Applied Biochemistry and Biotechnology (v.160, #8)

Glucose Oxidase-dextran Conjugates with Enhanced Stabilities Against Temperature and pH by Melda Altikatoglu; Yeliz Basaran; Candan Arioz; Ayse Ogan; Huriye Kuzu (2187-2197).
Multipoint covalent bonding of glucose oxidase (EC to hydrophilic natural polymer dextran and optimization of procedures to obtain, with enhanced temperature and pH stabilities, were studied. Purified enzyme was conjugated with various molecular weight dextrans (17.5, 75, and188 kD) in a ratio of 20:1, 10:1, 1:1, 1:5, 1:10, 1:15, and 1:20. After 1 h of incubation at pH 7, the activities of purified enzyme and conjugates were determined at different temperatures (25°C, 30°C, 35°C, 40°C, 50°C, 60°C, 70°C, and 80°C), and the results were evaluated for thermal resistance. Increases in temperature from 25°C to 50°C did not change the activities of the conjugates. The conjugate, which was prepared with 75 kDa dextran in a molar ratio of 1:5, showed the highest thermal resistance and even the activity still remains at 80°C at pH 7.0. This conjugate also displayed activity in a wide pH range (pH 4.0–7.0) at high temperatures. Conjugate, which was synthesized with 75 kDa dextran in a molar ratio of 1:5, appears to be feasible and useful for biotechnological applications.
Keywords: Thermal stabilization; Covalent conjugate; Glucose oxidase; Dextran; Fluorescence

Immobilization of Anti-Galectin-3 onto Polysiloxane–Polyvinyl Alcohol Disks for Tumor Prostatic Diseases Diagnosis by Mario Ribeiro de Melo-Júnior; Jorge Luiz Silva Araújo-Filho; Consuelo Antunes Barreto Lins; Nicodemos Teles de Pontes-Filho; Luiz Bezerra de Carvalho Jr (2198-2207).
This work aimed to immobilize the antibody anti-galectin-3 onto polysiloxane–polyvinyl alcohol (POS-PVA) support, to evaluate its capacity to capture the serum antigen galectin-3 and to quantify by ELISA the antigen levels in sera from patients with prostatic adenocarcinoma (PA) and benign prostatic hyperplasia (BPH) and healthy individuals. Also, for comparative effect, the galectin-3 expression in the prostate tissue through immunohistochemistry was evaluated. The optical density (galectin-3 level) values established for the sera from PA and BPH patients were lower compared with those found for the healthy individuals. Galectin-3 immunohistochemically showed a significant increase and reduction of the cytoplasmatic protein expression in BPH and PA, respectively, compared with the normal prostate. These results showed that POS-PVA disks could be used as solid phase to immobilize serum galectins and in immunoassays procedures for the correspondent IgG anti-galectins detection in human sera.
Keywords: Polysiloxane–polyvinyl alcohol; Antibody immobilization; Galectin-3; Prostatic tumor; ELISA

Rational Approaches for Drug Designing Against Leishmaniasis by Anil Kumar Shukla; Bishal Kumar Singh; Sanjukta Patra; Vikash Kumar Dubey (2208-2218).
Leishmaniasis has been ignored for many years mainly because it plagues remote and poor areas. However, recently, it has drawn attention of several investigators, and active research is going on for antileishmanial drug discovery. The current available drugs have high failure rates and significant side effects. Recently, liposomal preparations of amphotericin B are available and have proved to be a better drug, but they are very expensive. Miltefosine is one of the few orally administered drugs that are effective against Leishmania. However, it has exhibited teratogenicity, hence, should not be administered to pregnant women. Thus, the search for novel and improved antileishmanial drugs continue. A rational approach to design and develop new antileishmanials can be to identify several metabolic and biochemical differences between host and parasite that can be exploited as drug target. Moreover, many natural products also have significant antileishmanial activity and are yet to be exploited. In the current review, we aim to bring together various drug targets of Leishmania, recent development in the field, future prospects, and hope in the area.
Keywords: Leishmaniasis; Drug development; Drug targets; Metabolic pathways; Proteins

Microfiltration followed by concentration and diafiltration on ultrafiltration membranes (Biomax-100, Biomax-10 and Ultracel-10) was used to recover extracellular laccase (EC from culture broth of wood-rotting fungus Cerrena unicolor. Feed, permeate, retentate and membrane wash-out solutions were analysed for the presence of laccase, proteases, protein and brown impurities. An easy, cheap and short-term procedure was proposed to obtain retentates with low yields of total proteins (less than 14%), proteases activity (less than 15%) and brown impurities (from 2% to 29%) with a simultaneous laccase recovery above 73%. The degree of laccase purification varied from 6.7 to 11.0 and depended on the type of membrane used and content of brown pigments in the feed. Subsequent protein precipitation with cold acetone increased the degree of purification about twice and reduced proteases and brown impurities to some extent. Ultracel-10 membrane was recommended as the best solution to prevent fouling of membranes and to obtain laccase-enriched fraction with a very low content of proteases and brown pigments.
Keywords: Laccase; Membrane fractionation; Microfiltration; Ultrafiltration; Diafiltration; Precipitation

