Applied Biochemistry and Biotechnology (v.160, #1)

A novel end-point fluorimetric procedure based on the use of rhodamine-110-labeled specific substrate was developed to determine trypsin activities in biological samples. We evaluated the ability of trichloroacetic acid and acetic acid to stop the enzymatic reaction without hindering the detection of the fluorescence of rhodamine-110 released into the reaction mixture from the specific substrate (CBZ-l-alanyl-l-arginine)2-rhodamine-110. Trichloroacetic acid decreased markedly the fluorescence of rhodamine-110, even at low concentrations. On the other hand, the addition of 50 mmol/l acetic acid inactivated efficiently trypsin activity, causing minor effects on rhodamine-110 fluorescence. The proposed procedure was more sensitive than the spectrophotometric end-point method using N-α-benzoyl-dl-arginine-p-nitroanilide as substrate. The possibility of carrying out end-point fluorimetric assays improves the performance of monocell fluorimeters by setting specific conditions optimal for each enzyme activity independently of the fluorimeter. This method also allows replicate assays to be conducted simultaneously, resulting in considerable time saving and in increased performance of low-cost equipment.
Keywords: End-point assay; Fluorimetric assay; Protease activity; Rhodamine-110; Trypsin

Culture of Microalgae Chlamydomonas reinhardtii in Wastewater for Biomass Feedstock Production by Qing-xue Kong; Ling Li; Blanca Martinez; Paul Chen; Roger Ruan (9-18).
The objective of this research was to develop large-scale technologies to produce oil-rich algal biomass from wastewater. The experiments were conducted using Erlenmeyer flasks and biocoil photobioreactor. Chlamydomonas reinhardtii was grown in artificial media and wastewaters taken from three different stages of the treatment process, namely, influent, effluent, and centrate. Each of wastewaters contained different levels of nutrients. The specific growth rate of C. reinhardtii in different cultures was monitored over a period of 10 days. The biomass yield of microalgae and associated nitrogen and phosphorous removal were evaluated. Effects of CO2 and pH on the growth were also studied. The level of nutrients greatly influenced algae growth. High levels of nutrients seem to inhibit algae growth in the beginning, but provided sustained growth to a high degree. The studies have shown that the optimal pH for C. reinhardtii is in the range of 7.5. An injection of air and a moderate amount of CO2 promoted algae growth. However, too much CO2 inhibited algae growth due to a significant decrease in pH. The experimental results showed that algal dry biomass yield reached a maximum of 2.0 g L−1 day−1 in the biocoil. The oil content of microalgae of C. reinhardtii was 25.25% (w/w) in dry biomass weight. In the biocoil, 55.8 mg nitrogen and 17.4 mg phosphorus per liter per day were effectively removed from the centrate wastewater. Ferric chloride was found to be an effective flocculent that helps the algae settle for easy harvest and separation from the culture media.
Keywords: Microalgae culture; Biomass production; Feedstock; Oil yield; Biodiesel; Wastewater treatment; Nitrogen and phosphorus removal

A gene encoding a novel (S)-specific NADH-dependent alcohol dehydrogenase (LK-ADH) was isolated from the genomic DNA of Lactobacillus kefir DSM 20587 by thermal asymmetric interlaced-polymerase chain reaction. The nucleotide sequence of (S)-LK-ADH gene (adhS) was determined, which consists of an open reading frame of 1,044 bp, coding for 347 amino acids with a molecular mass of 37.065 kDa. After a BLAST similarity search in GenBank database, the amino acid sequence of (S)-LK-ADH showed some homologies to several zinc containing medium-chain alcohol dehydrogenases. This novel gene was deposited into GenBank with the accession number of EU877965. adhS gene was subcloned into plasmid pET-28a(+), and recombinant (S)-LK-ADH was successfully expressed in E. coli BL21(DE3) by isopropyl-β-d-1-thiogalactopyranoside induction. Purified enzyme showed a high enantioselectivity in the reduction of acetophenone to (S)-phenylethanol with an ee value of 99.4%. The substrate specificity and cofactor preference of recombinant (S)-LK-ADH were also tested.
Keywords: Lactobacillus kefir ; TAIL-PCR; (S)-specific alcohol dehydrogenase; NADH dependent

