Applied Biochemistry and Biotechnology (v.165, #1)

From Inulin to Fructose Syrups Using Sol–Gel Immobilized Inulinase by Gonçalo L. M. Santa; Susana M. S. A. Bernardino; Salomé Magalhães; Vanessa Mendes; Marco P. C. Marques; Luís P. Fonseca; Pedro Fernandes (1-12).
The present work aims to provide the basic characterization of sol–gel immobilized inulinase, a biocatalyst configuration yet unexploited, using as model system the hydrolysis of inulin to fructose. Porous xerogel particles with dimensions in slight excess of 10 μm were obtained, yielding an immobilization efficiency of roughly 80%. The temperature– and pH–activity profiles displayed a broader bell-shaped pattern as a result of immobilization. In the latter case, a shift of the optimal pH of 0.5 pH units was observed towards a less acidic environment. The kinetic parameters estimated from the typical Michaelis–Menten kinetics suggest that immobilization in sol–gel did not tamper with the native enzyme conformation, but on the other hand, entrapment brought along mass transfer limitations. The sol–gel biocatalyst displayed a promising operational stability, since it was used in more than 20 consecutive 24-hour batch runs without noticeable decay in product yield. The performance of sol–gel biocatalyst particles doped with magnetite roughly matched the performance of simple sol–gel particles in a single batch run. However, the operational stability of the former proved poorer, since activity decay was evident after four consecutive 24-hour batch runs.
Keywords: Sol–gel; Inulinase; Inulin hydrolysis; Enzyme immobilization; Applied biocatalysis

Biophysical and Folding Parameters of Trypanothione Reductase from Leishmania infantum by Anil Kumar Shukla; Sanjukta Patra; Vikash Kumar Dubey (13-23).
Out of various tropical diseases caused by trypanosomatids, leishmaniasis is a life-threatening disease caused by the leishmania parasite. We are targeting the thiol metabolic pathway of the parasite for drug development, and trypanothione reductase (TryR) is a key enzyme of this pathway. It is important to gather significant knowledge about biophysical and intrinsic properties of this enzyme which will be helpful in better understanding of this drug-target enzyme. We report here the modulation of activity and stability of TryR from Leishmania infantum in the presence of various denaturants and pHs. The enzyme is quite stable under high concentration of denaturants and showed better stability compared to TryR of Leishmania donovani, whose sequence differs at only on position (Ala363→Gly). Structural basis of the destabilizing effects is discussed.
Keywords: Protein stability; Protein; Folding; Leishmaniasis; Drug-target enzyme

Construction of a Standard Reference Plasmid for Detecting GM Cottonseed Meal by Qingfeng Guan; Xiumin Wang; Da Teng; Yalin Yang; Fang Tian; Qingqiang Yin; Jianhua Wang (24-34).
A plasmid was constructed for quantification of genetically modified (GM) cottonseed meal in the gene-specific level. The Cry1Ab/c gene was connected with the Sad1 gene by fusion PCR. The fusion gene was cloned into the pMD®19-T Simple Vector. The plasmid DNA was then digested with a restriction endonuclease SmaI to reduce the characteristic differences between the plasmid DNA and genomic DNA. For a rough quantitative analysis of GM cotton meal contents, a rapid method for measurement of the copy numbers of the transgenic Cry and cotton endogenous Sad1 gene using a real-time PCR system with the plasmid DNA as a calibrator was established. The inter-run and intra-run coefficients of variation were less than 1.48% and 2.36%, respectively. The limits of detection and quantitation of the Cry and Sad1 genes were 9 and 91 copies of pMDCS, respectively. These results prove that the standard plasmid represents a valuable alternative to genomic DNA as a certified reference material for the quantification of GM cotton and is a useful tool to establish a feasible identification management for GM cottonseed meal content in the feed industry.
Keywords: Bt cottonseed meal; Standard plasmid; Construction; Real-time PCR

A Xylanase Gene Directly Cloned from the Genomic DNA of Alkaline Wastewater Sludge Showing Application Potential in the Paper Industry by Yanyu Zhao; Huiying Luo; Kun Meng; Pengjun Shi; Guozeng Wang; Peilong Yang; Tiezheng Yuan; Bin Yao (35-46).
A xylanase gene, aws-2x, was directly cloned from the genomic DNA of the alkaline wastewater sludge using degenerated PCR and modified TAIL-PCR. The deduced amino acid sequence of AWS-2x shared the highest identity (60%) with the xylanase from Chryseobacterium gleum belonging to the glycosyl hydrolase GH family 10. Recombinant AWS-2x was expressed in Escherichia coli BL21 (DE3) and purified to electrophoretic homogeneity. The enzyme showed maximal activity at pH 7.5 and 55 °C, maintained more than 50% of maximal activity when assayed at pH 9.0, and was stable over a wide pH range from 4.0 to 11.0. The specific activity of AWS-2x towards hardwood xylan (beechwood and birchwood xylan) was significantly higher than that to cereal xylan (oat spelt xylan and wheat arabinoxylan). These properties make AWS-2x a potential candidate for application in the pulp and paper industry.
Keywords: Xylanase; Direct cloning and heteroexpression; Alkaline wastewater sludge; Hardwood xylan

