Applied Biochemistry and Biotechnology (v.152, #3)

Cytotoxic Activities of Extracts and Compounds from Viscum coloratum and its Transformation Products by Rhodobacter sphaeroides by Guan-E Yang; Bainian Chen; Zhaoming Zhang; Jun Gong; Hongjun Bai; Jiankuan Li; Yufen Wang; Baozhen Li (353-365).
The bioassay-oriented fractionation of mistletoe crude extracts (MCEE) using 75% ethanol and culture products of mistletoe transformed by Rhodobacter sphaeroides, a photosynthetic bacterium (PSBT), revealed that the high cytotoxic activities were due to the petroleum ether extracts (PEs) and the acid-precipitated proteins from the aqueous extracts (AQs) of MCEE and PSBT. The isolated triterpenes may account for the activities of the PEs of MCEE and PSBT, respectively. Extraction of MCEE using petroleum ether led to the isolation of 3-epi-betulinic acid (1), betulonic acid (2), oleanolic acid (3), and β-amyrin acetate (4), while petroleum ether extraction of PSBT led to the isolation of 1,3,4,betulinic acid (5), erythrodiol (6), and (3β)-olean-12-ene-3,23-diol (7). The PE of PSBT exerted higher cytotoxicity than the PE of MCEE, which was due to the different triterpene contents of these two extracts. The cytotoxic activities of all compounds were tested, and the results revealed that compounds 1, 2, 3, 5, 6, and 7 contributed significantly to the cytotoxicities of both PEs. The AQ of the PSBT exerted almost the same cytotoxic activity and lower toxicity compared to the AQ of the MCEE. These findings indicate that mistletoe products biotransformed by R. sphaeroides could be used to treat cancers, since they have lower toxicities and higher antitumor activities compared to standard treatments.
Keywords: Viscum coloratum (Kom.) Nakai; Mistletoe; Photosynthetic bacteria (PSB); Rhodobacter sphaeroides ; Cytotoxic activity; Biotransformation

A fed-batch culture system with constant feeding (glucose 80 g L−1, 0.25 ml min−1) was used to study the influence of glucose on cell dry weight and exopolysaccharides production from submerged Tremella fuciformis spores in a 5-L stirred-tank bioreactor. The results showed that high levels of cell mass (9.80 g L−1) and exopolysaccharides production (3.12 g L−1) in fed-batch fermentation were obtained after 1 h of feeding, where the specific growth rate (μ) and exopolysaccharides yield on substrate consumed (YP/S) were 0.267 d−1 and 0.14 g g−1. Unlike batch fermentation, maximal cell mass and exopolysaccharides production merely reached 7.11 and 2.08 g L−1; the specific growth rate (μ) and exopolysaccharides yield on substrate consumed (YP/S) were 0.194 d−1 and 0.093 g g−1, respectively. It is concluded that the synthesis of exopolysaccharides can be promoted effectively when feeding glucose at a late exponential phase.
Keywords: Cell dry weight; Constant glucose feeding; Exopolysaccharides; Tremella fuciformis spores

Variable Volume Fed-Batch Fermentation for Nisin Production by Lactococcus lactis subsp. lactis W28 by Zhaoliang Wu; Lin Wang; Yingjun Jing; Xueliang Li; Yanli Zhao (372-382).
A feeding technology that was suitable for improving the nisin production by Lactococcus lactis subsp. lactis W28 was established. The effects of initial sucrose concentration (ISC) in the fermentation broth, feeding time, and feeding rate on the fermentation were studied. It was observed that a fed-batch culture (ISC = 10 g l−1) with 100 ml sucrose solution (190 g l−1) being evenly fed (9–10 ml h−1) into the fermenter after 3-h fermentation gave the best performance in terms of biomass and nisin yield. Under these conditions, the total biomass and the total nisin yield were approximately 23% and 51% higher than those in batch fermentation, respectively. When the sucrose concentration was controlled at 5–10 g l−1 in variable volume intermittent fed-batch fermentation (VVIF) with ISC = 10 g l−1, the total biomass and the total nisin yield were 29% and 60% above those in batch fermentation, respectively. The VVIF proved to be effective to eliminate the substrate inhibition by maintaining sucrose at appropriate levels. It is also easy to be scaled up, since various parameters involved in industrial production were taken into account.
Keywords: Nisin; Fed-batch; Fermentation; Lactic acid bacteria; Biotechnology

