Applied Biochemistry and Biotechnology (v.165, #5-6)

Fermentation of Korean Red Ginseng by Lactobacillus plantarum M-2 and Its Immunological Activities by Bong-Gwan Kim; Kwang-Soon Shin; Taek Joon Yoon; Kwang-Won Yu; Kyung Soo Ra; Jin Man Kim; Sun Young Kim; Hyung Joo Suh (1107-1119).
We investigated ginsenoside transformation by fermentation of red ginseng with Lactobacillus plantarum M-2. We also examined the anti-metastasis and immune-stimulating activities of EtOH extracts of fermented red ginseng (FRG-E) in animal and human subjects. Total sugar decreased from 85.5 mg mL−1 to 44.1 mg mL−1 with increasing culture time during the fermentation with L. plantarum M-2. Uronic acid content reached a maximum level (534.3 μg mL−1) at 3 days of fermentation and decreased thereafter. Ginsenoside metabolites increased from 4,637.0 to 7,581.1 μg mL−1 after 4 days. The prophylactic intraperitoneal injection of FRG-E (500 μg mouse−1) inhibited lung metastasis about 81.1%, while the inhibitory effect against tumor metastasis by treatment of EtOH extract from non-fermented red ginseng (NFRG-E) was 66.9%. Immunoglobulin A (IgA) and G (IgG) levels in the serum of healthy subjects were higher after FRG-E administration than at baseline, whereas NFRG-E induced reductions of these variables related to immunity. At 1 week, the change in IgA level by FRG-E (5.14 mg mL−1) was significantly higher than that by NFRG-E (−14.50 mg mL−1; p < 0.05). It was concluded that the immunological activities of FRG-E were higher than those of NFRG-E, indicating that fermentation helped enhance the immunological activities of red ginseng.
Keywords: Korean red ginseng; Fermentation; Lactobacillus plantarum ; Immunological activity

In the present investigation, thermophilic fungus Rhizomucor pusillus was used to study biotransformation of antihelmintic drug albendazole to produce its active metabolite, albendazole sulfoxide and novel metabolites of commercial interest. A two-stage fermentation procedure was followed for biotransformation of albendazole. The transformation was identified and structures were confirmed by high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry analysis. Four metabolites albendazole sulfoxide, the active metabolite, albendazole sulfone, N-methyl metabolite of albendazole sulfoxide, and a novel metabolite were produced. The study demonstrates the biotransformation ability of thermophilic fungus R. pusillus NRRL28626 in the production of, the active metabolite of albendazole which has industrial and economic importance, other metabolites and a novel metabolite in an ecofriendly way.
Keywords: Biotransformation; ABZ; ABSO; LC-MS/MS; Rhizomucor pusillus NRRL28626

Kinetic Resolution of (R,S)-2-Butanol Using Enzymatic Synthesis of Esters by Maria Dolores Romero; Jose Maria Gomez; Beatriz Diaz-Suelto; Alicia Garcia-Sanz; Nicola Baster (1129-1140).
Kinetic resolution of (R,S)-2-butanol using enzymatic synthesis of esters has been studied. (R,S)-2-Butanol is commonly found as a racemic mixture, and the products of its esterification are racemic mixtures too. This work is of great significance in the field of the enzymatic kinetic resolution due to the little information found in literature about the resolution of (R,S)-2-butanol as pure compound. So, this article is a contribution about the enzymatic resolution of (R,S)-2-butanol. The reaction here studied is the esterification/transesterification of (R,S)-2-butanol in organic media (n-hexane) using as biocatalyst the lipase Novozym 435®. The main target of this study is to analyze the influence of certain variables in this reaction. Some of these variables are acyl donor (acids and esters), concentration of substrates, enzyme/substrate ratio, and temperature. The main conclusions of this study are the positive effect of higher substrates concentration (1.5 M) and larger amount of enzyme (13.8 g mol−1 substrate) on kinetic resolution rate but not a very noticeable effect on enantiomeric excesses. The longer the carboxylic acid chain is, the better results are obtained. Besides to achieve a satisfactory kinetic resolution, it is recommendable to select reaction times (180 min) at which the highest substrate enantiomeric excess is reached (~60%). The temperature has not an appreciable influence on the resolution in the range studied (40–60 °C). When an ester (vinyl acetate) is used as acyl donor, the resolution shows better results than when using a carboxylic acid as acyl donor (ees ~ 90% at 90 min). Moreover, Michaelis–Menten parameters, v max and K M, were determined, 0.04 mol 1−1 min−1 and 0.41 mol 1−1, respectively.
Keywords: Esterification; Kinetic resolution; Enzymatic; (R,S)-2-Butanol