Sterol Glycosyltransferases—The Enzymes That Modify Sterols by Pankaj Chaturvedi; Pratibha Misra; Rakesh Tuli (47-68).
Sterols are important components of cell membranes, hormones, signalling molecules and defense-related biotic and abiotic chemicals. Sterol glycosyltransferases (SGTs) are enzymes involved in sterol modifications and play an important role in metabolic plasticity during adaptive responses. The enzymes are classified as a subset of family 1 glycosyltransferases due to the presence of a signature motif in their primary sequence. These enzymes follow a compulsory order sequential mechanism forming a ternary complex. The diverse applications of sterol glycosides, like cytotoxic and apoptotic activity, anticancer activity, medicinal values, anti-stress roles and anti-insect and antibacterial properties, draws attention towards their synthesis mechanisms. Many secondary metabolites are derived from sterol pathways, which are important in defense mechanisms against pathogens. SGTs in plants are involved in changed sensitivity to stress hormones and their agrochemical analogs and changed tolerance to biotic and abiotic stresses. SGTs that glycosylate steroidal hormones, such as brassinosteroids, function as growth and development regulators in plants. In terms of metabolic roles, it can be said that SGTs occupy important position in plant metabolism and may offer future tools for crop improvement.
Keywords: Adaptive response; Brassinosteroids; Cellular homeostasis; Detoxification; Glycosyltransferases; Hormonal regulation; Insect resistance; Medicinal plant; Sterols stress

An inhibitor, 9-amino-1,2,3,4-tetra hydroacridine (tacrine), is a reversible inhibitor of esterases. The reversible inhibition of the enzyme activity is thought to be examined after separation and immobilization of the enzyme under non-denaturing conditions. Hydrolytic changes of phosphatidylcholine by carboxylesterase were obtained using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry after the esterase was separated by non-denaturing two-dimensional electrophoresis, was immobilized to membranes and was stained by Ponceau S. The changes were inhibited after the enzyme on the membrane was treated by tacrine. Furthermore, the hydrolytic activity of the esterase was recovered after the inhibitor was washed with aspartic acid solution. These results indicate that the phosphatidylcholine hydrolysis activity of the isolated and immobilized enzyme is reversibly inhibited under non-denaturing conditions. Furthermore, this method can be developed to the production of an enzyme reactor able to regulate amounts of lipids.
Keywords: Electrophoresis; Ponceau S; MALDI-TOF MS; Tacrine; Phosphatidylcholine

Studies on Immobilization and Partial Characterization of Lipases from Wheat Seeds (Triticum aestivum) by Morgana Karin Pierozan; Enrique Guillermo Oestreicher; J. Vladimir Oliveira; Débora Oliveira; Helen Treichel; Rogério Luís Cansian (75-86).
The objective of this study was to provide some features on immobilization and partial characterization of lipases from wheat seeds. The optimum pH and temperature were found to be 5.5 and 32–37 °C, respectively. The stability of the concentrated enzymatic extract to high temperatures (25, 35, 45, and 55 °C) showed that the incubation of the extract at 55 °C led to its complete inactivation. The concentrated enzymatic extract kept 90% of its hydrolytic and esterification activities until 70 and 40 days of storage at 4 °C, respectively. The extract presented higher hydrolytic specificity to substrates of medium and long chains and higher esterification affinity to fatty acids of short and medium chains and alcohols with two and three carbon atoms. After the immobilization process using activated coal and sodium alginate as supports, an enhancement of about threefold in lipase activity was observed. The development of the present work permitted us to point out some characteristics of lipases from wheat seeds necessary for the proposition of new future industrial applications for this important biocatalyst.
Keywords: Triticum aestivum ; Lipases; Partial characterization; Immobilization

The medicinal fungus Ganoderma lucidum was inoculated into the media with and without supplementation of medicinal insect extracts to screen stimulators from Chinese medicinal insects for mycelial growth and triterpenoids production in submerged fermentation. The methanol and ether extracts of the tested insects had no significant stimulatory effect on the mycelial biomass production (P > 0.05), and those of H. remigator and Mylabris phalerata markedly inhibited the mycelial growth. However, the ether extract of Catharsius molossus at a concentration of 200 mg l−1 led to a significant increase in triterpenoids concentration from 231.7 ± 9.77 to 313.7 ± 10.6 mg l−1 (P < 0.01). Analysis of fermentation kinetics of G. lucidum suggests that glucose concentration in the extract of C. molossus-added group decreased more quickly as compared to the control group from day 2 to day 7 of fermentation process, while the triterpenoids biosynthesis was promoted at the same culture period. However, the culture pH profile was not affected by the addition of the extract. Chemical study of the extract show that cis-9,10-methylenehexadecanoic acid (9,10-MEA) and hexadecanoic acid (especially 9,10-MEA) were the key active compounds of the extract responsible for the stimulatory effect on the triterpenoids production.
Keywords: Ganoderma lucidum ; Submerged fermentation; Catharsius molossus ; Triterpenoids; Medicinal insects

