Applied Biochemistry and Biotechnology (v.175, #6)

Exploring a New Serine Protease from Cucumis sativus L by Zohara Nafeesa; B. R. Shivalingu; H. K. Vivek; B. S. Priya; S. Nanjunda Swamy (2787-2794).
Coagulation is an important physiological process in hemostasis which is activated by sequential action of proteases. This study aims to understand the involvement of aqueous fruit extract of Cucumis sativus L. (AqFEC) European burp less variety in blood coagulation cascade. AqFEC hydrolyzed casein in a dose-dependent manner. The presence of protease activity was further confirmed by casein zymography which revealed the possible presence of two high molecular weight protease(s). The proteolytic activity was inhibited only by phenyl methyl sulphonyl fluoride suggesting the presence of serine protease(s). In a dose-dependent manner, AqFEC also hydrolysed Aα and Bβ subunits of fibrinogen, whereas it failed to degrade the γ subunit of fibrinogen even at a concentration as high as 100 μg and incubation time up to 4 h. AqFEC reduced the clotting time of citrated plasma by 87.65 %. The protease and fibrinogenolytic activity of AqFEC suggests its possible role in stopping the bleeding and ensuing wound healing process.
Keywords: Coagulation; Cucumis sativus ; Fibrinogenolytic activity; Procoagulating activity; Protease; Zymogram

Comparison of Two Different Reactive Dye Immobilized Poly(Hydroxyethyl Methacrylate) Cryogel Discs for Purification of Lysozyme by Işık Perçin; Gulsu Sener; Ahmet Hamdi Demirçelik; Nilay Bereli; Adil Denizli (2795-2805).
In this study, cibacron blue F3GA and alkali blue 6B immobilized poly(hydroxyethyl methacrylate) [PHEMA] cryogel discs were prepared. The cryogel discs were characterised by swelling tests, elemental analysis and scanning electron microscopy. Each cryogel disc was used for lysozyme adsorption from aqueous solutions. Maximum adsorption capacities were 103.3 and 106.7 mg/g for cibacron blue F3GA and alkali blue 6B immobilized cryogel discs, respectively. Equilibrium lysozyme concentration, pH, ionic strength and temperature were the factors of which effect on lysozyme adsorption was investigated. Reusability of the cryogel discs was tested and less than 5 % decrease in adsorption capacity was reported. In the last stage of this work, the cryogel discs were used for lysozyme purification from chicken egg white. Molecular weight and purity of the eluted lysozyme from cryogel discs were controlled by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In conclusion, cibacron blue F3GA and alkali blue 6B immobilized cryogel discs present a cheap and fast way for purification of lysozyme with a high purity.
Keywords: Cibacron blue F3GA; Alkali blue 6B; Lysozyme; Egg white; Cryogel; PHEMA

Genetic Diversity and Some Aspects of Antimicrobial Activity of Lactic Acid Bacteria Isolated from Goat Milk by Valéria Quintana Cavicchioli; Wesley dos Santos Dornellas; Luana Martins Perin; Fábio Alessandro Pieri; Bernadette Dora Gombossy de Melo Franco; Svetoslav Dimitrov Todorov; Luís Augusto Nero (2806-2822).
Lactic acid bacteria (LAB, n = 57) were previously obtained from raw goat milk, identified as Lactococcus spp. (n = 24) and Enterococcus spp. (n = 33), and characterized as bacteriocinogenic. Fingerprinting by pulsed field gel electrophoresis (PFGE) demonstrated high genetic diversity, and 30 strains were selected and exhibited strong antimicrobial activity against 46 target strains (LAB, spoilage, and foodborne pathogens). Six strains (Lactococcus lactis: GLc03 and GLc05; and Enterococcus durans: GEn09, GEn12, GEn14, and GEn17) were selected to characterize their bacteriocinogenic features, using Listeria monocytogenes ATCC 7644 as the target. The six strains produced bacteriocins at higher titer when incubated in MRS at 37 °C up to 12 h, when compared to growth at 25 and 30 °C. The produced bacteriocins kept their antimicrobial activity after exposure to 100 °C for 2 h and 121 °C for 20 min; the antimicrobial activity was also observed after treatment at pH 2.0 to 10.0, except for GLc03. L. monocytogenes populations were reduced approximately two logs after treatment with cell-free supernatants from the selected strains. These data show that goat milk can contain a diverse microbiota able to inhibit L. monocytogenes, a common pathogen found in dairy products, and can be potentially employed in biopreservation of food produced under different processing conditions.
Keywords: Goat milk; Lactic acid bacteria; Bacteriocins; PFGE; Inhibition; Listeria monocytogenes