Catalytic and Thermodynamic Properties of a Tannase Produced by Aspergillus niger GH1 Grown on Polyurethane Foam by Erika L. Ramos; Marco A. Mata-Gómez; Luis V. Rodríguez-Durán; Ruth E. Belmares; Raúl Rodríguez-Herrera; Cristóbal Noe Aguilar (1141-1151).
Tannase is an inducible enzyme with important applications in the food and pharmaceutical industries. This enzyme was produced by the fungus Aspergillus niger GH1 under solid-state fermentation using polyurethane foam as solid support and tannic acid as sole carbon source and tannase inducer. Physicochemical properties of A. niger tannase were characterized, and the kinetic and thermodynamics parameters on methyl gallate hydrolysis were evaluated. The enzyme was stable in a pH range of 2–8 and a functional temperature range of 25–65 °C. The highest k cat value was 2,611.10 s−1 at 65 °C. Tannase had more affinity for methyl gallate at 45 °C with a K M value of 1.82 mM and an efficiency of hydrolysis (k cat/K M) of 330.01 s−1 mM−1. The lowest E a value was found to be 21.38 kJ/mol at 4.4 mM of methyl gallate. The lowest free energy of Gibbs (ΔG) and enthalpy (ΔH) were found to be 64.86 and 18.56 kJ/mol, respectively. Entropy (ΔS) was −0.22 kJ/mol K. Results suggest that the A. niger GH1 tannase is an attractive enzyme for industrial applications due its catalytic and thermodynamical properties.
Keywords: Aspergillus niger GH1; Tannase; Thermodynamic parameters; Catalytical properties

The 1,3-dihydroxyacetone (DHA)-overproducing mutant of Gluconobacter oxydans was screened via UV mutagenesis to enhance the DHA production, and the DHA fermentation condition was optimized using the dissolved oxygen (DO) control strategy. The stable mutant G. oxydans ZJB11001 exhibits high DHA productivity and can tolerate high DHA concentrations. The optimal condition for DHA production by G. oxydans ZJB11001 in a 15-L fermentor required an initial medium containing 5 g/L yeast extract, 20 g/L glycerol, 0.5 g/L K2HPO4, 0.1 g/L MgSO4·7H2O. The glycerol feeding rate was manually controlled to maintain the glycerol concentration at 5–10 g/L range. The culture pH was maintained at 6.0 within the first 20 h, and then adjusted to 5.0 until the end of the fermentation. The DO concentration increased from 20% to 30% after 24 h of fermentation, and then to 40% after 60 h of fermentation. The maximum DHA concentration of 209.6 ± 6.8 g/L was achieved after 72 h of fed-batch fermentation at 30 °C.
Keywords: 1,3-Dihydroxyacetone; Gluconobacter oxydans ; UV irradiation; DO control strategy; Glycerol

3′-O-Acyl-trifluridines were prepared successfully through an enzymatic approach for the first time. Among the ten commercially available lipases tested, Pseudomonas cepacia lipase displayed the highest regioselectivity towards the acylation of 3′-hydroxyl of trifluridine. Furthermore, the effects of some crucial factors on the enzymatic myristoylation of trifluridine were examined. The optimal reaction medium, molar ratio of trifluridine to vinyl myristate and reaction temperature were found to be anhydrous THF, 1:7 and 50 °C, under which the reaction rate, substrate conversion, and 3′-regioselectivity were 63.9 mM/h, >99.0%, and 99%, respectively. Additionally, the enzyme recognition of the chain length of the acyl donors was investigated. The results showed that 3′-regioselectivity of the enzyme maintained 99% with the increment of acyl chain length (C6, C10, and C14). The reason might derive from the strong hydrophobic interaction between 5-CF3 group of the base moiety and Leu 287 located in the medium-sized pocket of the active site.
Keywords: Trifluridine; Enzymatic regioselective acylation; Substitute; Lipase

The human erythropoietin (HuEPO) structural gene was fused with the secretion signal of the Trichoderma reesei cellobiohydrolase I and controlled by a newly optimized cbh1 promoter in an integrated expression vector pTrCBH-EPO. The recombinant HuEPO construct was transformed into two different T. reesei strains, a protease-deficient strain RutC-30 M3, and a glycosylation-modified strain T108. After lactose induction, the heterologous rHuEPO was found to be stably expressed in the selected transformants T47 (derived from RutC-30 M3) and T112 (derived from T108), which were shown to have high genetic stability. Secretion of erythropoietin in these transformants was further confirmed by SDS-PAGE and western blot analysis. Moreover, the secreted rHuEPO from T112 had an apparent molecular weight of 32 kDa, which was higher than from T47 (28 kDa) and similar to that of mammals (more than 30 kDa). These results demonstrate the potential of using industrial filamentous fungi for the production of human-derived erythropoietin.
Keywords: Erythropoietin; Heterologous expression; Trichoderma reesei ; Glycosylation; cbh1 promoter

Nanotechnology: Emerging Tool for Diagnostics and Therapeutics by Mainak Chakraborty; Surangna Jain; Vibha Rani (1178-1187).
Nanotechnology is an emerging technology which is an amalgamation of different aspects of science and technology that includes disciplines such as electrical engineering, mechanical engineering, biology, physics, chemistry, and material science. It has potential in the fields of information and communication technology, biotechnology, and medicinal technology. It involves manipulating the dimensions of nanoparticles at an atomic scale to make use of its physical and chemical properties. All these properties are responsible for the wide application of nanoparticles in the field of human health care. Promising new technologies based on nanotechnology are being utilized to improve diverse aspects of medical treatments like diagnostics, imaging, and gene and drug delivery. This review summarizes the most promising nanomaterials and their application in human health.
Keywords: Nanotechnology; Drug delivery; Gene delivery; Imaging; Diagnostics