The great performance of a fibrous bed bioreactor (FBB) is mainly dependent on the cell adhesion and immobilization into the fibrous matrix. Therefore, understanding the mechanism and factors controling cell adhesion in the fibrous matrix is necessary to optimize the FBB setup and further improve the fermentability. The adhesion behavior of a strain of Clostridium tyrobutyricum isolated from an FBB was studied, which was proven to be affected by the different environmental conditions, such as growth phase of cells, pH, ionic strength, ionic species, and composition of media. Our results also suggested that electrostatic interactions played an important role on bacteria adhesion into the fibrous matrix. This study demonstrated that the compositions of fermentation broth would have a significant effect on cell adhesion. Consequently, a two-stage glucose supply control strategy was developed to improve the performance of FBB with higher viable cell density in the operation of the FBB setup.
Keywords: Clostridium tyrobutyricum ; Fibrous matrix; Adhesion; Electrostatic interaction; Fibrous bed bioreactor

Hepatitis B virus (HBV) X protein (HBx) is a 17-kDa transcriptional coactivator that plays a significant role in the regulation of genes involved in inflammation and cell survival. It has been known to be involved in the development of liver cancer and alteration of the cellular HBx level may influence the pathogenesis of HBV-induced liver diseases. The transcription factor GLI1, a member of the glioma-associated oncogene homologue (GLI) subfamily of Krüppel-like zinc finger proteins is involved in signal transduction within the hedgehog (Hh) signaling pathway, which is involved in the development of many human malignancies. GLI activation is important for cell proliferation and anti-apoptosis in various cancers. To investigate whether the transcriptional coactivator HBx binds to the zinc finger transcription factor GLI1, recombinant HBx and GLI1 were isolated. Expression and purification of the HBx and GLI1 proteins were successfully performed in Escherichia coli. The binding of HBx to GLI1 was detected by surface plasmon resonance spectroscopy (BIAcore), fluorescence measurement, and a His-tagged pull-down experiment. After measuring the fluorescence emission spectra of purified HBx and GLI1, it was found that the interaction of these proteins is accompanied by significant conformational changes in one or both. This study provides important clues for the structural identification of signal transduction pathways involving the HBx and GLI1 proteins.
Keywords: HBx; GLI1; Interaction; Hepatocellular carcinogenesis

Cultivating Chlorella sp. in a Pilot-Scale Photobioreactor Using Centrate Wastewater for Microalgae Biomass Production and Wastewater Nutrient Removal by Min Min; Liang Wang; Yecong Li; Michael J. Mohr; Bing Hu; Wenguang Zhou; Paul Chen; Roger Ruan (123-137).
This study is concerned with a novel mass microalgae production system which, for the first time, uses “centrate”, a concentrated wastewater stream, to produce microalgal biomass for energy production. Centrate contains a high level of nutrients that support algal growth. The objective of this study was to investigate the growth characteristics of a locally isolated microalgae strain Chlorella sp. in centrate and its ability to remove nutrients from centrate. A pilot-scale photobioreactor (PBR) was constructed at a local wastewater treatment plant. The system was tested under different harvesting rates and exogenous CO2 levels with the local strain of Chlorella sp. Under low light conditions (25 μmol·m-2s-1) the system can produce 34.6 and 17.7 g·m-2day-1 biomass in terms of total suspended solids and volatile suspended solids, respectively. At a one fourth harvesting rate, reduction of chemical oxygen demand, total Kjeldahl nitrogen, and soluble total phosphorus were 70%, 61%, and 61%, respectively. The addition of CO2 to the system did not exhibit a positive effect on biomass productivity or nutrient removal in centrate which is an organic carbon rich medium. The unique PBR system is highly scalable and provides a great opportunity for biomass production coupled with wastewater treatment.
Keywords: Centrate wastewater treatment; CO2 ; Harvest rate; Microalgae

Influence of Osmotic Stress on Fermentative Production of Succinic Acid by Actinobacillus succinogenes by Xiaojiang Fang; Jian Li; Xiaoyu Zheng; Yonglan Xi; Kequan Chen; Ping Wei; Ping-Kai Ouyang; Min Jiang (138-147).
This study investigated the influence of osmotic stress on succinic acid production by Actinobacillus succinogenes NJ113. Both cell growth and succinic acid production were inhibited with the increase in osmotic stress of the medium. The use of three different osmoprotectants in the production of succinic acid was studied in order to decrease the inhibitory effects of osmotic stress during fermentation. Results indicated that proline offers optimal osmoprotection in the production of succinic acid by A. succinogenes NJ113. In tests of batch fermentation, the maximum cell concentration was observed to be 5.36 g DCW/L after the addition of 25 mmol/L proline to the fermentation medium. The cell concentration was 24% higher than that noted for the control. A total quantity of 56.2 g/L of succinic acid was produced, with a production rate of 1 g/L per hour, after 56 h of fermentation. The concentration and productivity of succinic acid was observed to be increased by 22.2% and 22%, respectively, as compared with the control. The specific activity levels of key enzymes in the metabolic network was noted to be higher following the addition of proline, particularly in the later stages of fermentation. This method of enhancing succinic acid production by the addition of an osmoprotectant may potentially provide an alternative approach for enhanced production of other organic acids.
Keywords: Osmotic stress; Proline; Actinobacillus succinogenes ; Succinic acid

