation period, the specific degradation rate decreased from 30.5 to 28.5 and 20 mg 4CP/gVSS/h, when starvation periods of 24 and 48 h were applied, respectively. When two concentration peaks of 500 and 1,050 mg/L were applied, a loss of 6% and 8% on the specific degradation rate, respectively, was also observed. The moving bed thus showed great robustness against starvation periods and shock loads. Suspended biomass presented higher specific degradation rates, but attached biomass did not generate a metabolite that is inhibitory when it accumulates.

Keywords: Moving bed; Biofilm; SBR; Shock loads; Starvation; 4-chlorophenol

Characteristics of Sulfobacin A from a Soil Isolate Chryseobacterium gleum by Priti N. Chaudhari; Kishor S. Wani; Bhushan Liladhar Chaudhari; Sudhir B. Chincholkar (pp. 231-241).

A nonmotile, nonspore-forming, Gram-negative, aerobic, small rod-shaped bacterium, isolated from soil, was identified as Chryseobacterium gleum on the basis of 16S rRNA gene sequence analysis. It was observed to grow luxuriously at pH 9 and tolerate highly alkaline environment up to pH 12. Orange red color was a peculiar character of these cells which on purification obtained 60–80 mg/l and found to be sphingosine type of sulfonolipid “sulfobacin A” on the basis of infrared, nuclear magnetic resonance, and mass spectral data. Inhibition of sulfobacin A synthesis by incorporation of l-cycloserine in culture growth medium suggested presence of serine palmitoyl transferase which is one of the important enzymes involved in its biosynthesis. Sulfobacin A from C. gleum LMG P-22264 exhibited cytotoxicity against four cell lines tested. Maximum activity against human mammary adenocarcinoma cells was indicative of its potential as an anticancer agent.

Keywords: Chryseobacterium gleum ; Sulfonolipid; Sulfobacin A; l-cycloserine; Serine palmitoyl transferase; Cytotoxicity

Extraction of Co-Products from Biomass: Example of Thermal Degradation of Silymarin Compounds in Subcritical Water by Lijun Duan; Sunny N. Wallace; Abigail Engelberth; Justin K. Lovelady; Edgar C. Clausen; Jerry W. King; Danielle Julie Carrier (pp. 242-242).

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.158, #1)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.158, #1)