Currently, fumaric acid is produced by catalytic isomerization of maleic acid in aqueous solutions at low pH. Being petroleum based, requiring catalyst, and producing vast amounts of by-products and wastewater, the production of fumaric acid from renewable resources by a “green” process is increasingly attractive. In an aqueous solution, the reaction equilibrium constant of the fumarase-mediated conversion of l-malic acid to fumaric acid is 1:4.2 (fumaric acid to l-malic acid). To shift the reaction equilibrium to fumaric acid, solvent engineering was carried out by varying hydrophilic solvents and their concentrations. Generally, organic solvents may denature fumarase. Therefore, fumarase from Thermus thermophilus was employed to overcome this problem. Ethylene glycol was found more suitable than other solvents. This fumarase was shown to be more stable in 50 % than in 70 % ethylene glycol. Therefore, a preparation was carried out in 50 % ethylene glycol. Under this condition, 54.7 % conversion was observed using fumarase for transforming 1 mmol l-malic acid. After precipitation by adapting the pH, and washing to remove residual solvent and substrate, 27 % total yield was obtained with 99 % purity. The results demonstrated that the alternative green route to produce bio-based fumaric acid via l-malic acid is feasible and viable.
Keywords: Fumarase; Fumaric acid; Solvent engineering; Thermostable

Most biobutanol-producing Clostridium strains are unable to ferment polysaccharides such as cellulose and xylan due to the lack of hydrolyzing enzymes. In this study, we show that Clostridium beijerinckii G117, a newly isolated biobutanol-producing strain, expresses xylanase enzyme in the presence of 1 % beechwood xylan. The xylanase activity in the medium containing actively growing culture and 1 % of beechwood xylan can reach up to 2.66 U/ml after 14 h of fermentation. Using salting-out and size-exclusion chromatography, we purify the crude xylanase by 8.7-fold from the supernatant with a yield of 32.2 %. This purified xylanase has a molecular weight of 22.6 kDa, making it one of the smallest reported clostridial xylanases. Conserved domain analysis reveals that the xylanase belongs to glycoside hydrolase family 11 (GH11) but lacks a carbohydrate binding domain. When beechwood xylan is used as substrate for the xylanase, majority of the products are xylo-oligosaccharide (~98 %), suggesting that this is an endo-1,4-β-xylanase.
Keywords: Xylanase; Clostridium ; Xylanase purification; Biobutanol; Lignocellulosic biomass

Improvement of Ansamitocin P-3 Production by Actinosynnema mirum with Fructose as the Sole Carbon Source by Tinglan Li; Yuxiang Fan; Komi Nambou; Fengxian Hu; Tadayuki Imanaka; Liujing Wei; Qiang Hua (2845-2856).
Ansamitocin P-3 (AP-3) is an active and potent anti-tumor maytansinoid, which is usually produced by Actinosynnema spp. In this study, the effects of different carbon sources on biomass and AP-3 production by Actinosynnema mirum were investigated. The results showed great biomass production behavior of A. mirum in glucose medium comparatively to other carbon sources. Interestingly, when fructose was used as the sole carbon source, the highest yield of AP-3 was obtained, which was about fourfold than that of strain cultured in glucose after 168 h. Further analysis conducted in regard to better understanding of such observations in glucose and fructose defined media showed that fructose improves AP-3 production through the stimulation of the key genes of the secondary metabolism pathways. It was concluded that fructose could be a potential carbon source for cost-effective production of AP-3 from an industrial point of view.
Keywords: Actinosynnema mirum ; Ansamitocin P-3; Fructose; Glucose; Metabolite analysis