Purification, Characterization, and Preliminary X-Ray Diffraction Analysis of a Lactose-Specific Lectin from Cymbosema roseum Seeds by Bruno A. M. Rocha; Frederico B. M. B. Moreno; Plínio Delatorre; Emmanuel P. Souza; Emmanuel S. Marinho; Raquel G. Benevides; Joane Kathelen Rodrigues Rustiguel; Luis A. G. Souza; Celso S. Nagano; Henri Debray; Alexandre H. Sampaio; Walter F. de Azevedo Jr.; Benildo S. Cavada (383-393).
The unique carbohydrate-binding property of lectins makes them invaluable tools in biomedical research. Here, we report the purification, partial primary structure, carbohydrate affinity characterization, crystallization, and preliminary X-ray diffraction analysis of a lactose-specific lectin from Cymbosema roseum seeds (CRLII). Isolation and purification of CRLII was performed by a single step using a Sepharose-4B-lactose affinity chromatography column. The carbohydrate affinity characterization was carried using assays for hemagglutination activity and inhibition. CRLII showed hemagglutinating activity toward rabbit erythrocytes. O-glycoproteins from mucine mucopolysaccharides showed the most potent inhibition capacity at a minimum concentration of 1.2 µg mL−1. Protein sequencing by mass spectrometry was obtained by the digestion of CRLII with trypsin, Glu-C, and AspN. CRLII partial protein sequence exhibits 46% similarity with the ConA-like α chain precursor. Suitable protein crystals were obtained with the hanging-drop vapor-diffusion method with 8% ethylene glycol, 0.1 M Tris-HCl pH 8.5, and 11% PEG 8,000. The monoclinic crystals belong to space group P21 with unit cell parameters a = 49.4, b = 89.6, and c = 100.8 Å.
Keywords: Cymbosema roseum ; Crystallization; Tandem mass spectrometry; Lectins; Lactose-specific lectin

Improved Enzyme Stability in Lipase-Catalyzed Synthesis of Fatty Acid Ethyl Ester from Soybean Oil by Rafael Costa Rodrigues; Giandra Volpato; Keiko Wada; Marco Antônio Záchia Ayub (394-404).
In this work, we describe the optimization of the ethanolysis of soybean oil by the enzyme Lipozyme™ TL-IM in the lipase-catalyzed biodiesel synthesis and the improvement of the enzyme stability over repeated batches. The studied process variables were: reaction temperature, substrate molar ratio, enzyme content, and volume of added water. Fractional factorial design was used to analyze the variables so as to select those with higher influence on the reaction and then perform a central composite design to find the optimal reaction conditions. The optimal conditions found were: temperature, 26 °C; substrate molar ratio, 7.5:1 (ethanol/oil); enzyme content, 25% in relation to oil weight; and added water, 4% in relation to oil weight. Under these conditions, the yield conversion obtained was 69% in 12 h. The enzyme stability assessment in repeated batches was carried out by washing the immobilized enzyme with different solvents (n-hexane, water, ethanol, and propanol) after each batch. In the treatment with n-hexane, around 80% of the enzyme activity still remains after seven cycles of synthesis, suggesting its economical application on biodiesel production.
Keywords: Biodiesel; Lipases; Response surface methodology; Enzyme stability; Organic solvents

Reactive Blue 4 Decolorization under Mesophilic and Thermophilic Anaerobic Treatments by A. Boonyakamol; T. Imai; P. Chairattanamanokorn; T. Higuchi; M. Sekine; M. Ukita (405-417).
Anaerobic decolorization of anthraquinone dye represented by Reactive Blue 4 (RB4) was studied to evaluate the factors involved in dye-reducing behaviors such as dye concentration, co-substrate, treatment temperature, salt content, and dye-reducing microbial consortia. The experiment was conducted using digested sludge treated under mesophilic (35 °C) and thermophilic (55 °C) conditions. The results indicated that the thermophilic treatment gave higher potential for this dye decolorization compared with the mesophilic one. A reduced form of RB4 did not show an auto-oxidizing reaction but treated RB4 dye was shown in light yellow color, the intensity of which was related to the initial concentration of the dye used in the treatments. Starch, which showed similar decolorizing efficiency under thermophilic conditions, could be used as a co-substrate instead of glucose for the purpose of operating cost reduction. Due to the high content of salt contained in dye wastewater, the effect of salt (NaCl) was investigated. Results showed that decolorization could be accelerated with a concentration of NaCl lower than 200 mM, but the decolorization was inhibited by high concentrations of salt. The presence of RB4 inhibited methane productivity, while total organic carbon (TOC) reduction was similar to control, without dye addition. Increasing the temperature accelerated the decolorizing potential and TOC reduction. The evaluation of dye-reducing microbial consortia was done with acidogen and methanogen inhibitors which acidogenesis microorganism was dominant in RB4 decolorization.
Keywords: Dye decolorization; Mesophilic; Thermophilic; Dye-reducing microbial consortia