Pancreatic cancer is a malignant tumor with the worst prognosis among all cancers. At the time of diagnosis, surgical cure is no longer a feasible option for most patients, thus early detection of pancreatic cancer is crucial for its treatment. Metabolomics is a powerful new analytical approach to detect the metabolome of cells, tissue, or biofluids. Here, we report the application of 1H nuclear magnetic resonance (NMR) combined with principal components analysis to discriminate pancreatic cancer patients from healthy controls based on metabolomic profiling of the serum. The metabolic analysis revealed significant lower of 3-hydroxybutyrate, 3-hydroxyisovalerate, lactate, and trimethylamine-N-oxide as well as significant higher level of isoleucine, triglyceride, leucine, and creatinine in the serum from pancreatic cancer patients compared to that of healthy controls. Our data demonstrate that the subtle differences in metabolite profiles in serum of pancreatic cancer patients and that of healthy subjects as a result of physiological and pathological variations could be identified by NMR-based metabolomics and exploited as metabolic markers for the early detection of pancreatic cancer.
Keywords: Metabolomic profiling; Serum; Pancreatic cancer; 1H NMR spectroscopy; Principal component analysis

Identification and Quantitation of Reaction Intermediates and Residuals in Lipase-Catalyzed Transesterified Oils by HPLC by Alberta N. A. Aryee; Leroy E. Phillip; Roger I. Cue; Benjamin K. Simpson (155-177).
A high-performance liquid chromatography (HPLC) unit equipped with size exclusion column and a refractive index detector was used for simultaneous monitoring, identification, and quantitation of the reaction components from lipase-catalyzed transesterification of three oils. The procedure simultaneously separated and detected the unreacted triacylglycerols (TAG), diacyl-, and monoacyl-glycerol (DAG and MAG) co-products, residual alcohol as well as free fatty acid (FFA) based on retention times. The chromatograms showed well separated and resolved peaks. The elution of the components from the transesterification reaction in increasing order was: TAG < DAG < FFA < MAG. Generally, higher alcohol ratios decreased the conversion of TAG in all the oils studied with between 14% and 94% of TAG remaining at all the treatment combinations. Higher amount of salmon skin oil (SSO) TAG was generally converted to DAG than Rothsay composite (RC) and olive oil (OO) TAG. Relatively higher amount of OO DAG was converted to MAG than SSO and RC with only 5–14% DAG remaining in OO. RC and OO generally accumulated less MAG, and this was reflected as lower MAG levels in RC (<6%) and OO (<14%) compared with SSO (<27%). For the various treatment combinations and the three oils used in this study, the least amount of FFA was recorded in transesterified OO with a maximum of approximately 4%. This HPLC method can be used as a simple and fast technique to analyze the reaction components and products of transesterification reactions without the need for additional derivatization steps.
Keywords: Acylglycerols; Lipase; Isocratic elution; Refractive index detection; HPLC

Cellulose Hydrolysis by Cellobiohydrolase Cel7A Shows Mixed Hyperbolic Product Inhibition by Rui Manuel Furtado Bezerra; Albino A. Dias; Irene Fraga; António Nazaré Pereira (178-189).
In order to establish which are the contribution of linear (total), hyperbolic (partial) or parabolic inhibitions by cellobiose, and also a special case of substrate inhibition, the kinetics of cellobiohydrolase Cel7A obtained from Trichoderma reesei was investigated. Values of kinetic parameters were estimated employing integrated forms of Michaelis–Menten equations through the use of non-linear regression, and criteria for selecting inhibition models are discussed. With cellobiose added at the beginning of the reaction, it was found that cellulose hydrolysis follows a kinetic model, which takes into account a mixed hyperbolic inhibition, by cellobiose with the following parameter values: K m 5.0 mM, K ic 0.029 mM, K iu 1.1 mM, k cat 3.6 h−1 and k cat′ 0.2 h−1. Cellulose hydrolysis without initial cellobiose added also follows the same inhibition model with similar values (4.7, 0.029 and 1.5 mM and 3.2 and 0.2 h−1, respectively). According to Akaike information criterion, more complex models that take into account substrate and parabolic inhibitions do not increase the modulation performance of cellulose hydrolysis.
Keywords: Cellulase kinetics; Cellobiohydrolase Cel7A; Integrated Michaelis–Menten equations; Hyperbolic, parabolic and substrate inhibitions; Diagnosis of enzyme inhibition