A Bacillus subtilis (MTCC9102) isolate was shown to produce significant amount of keratinase under optimized conditions in solid-state fermentation using Horn meal as a substrate. Optimized value for moisture, inoculum, and aeration were found to be 100% (v/w), 50% (v/w), and 150% (w/w), respectively, and the optimum nitrogen source was peptone and carbon source was dextrose. Maximum keratinolytic activity was observed at 48 h after incubation, and the optimum age (24 h) of inoculum was significant. The influence of cultivation temperature and initial pH of the medium on keratinase production revealed the optimum values for the temperature and pH as 37 °C and 7, respectively. Maximum keratinase activity of the crude extract was 15,972 U/mg/ml. These results indicate that this bacterial strain shows a high biotechnological potential for keratinase production in solid-state fermentation, and use of the horn meal as the substrate can be implemented for keratinous solid wastes management.
Keywords: Solid bio-wastes; Horn meal; Keratinase; SSF

Purification and Characterization of Enterocin LR/6, a Bacteriocin from Enterococcus faecium LR/6 by Manoj Kumar; Santosh Kumar Tiwari; Sheela Srivastava (40-49).
Enterocin LR/6, a bacteriocin obtained from the culture filtrate of Enterococcus faecium strain LR/6, has been purified to homogeneity using ammonium sulfate precipitation, cation-exchange chromatography, gel-filtration, and checked on reverse-phase high-performance liquid chromatography. It is active at high temperatures (boiling as well as autoclaving) and over a wide range of pH (2.0–8.0). Also, it is sensitive to a number of proteolytic enzymes but is stable in the presence of surfactants and organic solvents. The protein could be stored at least up to 1 year at low temperatures (4 °C and −20 °C) without any loss of activity. The N-terminal sequence of enterocin LR/6 showed no homology with known enterocins or other bacteriocins present in the database, suggesting it to be a novel enterocin. Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed its mass to be ∼6.1 kDa. It showed a bactericidal mode of action against indicator strain, Micrococcus luteus.
Keywords: Enterococcus faecium ; Enterocin LR/6; Purification; Characterization; Mode of action

Xylanase Production by Penicillium canescens on Soya Oil Cake in Solid-State Fermentation by Assamoi Allah Antoine; Destain Jacqueline; Philippe Thonart (50-62).
There is an increasing interest for the organic residues from various sectors of agriculture and industries over the past few decades. Their application in the field of fermentation technology has resulted in the production of bulk chemicals and value-added products such as amino acid, enzymes, mushroom, organic acids, single-cell protein, biologically active secondary metabolites, etc. (Ramachandran et al., Bioresource Technology 98:2000–2009, 2007). In this work, the production of extracellular xylanase by the fungus Penicillium canescens was investigated in solid-state fermentation using five agro-industrial substrates (soya oil cake, soya meal, wheat bran, whole wheat bran, and pulp beet). The best substrate was the soya oil cake. In order to optimize the production, the most effective cultivation conditions were investigated in Erlenmeyer flasks and in plastic bags with 5 and 100 g of soya oil cake, respectively. The initial moisture content, initial pH, and temperature of the culture affected the xylanase synthesis. The optimal fermentation medium was composed by soya oil cake crushed to 5 mm supplemented with 3% and 4% (w/w) of casein peptone and Na2HPO4.2H2O. After 7 days of incubation at 30 °C and under 80% of initial moisture, a xylanase production level of 18,895 ± 778 U/g (Erlenmeyer flasks) and 9,300 ± 589 U/g (plastic bags) was reached. The partially purified enzyme recovered by ammonium sulfate fractionation was completely stable at freezing and refrigeration temperatures up to 6 months and reasonably stable at room temperature for more than 3 months.
Keywords: Agro-industrial substrates; Penicillium canescens ; Xylanase; Solid-state fermentation; Soya oil cake; Storage stability

Identification and Purification of Metalloprotease from Dry Grass Pea (Lathyrus sativus L.) Seeds by Vadde Ramakrishna; Sake Rajasekhar; Lokireddy Sudarsana Reddy (63-71).
Proteolytic enzymes play a central role in the biochemical mechanism of germination. The present study reported the presence of Zn2+-dependent endoproteases in the dry seeds of grass pea (Lathyrus sativus L.) with maximum caseinolytic activity observed at pH 8.0. Studies with class-specific inhibitors (specific for cysteine, serine, aspartate, and metalloproteases) on crude extract identified the inhibitory effect of 1,10-phenanthroline. This inhibitory effect was overcome by addition of Zn2+, not with Fe, Ca, Cu, Mg, or Co and indicates that the protease is Zn2+ dependent. This metalloprotease was further characterized by attempting gelatin-PAGE zymography and observed three distinct zones of proteolytic activity with higher mobility. The protease fraction consisted of three isoforms as evidenced by the appearance of three different bands on gelatin-PAGE zymogram. We also purified these proteases to 110-fold by a three-step procedure comprising crude extract from dry seeds, (NH4)2SO4 fractionation, and casein–alginate affinity chromatography. The molecular mass of isoforms of metalloproteases is 25, 18, and 14 kDa.
Keywords: Metalloprotease; Purification; Partial characterization; Legume seeds; Grass pea