In this study, the effect of three operating parameters, i.e., the first/second volumetric feeding ratio (milliliters/milliliters), the first anaerobic/aerobic (an/oxic) time ratio (minute/minute), and the second an/oxic time ratio (minute/minute), on the performance of a two-step fed sequencing batch reactor (SBR) system to treat swine wastewater for nutrients removal was examined. Central Composite Design, coupled with Response Surface Methodology, was employed to test these parameters at five levels in order to optimize the SBR to achieve the best removal efficiencies for six response variables including total nitrogen (TN), ammonium nitrogen (NH4-N), total phosphorus (TP), dissolved phosphorus (DP), chemical oxygen demand (COD), and biochemical oxygen demand (BOD). The results showed that the three parameters investigated had significant impact on all the response variables (TN, NH4-N, TP, DP, COD, and BOD), although the highest removal efficiency for each individual responses was associated with different combination of the three parameters. The maximum TN, NH4-N, TP, DP, COD, and BOD removal efficiencies of 96.38 %, 95.38 %, 93.62 %, 94.3 %, 95.26 %, and 92.84 % were obtained at the optimal first/second volumetric feeding ratio, first an/oxic time ratio, and second an/oxic time ratio of 3.23, 0.4, and 0.8 for TN; 2.64, 0.72, and 0.76 for NH4-N; 3.08, 1.16, and 1.07 for TP; 1.32, 0.81, and 1.0 for DP; 2.57, 0.96, and 1.12 for COD; and 1.62, 0.64, and 1.61 for BOD, respectively. Good linear relationships between the predicted and observed results for all the response variables were observed.
Keywords: Two-step fed SBR; Operating parameters optimization; Swine manure nutrients removal; Response surface methodology

Impact of Enzyme Loading on the Efficacy and Recovery of Cellulolytic Enzymes Immobilized on Enzymogel Nanoparticles by Ashani Samaratunga; Olena Kudina; Nurun Nahar; Andrey Zakharchenko; Sergiy Minko; Andriy Voronov; Scott W. Pryor (2872-2882).
Cellulase and β-glucosidase were adsorbed on a polyacrylic acid polymer brush grafted on silica nanoparticles to produce enzymogels as a form of enzyme immobilization. Enzyme loading on the enzymogels was increased to a saturation level of approximately 110 μg (protein) mg−1 (particle) for each enzyme. Enzymogels with varied enzyme loadings were then used to determine the impact on hydrolysis rate and enzyme recovery. Soluble sugar concentrations during the hydrolysis of filter paper and Solka-Floc with the enzymogels were 45 and 53 %, respectively, of concentrations when using free cellulase. β-Glucosidase enzymogels showed lower performance; hydrolyzate glucose concentrations were just 38 % of those using free enzymes. Increasing enzyme loading on the enzymogels did not reduce net efficacy for cellulase and improved efficacy for β-glucosidase. The use of free cellulases and cellulase enzymogels resulted in hydrolyzates with different proportions of cellobiose and glucose, suggesting differential attachment or efficacy of endoglucanases, exoglucanases, and β-glucosidases present in cellulase mixtures. When loading β-glucosidase individually, higher enzyme loadings on the enzymogels produced higher hydrolyzate glucose concentrations. Approximately 96 % of cellulase and 66 % of β-glucosidase were recovered on the enzymogels, while enzyme loading level did not impact recovery for either enzyme.
Keywords: Cellulase; Enzyme immobilization; Enzyme recovery; Enzymatic hydrolysis; Enzymogels

Inflammasomes are intracellular macromolecular complexes assembled to activate inflammatory caspases such as caspase-1 and caspase-5, which perform critical roles during innate immune response. The NALP3 inflammasome comprises three protein components, NALP3, ASC, and caspase-1. ASC, which contains both a pyrin domain (PYD) and a caspase recruitment domain (CARD), acts as a bridge to recruit NALP3 using the PYD/PYD interaction and to recruit caspase-1 via the CARD/CARD interaction. In this study, we successfully purified and characterized ASC CARD and caspase-1 CARD. The results showed that ASC CARD was unable to interact with caspase-1 CARD in vitro; therefore, we proposed an interaction mode between ASC CARD and caspase-1 CARD from a structural based modeling study.
Keywords: Inflammation; Inflammasome; Caspase-1; ASC; CARD

Effect of Dissolved Inorganic Carbon on β-Carotene and Fatty Acid Production in Dunaliella sp by Ramachandran Srinivasan; Velayutham Arumuga Kumar; Dileep Kumar; Nachimuthu Ramesh; Subramanian Babu; Kodiveri Muthukalianan Gothandam (2895-2906).
This study aimed to explore the effect of sodium bicarbonate (0–200 mM) on the production of β-carotene and lipid content in Dunaliella salina and Dunaliella bardawil. Total carotenoid and chlorophyll content were determined at regular intervals by a UV–VIS spectrophotometer. The β-carotene and lipid contents were analyzed using high-performance liquid chromatography (HPLC) and gas chromatography coupled with mass spectrometry (GC-MS). The HPLC results revealed a twofold increase of β-carotene in D. salina and D. bardawil cultures grown with sodium bicarbonate. Moreover, total fatty acid profiles from GC-MS indicated a maximum relative percentage of saturated fatty acids (tetradecanoic acid, 10,13-diethyl, methyl ester and methyl 16-methyl-heptadecanoate) compared to polyunsaturated fatty acids in both algae. Our results indicate that the optimum concentration of bicarbonate (100 to 150 mM) was required to stimulate a positive effect on β-carotene production as well as the lipid profile in Dunaliella sp.
Keywords: β-Carotene; Chlorophyll; Dunaliella salina ; Dunaliella bardawil ; Bicarbonate; Saturated fatty acids