Biotechnological Production of 20-alpha-Dihydrodydrogesterone at Pilot Scale by Julia Maria Naumann; Andy Zöllner; Călin-Aurel Drăgan; Josef Messinger; Joachim Adam; Matthias Bureik (190-203).
The human sex hormone progesterone plays an essential and complex role in a number of physiological processes. Progesterone deficiency is associated with menstrual disorders and infertility as well as premature birth and abortion. For progesterone replacement therapy, the synthetic progestogen dydrogesterone is commonly used. In the body, this drug is metabolized to 20α-dihydrodydrogesterone (20α-DHD), which also shows extensive pharmacological effects and hence could act as a therapeutic agent itself. In this study, we describe an efficient biotechnological production procedure for 20α-DHD that employs the stereo- and regioselective reduction of dydrogesterone in a whole-cell biotransformation process based on recombinant fission yeast cells expressing the human enzyme AKR1C1 (20α-hydroxysteroid dehydrogenase, 20α-HSD). In a fed-batch fermentation at pilot scale (70 L) with a genetically improved production strain and under optimized reaction conditions, an average 20α-DHD production rate of 190 μM day−1 was determined for a total biotransformation time of 136 h. Combined with an effective and reliable downstream processing, a continuous production rate of 12.3 ± 1.4 g 20α-DHD per week and fermenter was achieved. We thus established an AKR-dependent whole-cell biotransformation process that can also be used for the production of other AKR1C1 substrates (as exemplarily shown by the production of 20α-dihydroprogesterone in gram scale) and is in principle suited for the production of further human AKR metabolites at industrial scale.
Keywords: 20α-hydroxysteroid dehydrogenase; AKR1C1; Aldo-keto reductase; Dydrogesterone; Schizosaccharomyces pombe ; Whole-cell biotransformation

In solid-state fermentation, among various solid supports evaluated, banana peel was found to be an ideal support and resulted into higher levels of laccase (6281.4 ± 63.60 U l−1) along with notable levels of manganese peroxidase production (1339.0 ± 131.23 U l−1) by Aspergillus fumigatus VkJ2.4.5. Maximum levels of laccase was achieved under derived conditions consisting of 80% of moisture level, 6 days of incubation period, 6% inoculum level, and an aeration level of 2.5 l min−1. A column-tray bioreactor was designed to scale up and economize the enzyme production in three successive cycles of fermentation using the same fungal biomass. Thermal and pH stability profiles revealed that enzyme was stable up to 50°C and at varying pH range from 5–9 for up to 2 h. The apparent molecular weight of laccase was found to be 34 ± 1 kDa. MALDI-TOF/TOF analysis of the protein showed significant homology with maximum identity of 67% to other laccases reported in database.
Keywords: Solid-state fermentation; Aspergillus fumigatus ; Banana peel; Laccase; Manganese peroxidase; Bioreactor

Studies on Silver Accumulation and Nanoparticle Synthesis By Cochliobolus lunatus by Rahul B. Salunkhe; Satish V. Patil; Bipinchandra K. Salunke; Chandrashekhar D. Patil; Avinash M. Sonawane (221-234).
Development of reliable and eco-friendly processes for synthesis of metallic nanoparticles is an important step in the field of application of nanotechnology. Biological systems provide a useful option to achieve this objective. In this study, potent fungal strain was selectively isolated from soil samples on silver supplemented medium, followed by silver tolerance (100–1,000 ppm) test. The isolated fungus was subjected to morphological, 18S rRNA gene sequencing and phylogenic studies and confirmed as Cochliobolus lunatus. The silver accumulation and nanoparticle formation potential of wet cell mass of C. lunatus was investigated. The accumulation and nanoparticle formation by wet fungal cell mass with respect to pH change was also studied. The desorbing assay was used to recover accumulated silver from cell mass. C. lunatus was found to produce optimum biomass (0.94 g%) at 635 ppm of silver. Atomic absorption spectroscopy study showed that at optimum pH (6.5 ± 0.2), cell mass accumulates 55.6% of 100 ppm silver. SEM and FTIR studies revealed that the cell wall of C. lunatus is the site of silver sorption, and certain organic groups such as carbonyl, carboxyl, and secondary amines in the fungal cell wall have an important role in biosorption of silver in nanoform. XRD determined the FCC crystalline nature of silver nanoparticles. TEM analysis established the shape of the silver nanoparticles to be spherical with the presence of very small-sized nanoparticles. Average size of silver nanoparticles (14 nm) was confirmed by particle sizing system. This study reports the synthesis and accumulation of silver nanoparticles through reduction of Ag+ ions by the wet cell mass of fungus C. lunatus.
Keywords: Cochliobolus lunatus ; Biosorption; Silver accumulation; Silver nanoparticles

This study was conducted to investigate the antibacterial effect of BSAP-254 on Bacillus cereus with the induced stress proteins. The BSAP-254 is an antimicrobial peptide isolated from soybean-fermenting bacteria, Bacillus subtilis SC-8. It had a narrow spectrum of activity against B. cereus group. The growth inhibitory effect of BSAP-254 (50 μg/mL) reduced the population of B. cereus from >108 to 104 colony-forming units per milliliter within 30 min. In B. cereus exposed to BSAP-254, 14 intracellular proteins were differentially expressed as determined by 2-DE coupled with MS. Of the differentially expressed proteins identified, the stress protein GroEL, which is heat shock protein, was induced in B. cereus exposed to antibacterial peptide.
Keywords: Antibacterial effect; Bacillus subtilis ; Bacillus cereus ; Soybean-fermenting bacteria; Food-borne pathogenic bacteria; Heat shock protein GroEL