Evaluation of Antineoplastic Activity of Extracellular Asparaginase Produced by Isolated Bacillus circulans by R. S. Prakasham; M. Hymavathi; Ch Subba Rao; S. K. Arepalli; J. Venkateswara Rao; P. Kavin Kennady; K. Nasaruddin; J. B. Vijayakumar; P. N. Sarma (72-80).
l-Asparaginase is an important component in the treatment of acute lymphoblastic leukemia in children. Its antineoplastic activity toward malignant cells is due to their characteristic nature in slow synthesis of l-asparagine (Asn), which causes starvation for this amino acid, while normal cells are protected from Asn starvation due to their ability to produce this amino acid. The relative selectivity with regard to the metabolism of malignant cells forces to look for novel asparaginase with little glutaminase-producing systems compared to existing enzyme. In this investigation, the role of the extracellular asparaginase enzyme produced by an isolated bacterial strain was studied. Biochemical characterization denoted that this isolated bacterial strain belongs to the Bacillus circulans species. The strain was tested for l-asparaginase production, and it was observed that, under an optimized environment, this isolate produces a maximum of 85 IU ml−1 within 24-h incubation. This enzyme showed less (60%) glutaminase activity compared to commercial Erwinia sp. l-asparaginase. The partially purified enzyme showed an approximate molecular weight of 140 kDa. This enzyme potency in terms of antineoplastic activity was analyzed against the cancer cells, CCRF-CEM. Flow cytometry experiments indicated an increase of sub-G1 cell population when the cells were treated with l-asparaginase.
Keywords: l-Asparaginase; Antineoplastic activity; CCRF-CEM cells; Enzyme production

In the present study, hexavalent chromium (Cr(VI)) reduction potential of chromium reductase associated with the cell-free extracts (CFE) of Arthrobacter rhombi-RE species was evaluated. Arthrobacter rhombi-RE, an efficient Cr(VI) reducing bacterium, was enriched and isolated from a chromium-contaminated site. Chromium reductase activity of Arthrobacter rhombi-RE strain was associated with the cell-free extract and the contribution of extracellular enzymes to Cr(VI) reduction was negligible. NADH enhanced the chromium reductase activity. The enzyme activity was optimal at a pH of 5.5 and a temperature of 30 °C. Among the ten electron donors screened, sodium pyruvate was the most effective one followed by NADH and propionic acid. Michaelis–Menten constant, K m, and maximum reaction rate, V max, obtained from the Lineweaver–Burk plot were 48 μM and 4.09 nM/mg protein/min, respectively, in presence of NADH as electron donor and 170.5 μM and 4.29 nM/mg protein/min, respectively, in presence of sodium pyruvate as electron donor. Ca2+ enhanced the enzyme activity while Hg2+, Cd2+, Ba2+, and Zn2+ inhibited the enzyme activity. Among the various immobilization matrices screened, calcium alginate beads seemed to be the most effective one. Though immobilized enzyme system was able to reduce Cr(VI), the performance was not very encouraging in continuous mode of operation.
Keywords: Arthrobacter rhombi-RE ; Chromium reductase; Cr(VI) bioremediation; Immobilized enzyme

Cell Disruption Optimization and Covalent Immobilization of β-D-Galactosidase from Kluyveromyces marxianus YW-1 for Lactose Hydrolysis in Milk by Munish Puri; Shivani Gupta; Parveen Pahuja; Aneet Kaur; J. R. Kanwar; J. F. Kennedy (98-108).
β-D-galactosidase (EC 3.2.1.23) from Kluyveromyces marxianus YW-1, an isolate from whey, has been studied in terms of cell disruption to liberate the useful enzyme. The enzyme produced in a bioreactor on a wheat bran medium has been successfully immobilized with a view to developing a commercially usable technology for lactose hydrolysis in the food industry. Three chemical and three physical methods of cell disruption were tested and a method of grinding with river sand was found to give highest enzyme activity (720 U). The enzyme was covalently immobilized on gelatin. Immobilized enzyme had optimum pH and temperature of 7.0 and 40 °C, respectively and was found to give 49% hydrolysis of lactose in milk after 4 h of incubation. The immobilized enzyme was used for eight hydrolysis batches without appreciable loss in activity. The retention of high catalytic activity compared with the losses experienced with several previously reported immobilized versions of the enzyme is significant. The method of immobilization is simple, effective, and can be used for the immobilization of other enzymes.
Keywords: β-D-galactosidase; Cell-disruption; Wheat bran (WB); Immobilization; Lactose hydrolysis; Klyuveromyces marxianus YW-1