A novel hydrogen peroxide (H2O2) biosensor was developed by immobilizing hemoglobin on the gold colloid modified electrochemical pretreated glassy carbon electrode (PGCE) via the bridging of an ethylenediamine monolayer. This biosensor was characterized by UV-vis reflection spectroscopy (UV-vis), electrochemical impendence spectroscopy (EIS) and cyclic voltammetry (CV). The immobilized Hb exhibited excellent electrocatalytic activity for hydrogen peroxide. The Michaelis–Menten constant (K m) was 3.6 mM. The currents were proportional to the H2O2 concentration from 2.6 × 10−7 to 7.0 × 10−3 M, and the detection limit was as low as 1.0 × 10−7 M (S/N = 3).
Keywords: Biosensor; Glassy carbon electrode; Gold colloid; Hemoglobin; Hydrogen peroxide

Hydrolytic Properties of a Hybrid Xylanase and Its Parents by Jian-Yi Sun; Ming-Qi Liu; Xiao-Yan Weng (428-439).
The hydrolytic properties of a hybrid xylanase (ATx) and its parents (reAnxA and reTfxA) were studied using xylans and xylooligosaccharides as substrates. Analysis of reaction mixtures by high-performance liquid chromatograph revealed that xylotriose (X3) was the main product released from birchwood xylan and wheat bran insoluble xylan by ATx and reAnxA, respectively. Xylobiose (X2) was the main product separately released from birchwood xylan and wheat bran insoluble xylan by reTfxA. Xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6) could be hydrolyzed by ATx, which showed no activity on X2 and X3. Therefore, X4 might be the minimum oligomer hydrolyzed by ATx. X2–X6 could be hydrolyzed by reAnxA and reTfxA, respectively. All of ATx, reAnxA, and reTfxA showed transglycosylation activity.
Keywords: Xylanase; Hydrolysis; Transglycosylation; HPLC

In this paper, a new spinosad-producing mutant UV-42-13 was obtained by employing rhamnose and sodium propionate resistant selection strategies in series with UV irradiation. Spinosad production of the mutant was 125.3 mg/L, improved 285.5% compared with that of the wild strain (32.5 mg/L).The results of experiment on tolerance of propyl alcohol addition showed that the tolerant ability to precursor was higher. The precursor-resistant ability of the mutant improved through tolerance experiment by adding propyl alcohol, and the spinosad production was greatly increased. The kinetic models for biomass, substrate consumption, and spinosad production of mutant strain and wild strain were studied by conducting batch fermentation in the shaking flask. The result showed that the kinetic models could describe the fermentation process of spinosad producing well.
Keywords: Saccharopolyspora spinosa ; Spinosad; Fermentation; Rational selection; Kinetic models

Gastroprotective Effect of Nicorandil in Indomethacin and Alcohol-Induced Acute Ulcers by M. A. El-Moselhy; N. M. Abdel-Hamid; S. R. Abdel-Raheim (449-459).
Despite the fact that dietary habits and lifestyles are incredibly advancing, gastric ulceration is still a terrible complaint. Extensive use of non-steroidal anti-inflammatory drugs (NSAIDs) and alcohol, in addition to stress, are all predisposing factors for ulcers. Most medical treatments are always time consuming and not efficient or satisfactory to the patients. Cardiovascular patients always need NSAIDs, or mostly cannot quit alcohols, while using many cardiovascular drugs. We aim to study a possible benefit of a common nitrogen oxide donor, anti-anginal drug, nicorandil [N-(2-hydroxyethyl) nicotinamide nitrate ester], in managing acute gastric ulcers through studying its effect on some relevant intermediates to ulcerogenesis as lipid peroxidation, tumor necrosis factor-alpha (TNF-α), and nitric oxide (NO). In addition, gastric mucosal histology was studied to pursue the drug effects on tissue level. Our study revealed that both indomethacin and alcohol induced gastric ulcer mainly through up-regulation of gastric mucosal lipid peroxidation, local tissue inflammation, leukocytic infiltration, and necrosis. Both ulcerogens significantly elevated TNF-α and decreased NO, initiating ulcer formation. Nicorandil pretreatment depicted a higher preventive index in indomethacin- (89.8%) and alcohol-induced (77.7%) acute ulceration. On the tissue level, it also protected the gastric mucosa combating leukocyte infiltration and tissue congestion. Nicorandil protected tissue necrosis through decreasing oxidative stress, elevating NO levels, and down-regulating the ulcerogen-induced TNF-α elevation and improved sub-mucosal blood supply. We conclude that nicorandil may be a suitable bimodal treatment for cardiovascular patients who are at high risk of gastric ulcers by using variable analgesics to alleviate possible cardiac pain episodes, and probably frequent doses will offer a more established and long-lasting protection.
Keywords: Gastric ulcer; Indomethacin; Alcohol; Nicorandil; Lipoperoxidation; Nitric oxide; TNF-α