We expressed a putative β-galactosidase from Sulfolobus acidocaldarius in Escherichia coli and purified the recombinant enzyme using heat treatment and Hi-Trap ion-exchange chromatography. The resultant protein gave a single 57-kDa band by SDS-PAGE and had a specific activity of 58 U/mg. The native enzyme existed as a dimer with a molecular mass of 114 kDa by gel filtration. The maximum activity of this enzyme was observed at pH 5.5 and 90 ºC. The half-lives of the enzyme at 70, 80, and 90 ºC were 494, 60, and 0.2 h, respectively. The hydrolytic activity with p-nitrophenyl(pNP) substrates followed the order p-nitrophenyl-β-d-fucopyranoside > pNP-β-d-glucopyranoside > pNP-β-d-galactopyranoside > pNP-β-d-mannopyranoside > pNP-β-d-xylopyranoside, but not toward aryl-α-glycosides or pNP-β-l-arabinofuranoside. Thus, the enzyme was actually a β-glycosidase. The β-glycosidase exhibited transglycosylation activity with pNP-β-d-galactopyranoside, pNP-β-d-glucopyranoside, and pNP-β-d-fucopyranoside in decreasing order of activity, in the reverse order of its hydrolytic activity. The hydrolytic activity was higher toward cellobiose than toward lactose, but the transglycosylation activity was lower with cellobiose than with lactose.
Keywords: β-Glycosidase; Sulfolobus acidocaldarius ; Hydrolytic activity; Transglycosylation activity; Thermostable enzyme

Proteomic Analysis of the Interactions between Mycoplasma hyopneumoniae and Porcine Tracheal Ciliated Cells by Yuan-Zuo Li; Yen-Peng Ho; Shui-Tein Chen; David Shiuan (2248-2255).
Mycoplasma hyopneumoniae colonizes at the porcine respiratory-ciliated epithelial cells and causes the enzootic pneumonia of swine. The adhesion step is crucial in the colonization process. A few adhesion molecules have been characterized, and the concurrent receptors from the porcine ciliated cells have also been suggested to recognize the adhesion molecules. In the present study, the interactions between M. hyopneumoniae and porcine tracheal ciliated cells were investigated by employing the Far-Western blotting method. The results indicate that aconitase, lamin A/C, and peroxiredoxin of the porcine tracheal ciliated cell may interact specifically with the mycoplasmal proteins. We speculate that these mycoplasmal proteins could be secreted cleavage products, and their relative small size may enable them to penetrate into ciliated cells interfering with important metabolic pathways and other critical cellular processes.
Keywords: Mycoplasma hyopneumoniae ; Pathogen-host interactions; Porcine tracheal ciliated cells; Proteomics; Virulence factors

Effect of Additives on the Activity of Tannase from Aspergillus awamori MTCC9299 by Vinod Chhokar; Meenakshi Sangwan; Vikas Beniwal; Kiran Nehra; Kaur S. Nehra (2256-2264).
Tannase from Aspergillus awamori MTCC 9299 was purified using ammonium sulfate precipitation followed by ion-exchange chromatography. A purification fold of 19.5 with 13.5% yield was obtained. Temperature of 30 °C and pH of 5.5 were found optimum for tannase activity. The effects of metals and organic solvents on the activity of tannase were also studied. Metal ions Mg+2, Mn+2, Ca+2, Na+, and K + stimulated the tannase activity, while Cu+2, Fe+3, and Co+2 acted as inhibitors of the enzyme. The addition of organic solvents like acetic acid, isoamylalcohol, chloroform, isopropyl alcohol, and ethanol completely inhibited the enzyme activity. However, butanol and benzene increased the enzyme activity.
Keywords: Tannase; DEAE-cellulose; Organic solvents; Metal ions; Aspergillus awamori MTCC 9299