Immobilization of Modified Papain with Anhydride Groups on Activated Cotton Fabric by Yong Xue; HuaLi Nie; Limin Zhu; Shubai Li; Haitao Zhang (109-121).
Papain (EC 3.4.22.2) has been chemically modified using two novel reagents including different anhydrides of 1,2,4-benzenetricarboxylic and pyromellitic acids. Then, the modified papain was immobilized on the activated cotton fabric by a two-step method. The number of free amino groups in the modified protein was investigated through the 2,4,6-trinitrobenzenesulfonic acid method. Energy dispersive spectrometer was used to characterize papain immobilization. Some parameters of both modified and native papain immobilized on cotton fabric, such as optimum temperature, optimum pH, and the stabilities for reservation in various detergents were studied and compared. The resultant papain had its optimum pH shifted from 6.0 to 9.0. Compared with immobilized native papain, the thermal stability and the resistance to alkali and washing detergent of immobilized modified enzyme were improved considerably. When the concentration of detergent was 20 mg/ml, the activity of the immobilized pyromellitic papain retained about 40% of its original activity, whereas the native papain was almost inhibited. This work demonstrated that the cotton fabric immobilized modified papain has potential applications in the functional textiles field.
Keywords: Papain; 1,2,4-Benzenetricarboxylic anhydride; Pyromellitic anhydride; Modification; Immobilization; Cotton fabric; Detergent

Characterization of Capsicum annuum Recombinant α- and β-Tubulin by Bon-Sung Koo; Myung-Hyun Jang; Haechul Park; Satish Kalme; Hey-Yeon Park; Jin-Wook Han; Yun-Soo Yeo; Sang-Hong Yoon; Soo-Jin Kim; Chang-Muk Lee; Moon-Young Yoon (122-128).
There are several conditions which might modulate polymerization to produce polymers having normal lattice structure. In the absence of 1 mM MgCl2 the assembly was reduced by 36% in Capsicum annuum tubulin (CAnm tubulin). There was no significant difference in the final assembly formation in the presence of 5% to 10% glycerol. However, nucleation rate was slow and apparent study state was achieved lately in the presence of 10% glycerol. Taxol at 100 μM concentration increased 23% tubulin assembly. One millimolar CaCl2, ≥1% dimethyl sulfoxide (DMSO) and physiologically low temperature reduced CAnm tubulin assembly. A value of 0.089 mg/ml was obtained as critical concentration for polymerization. Benomyl significantly reduced the number of cysteine residues accessible to 5,5’-dithiobis-(2-nitrobenzoic acid); there were 4.77 ± 0.21 and 3.49 ± 0.35 residues accessible per tubulin dimer in the presence of 50 and 100 μM benomyl respectively.
Keywords: Benomyl; Capsicum annuum ; Polymerization; Sulfhydryl group; Taxol

Purification and Characterization of a Novel Collagenase from Bacillus pumilus Col-J by Qi Wu; Chen Li; Chenglei Li; Hui Chen; Liu Shuliang (129-139).
The collagenase, produced extracellular by Bacillus pumilus Col-J, was purified by ammonium sulfate precipitation followed by two gel filtrations, involving Sephadex G-100 column and Sepharose Fast Flow column. Purified collagenase has a 31.53-fold increase in specific activity of 87.33 U/mg and 7.00% recovery. The collagenase has a relative molecular weight of 58.64 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimal temperature for the enzyme reaction was 45 °C. More than 50% of the original activity still remained after 5 min of incubation at 70 °C or 10 min at 60 °C. The maximal enzyme activity of collagenase was obtained at pH 7.5, and it was stable over a pH range of 6.5–8.0. The collagenase activity was strongly inhibited by Mn2+, Pb2+, ethylenediamine tetraacetic acid, ethylene glycol tetraacetic acid, and β-mercaptoethanol. However, Ca2+ and Mg2+ greatly increased its activity. The collagenase from B. pumilus Col-J showed highly specific activity towards the native collagen from calf skin. The K m and V max of the enzyme for collagen were 0.79 mg/mL and 129.5 U, respectively.
Keywords: Collagenase; Bacillus pumilus ; Purification; Characterization