The complete coding sequences of the polA genes from seven thermophilic Geobacillus species, isolated from hot springs of Gönen and Hisaralan in Turkey, were cloned and sequenced. The polA genes of these Geobacillus species contain a long open reading frame of 2,637 bp encoding DNA polymerase I with a calculated molecular mass of 99 kDa. Amino acid sequences of these Geobacillus DNA polymerases are closely related. The multiple sequence alignments show all include the conserved amino acids in the polymerase and 5′-3′ exonuclease domains, but the catalytic residues varied in 3′-5′ exonuclease domain of these Geobacillus DNA polymerases. One of them, DNA polymerase I from Geobacillus kaue strain NB (Gkaue polI) is purified to homogeneity and biochemically characterized in vitro. The optimum temperature for enzymatic activity of Gkaue polI is 70 °C at pH 7.5–8.5 in the presence of 8 mM Mg2+ and 80–100 mM of monovalent ions. The addition of polyamines stimulates the polymerization activity of the enzyme. Three-dimensional structure of Gkaue polI predicted using homology modeling confirmed the conservation of all the functionally important regions in the polymerase active site.
Keywords: Geobacillus sp.; DNA polymerase I; Geobacillus kaue strain NB polI; DNA polymerization in vitro; Homology modeling

The specificities of carboxypeptidases from Actinomucor elegans were investigated by determining enzymatic activities at pH 7.0 and pH 4.0 with 16 Z-dipeptides and three Z-tripeptides as substrates. The debittering effect was evaluated and the free amino acid compositions of the soybean protein hydrolysates were analyzed before and after treatment with A. elegans extract at pH 7.0 and pH 4.0, with carboxypeptidases from Aspergillus oryzae as control. The results of the enzyme activity determinations indicated that carboxypeptidases from A. elegans prefer hydrophobic substrates, such as Z-Phe-Leu, Z-Phe-Tyr-Leu, and Z-Phe-Tyr. The sensory evaluation and free amino acid composition analysis showed that these carboxypeptidases are efficient tools for decreasing the bitterness of peptides because they liberated the fewest free amino acids, which consisted of 73% hydrophobic amino acids, under acidic conditions. Carboxypeptidases from A. elegans display promising prospects for future applications in the protein hydrolysate industry.
Keywords: A. elegans ; A. oryzae ; Bitter taste; Carboxypeptidase; Protein hydrolysate; Sufu

Analysis of Differentially Expressed Proteins in Colorectal Cancer Using Hydroxyapatite Column and SDS-PAGE by Shi-Rou Lim; Boon-Hui Gooi; Manjit Singh; Lay-Harn Gam (1211-1224).
Limitation on two dimensional (2D) gel electrophoresis technique causes some proteins to be under presented, especially the extreme acidic, basic, or membrane proteins. To overcome the limitation of 2D electrophoresis, an analysis method was developed for identification of differentially expressed proteins in normal and cancerous colonic tissues using self-pack hydroxyapatite (HA) column. Normal and cancerous colon tissues were homogenized and proteins were extracted using sodium phosphate buffer at pH 6.8. Protein concentration was determined and the proteins were loaded unto the HA column. HA column reduced the complexity of proteins mixture by fractionating the proteins according to their ionic strength. Further protein separation was accomplished by a simple and cost effective sodium dodecyl sulfate–polyacrylamide gel electrophoresis method. The protein bands were subjected to in-gel digestion and protein analysis was performed using electrospray ionization (ESI) ion trap mass spectrometer. There were 17 upregulated proteins and seven downregulated proteins detected with significant differential expression. Some of these proteins were low abundant proteins or proteins with extreme pH that were usually under presented in 2D gel analysis. We have identified brain mitochondrial carrier protein 1, T-cell surface glycoprotein CD1a, SOSS complex subunit B2, and Protein Jade 1 which were previously not detected in 2D gel analysis method.
Keywords: Colon cancer; Hydroxyapatite chromatography; Proteomics; Tissue specimens; Total phosphate extraction

Optimization of a Multiplex PCR Assay for Detecting Transgenic Soybean Components in Feed Products by Fang Tian; Xiumin Wang; Da Teng; Yalin Yang; Qingfeng Guan; Changjin Ao; Jianhua Wang (1225-1234).
A multiplex polymerase chain reaction (m-PCR) assay was developed for the simultaneous detection of multiple components of genetically modified (GM) soybean. It uses two sets of primers (I, lectin1/35S/CP4; II, lectin2/35S/CP4) specific for a soybean reference gene, the 35S promoter, and an event-specific gene. Amplified fragments of 118, 414, 195, and 320 bp were easily detected by agarose gel electrophoresis and were positively confirmed by sequencing. Primer set concentrations and annealing temperatures in the m-PCR were optimized. The optimized m-PCR conditions were obtained for primer set I at a ratio of 1:2:3 and a 59.2 °C annealing temperature and set II at the same ratio and 58.6 °C, 60.3 °C, and 61.2 °C annealing temperatures. The sensitivities of the two m-PCR primer sets (I and II) were 0.25% and 0.5%, respectively. The results showed that this m-PCR assay provides rapid, reliable, and effective identification of multiple components of GM soybean in feed.
Keywords: Genetically modified soybean; Feed; Multiplex PCR