Characterization and Antineoplasic Effect of Extracts Obtained from Pleurotus sajor-caju Fruiting Bodies by Nicole Dalonso; Rafaela Souza; Márcia Luciane Lange Silveira; Ângelo Adolfo Ruzza; Theodoro Marcel Wagner; Elisabeth Wisbeck; Sandra Aparecida Furlan (2265-2274).
Fungi of the Pleurotus genus present a great industrial interest due to their possibility of producing pharmacological compounds, pigments, aromas, organic acids, polysaccharides, enzymes, vitamins, amino acids, etc. Among the therapeutic products, we can highlight those with antineoplasic activity, attributed to the fungi cell wall components. Based on this, the objective of this work was to study the antineoplasic capacity of the polysaccharidic fractions obtained from Pleurotus sajor-caju fruiting bodies. Female Swiss mice were inoculated with the Ehrlich ascitic tumor (5 × 106 cells/animal) in ascitic form. The polysaccharidic fractions were administered intraperitoneally, during a 6-day period. Fractions FI and FII presented a lower volume of ascitic liquid (3.1 and 1.8 mL, respectively) and a higher reduction in the number of neoplasic cells present in the ascitic liquid (86.2% and 85%, respectively), when compared to the positive control (group inoculated with the tumor but without treatment). These fractions were characterized in terms of monosaccharide composition. Glucose was the major component detected, followed by galactose and mannose. The anomeric carbon configuration of the β-glucan was confirmed by the 13C NMR (δ 103.7). Substituted and free C3 and C6 were also detected. Protein bands were confirmed through infrared analysis.
Keywords: Antineoplasic activity; Bioactive molecules; Characterization; Pleurotus sajor-caju ; Polysaccharidic fractions

High-Yield Hypocrellin A Production in Solid-State Fermentation by Shiraia sp. SUPER-H168 by Yujie Cai; Xiaohui Liang; Xiangru Liao; Yanrui Ding; Jun Sun; Xiaohui Li (2275-2286).
Hypocrellin A production by Shiraia sp. SUPER-H168 was studied under solid-state fermentation. Corn was found to be the best substrate after evaluating eight kinds of agro-industrial crops and residues. The optimized solid-state fermentation conditions were as follows: inoculum size 3 × 106 spores, substrate particle size 0.8–1 mm, initial moisture content 50%, and temperature 30 °C. Six kinds of external carbon source and seven kinds of external nitrogen source were evaluated, respectively, for HA production. Glucose and NaNO3 were the best. The combination of them was optimized by the response surface method. The optimum compositions of the supplementary glucose and NaNO3 were 1.65 g/100 g and 0.43 g/L, respectively. Hypocrellin A production reached 4.7 mg/g.
Keywords: Hypocrellin A; Solid state fermentation; Optimization; C/N ratio; Response surface method

Alcohol dehydrogenase (ADH) from permeabilized brewer's yeast was immobilized on derived attapulgite nanofibers via glutaraldehyde covalent binding. The effect of immobilization on ADH activity, optimum temperature and pH, thermal, pH and operational stability, reusability of immobilized ADH, and bioreduction of ethyl 3-oxobutyrate (EOB) to ethyl(S)-3-hydroxybutyrate ((S)-EHB) by the immobilized ADH were investigated. The results show the immobilized ADH retained higher activity over wider ranges of pH and temperature than those of the free. The optimum temperature and pH were 7.5 and 35 °C, respectively, and 58% of the original activity was retented after incubation at 35 °C for 32 h. More importantly, in bioreduction of EOB mediated by immobilized ADH, the conversion of substrate and enantiomeric excess (ee) of product reached 88% and 99.2%, respectively, within 2 h and retained about 42% of the initial activity after eight cycles.
Keywords: Alcohol dehydrogenase(ADH); Ethyl(S)-3-hydroxybutyrate((S)-EHB); Permeabilized brewer's yeast cells; Immobilization; Attapulgite nano-fibrils

A Multistage Process to Enhance Cellobiose Production from Cellulosic Materials by Caroline Vanderghem; Pascal Boquel; Christophe Blecker; Michel Paquot (2300-2307).
Cellobiose, a disaccharide, is a valuable product that can be obtained from cellulose hydrolysis. In this study, a simple methodology is presented to enhance the production and improve the selectivity of cellobiose during enzymatic hydrolysis of cellulose. The approach consisted of a multistage removal of filtrate via vacuum filtration and resuspension of the retentate. By this process, the remaining solid was further hydrolyzed without additional enzyme loading. Compared to the continuous hydrolysis process, the production of cellobiose increased by 45%. Increased selectivity of cellobiose is due to the loss of β-glucosidases in the filtrate, while enhanced productivity is likely due to mitigated product inhibition.
Keywords: Cellobiose; Cellulose hydrolysis; Multistage process; Cellulase binding; β-glucosidases