Evaluation of Probiotic Characteristics of Siderophoregenic Bacillus spp. Isolated from Dairy Waste by Anil K. Patel; Jayesh J. Ahire; Shrikant P. Pawar; Bhushan L. Chaudhari; Yogesh S. Shouche; Sudhir Bhaskarrao Chincholkar (140-155).
Siderophoregenic Bacillus strain DET9 has been selectively isolated from dairy waste. It was evaluated for probiotic characteristics and susceptibility pattern against antibiotics. Its spores showed excellent tolerance to simulated gastrointestinal tract conditions and exhibited antimicrobial activity against organisms such as Escherichia coli, Micrococcus flavus, and Staphylococcus aureus. Its susceptibility to antibiotics reduces the prospect to donate resistance determinants if administered in the form of probiotic preparations. It was observed to produce ∼60 mg/l catecholate type of siderophore under iron stressed conditions, identified as a 2,3-dihydroxy benzoic acid by high-performance liquid chromatography, infrared spectroscopy, nuclear magnetic resonance, and mass spectral analysis. Partial 16S-rRNA gene sequencing analysis shows that the isolate exhibited homology with Bacillus thuringiensis and Bacillus weihenstephanensis, whereas biochemical characterization revealed its novelty. DET9 exhibited no mortality of fishes in a 60-day trial, when fishes (surfi tetra) were challenged up to 100 ppm cell concentration, with their daily diet.
Keywords: Probiotic; Bacillus ; Siderophore; Catecholate; 2,3-Dihydroxybenzoic acid

As an energy-efficient alternative to distillation, pervaporation has been widely combined with fermentation to remove organic compounds from their dilute solutions in a fermentation broth. In this work, the organic permselective composite membrane is prepared by coating polydimethylsiloxane (PDMS) cross-linked with n-heptane on the substrate of polytetrafluoroethylene(PTFE) membrane. The separation behavior is studied in different dilute organic solutions, which include acetone dilute solution, butanone dilute solution, cyclohexanone dilute solution, ethanol dilute solution, isopropanol dilute solution, n-butyl alcohol dilute solution, acetic acid dilute solution, and ethyl acetate dilute solution. Most of these solutions are main reaction products or by-products from fermentation process. The effects of solubility of organics in the membrane, molecular weight, and polarity of the organics on the pervaporation performance are investigated. The effects of operating temperature and organic concentration in the feed solutions on the performance of composite membrane are studied as well. The experimental results show that molecular volume has less influence than solubility and molecular polarity for these organic solvent. The selectivity of PDMS membrane to ethyl acetate is relative high due to good solubility and diffusion of ethyl acetate molecules in polymer.
Keywords: Pervaporation; Dilute organic solution; Molecular volume; Molecular polarity; Solubility

This objective of this study is to conduct a systematic investigation of the effects of configurations, electrolyte solutions, and electrode materials on the performance of microbial fuel cells (MFC). A comparison of voltage generation, power density, and acclimation period of electrogenic bacteria was performed for a variety of MFCs. In terms of MFC configuration, membrane-less two-chamber MFCs (ML-2CMFC) had lower internal resistance, shorter acclimation period, and higher voltage generation than the conventional two-chamber MFCs (2CMFC). In terms of anode solutions (as electron donors), the two-chamber MFCs fed with anaerobic treated wastewater (AF-2CMFCs) had the power density 19 times as the two-chamber MFCs fed with acetate (NO3 2CMFCs). In terms of cathode solutions (as electron acceptors), AF-2CMFCs with ferricyanide had higher voltage generation than that of ML-2CMFCs with nitrate (NO3 ML-2CMFCs). In terms of electrode materials, ML-2CMFCs with granular-activated carbon as the electrode (GAC-ML-2CMFCs) had a power density 2.5 times as ML-2CMFCs with carbon cloth as the electrode. GAC-ML-2CMFCs had the highest columbic efficiency and power output among all the MFCs tested, indicating that the high surface area of GAC facilitate the biofilm formation, accelerate the degradation of organic substrates, and improve power generation.
Keywords: Electrogenic bacteria; Microbial fuel cell; Power generation; Internal resistance; Wastewater treatment