Surface Interactions and Fouling Properties of Micrococcus luteus with Microfiltration Membranes by Lei Feng; Xiufen Li; Ping Song; Guocheng Du; Jian Chen (1235-1244).
This study was conducted to investigate microbial adhesion of Micrococcus luteus to polypropylene (PP) and polyvinylidene fluoride (PVDF) membranes in relation to the variation of the interfacial energies in the membrane–bacteria systems, for revealing effects of short-range surface interactions on filtration behavior. Both the membranes and M. luteus showed typical strong electron donors and hydrophilic properties. The AB component was dominant in the interfacial energies of the two membrane–bacteria systems. M. luteus presented larger negative $$ U_{ ext{mlb}}^{ ext{XDLVO}} $$ to the PP membrane than to the PVDF membrane. The adhesion experiments also proved that M. luteus had higher adhesion percentage to the PP membrane. This study demonstrated that the adhesion potentials of M. luteus to the PP and PVDF membranes might be explained in terms of bacterium, membrane, and intervening medium surface properties, which are mainly determined by the interfacial energies in the systems according to the XDLVO theory.
Keywords: Micrococcus luteus ; Biofouling microfiltration; Interfacial energy; The XDLVO theory

Expression of MMP2 and MMP9 (Gelatinases A and B) in Human Colon Cancer Cells by Umadevi Damodharan; Ravikumar Ganesan; Uma C. Radhakrishnan (1245-1252).
Matrix metalloproteinases (MMPs) are a family of zinc-dependent neutral endopeptidases, collectively capable of degrading essentially all matrix components. Elevated levels of distinct MMPs are detected in tumor tissue or serum of patients with advanced cancer, and they are the major prognostic indicators in cancer. Inhibition of MMPs has been explored as a therapeutic goal for almost two decades. Nitric oxide (NO), a free radical plays an important role in signaling pathways in regulation of MMP expression. In the present study, we demonstrated the role of exogenous NO levels in the regulation of MMP2 and MMP9 (gelatinases A and B) in colon cancer cell line WiDr and its inhibition with emodin (a naturally occurring anthraquinone).
Keywords: Matrix metalloproteinase; Colon cancer; Emodin; Sodium nitroprusside; Nitric oxide

Cloning and Characterization of a Heme Oxygenase-2 Gene from Alfalfa (Medicago sativa L.) by Guang-Qing Fu; Qi-Jiang Jin; Yu-Ting Lin; Jian-Fei Feng; Li Nie; Wen-Biao Shen; Tian-Qing Zheng (1253-1263).
Heme oxygenase (HO, EC catalyzes the oxidation of heme and performs vital roles in plant development and stress responses. Two HO isozymes exist in plants. Between these, HO-1 is an oxidative stress-response protein, and HO-2 usually exhibited constitutive expression. Although alfalfa HO-1 gene (MsHO1) has been investigated previously, HO2 is still poorly understood. In this study, we report the cloning and characterization of HO2 gene, MsHO2, from alfalfa (Medica sativa L.). The full-length cDNA of MsHO2 contains an ORF of 870 bp and encodes for 290 amino acid residues with a predicted molecular mass of 33.3 kDa. Similar to MsHO1, MsHO2 also appears to have an N-terminal transit peptide sequence for chloroplast import. Many conserved residues in plant HO were also conserved in MsHO2. However, unlike HO-1, the conserved histidine (His) required for heme-iron binding and HO activity was replaced by tyrosine (Tyr) in MsHO2. Further biochemical activity analysis of purified mature MsHO2 showed no HO activity, suggesting that MsHO2 may not be a true HO in nature. Semi-quantitative RT-PCR confirmed its maximum expression in the germinating seeds. Importantly, the expression levels of MsHO2 were up-regulated under sodium nitroprusside (SNP) and H2O2 (especially) treatment, respectively.
Keywords: Alfalfa; Heme oxygenase 2; Prokaryotic expression in E. coli ; Gene expression

Characterization and Identification of a Chymotryptic Hydrolysate of Alpha-Lactalbumin Stimulating Cholecystokinin Release in STC-1 Cells by Lucie Catiau; Véronique Delval-Dubois; Didier Guillochon; Naïma Nedjar-Arroume (1264-1273).
Alpha-lactalbumin hydrolysate is of significant interest, due to its potential application as a source of bioactive peptides in nutraceutical and pharmaceutical domains. This study was focused on the cholecystokinin (CCK) family compounds which are small peptides involved in the satiety control. The action of chymotryptic hydrolysate of alpha-lactalbumin on cholecystokinin release from intestinal endocrine STC-1 cells was investigated. We demonstrated for the first time that a chymotryptic hydrolysate of alpha-lactalbumin was able to highly stimulate CCK-releasing activity from STC-1 cells. The peptidic hydrolysate was characterized by LC/MS and MS/MS, thus highlighting the presence of 11 fractions containing 21 peptides, each potentially having the desired activity.
Keywords: Alpha-lactalbumin hydrolysate; Cholecystokinin; Enteroendocrine STC-1 cells; Peptides