Bacterial porins are major outer membrane proteins that function as essential solute transporters between the bacteria and the extracellular environment. Structural features of porins are also recognized by eukaryotic cell receptors involved in innate and adaptive immunity. To better investigate the function of porins, proper refolding is necessary following purification from inclusion bodies [1, 2]. Using a single-step size exclusion chromatographic method, we have purified three major porins from pathogenic bacteria, the OmpP2 (P2) from Haemophilus influenzae, FomA from Fusobacterium nucleatum and PorB from Neisseria meningitidis, at high yield and report their unique solute transport activity with size exclusion limit. Furthermore, we have optimized their purification method and achieved improvement of their thermostability for facilitating functional and structural analyses.
Keywords: Outer membrane protein (OMP); Porin; β-Barrel; Inclusion body; Solute transport

Adenosylmethionine synthetase plays a key role in the biogenesis of the sulfonium compound S-adenosylmethionine, the principal widely used methyl donor in the biological methylations. We report here, for the first time, the characterization of adenosylmethionine synthetase from the hyperthermophilic archaeon Pyrococcus furiosus (PfMAT). The gene PF1866 encoding PfMAT was cloned and expressed, and the recombinant protein was purified to homogeneity. PfMAT shares 51, 63, and 82 % sequence identity with the homologous enzymes from Sulfolobus solfataricus, Methanococcus jannaschii, and Thermococcus kodakarensis, respectively. PfMAT is a homodimer of 90 kDa highly thermophilic with an optimum temperature of 90 °C and is characterized by remarkable thermodynamic stability (Tm, 99 °C), kinetic stability, and resistance to guanidine hydrochloride-induced unfolding. The latter process is reversible as demonstrated by the analysis of the refolding process by activity assays and fluorescence measurements. Limited proteolysis experiments indicated that the proteolytic cleavage site is localized at Lys148 and that the C-terminal peptide is necessary for the integrity of the active site. PfMAT shows kinetic features that make it the most efficient catalyst for S-adenosylmethionine synthesis among the characterized MAT from Bacteria and Archaea. Molecular and structural characterization of PfMAT could be useful to improve MAT enzyme engineering for biotechnological applications.
Keywords: S-Adenosylmethionine; Adenosylmethionine synthetase; Methionine adenosyltransferase; Pyrococcus furiosus ; Hyperthermostability; Archaea

Enhanced Production of Nargenicin A1 and Generation of Novel Glycosylated Derivatives by Dipesh Dhakal; Tuoi Thi Le; Ramesh Prasad Pandey; Amit Kumar Jha; RitBahadur Gurung; Prakash Parajuli; Anaya Raj Pokhrel; Jin Cheol Yoo; Jae Kyung Sohng (2934-2949).
Nargenicin A1, an antibacterial polyketide macrolide produced by Nocardia sp. CS682, was enhanced by increasing the pool of precursors using different sources. Furthermore, by using engineered strain Nocardia sp. ACC18 and supplementation of glucose and glycerol, enhancement was ~7.1 fold in comparison to Nocardia sp. CS682 without supplementation of any precursors. The overproduced compound was validated by mass spectrometry and nuclear magnetic resonance analyses. The novel glycosylated derivatives of purified nargenicin A1 were generated by efficient one-pot reaction systems in which the syntheses of uridine diphosphate (UDP)-α-D-glucose and UDP-α-D-2-deoxyglucose were modified and combined with glycosyltransferase (GT) from Bacillus licheniformis. Nargenicin A1 11-O-β- D-glucopyranoside, nargenicin A1 18-O-β-D-glucopyranoside, nargenicin A111 18-O-β-D- diglucopyranoside, and nargenicin 11-O-β-D-2-deoxyglucopyranoside were generated. Nargenicin A1 11-O-β-D-glucopyranoside was structurally elucidated by ultra-high performance liquid chromatography-photodiode array (UPLC-PDA) conjugated with high-resolution quantitative time-of-flight-electrospray ionization mass spectroscopy (HR-QTOF ESI-MS/MS), supported by one- and two-dimensional nuclear magnetic resonance studies, whereas other nargenicin A1 glycosides were characterized by UPLC-PDA and HR-QTOF ESI-MS/MS analyses. The overall conversion studies indicated that the one-pot synthesis system is a highly efficient strategy for production of glycosylated derivatives of compounds like macrolides as well. Furthermore, assessment of solubility indicated that there was enhanced solubility in the case of glycoside, although a substantial increase in activity was not observed.
Keywords: Nocardia sp. CS682; Nargenicin A1 ; One-pot glycosylation; Novel nargenicin A1 ; Glucopyranoside