Effect of Inoculum Types on Bacterial Adhesion and Power Production in Microbial Fuel Cells by Daqian Jiang; Baikun Li; Wenzhao Jia; Yu Lei (182-196).
Microbial fuel cell (MFC) is an emerging biotechnology to convert the organic substrates in wastewater to electricity by anaerobic electrogenic bacteria. The main challenge for MFC research is to elucidate the fundamental mechanisms of electron generation and transfer and to apply these mechanisms to improve the power production in the engineering operation. This study extensively investigated the effects of three inocula (Geobacter sulfurreducens, soil, and wastewater) on the power production and electrochemical characteristics (i.e., internal resistances, Coulombic Efficiency) of MFCs. The results showed that the extents of bacterial adhesion varied between mixed cultures (soil) and pure cultures (G. sulfurreducens). The voltage output increased 30% when bacterial adhesion was well-developed in the soil inocula. Meanwhile, the inoculum types clearly affected the internal resistance (R in) and power production of MFCs. Pure culture inoculum (G. sulfurreducens) had the lowest R in (165 Ω) and the highest Coulombic Efficiency (CE, 25.8%) and Energy Conversion Efficiency (ECE, 7.2%), while the mixed culture inocula (soil) with the high concentration of nonelectrogenic bacteria, exhibited the highest R in (620 Ω), lowest CE (9.2%) and lowest ECE (2.4%). Additionally, a second-order correlation was established between the anode potential (P A) and power output while an exponential correlation was established between the difference between anode and cathode potentials (ΔP C−A) and power output.
Keywords: Microbial fuel cells (MFCs); Bacterial adhesion; Internal resistance; Redox potential (ORP); Open circuit potential (OCP); Coulombic efficiency (CE); Energy conversion efficiency (ECE)

Effect of Ceramide on Mesenchymal Stem Cell Differentiation Toward Adipocytes by F. Xu; C.-C. Yang; C. Gomillion; K. J. L. Burg (197-212).
Proinflammatory cytokines such as tumor necrosis factor (TNF) α are well known to inhibit adipocyte differentiation. TNF-α triggers ceramide synthesis through binding of TNF-α to its p55 receptor. Therefore, ceramide is implicated in many of the multiple signaling pathways initiated by TNF-α. In breast tissue engineering, it is important to know how to modulate adipocyte differentiation of the stem cells with exogenous additives like ceramide in vitro. We hypothesized that stem cell adipogenesis could be retained in TNF-α-treated preadipocytes in which ceramide synthesis was blocked and that exogenous ceramide could inhibit adipocyte differentiation. We first studied the effect of ceramide synthase inhibitor, Fumonisin B2, on the adipogenesis of murine mesenchymal stem cells (D1 cells), treated with TNF-α. We then studied the effect of specific exogenous C6-ceramide on D1 cell viability and differentiation. It was found that 1 ng/ml of TNF-α significantly inhibited D1 cell adipogenesis. Cells treated with 5 μM of Fumonisin B2 were able to undergo adipogenesis, even when treated with TNF-α. High concentrations of exogenous C6-ceramide (>50 μM) had an inhibitory effect, not only on the pre-confluent proliferation of the D1 cells but also on the post-confluent cell viability. High concentrations of C6-ceramide (>50 μM) also inhibited mitotic clonal expansion when D1 cell differentiation was induced by the addition of an adipogenic hormonal cocktail. C6-ceramide at low concentrations (10–25 μM) inhibited lipid production in D1 cells, demonstrated by decreased levels of both total triglyceride content and specific fatty acid composition percentages. Genetic expression of peroxisome proliferator-activated receptor (PPAR) γ and aP2 in D1 cells was reduced by C6-ceramide treatment. CCAAT/enhancer-binding protein (C/EBP) β levels in D1 cells were reduced by C6-ceramide treatment during early differentiation; PPARγ and aP2 protein levels were reduced at terminal differentiation. C6-ceramide at lower concentrations also decreased lipid accumulation of differentiating D1 cells. Our results suggest that ceramide synthase inhibitor retains the adipogenic potential of TNF-α-treated mesenchymal stem cells, while exogenous ceramide at lower concentrations inhibit the adipogenesis of mesenchymal stem cells. Ceramide, therefore, could be a modulator candidate in breast tissue engineering strategies.
Keywords: Adipocyte; Adipogenesis; Ceramide; Differentiation; Fumonisin B2; Inhibition; Proliferation; Stem cells; TNF-α