Oxidative Lime Pretreatment of Dacotah Switchgrass by Matthew Falls; Rocio Sierra-Ramirez; Mark T. Holtzapple (243-259).
Oxidative lime pretreatment increases the enzymatic digestibility of lignocellulosic biomass primarily by removing lignin. In this study, recommended pretreatment conditions (reaction temperature, oxygen pressure, lime loading, and time) were determined for Dacotah switchgrass. Glucan and xylan overall hydrolysis yields (72 h, 15 FPU/g raw glucan) were measured for 105 different reaction conditions involving three different reactor configurations (very short term, short term, and long term). The short-term reactor was the most productive. At the recommended pretreatment condition (120 °C, 6.89 bar O2, 240 min), it achieved an overall glucan hydrolysis yield of 85.2 g glucan hydrolyzed/100 g raw glucan and an overall xylan yield of 50.1 g xylan hydrolyzed/100 g raw xylan. At this condition, glucan oligomers (1.80 g glucan recovered/100 g glucan in raw biomass) and xylan oligomers (25.20 g xylan recovered/100 g xylan in raw biomass) were recovered from the pretreatment liquor, which compensate for low pretreatment yields.
Keywords: Switchgrass; Pretreatment; Lime; Enzymatic digestion; Biofuels

Comparison of Denitrification Between Paracoccus sp. and Diaphorobacter sp. by Srinandan S. Chakravarthy; Samay Pande; Ashish Kapoor; Anuradha S. Nerurkar (260-269).
Denitrification was compared between Paracoccus sp. and Diaphorobacter sp. in this study, both of which were isolated from activated sludge of a denitrifying reactor. Denitrification of both isolates showed contrasting patterns, where Diaphorobacter sp. showed accumulation of nitrite in the medium while Paracoccus sp. showed no accumulation. The nitrate reduction rate was 1.5 times more than the nitrite reduction in Diaphorobacter sp., as analyzed by the resting state denitrification kinetics. Increasing the nitrate concentration in the medium increased the nitrite accumulation in Diaphorobacter sp., but not in Paracoccus sp., indicating a branched electron transfer during denitrification. Diaphorobacter sp. was unable to denitrify efficiently at high nitrate concentrations from 1 M, but Paracoccus sp. could denitrify even up to 2 M nitrate. Paracoccus sp. was found to be an efficient denitrifier with insignificant amounts of nitrite accumulation, and it could also denitrify high amounts of nitrate up to 2 M. Efficient denitrification without accumulation of intermediates like nitrite is desirable in the removal of high nitrates from wastewaters. Paracoccus sp. is shown to suffice this demand and could be a potential organism to remove high nitrates effectively.
Keywords: Denitrification; Wastewater treatment; Paracoccus sp.; Diaphorobacter sp.; High nitrate removal

We propose that microaerobic composting (MC) can be used to decompose vegetal matter with a short turnover time and large carbon (C) recycling potential. We used a novel method for measuring the degree of fragmentation of water-insoluble acid-soluble (WIAS) polysaccharides as a proxy in tracking their relative degree of degradation (i.e., fragmentation endpoint index). Oak leaves and food scrap processed by MC reached a fragmentation end point within 2 weeks. After amending the MC products into soil, the half-life of the polysaccharide residues was ~6–7 times longer (~100–110 days) than that measured during MC. The main products given up during MC were volatile organic acids (VOAs), alcohols and soluble carbohydrates in the compost tea, and CO2. These products accounted for about 2% of the initial carbon in the feedstock. Very small amounts of VOAs, particularly butyric acid, were formed in the amended soil. Based on a residence time of materials in fermentors of 2 weeks, a ~100-m3 capacity MC facility could process 2,000–4,000 metric tons of vegetable matter amended in ten hectares of arable land per year.
Keywords: Microaerobic composting; Carbon recycling; Carbon sequestration; EPF index; Polysaccharide turnover; Soil

Chemical Modification and Structural Analysis of Protein Isolates to Produce Hydrogel using Whitemouth Croaker (Micropogonias furnieri) Wastes by Vilásia Guimarães Martins; Jorge Alberto Vieira Costa; Srinivasan Damodaran; Carlos Prentice (279-289).
Recovery and alteration of fish protein from wastes and its use has been regarded as a promising alternative to develop useful products once polymer gels have a high capacity of water uptake. This study aims to produce hydrogel, a super absorbent biopolymer from modified fish protein, in order to evaluate the protein structure. In the modified proteins, analyses of the extent of modification of the lysine residues, electrophoresis, and electrometric titration were performed. In the hydrogels were realized assays of swelling water. The proteins with more modifications were shown as 63.5% and 75.9% of lysine residues, from fish protein isolate obtained with alkaline and acid solubilization, respectively. The modified protein in that same rate presented 332.0 and 311.4 carboxyl groups. Accordingly, the hydrogel produced from alkaline and acid isolates reached a maximum water uptake in 24 h of 79.42 and 103.25 gwater/gdry gel, respectively.
Keywords: Fish; Waste; Hydrogel; Biopolymer; Super absorbent; Protein isolate