An Oxidant- and Solvent-Stable Protease Produced by Bacillus cereus SV1: Application in the Deproteinization of Shrimp Wastes and as a Laundry Detergent Additive by Laila Manni; Kemel Jellouli; Olfa Ghorbel-Bellaaj; Rym Agrebi; Anissa Haddar; Alya Sellami-Kamoun; Moncef Nasri (2308-2321).
The current increase in amount of shrimp wastes produced by the shrimp industry has led to the need in finding new methods for shrimp wastes disposal. In this study, an extracellular organic solvent- and oxidant-stable metalloprotease was produced by Bacillus cereus SV1. Maximum protease activity (5,900 U/mL) was obtained when the strain was grown in medium containing 40 g/L shrimp wastes powder as a sole carbon source. The optimum pH, optimum temperature, pH stability, and thermal stability of the crude enzyme preparation were pH 8.0, 60 °C, pH 6–9.5, and <55 °C, respectively. The crude protease was extremely stable toward several organic solvents. No loss of activity was observed even after 60 days of incubation at 30 °C in the presence of 50% (v/v) dimethyl sulfoxide and ethyl ether; the enzyme retained more than 70% of its original activity in the presence of ethanol and N,N-dimethylformamide. The protease showed high stability toward anionic (SDS) and non-ionic (Tween 80, Tween 20, and Triton X-100) surfactants. Interestingly, the activity of the enzyme was significantly enhanced by oxidizing agents. In addition, the enzyme showed excellent compatibility with some commercial liquid detergents. The protease of B. cereus SV1, produced under the optimal culture conditions, was tested for shrimp waste deproteinization in the preparation of chitin. The protein removal with a ratio E/S of 20 was about 88%. The novelties of the SV1 protease include its high stability to organic solvents and surfactants. These unique properties make it an ideal choice for application in detergent formulations and enzymatic peptide synthesis. In addition, the enzyme may find potential applications in the deproteinization of shrimp wastes to produce chitin.
Keywords: Shrimp wastes; Bacillus cereus SV1; Enzymatic deproteinization; Organic solvent-stable protease; Oxidant stable

Management of rotavirus diarrhoea cases and prevention of nosocomial infection require rapid diagnostic method at the patient care level. Diagnostic tests currently available are not routinely used due to economic or sensitivity/specificity constraints. Agarose-based sieving media and running conditions were modulated by using central composite design and response surface methodology for screening and electropherotyping of rotaviruses. The electrophoretic resolution of rotavirus genome was calculated from input parameters characterising the gel matrix structure and running conditions. Resolution of rotavirus genome was calculated by densitometric analysis of the gel. The parameters at critical values were able to resolve 11 segmented rotavirus genome. Better resolution and electropherotypic variation in 11 segmented double-stranded RNA genome of rotavirus was detected at 1.96% (w/v) agarose concentration, 0.073 mol l−1 ionic strength of Tris base–boric acid–ethylenediamine tetraacetic acid buffer (1.4×) and 4.31 h of electrophoresis at 4.6 V cm−1 electric field strength. Modified agarose gel electrophoresis can replace other methods as a simplified alternative for routine detection of rotavirus where it is not in practice.
Keywords: Central composite design; Electropherotyping; Resolution; Rotavirus genome; Screening

Analysis of Carbon Metabolism and Improvement of γ-Polyglutamic Acid Production from Bacillus subtilis NX-2 by Jun Yao; Hong Xu; Ningning Shi; Xin Cao; Xiaohai Feng; Sha Li; Pingkai Ouyang (2332-2341).
Bacillus subtilis NX-2 produces γ-polyglutamic acid (γ-PGA) when using glucose and l-glutamate as carbon sources. The conversion of carbon sources into γ-PGA was analyzed with the 13C-NMR method after enriching the media with 13C-labeled glucose. The results showed that the percentage of γ-PGA monomers derived from glucose was relatively low, approximately 6% and 9%, respectively, with an initial glucose concentration of 30 and 40 g L−1. It was concluded that glucose was utilized mainly as the growth-limiting substrate for cell growth and supplied the required energy during γ-PGA biosynthesis, while l-glutamate was preferred as the main substrate for γ-PGA formation. To achieve an efficient conversion of l-glutamate and enhance the γ-PGA production, a fed-batch culture was proposed by feeding of glucose. By this method, supplied l-glutamate (40 g L−1) was completely depleted, and γ-PGA yield was attained 42 g L−1.
Keywords: Bacillus subtilis ; Batch culture; γ-Polyglutamic acid; Metabolic pathway; Isotope; NMR

The esterification of propionic acid was investigated using three different alcohols, namely, isopropyl alcohol, isobutyl alcohol, and isoamyl alcohol. The variation of conversion with time for the synthesis of isoamyl propionate was investigated in the presence of five enzymes. Novozym 435 showed the highest activity, and this was used as the enzyme for investigating the various parameters that influence the esterification reaction. The Ping-Pong Bi-Bi model with inhibition by both acid and alcohol was used to model the experimental data and determine the kinetics of the esterification reaction.
Keywords: Supercritical carbon dioxide; Novozym 435; Esterification; Primary alcohol; Secondary alcohol; Ping-Pong Bi-Bi mechanism