High-Level Expression and Novel Antifungal Activity of Mouse Beta Defensin-1 Mature Peptide in Escherichia coli by Yueling Wang; Yan Jiang; Tianxiang Gong; Xiaoyan Cui; Wanyi Li; Yan Feng; Baoning Wang; Zhonghua Jiang; Mingyuan Li (213-221).
Mouse beta defensin-1 (mBD-1) is a cationic 37-amino acid antimicrobial peptide with three conserved cysterine disulfied bonds. It exhibits a broad antimicrobial spectrum, but mBD-1 against Candida albicans (C. albicans) and Cryptococcus neoformans (C. neoformans) is poorly understood. This study describes the mBD-1 gene, the heterologous fusion expression of the peptide in Escherichia coli, and the bioactive assay of released mature mBD-1. By constructing the expression plasmid (pET32a-mBD1), high yields of soluble mBD-1 fusion protein (0.67 g/L) could be obtained in E. coli and cleaved by enterokinase. The digested product was further purified and desalted with the final amount of pure mature mBD-1 being 0.14 g/L. Classical fungi growth inhibition assay showed clear antifungal activity against C. albicans and C. neoformans with IC50 of 5 and 2 μM, respectively. The results show that the mBD-1 control fungal colonization through hyphal induction, direct fungicidal activity, and the activity is suppressed by increasing NaCl concentration. Successful expression of the mBD-1 peptide in E. coli offers a basis for further studying its antifungal mechanisms and may provide significance in developing this peptide to an antifungal drug.
Keywords: Mouse beta defensin-1; Soluble expression; Fusion protein; Purification; Antifungal activity; Candida albicans ; Cryptococcus neoformans

Inferring Minimal Feasible Metabolic Networks of Escherichia coli by Da Jiang; Shuigeng Zhou; Hui Liu; Yi-Ping Phoebe Chen (222-231).
Since the organism contains many redundant reactions, the minimal feasible metabolic network that contains the basic growth function is not the collection of reactions that associate the essential genes. To identify minimal metabolic reaction set is a challenging work in theoretical approach. A new method is presented here to identify the smallest required reaction set of growth-sustaining metabolic networks. The content and number of the minimal reactions for growth are variable in different random processes. Though the different carbon sources also vary the content of the reactions in the minimal metabolic networks, most essential reactions locate in the same metabolic subsystems, such as cofactor and prosthetic group biosynthesis, cell envelope biosynthesis, and membrane lipid metabolism.
Keywords: Metabolic networks; Minimal reaction sets; Structure analysis

Genetic Engineering of Caulobacter crescentus for Removal of Cadmium from Water by Jigar Patel; Qiong Zhang; R. Michael L. McKay; Robert Vincent; Zhaohui Xu (232-243).
Hexa-histidine (6His) peptide was inserted to a permissive site of the surface layer (S-layer) protein RsaA of Caulobacter crescentus. The recombinant strain JS4022/p723–6H, expressing RsaA-6His fusion protein was examined for its ability to sequester Cd(II) from the bacterial growth medium. When mixed with 1 ppm CdCl2, JS4022/p723–6H removed 94.3 ∼ 99.9% of the Cd(II), whereas the control strain removed only 11.4 ∼ 37.0%, depending on experimental conditions. The effective contact time of the cells and Cd(II) was as short as 15 min. When higher concentrations of CdCl2 were tested, JS4022/p723–6H consistently demonstrated enhanced binding capacity over the control strain. At 15 ppm of Cd(II), each gram of JS4022/p723–6H dry cells retrieved 16.0 mg of Cd(II), comparing to 11.6 mg g−1 achieved by the control strain. This work provides a potential cost-effective solution toward bioremediation of heavy metals from aqueous systems.
Keywords: Caulobacter crescentus ; S-layer; Cadmium; Heavy metals; Bioremediation

Succinic Acid Production by Actinobacillus succinogenes Using Spent Brewer's Yeast Hydrolysate as a Nitrogen Source by Min Jiang; Kequan Chen; Zhongmin Liu; Ping Wei; Hanjie Ying; Honam Chang (244-254).
To develop a cost-effective fermentation medium, spent brewer's yeast hydrolysate was evaluated as a nitrogen source for succinic acid production by Actinobacillus succinogenes NJ113 in glucose-containing media. Autolysis and enzymatic hydrolysis were used to hydrolyze the spent brewer's yeast cells to release the nutrients. The results showed that enzymatic hydrolysis was a more effective method due to the higher succinic acid yield and cell growth. However, the incomplete glucose consumption indicated existence of nutrient limitation. Vitamins were subsequently identified as the main limiting factors for succinic acid production using enzymatically hydrolyzed spent brewer's yeast as a nitrogen source. After the addition of vitamins, cell growth and succinic acid concentration both improved. As a result, 15 g/L yeast extract could be successfully replaced with the enzymatic hydrolysate of spent brewer's yeast with vitamins supplementation, resulting in a production of 46.8 g/L succinic acid from 68 g/L glucose.
Keywords: Succinic acid; Actinobacillus succinogenes NJ113; Brewer's yeast hydrolysate; Nitrogen source