Enzymatic Treatment and Detoxification of Acid Orange 7 from Textile Wastewater by Fathollah Gholami-Borujeni; Amir Hossein Mahvi; Simin Nasseri; Mohammad Ali Faramarzi; Ramin Nabizadeh; Mahmood Alimohammadi (1274-1284).
A crude preparation of horseradish roots was used as a low-purity source of horseradish peroxidase (HRP) in dye decolorization experiments. The technical feasibility of the process was studied in bench scale for enzymatic removal of acid orange 7 (AO7), a synthetic dye. Further studies were carried out to understand the effects of process parameters such as pH value, H2O2 level, concentrations of the synthetic dye, and HRP during enzyme-mediated dye degradation. Experimental data revealed that the concentration of AO7, pH of the aqueous phase, amount of the enzyme, and H2O2 level played significant roles on the overall enzymatic reaction. Polyethylene glycol, as an anti-inactivation of HRP, in various concentrations showed no significant effect on the decolorization. The experimental data of initial reaction rates were fitted using an analytical equation proposed by Michaelis–Menten. The acute toxicity tests using Daphnia magna exhibited that the enzymatic treatment significantly decreased the toxicity of the dye solution.
Keywords: Acid orange 7; Enzymatic treatment; Horseradish peroxidase; Horseradish root; Removal

Purification and Properties of a New Thermostable Cyclodextrin Glucanotransferase from Bacillus pseudalcaliphilus 8SB by Tsvetina Kitayska; Penka Petrova; Viara Ivanova; Alexandra Ivanova Tonkova (1285-1295).
A new cyclodextrin glucanotransferase (CGTase, EC from an alkaliphilic halotolerant Bacillus pseudalcaliphilus 8SB was studied in respect to its γ-cyclizing activity. An efficient conversion of a raw corn starch into only two types of cyclodextrins (β- and γ-CD) was achieved by the purified enzyme. Crude enzyme obtained by ultrafiltration was purified up to fivefold by starch adsorption with a recovery of 62% activity. The enzyme was a monomer with a molecular mass 71 kDa estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE. The CGTase exhibited two pH optima, at pH 6.0 and 8.0, and was at most active at 60 °C and pH 8.0. The enzyme retained more than 80% of its initial activity in a wide pH range, from 5.0 to 11.0. The CGTase was strongly inhibited by 15 mM Cu2+, Fe2+, Ag+, and Zn2+, while some metal ions, such as Ca2+, Na+, K+, and Mo7+, exerted a stimulating effect in concentration of 5 mM. The important feature of the studied CGTase was its high thermal stability: the enzyme retained almost 100% of its initial activity after 2 h of heating at 40–60 °C; its half-life was 2 h at 70 °C in the presence of 5 mM Ca2+. The achieved 50.7% conversion of raw corn starch into 81.6% β- and 18.4% γ-CDs after 24 h enzyme reaction at 60 °C and pH 8.0 makes B. pseudalcaliphilus 8SB CGTase industrially important enzyme for cyclodextrin production.
Keywords: Cyclodextrin glucanotransferase; Starch conversion; Cyclodextrins; Bacillus pseudalcaliphilus ; Halotolerant alkaliphilic bacilli

Isolation of Phlorotannins from Eisenia bicyclis and Their Hepatoprotective Effect against Oxidative Stress Induced by tert-Butyl Hyperoxide by Sang Min Kim; Kyungsu Kang; Je-Seung Jeon; Eun Hye Jho; Chul Young Kim; Chu Won Nho; Byung-Hun Um (1296-1307).
Eisenia bicyclis (Kjellman) Setchell is a common brown alga that inhabits the middle Pacific coast around Korea and Japan. In this study, the ethanol extract and its serial solvent fractions were prepared from fresh E. bicyclis, and their hepatoprotective effects were investigated against hepatotoxicity in tert-butyl hyperoxide(t-BHP)-injured HepG2 cells. When these samples were used at a dose of 10–40 μg/mL, they significantly protected the t-BHP-induced cell death in HepG2 cells. Among fractions, ethyl acetate fraction (EF) and n-butanol extract (BF) exhibited potent hepatoprotective activities (62.60% for EF and 64.86% for BF) in t-BHP-injured HepG2 cells at a concentration of 10 μg/mL. To find the potential factors for this activity, the samples were characterized on total phenolics, chlorophylls, carotenoids, and radical scavenging activity. Among them, EF showed the highest content of total phenolics and the strongest antioxidant activity both in on- and offline assays. Five phlorotannin compounds, oligomers of phloroglucinol, were isolated chromatographically from this fraction and structurally identified by 1H-NMR and liquid chromatography–electrospray ionization–mass spectrometry analyses as eckol(1), 6,6′-bieckol(2), 8,8′-bieckol(3), dieckol(4), and phlorofucofuroeckol A(5). Compound 5 among five purified compounds showed the strongest protective activity (45.54%) at a concentration of 10 μM. At the high dose (40 μM), the protective activities of three compounds (compound 2, 4, and 5) were higher than that of quercetin treated with 10 μM concentration. Therefore, we can speculate that they can be developed as potential candidates for natural hepatoprotective agents.
Keywords: Eisenia bicyclis ; Hepatoprotective effect; Oxidative stress; Phlorotannins; Radical scavenging activity