Engineering Ecotin for Identifying Proteins with a Trypsin Fold by Plínio C. Sathler; Charles S. Craik; Toshihiko Takeuchi; Russolina B. Zingali; Helena C. Castro (2355-2365).
Ecotin is a bidentate, fold-specific inhibitor of mammalian serine-proteases produced by Escherichia coli. This molecule may be engineered to increase and/or change its affinity and specificity providing significant biotechnological potential. Since ecotin binds tightly to serine proteases of the trypsin fold, it may help to identify the role of these enzymes in different biological processes. In this work, we tested ecotin variants as an affinity purification reagent for identifying enzymes in samples of tumor progression and mammary gland involution. Initially, we used a commercial source of urokinase-type plasminogen activator (u-PA) that remained fully active after elution from an affinity column of the ecotin variant (M84R, M85R). We then successfully identified u-PA from more complex mixtures including lysates from a prostate cancer cell line and involuting mouse mammary glands. Interestingly, a membrane-type serine protease 1 was isolated from the Triton X-100-solubilized PC-3 cell lysates, and surprisingly, haptoglobin, a serine-protease homolog protein, was also identified in mammary gland lysates and in blood. Haptoglobin does not prevent ecotin inhibition of u-PA, but it may act as a carrier within blood when ecotin is used in vivo. Finally, this affinity purification matrix was also able to identify a thrombin-like enzyme from snake venom using an ecotin variant directed against thrombin. Overall, the ecotin variants acted as robust tools for the isolation and characterization of proteins with a trypsin fold. Thus, they may assist in the understanding of the role of these serine proteases and homologous proteins in different biological processes.
Keywords: Ecotin; Inhibitor; Affinity chromatography; Dimeric inhibitor; Urokinase-type plasminogen activator; Haptoglobin

Enhancement of Human γ-Interferon Production in Recombinant E. coli Using Batch Cultivation by Valiollah Babaeipour; Seyed Abbas Shojaosadati; Rasoul Khalilzadeh; Nader Maghsoudi; Amir Mohammad Farnoud (2366-2376).
Development of inexpensive and simple culture media and appropriate induction conditions are always favorable for industry. In this research, chemical composition and stoichiometric data for γ-interferon production and recombinant Escherichia coli growth were used in order to achieve a simple medium and favorable induction conditions. To achieve this goal, the effects of medium composition and induction conditions on the production of γ-interferon were investigated in batch culture of E. coli BL21 (DE3) [pET3a-ifnγ]. These conditions were considered as suitable conditions for the production of γ-interferon: 2.5× M9 medium, supplemented with a mixture of amino acids (milligram per liter), including glutamic acid 215, aspartic acid 250, lysine 160, and phenylalanine 90, and induction at late-log phase (OD600 = 4.5). Under these conditions, dry cell weight of 6 ± 0.2 g/l and γ-interferon concentration of 2.15 ± 0.1 g/l were obtained. Later, without changing the concentration ratio of amino acids and glucose, the effect of increase in the primary glucose concentration on productivity of γ-interferon was investigated. It was found that 25 g/l glucose will result in maximum attainable biomass and recombinant human γ-interferon. At improved conditions, a dry cell weight of 14 ± 0.2 g/l, concentration and overall productivity of γ-interferon 4.2 ± 0.1 g/l and 420 ± 10 mg/l h, respectively, were obtained.
Keywords: γ-Interferon; Induction condition; Productivity; E. coli ; Batch culture

Expression and Purification of an Antimicrobial Peptide by Fusion with Elastin-like Polypeptides in Escherichia coli by Fan Hu; Tao Ke; Xin Li; Pei Hong Mao; Xiang Jin; Feng Li Hui; Xiang Dong Ma; Li Xin Ma (2377-2387).
Different carrier molecules have been fused to antimicrobial polypeptides (AMPs) to facilitate recombinant protein expression and purification. Some of them have improved the stability of AMPs and reduced the toxicity to host cells, but most current strategies still have some problems to be solved such as poor yield, low purity, high expense, time-consumption, and difficulty in scaling-up. Here, we introduced the elastin-like polypeptides (ELPs) as a fusion partner to express an antimicrobial polypeptide halocidin18 (Hal18). By the reversible soluble–insoluble phase transition, 69 mg of the fusion protein were purified from 1 l of culture medium with the purity of nearly 95%. After cleavage with hydroxylamine, the ELP’s tag was easily separated from Hal18 in the next round of inverse transition cycle and Hal18 (1.7 mg, ∼1.9 kDa) was mainly found in the supernatant with a recovery of about 47% and purity of 60%. Antimicrobial activity showed that Hal18 had strong antimicrobial activity against Escherichia coli and Micrococcus luteus but weak activity against Pichia pastoris.
Keywords: Antimicrobial polypides; Elastin-like polypeptide; Soluble expression; Halocidin; Fusion protein