Bleach Enhancement of Mixed Wood Pulp by Xylanase–Laccase Concoction Derived Through Co-culture Strategy by Pallavi Dwivedi; V. Vivekanand; Nidhi Pareek; Amit Sharma; Rajesh P. Singh (255-268).
Mixed enzyme preparation having both xylanase and laccase activity was evaluated for its bleach enhancing ability of mixed wood pulp. The enzyme was produced through co-cultivation of mutant Penicillium oxalicum SAUE-3.510 and Pleurotus ostreatus MTCC 1804 under solid-state fermentation. Bleaching of pulp with mixed enzyme had resulted into a notable decrease in kappa number and increased brightness as compared to xylanase alone. Analysis of bleaching conditions had denoted that 8 IU g−1 of mixed enzyme preparation (xylanase/laccase, 22:1) had led into maximal removal of lignin from pulp when bleaching was performed at 10% pulp consistency (55 °C, pH 9.0) for 3 h. An overall improvement of 21%, 8%, 3%, and 5% respectively in kappa number, brightness, yellowness, and viscosity of pulp was achieved under derived bleaching conditions. Process of enzymatic bleaching was further ascertained by analyzing the changes occurring in polysaccharide and lignin by HPLC and FTIR. The UV absorption spectrum of the compounds released during enzymatic treatment had denoted a characteristic peak at 280 nm, indicating the presence of lignin in released coloring matter. The changes in fiber morphology following enzymatic delignification were studied by scanning electron microscopy.
Keywords: Biobleaching; Penicillium oxalicum ; Co-cultivation; Xylanase; Laccase; Pleurotus ostreatus

Polymerase chain reaction (PCR) is one of the most powerful techniques in a variety of clinical and biological research fields. In this paper, a chemometrics approach, combining experimental design (ED) and artificial neural network (ANN), was proposed for optimization of PCR amplification of lycopene cyclase gene carRA in Blakeslea Trispora. Five-level star design was carried out to obtain experimental information and provide data source for ANN modeling. Nine variables were used as inputs in ANN, including the added amount of template, primer, dNTP, polymerase and magnesium ion, the temperature of denaturating, annealing and extension, and the number of cycles. The output variable was the efficiency (yield) of the PCR. Based on the developed model, the effects of each parameter on PCR efficiency were predicted and the most suitable operation condition for present system was determined. At last, the validation experiment was performed under the optimized condition, and the expectant results were produced. The results obtained in this paper showed that the combination of ANN and ED provided a satisfactory optimization model with good descriptive and predictive abilities, indicating that the method of combining ANN and ED can be a useful tool in PCR optimization and other biological applications.
Keywords: Artificial neural network; Experimental design; Optimization; PCR

The enzymatic hydrolysis of lignocellulosic biomass is known to be product-inhibited by glucose. In this study, the effects on cellulolytic glucose yields of glucose inhibition and in situ glucose removal were examined and modeled during extended treatment of heat-pretreated wheat straw with the cellulolytic enzyme system, Celluclast® 1.5 L, from Trichoderma reesei, supplemented with a β-glucosidase, Novozym® 188, from Aspergillus niger. Addition of glucose (0–40 g/L) significantly decreased the enzyme-catalyzed glucose formation rates and final glucose yields, in a dose-dependent manner, during 96 h of reaction. When glucose was removed by dialysis during the enzymatic hydrolysis, the cellulose conversion rates and glucose yields increased. In fact, with dialytic in situ glucose removal, the rate of enzyme-catalyzed glucose release during 48–72 h of reaction recovered from 20–40% to become ≈70% of the rate recorded during 6–24 h of reaction. Although Michaelis–Menten kinetics do not suffice to model the kinetics of the complex multi-enzymatic degradation of cellulose, the data for the glucose inhibition were surprisingly well described by simple Michaelis–Menten inhibition models without great significance of the inhibition mechanism. Moreover, the experimental in situ removal of glucose could be simulated by a Michaelis–Menten inhibition model. The data provide an important base for design of novel reactors and operating regimes which include continuous product removal during enzymatic hydrolysis of lignocellulose.
Keywords: Cellulases; Lignocellulose; Product inhibition; Product removal; Reactor design

Use of a Bacterially Expressed Human Factor IX Light Chain to Develop Polyclonal Antibody Anti-hFIX by Sedigheh Safari; Alireza Zomorodipour; Nour Amirmozaffari; Morteza Daliri Choopari (298-298).