New Urea Biosensor Based on Urease Enzyme Obtained from Helycobacter pylori by Bahar Dindar; Emine Karakuş; Fatih Abasıyanık (1308-1321).
The urease enzyme of Helicobacter pylori was isolated from biopsy sample obtained from antrum big curvature cell extracts. A new urea biosensor was prepared by immobilizing urease enzyme isolated from Helicobacter pylori on poly(vinylchloride) (PVC) ammonium membrane electrode by using nonactine as an ammonium ionophore. The effect of pH, buffer concentration, and temperature for the biosensor prepared with urease from H. pylori were obtained as 6.0, 5 mM, and 25 °C, respectively. We also investigated urease concentration, stirring rate, and enzyme immobilization procedures in response to urea of the enzyme electrode. The linear working range of the biosensor extends from 1 × 10−5 to 1 × 10−2 M and they showed an apparent Nernstian response within this range. Urea enzyme electrodes prepared with urease enzymes obtained from H. pylori and Jack bean based on PVC membrane ammonium-selective electrode showed very good analytical parameters: high sensitivity, dynamic stability over 2 months with less decrease of sensitivity, response time 1–2 min. The analytical characteristics were investigated and were compared those of the urea biosensor prepared with urease enzyme isolated from Jack bean prepared at the same conditions. It was observed that rapid determinations of human serum urea amounts were also made possible with both biosensors.
Keywords: Helicobacter pylori ; Urease enzyme; Isolation; Urea; Polyvinylchloride; Carboxylated polyvinylchloride; Palmitic acid; Nonactin

The Application of Ultrasound in the Enzymatic Hydrolysis of Switchgrass by Michael W. Easson; Brian Condon; Bruce S. Dien; Loren Iten; Ryan Slopek; Megumi Yoshioka-Tarver; Allan Lambert; Jade Smith (1322-1331).
In a series of experiments, untreated and ammonium hydroxide pretreated Klenow lowland variety switchgrasses are converted to reducing sugars using low-frequency (20 kHz) ultrasound and commercially available cellulase enzyme. Results from experiments using untreated and pretreated switchgrasses with and without ultrasound are presented and discussed. In untreated switchgrass experiments, the combination of ultrasound and enzymes resulted in an increase of 7.5% in reducing sugars compared to experiments using just enzymes. In experiments using ammonium hydroxide pretreated switchgrass, the combination of ultrasound and enzymes resulted in an increase of 9.3% in reducing sugars compared to experiments using just enzymes. Experimental evidence indicates that there is a synergistic effect from the combination of ultrasound and enzymes which lowers the diffusion-limiting barrier to enzyme/substrate binding and results in an increase in reaction rate. Scanning electron microscopic images provide evidence that ultrasound-induced pitting increases substrate surface area and affects reaction rate and yield.
Keywords: Enzyme; Ultrasound; Switchgrass; Synergism; Pretreatment; Biofuels

Lipase from Pseudomonas stutzeri PL-836 was immobilized on hydrophobic supports and evaluated in the transesterification of wood sterols in solvent-free and solvent-containing media. Triton X-100 was used as additive during immobilization in butyl and octadecyl sepabeads increasing enzyme activity yield by 5% and 60%, respectively. Hyperactivation was observed during immobilization in EC octadecyl sepabeads with enzyme activity yield of 200% and protein immobilization yield of 93%. Thermostability of the immobilized enzyme was assessed at 50 °C in different media in the absence and presence of exogenous solvents. The presence of Triton X-100 during immobilization reduced enzyme stability while tert-butanol increased it. Transesterification in solvent-free and solvent-containing medium with lipase immobilized in EC octadecyl sepabeads showed that the presence of exogenous solvent increased both conversion yield and productivity. At rather high levels of biocatalyst hydration (40% on wet basis) the presence of tert-butanol in the reaction medium more than doubled conversion yield and productivity.
Keywords: Lipase; Hydrophobic supports; Enzyme immobilization; Transesterification; Wood sterols; Fatty acid esters

A wide range of external stress stimuli triggers a plant cell to undergo a complex network of reactions that ultimately lead to the synthesis and accumulation of secondary metabolites. These secondary metabolites help the plant to survive under stress challenge. The potential of biotic and abiotic elicitors for the induction and enhancement of secondary metabolite production in various culture systems including hairy root (HR) cultures is well-known. The elicitor-induced defense responses involves signal perception of elicitor by a cell surface receptor followed by its transduction involving some major cellular and molecular events including activation of major secondary message signaling pathways. This result in induction of gene expressions escorting to the synthesis of various proteins mainly associated with plant defense responses and secondary metabolite synthesis and accumulation. The review discusses the elicitor-induced various cellular and molecular events and correlates them with enhanced secondary metabolite synthesis in HR systems. Further, this review also concludes that combining elicitation with in-silico approaches enhances the usefulness of this practice in better understanding and identifying the rate-limiting steps of biosynthetic pathways existing in HRs which in turn can contribute towards better productivity by utilizing metabolic engineering aspects.
Keywords: Agrobacterium rhizogenes ; Elicitor(s); Elicitation; Hairy roots; Secondary metabolites; Signal transduction