First Detection of Shiga Toxin-Producing Escherichia coli in Shellfish and Coastal Environments of Morocco by Mohamed Bennani; Samira Badri; Tarik Baibai; Nadia Oubrim; Mohammed Hassar; Nozha Cohen; Hamid Amarouch (290-299).
Shiga toxin Escherichia coli (STEC), also called verotoxin-producing E. coli, is a major cause of food-borne illness, capable of causing hemorrhagic colitis and hemolytic–uremic syndrome (HUS). This study was carried out to evaluate the presence of (STEC) and E. coli O157:H7 in shellfish and Mediterranean coastal environments of Morocco. The contamination of shellfish and marine environment with Shiga toxin-producing E. coli (STEC) and E. coli O157:H7, was investigated during 2007 and 2008. A total of 619 samples were analyzed and 151 strains of E. coli were isolated. The presence of the stx1, stx2, and eae genes was tested in E. coli isolates strains using a triplex polymerase chain reaction. STEC was detected in three positives samples (1.9%), corresponding to the serotype O157:H7, the others Shiga toxin-producing E. coli non-O157 were also detected.
Keywords: Coastal environment; Escherichia coli ; Shiga-like toxin-producing E. coli ; STEC; PCR triplex

Purification and Characterization of a New Bacillus thuringiensis Bacteriocin Active Against Listeria monocytogenes, Bacillus cereus and Agrobacterium tumefaciens by Fakher Kamoun; Ines Ben Fguira; Najeh Belguith Ben Hassen; Hafedh Mejdoub; Didier Lereclus; Samir Jaoua (300-314).
This study reports on the identification, characterization and purification of a new bacteriocin, named Bacthuricin F103, from a Bacillus thuringiensis strain BUPM103. Bacthuricin F103 production began in the early exponential phase and reached a maximum in the middle of the same phase. Two chromatographic methods based on high performance liquid chromatography and fast protein liquid chromatography systems were used to purify Bacthuricin F103. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that this bacteriocin had a molecular weight of approximately 11 kDa. It also showed a wide range of thermostability of up to 80 °C for 60 min and a broad spectrum of antimicrobial activity over a pH range of 3.0–10.0. This bacteriocin was noted, and for the first time, to exhibit potent antimicrobial activity against Agrobacterium subsp. strains, the major causal agents of crown gall disease in tomato and vineyard crops, and against several challenging organisms in food, such as Listeria monocytogenes and Bacillus cereus. Complete killing with immediate impact on cells was observed within a short period of time. The sequence obtained for Bacthuricin F103 by direct N-terminal sequencing shared considerable homology with hemolysin. Bacthuricin F103 was noted to act through the depletion of intracellular ions, which suggest that the cell membrane was a possible target to Bacthuricin F103.
Keywords: Bacillus thuringiensis ; Bacthuricin F103; Chromatographic methods; Antimicrobial activity; N-terminal sequencing

Molasses as a Whole Medium for Biosurfactants Production by Bacillus Strains and Their Application by Atipan Saimmai; Vorasan Sobhon; Suppasil Maneerat (315-335).
Two types of biosurfactant (BS)-producing bacteria, Bacillus licheniformis TR7 and Bacillus subtilis SA9, were isolated from mangrove sediment in the south of Thailand. The BS production was done by using only molasses as a whole medium for growth and production. Under optimized conditions, the yields of TR7 and SA9 BS were found to be 3.30 and 3.78 g/l, respectively. It could reduce the surface tension of pure water to 28.5 and 29.5 mN/m, with the critical micelle concentrations of about 10 and 30 mg/l, respectively. Good thermal, pH, and salt stability were exhibited. Both BSs could recover oil more effectively than the two synthetic surfactants. In addition, TR7 and SA9 BS could enhance the solubility of polyaromatic hydrocarbons (PAHs). Thus, these BSs have the potential for the removal of oil and PAHs from the combined contaminated environment and facilitate its bioremediation. These studies indicate that molasses, as a renewable, relatively inexpensive and easily available resource, can be used for important biotechnological processes.
Keywords: Biosurfactant; Mangrove sediment; Bacillus spp.; Molasses; Oil recovery; Polyaromatic hydrocarbon

Purification of Soluble Acetylcholinesterase from Sheep Liver by Affinity Chromatography by Kasim Abass Askar; A. Caleb Kudi; A. John Moody (336-346).
The purpose of this study was to develop a protocol for the purification of acetylcholinesterase (AChE, acetylcholine acetylhydrolase, E.C. enzyme and to extend a purification method for further enzyme characterization. A further aim was to study whether the edrophonium’s pharmacologic action is due primarily to the inhibition or inactivation of AChE at sites of cholinergic transmission. The purification of a soluble AChE from sheep liver using affinity chromatography on Concanavalin A–Sepharose 4B and edrophonium–Sepharose 6B is studied. The affinity matrix was synthesized by coupling an inhibitor edrophonium to epoxy-activated Sepharose at flow rate of 0.5 ml/min. AChE is a pivotal enzyme in the cholinergic nervous system. Its primary function is to catalyze hydrolysis of released acetylcholine (ACh) and thus maintain homeostasis of this neurotransmitter in the central and peripheral nervous systems. Hence, AChE is important in both pharmacological and toxicological mechanisms. It was purified 842-fold with a specific activity of 21 U/mg protein. Sodium dodecyl sulfate (SDS) electrophoresis resulted in a monomeric molecular weight of 67.04 kDa, while on gel chromatography using Sephacryl S-200 under nondenaturing conditions to be 201.5 kDa. Based on the molecular weight obtained by gel filtration, the purified AChE was assumed to be a tetrameric form.
Keywords: Purification; Soluble acetylcholinesterase; Affinity chromatography