In present study, we investigated hypoglycemic and antihyperglycemic potential of five extracts (water, ethanol, methanol, hexane, and chloroform) of four plants (i.e., seeds of Eugenia jambolana, fruits of Momordica charantia, leaves of Gymnema sylvestre, and seeds of Trigonella foenum graecum) alone and/or in combination with glimepiride in rats. Ethanol extract of E. jambolana, water extract of M. charantia, ethanol extract of G. sylvestre, and water extract of T. graecum exhibited highest hypoglycemic and antihyperglycemic activity (most active) in rats among all the extracts, while hexane extracts exhibited least activities. Most active extracts were further studied to dose-dependent (200, 100, and 50 mg/kg body weight (bw)) hypoglycemic and antihyperglycemic effects alone and in combination with glimepiride (20, 10, and 5 mg/kg bw). The combination of most active extracts (200 mg/kg bw) and lower dose of glimepiride (5 mg/kg bw) showed safer and potent hypoglycemic as well as antihyperglycemic activities without creating severe hypoglycemia in normal rats, while higher doses (200 mg/kg bw of most active extracts, and 10 and 20 mg/kg bw of glimepiride) were generated lethal hypoglycemia in normal rats. From this study, it may be concluded that the ethanol extract of E. jambolana seeds, water extract of M. charantia fruits, ethanol extract of G. sylvestre leaves, and water extract of T. graecum seeds have higher hypoglycemic and antihyperglycemic potential and may use as complementary medicine to treat the diabetic population by significantly reducing dose of standard drugs.
Keywords: Eugenia jambolana ; Momordica charantia ; Gymnema sylvestre ; Trigonella foenum graecum ; Hypoglycemia; Glucose-infused diabetes; Herbal; Glimepiride

α-Amylase: An Ideal Representative of Thermostable Enzymes by Om Prakash; Nivedita Jaiswal (2401-2414).
The conditions prevailing in the industrial applications in which enzymes are used are rather extreme, especially with respect to temperature and pH. Therefore, there is a continuing demand to improve the stability of enzymes and to meet the requirements set by specific applications. In this respect, thermostable enzymes have been proposed to be industrially relevant. In this review, α-amylase, a well-established representative of thermostable enzymes, providing an attractive model for the investigation of the structural basis of thermostability of proteins, has been discussed.
Keywords: α-Amylase; Thermostability; Structural flexibility; Rigidity; Unfolding state

The adaptive response of the fungus Blakeslea trispora to the oxidative stress induced by butylated hydroxytoluene (BHT) during carotene production in shake flask culture was investigated. The culture response to oxidative stress was studied by measuring the specific activities of catalase (CAT) and superoxide dismutase (SOD) and the micromorphology of the fungus using a computerized image analysis system. The addition of exogenous BHT to the medium caused changes of the morphology of microorganism from aggregates with large projected area to aggregates with small projected area. This morphological differentiation of the fungus was associated with high oxidative stress as evidenced by remarkable increase of the specific activities of CAT and SOD. The oxidative stress in B. trispora resulted in a fivefold increase of carotene production. The highest concentration of carotenes (125.0 mg/g dry biomass) was obtained in culture grown in medium supplemented with 20 mM of BHT.
Keywords: Butylated hydroxytoluene; Oxidative stress; Morphology; Blakeslea trispora ; Carotenes

Dynamics and Control Strategies for a Butanol Fermentation Process by Adriano Pinto Mariano; Caliane Bastos Borba Costa; Maria Regina Wolf Maciel; Francisco Maugeri Filho; Daniel Ibraim Pires Atala; Dejanira de Franceschi de Angelis; Rubens Maciel Filho (2424-2448).
In this work, mathematical modeling was employed to assess the dynamic behavior of the flash fermentation process for the production of butanol. This process consists of three interconnected units as follows: fermentor, cell retention system (tangential microfiltration), and vacuum flash vessel (responsible for the continuous recovery of butanol from the broth). Based on the study of the dynamics of the process, suitable feedback control strategies [single input/single output (SISO) and multiple input/multiple output (MIMO)] were elaborated to deal with disturbances related to the process. The regulatory control consisted of keeping sugar and/or butanol concentrations in the fermentor constant in the face of disturbances in the feed substrate concentration. Another objective was the maintenance of the proper operation of the flash tank (maintenance of the thermodynamic equilibrium of the liquid and vapor phases) considering that oscillations in the temperature in the tank are expected. The servo control consisted of changes in concentration set points. The performance of an advanced controller, the dynamic matrix control, and the classical proportional-integral controller was evaluated. Both controllers were able to regulate the operating conditions in order to accommodate the perturbations with the lowest possible alterations in the process outputs. However, the performance of the PI controller was superior because it showed quicker responses without oscillations.
Keywords: Flash fermentation; Biobutanol; Dynamics; Control; PI; DMC