Assessment of Genetic Stability and Instability of Tissue Culture-Propagated Plantlets of Aloe vera L. by RAPD and ISSR Markers by Mangal Singh Rathore; J. Chikara; Shaik G. Mastan; H. Rahman; K. G. V. Anand; N. S. Shekhawat (1356-1365).
Efficient plantlet regeneration with and without intermediate callus phase was achieved for a selected genotype of Aloe vera L. which is sweet in test and used as a vegetable and source of food. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) marker assays were employed to evaluate genetic stability of plantlets and validate the most reliable method for true-to-type propagation of sweet aloe, among two regeneration systems developed so far. Despite phenotypic similarities in plantlets produced through both regeneration systems, the differences in genomic constituents of plantlets produced through intermediate callus phase using soft base of inflorescence have been effectively distinguished by RAPD and ISSR markers. No polymorphism was observed in regenerants produced following direct regeneration of axillary buds, whereas 80% and 73.3% of polymorphism were observed in RAPD and ISSR, respectively, in the regenerants produced indirectly from base of the inflorescence axis via an intermediate callus phase. Overall, 86.6% of variations were observed in the plantlets produced via an intermediate callus phase. The occurrence of genetic polymorphism is associated with choice of explants and method used for plantlet regeneration. This confirms that clonal propagation of sweet aloe using axillary shoot buds can be used for commercial exploitation of the selected genotype where a high degree of fidelity is an essential prerequisite. On the other hand, a high degree of variations were observed in plantlets obtained through indirect regeneration and thus cannot be used for the mass multiplication of the genotype; however, it can be used for crop improvement through induction of somaclonal variations and genetic manipulations.
Keywords: Aloe tissue culture; Direct regeneration; Indirect regeneration; Genetic stability; Genetic instability

Direct and Indirect Organogenesis of Alpinia galanga and the Phytochemical Analysis by Kiranmayee Rao; Bhuvaneswari Chodisetti; Suryakala Gandi; Lakshmi Narasu Mangamoori; Archana Giri (1366-1378).
Alpinia galanga is a rhizomatous herb rich in essential oils and various other significant phytoconstituents. Rapid direct regeneration was obtained from the rhizome explants (15.66 ± 0.57 shoots) on MS media supplemented with zeatin at a concentration of 2 mg/l. The callus cultures of A. galanga were initiated from the rhizome explants on MS media supplemented with 2 mg/l each of BAP, 2,4-D, and NAA. The callus was analyzed for the presence of a vital phytoconstituent—acetoxychavicol acetate (ACA) associated with various biological properties. ACA was detected in the young friable callus as well as the stationary phase callus. Moreover, the induction of morphogenetic response in callus resulted in higher accumulation of ACA. The phytohormone withdrawal from the propagation media and the subsequent transfer of callus to BAP (2 mg/l) containing MS media has resulted in multiple shoot induction. The regenerated (indirect) plants have shown 1.6-fold higher ACA content (1.253%) when compared to the control plant (0.783%). Micropropagation of such conventionally propagated plants is very essential to meet the commercial demand as well as to ensure easy storage and transportation of disease free stocks.
Keywords: Alpinia galanga ; Rhizome; Callus; Micropropagation; Acetoxychavicol acetate; HPLC

Artemin (ARTN) is a neurotrophic growth factor of the GDNF ligand family that signals through the specific GFRα-3 coreceptor/cRet tyrosine kinase-mediated signaling cascade. Its expression and signaling action in adults are restricted to nociceptive sensory neurons in the dorsal root ganglia. Consequently, Artemin supports survival and growth of sensory neurons and has been studied as a possible treatment for neuropathic pain. We have developed a robust and sensitive cellular assay to measure ARTN biological activity. Using recombinant Artemin produced in Escherichia coli bacteria together with this specific assay, we demonstrate that ARTN is an exceptionally stable polypeptide. Multiple freeze–thaw cycles, incubation at elevated temperatures (up to 90 °C) for 0.5 h, prolonged storage at 4 °C, and exposure to conditions of different pH, salt concentration, and additives had no measurable effect on the biological activity of ARTN. In some of the tested conditions, partial removal of nine NH2-terminal amino acids of the ARTN protein occurred, but this truncation had no important effect on the ARTN signaling response. Consequently, we postulate that formulation and storage for in vivo testing of ARTN in neuropathic pain paradigms in animals and humans should be straightforward.
Keywords: Cellular assay; ERK; Growth factor; Neurological disease; Neurotrophic factor; Protein stability