Effect of Organic Load on the Performance and Methane Production of an AnSBBR Treating Effluent from Biodiesel Production by Roberto Antonio Bezerra; José Alberto Domingues Rodrigues; Suzana Maria Ratusznei; Catarina Simone Andrade Canto; Marcelo Zaiat (347-368).
Currently, there is an increasing demand for the production of biodiesel and, consequently, there will be an increasing need to treat wastewaters resulting from the production process of this biofuel. The main objective of this work was, therefore, to investigate the effect of applied volumetric organic load (AVOL) on the efficiency, stability, and methane production of an anaerobic sequencing batch biofilm reactor applied to the treatment of effluent from biodiesel production. As inert support, polyurethane foam cubes were used in the reactor and mixing was accomplished by recirculating the liquid phase. Increase in AVOL resulted in a drop in organic matter removal efficiency and increase in total volatile acids in the effluent. AVOLs of 1.5, 3.0, 4.5 and 6.0 g COD L−1 day−1 resulted in removal efficiencies of 92%, 81%, 67%, and 50%, for effluent filtered samples, and 91%, 80%, 63%, and 47%, for non-filtered samples, respectively, whereas total volatile acids concentrations in the effluent amounted to 42, 145, 386 and 729 mg HAc L−1, respectively. Moreover, on increasing AVOL from 1.5 to 4.5 g COD L−1 day−1 methane production increased from 29.5 to 55.5 N mL CH4 g COD−1. However, this production dropped to 36.0 N mL CH4 g COD−1 when AVOL was increased to 6.0 g COD L−1 day−1, likely due to the higher concentration of volatile acids in the reactor. Despite the higher concentration of volatile acids at the highest AVOL, alkalinity supplementation to the influent, in the form of sodium bicarbonate, at a ratio of 0.5–1.3 g NaHCO3 g COD fed −1 , was sufficient to maintain the pH near neutral and guarantee process stability during reactor operation.
Keywords: AnSBBR; Applied volumetric organic load; Methane production; Biodiesel effluent

To determine the variations of growth, some key enzyme activities such as glucose kinase (GK), glucose-6-phosphate dehydrogenase (G6PDH), α-ketoglutarate dehydrogenase (KGDH), and isocitrate lyase (ICL) besides metabolite levels of pyruvate and antibiotic production of newly isolated Streptomyces sp. M3004 were grown in culture media which contain 10–20 g/l concentration with either glucose or glycerol as carbon source. Biomass and intracellular glucose and glycerol levels of Streptomyces sp. M3004 showed positive correlation with the concentration of these carbon sources, and these levels were higher in glucose compared with the glycerol-supplemented mediums. GK, G6PDH, and KGDH activities showed marked correlation with the concentration of both glucose and glycerol, and the activity levels were 4.14-, 1.47-, and 1.27-fold higher in glucose than glycerol. A key enzyme of the glyoxalate cycle, ICL activities decreased with increasing glucose concentrations from 10 to 20 g/l, but increased up to 15 g/l of glycerol. The positive correlations were also determined between intracellular glucose and glycerol levels besides pyruvate and protein variations with respect to concentrations of the carbon sources. Antibacterial activities of Streptomyces sp. M3004 reached maximum on the stationary phase, while it did not change significantly with respect to glucose and glycerol.
Keywords: Streptomyces sp.; Carbon sources; Carbohydrate metabolism; Protein; Antibiotic

Differential Properties of Aspergillus niger Tannase Produced Under Solid-State and Submerged Fermentations by Jaqueline Renovato; Gerardo Gutiérrez-Sánchez; Luis V. Rodríguez-Durán; Carl Bergman; Raúl Rodríguez; Cristóbal Noe Aguilar (382-395).
Significant differences on structure, stability, and catalytic properties of tannase were found when this enzyme was produced under solid-state and submerged fermentations (SSF and SmF) by Aspergillus niger. The specific activity was 5.5 times higher on SSF than in SmF. Significant differences in isoelectric points of tannases were found. The pH optima for both types of enzyme was found at 6 and the pH stability of SSF and SmF tannase were at 6 and 5–8, respectively. The optimal temperature range was from 50 to 60 °C for SmF tannase and 60 °C for SSF tannase, and both enzyme types showed tolerance to high temperatures (60–70 °C). The SSF tannase showed a major specificity for methyl gallate substrate while SmF tannase for tannic acid. All metal ions tested, had an activity inhibition from 30–46% on SSF tannase. SDS-PAGE analysis as well as gel localization studies of both SSF and SmF purified tannases showed a single band with a molecular weight of 102 and 105 kDa, respectively. Different levels of glycosylation were found among SSF and SmF purified tannases. This is the first report about structural differences among tannase produced under SSF and SmF and this study provides basis for explanation of the stability and catalytic differences observed previously for this two tannase types.
Keywords: Tannase; Catalysis; Stability; Glycosylation; Aspergillus niger