Enhancing the Enzymatic Hydrolysis of Corn Stover by an Integrated Wet-milling and Alkali Pretreatment by Xun He; Yelian Miao; Xuejian Jiang; Zidong Xu; Pingkai Ouyang (2449-2457).
An integrated wet-milling and alkali pretreatment was applied to corn stover prior to enzymatic hydrolysis. The effects of NaOH concentration in the pretreatment on crystalline structure, chemical composition, and reducing-sugar yield of corn stover were investigated, and the mechanism of increasing reducing-sugar yield by the pretreatment was discussed. The experimental results showed that the crystalline structure of corn stover was disrupted, and lignin was removed, while cellulose and hemicellulose were retained in corn stover by the pretreatment with 1% NaOH in 1 h. The reducing-sugar yield from the pretreated corn stovers increased from 20.2% to 46.7% when the NaOH concentration increased from 0% to 1%. The 1% NaOH pretreated corn stover had a holocellulose conversion of 55.1%. The increase in reducing-sugar yield was related to the crystalline structure disruption and delignification of corn stover. It was clarified that the pretreatment significantly enhanced the conversion of cellulose and hemicellulose in the corn stover to sugars.
Keywords: Lignocellulosic biomass; Corn stover; Pretreatment; Enzymatic hydrolysis; Sugar

Utilization of Salmon Milt DNA Against UV Damage by Yoshiharu Sasaki; Daisuke Miyoshi; Naoki Sugimoto (2458-2466).
We examined the effect of ultraviolet (UV) irradiation on the UV spectra and radical scavenging activity of DNA strands and found that the absorption spectra of salmon milt DNA was extended up to about 350 nm after ultraviolet C (UVC, 100–280 nm) irradiation with 300 kJ/m2. The UV B (UVB, 280–315 nm) protection ability of UVC-irradiated salmon milt DNA for a single-stranded target DNA (19-mer) was further studied. The percentage of damaged target DNA after 50 kJ/m2 of UVB irradiation in the presence of UVC-irradiated salmon milt DNA, UVC-unirradiated salmon milt DNA, and 2-phenylbenzimidazole sulfonic acid was estimated to be 24.6%, 27.0%, and 18.9%, respectively. Moreover, the ultraviolet A (UVA, 315–400 nm)/UVB ratio and critical wavelength of natural (UVC-unirradiated) salmon milt DNA were estimated to be 0.13 and 313 nm, respectively, whereas those of the UVC-irradiated salmon milt DNA were 0.34 and 375 nm, respectively. Interestingly, the value of 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity in UVC-irradiated salmon milt DNA was about five times higher than that of UVC-unirradiated salmon milt DNA. These results indicate that the UVC-irradiated salmon milt DNA could be useful as a protector against a wide range of UV light from UVC∼UVA.
Keywords: Ultraviolet C-irradiated salmon milt DNA; Ultraviolet C-unirradiated salmon milt DNA; Ultraviolet absorption; Ultraviolet B protection ability; 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity

Antibacterial Activity of Hylomecon hylomeconoides Against Methicillin-Resistant Staphylococcus aureus by Jang-Gi Choi; Ok-Hwa Kang; Hee-Sung Chae; Brice Obiang-Obounou; Young-Seob Lee; You-Chang Oh; Min-San Kim; Dong-Won Shin; Jeong-Ah Kim; Young-Ho Kim; Dong-Yeul Kwon (2467-2474).
Methicillin-resistant Staphylococcus aureus (MRSA) is serious clinical urgent problems worldwide. In the present study, the antibacterial activity of Hylomecon hylomeconoides was investigated. The EtOH extract and its fraction (n-hexane, CH2Cl2, EtOAc, and H2O) were investigated against MRSA. The most active extract (CH2Cl2) led to the isolation of 6-methoxydihydrosanguinarine (6-MS), 6-acetonylhydrosanguinarine, and dihydrosanguinarine. These compounds were very active against MRSA strains with minimum inhibitory concentrations (MICs) ranging from 1.95 to 250 μg/ml. Our study did however focus on 6-MS as it appeared to be the most active with MICs in the range of 1.9 to 3.9 μg/ml. These results encourage us to think that 6-MS can be used as a natural antibacterial agent.
Keywords: Antibacterial; MRSA; Hylomecon hylomeconoides ; EtOH extract; 6-Methoxydihydrosanguinarine