Dilute-acid pretreatment liquor (PL) produced at NREL through a continuous screw-driven reactor was analyzed for sugars and other potential inhibitory components. Their inhibitory effects on enzymatic hydrolysis of Solka Floc were investigated. When the PL was mixed into the enzymatic hydrolysis reactor at 1:1 volume ratio, the glucan and xylan digestibility decreased by 63% and 90%, respectively. The tolerance level of the enzyme for each inhibitor was determined. Of the identified degradation components, acetic acid was found to be the strongest inhibitor for cellulase activity, as it decreased the glucan yield by 10% at 1 g/L. Among the sugars, cellobiose and glucose were found to be strong inhibitors to glucan hydrolysis, whereas xylose is a strong inhibitor to xylan hydrolysis. Xylo-oligomers inhibit xylan digestibility more strongly than the glucan digestibility. Inhibition by the PL was higher than that of the simulated mixture of the identifiable components. This indicates that some of the unidentified degradation components, originated mostly from lignin, are potent inhibitors to the cellulase enzyme. When the PL was added to a simultaneous saccharification and co-fermentation using Escherichia coli KO11, the bioprocess was severely inhibited showing no ethanol formation or cell growth.
Keywords: Inhibition; Cellulase; Hydrolysate; Tolerance; Xylo-oligomers; Lignin; Dilute-acid pretreatment; Degradation; Phenolic; Enzymatic hydrolysis

Production, Purification, and Biochemical Characterization of a Fibrinolytic Enzyme from Thermophilic Streptomyces sp. MCMB-379 by Ratnakar Ravindra Chitte; Siddharath V. Deshmukh; Pradnya Pralhad Kanekar (1406-1413).
Production of the fibrinolytic enzyme was carried out using 2.5-L glass fermentor, culture of thermophilic Streptomyces sp., and glucose yeast extract peptone medium of pH 8.0. Five successive batches were carried out under controlled fermentation conditions viz., agitation 140 rpm, aeration 0.5 vvm, 55 °C, and 18 h. The total protein extracellularly produced in the cell-free broth was ~300–500 mg/L. The enzyme belongs to serine endopeptidase type. Studies on the fibrin degradation indicate that the enzyme degrades the fibrin into small molecular weight products as seen from HPLC profile. Phase-contrast microscopic structure of fibrin showed that enzyme cleaves the fibrin filaments. The ex vivo activity of the actinokinase was compared with 500 IU of urokinase and 350 IU of streptokinase. The ex vivo clot lysis was found to be faster as compared to the commercial available enzymes.
Keywords: Streptomyces sp.; Thermophiles; Fibrinolytic enzyme

Purification and Characterization of the Enzyme Cholesterol Oxidase from a New Isolate of Streptomyces sp. by Vandana Praveen; Akanksha Srivastava; C. K. M. Tripathi (1414-1426).
An extracellular cholesterol oxidase (cho) enzyme was isolated from the Streptomyces parvus, a new source and purified 18-fold by ion exchange and gel filtration chromatography. Specific activity of the purified enzyme was found to be 20 U/mg with a 55 kDa molecular mass. The enzyme was stable at pH 7.2 and 50 °C. The enzyme activity was inhibited in the presence of Pb2+, Ag2+, Hg2+, and Zn2+ and enhanced in the presence of Mn2+. The enzyme activity was inhibited by the thiol-reducing reagents (DTT, β-mercaptoethanol), suggesting that disulfide linkage is essential for the enzyme activity. The enzyme activity was found to be maximum in the presence of Triton X-100 and X-114 detergents whereas sodium dodecyl sulfate fully inactivated the enzyme. The enzyme showed moderate stability towards all organic solvents except acetone, benzene, chloroform and the activity increased in the presence of isopropanol and ethanol. The K m value for the oxidation of cholesterol by this enzyme was 0.02 mM.
Keywords: Cholesterol oxidase; Streptomyces parvus ; Ion exchange; Gel filtration chromatography; Isopropanol; β-mercaptoethanol

The present study examines the effect of p-coumaric acid (CA), a precursor of stilbenes and isoflavonoids, on biosynthesis of resveratrol in cell cultures of Vitis amurensis. Earlier, we transformed V. amurensis with the rolB gene of Agrobacterium rhizogenes and showed increased level of resveratrol production in the rolB transgenic cell culture. We used control and the rolB-transgenic cell culture of V. amurensis as a model system in this study. CA was capable of increasing resveratrol production in the control and the rolB-transgenic cell cultures in 10.3 and 1.5 times, respectively. The CA-treated control and rolB transgenic calli produced up to 0.06% and 1.1% DW of resveratrol. Using quantitative real-time RT-PCR, we characterized the expression of phenylalanine ammonia-lyase (PAL) and stilbene synthase (STS) genes in the CA-treated control and rolB transgenic cell cultures. The expression of PAL genes remained essentially unchanged under 0.1 mM of CA, while expression of VaPAL1, VaPAL2, VaPAL3, and VaPAL5 genes was considerably decreased under 0.5 and 2 mM CA compared with the untreated cells. In the CA-treated calli, expression of VaSTS2 and VaSTS3 was increased, while expression of VaSTS5, VaSTS8, VaSTS9, and VaSTS10 was significantly decreased. These results indicate that CA increased resveratrol accumulation in V. amurensis calli via selective enhancement of expression of individual STS genes.
Keywords: p-Coumaric acid; Callus culture; Resveratrol; STS ; Vitis amurensis