Sorption of Paraquat and 2,4-D by an Oscillatoria Sp.-Dominated Cyanobacterial Mat by Dhananjay Kumar; Bhanu Prakash; Lalit K. Pandey; J. P. Gaur (2475-2485).
The present study characterises sorption of two pesticides, namely, paraquat (PQ) and 2,4-dichlorophenoxyacetic acid (2,4-D) by an Oscillatoria sp.-dominated cyanobacterial mat. Sorption of PQ onto the test mat was not significantly affected by the pH of the solution within the pH range 2–7. However, 2,4-D sorption was strongly influenced by the solution pH and was maximum at pH 2. Whereas PQ sorption increased with increase in temperature, 2,4-D sorption showed an opposite trend. The sorption of PQ and 2,4-D achieved equilibrium within 1 h of incubation, independent of concentration of pesticide and mat biomass in the solution. The pseudo-second-order kinetic model better defined PQ sorption than the pseudo-first-order model, whereas 2,4-D sorption was well defined by both the models. Sorption isotherms of both the pesticides showed L-type curve. Freundlich model more precisely defined PQ sorption than Langmuir model, thereby suggesting heterogeneous distribution of PQ binding sites onto the biomass surface. However, the Langmuir model more correctly defined 2,4-D sorption, thus, indicating homogeneous distribution of 2,4-D binding sites onto the biomass surface. The test biomass is a good sorbent for the removal of PQ because it could, independent of pH of the solution, sorb substantial amount of PQ (q max = 0.13 mmol g−1).
Keywords: Sorption; Paraquat; 2,4-D; Oscillatoria ; Cyanobacterial mat

Antibacterial Functionalization of Wool via mTGase-Catalyzed Grafting of ε-Poly-l-lysine by Qiang Wang; Guibiao Jin; Xuerong Fan; Xianfei Zhao; Li Cui; Ping Wang (2486-2497).
ε-Poly-l-lysine (ε-PL), a natural biomacromolecule having a broad spectrum of antibacterial activity, was grafted on the wool fiber via the acyl transfer reaction catalyzed by microbial transglutaminase (mTGase) to develop a new strategy for antibacterial functionalization of proteinous materials. The effects of the concentrations of ε-PLs and mTGases on the graft yields were investigated. A coating of ε-PL that almost completely covered the scale profile on the wool surface was visualized by scanning electron microscopy (SEM) and further demonstrated in terms of Allwörden’s reaction characteristic of wool. Identifiable differences in lysine content and color depth among the stained wool samples reveal the changes in the surface composition and polarity caused by the incorporation of ε-PL onto the wool substrate, respectively. The ratio of bacteriostasis to Escherichia coli of the wool fabric grafting ε-PL reached 96.6 %, indicating an excellent antibacterial effect. The application of ε-PL and corresponding mTGase-catalyzed grafting reaction would provide a worthwhile reference for antibacterial functionalization of proteinous materials in various forms.
Keywords: Microbial transglutaminase (mTGase); Antibacterial functionalization; Enzyme-catalyzed grafting; ε-Poly-l-lysine; Wool

Optimization of 2-ethylhexyl Palmitate Production Using Lipozyme RM IM as Catalyst in a Solvent-Free System by Aline Richetti; Selma G. F. Leite; Octávio A. C. Antunes; Andrea L. F. de Souza; Lindomar A. Lerin; Rogério M. Dallago; Natalia Paroul; Marco Di Luccio; J. Vladimir Oliveira; Helen Treichel; Débora de Oliveira (2498-2508).
This work reports the application of a lipase in the 2-ethylhexyl palmitate esterification in a solvent-free system with an immobilized lipase (Lipozyme RM IM). A sequential strategy was used applying two experimental designs to optimize the 2-ethylhexyl palmitate production. An empirical model was then built so as to assess the effects of process variables on the reaction conversion. Afterwards, the operating conditions that optimized 2-ethylhexyl palmitate production were established as being acid/alcohol molar ratio 1:3, temperature of 70°C, stirring rate of 150 rpm, 10 wt.% of enzyme, leading to a reaction conversion as high as 95%. From this point, a kinetic study was carried out evaluating the effect of acid:alcohol molar ratio, the enzyme concentration and the temperature on product conversion. The results obtained in this step permit to verify that an excess of alcohol (acid to alcohol molar ratio of 1:6), relatively low enzyme concentration (10 wt.%) and temperature of 70°C, led to conversions next to 100%.
Keywords: 2-ethylhexyl palmitate; Esterification; Lipozyme RM IM; Solvent-free system; Kinetic evaluation