Physiological and Molecular Changes in Barley and Wheat Under Salinity by Aslihan Temel; Nermin Gozukirmizi (2950-2960).
In this study, it was aimed to compare salinity-induced changes in barley (Hordeum vulgare L. cv. Bornova-92) and bread wheat (Triticum aestivum L. cv. Gerek-79). Seeds were germinated under saline conditions (0, 50, 100, 250, and 500 mM NaCl) for 2 days and recovered under non-saline conditions for 2 days. At the end of the salt treatment, germination, water content (WC), total soluble protein content, and catalase (CAT, EC activity were affected in both species, while superoxide dismutase (SOD, EC activity was affected in barley. Salinity affected WC, protein content, and CAT activity in both species, while it affected germination in barley and affected fresh weight and SOD activity in wheat after recovery. Physiological responses of both species were correlated. Expression of α-tubulin, Atls1, and Lls1 genes was down-regulated in barley after 250 mM NaCl treatment. HVA1 gene was highly (more than 50-fold) stimulated by salinity in barley. However, α-tubulin and Atls1 genes were down-regulated, and Lls1 gene was up-regulated in wheat after recovery from 250-mM NaCl treatment. Increase in HVA1 expression was not significant in wheat. The expression profiles of barley and wheat under salinity are different, and barley tended to regulate gene expression faster than wheat.
Keywords: α-tubulin ; Hordeum vulgare L; HVA ; L1s1 ; Salt stress; Triticum aestivum L

Kinetic Study of Candida antarctica Lipase B Immobilization Using Poly(Methyl Methacrylate) Nanoparticles Obtained by Miniemulsion Polymerization as Support by Alexsandra Valério; Gabrieli Nicoletti; Eliane P. Cipolatti; Jorge L. Ninow; Pedro H. H. Araújo; Cláudia Sayer; Débora de Oliveira (2961-2971).
With the objective to obtain immobilized Candida antarctica lipase B (CalB) with good activity and improved utilization rate, this study evaluated the influence of enzyme and crodamol concentrations and initiator type on the CalB enzyme immobilization in nanoparticles consisting of poly(methyl methacrylate) (PMMA) obtained by miniemulsion polymerization. The kinetic study of immobilized CalB enzyme in PMMA nanoparticles was evaluated in terms of monomer conversion, particle size, zeta potential, and relative activity. The optimum immobilization condition for CalB was compared with free enzyme in the p-NPL hydrolysis activity measurement. Results showed a higher CalB enzyme stability after 20 hydrolysis cycles compared with free CalB enzyme; in particular, the relative immobilized enzyme activity was maintained up to 40 %. In conclusion, PMMA nanoparticles proved to be a good support for the CalB enzyme immobilization and may be used as a feasible alternative catalyst in industrial processes.
Keywords: Poly(methyl methacrylate); Miniemulsion; Nanoparticles; CalB enzyme; Lipase

This study investigated the effects of acidity and metal ion on the antibacterial activity of α- and β-chitosan at different molecular weights (Mw, 22–360 kDa) against Escherichia coli and Listeria innocua through agar well diffusion assay. Spectrophotometric, electrophoretic, and confocal fluorescence microscopy analysis were further employed to evaluate the antibacterial mechanisms probably involved. Increasing pH from 4.0 to 5.0 weakened the antibacterial ability of chitosan as shown by the decreased bacteria growth inhibition zone (BGIZ) from 0.63 to 0.57 cm for β-chitosan (61 kDa) and from 0.62 to 0.57 cm for α-chitosan (30 kDa) against E. coli. All β- and α-chitosan samples showed antibacterial activity against L. innocua, in which 22 kDa β-chitosan and 30 kDa α-chitosan at pH 4.0 had the highest antibacterial activity with BGIZ of 1.22 and 0.98 cm, respectively. Interactive effect between pH and Mw on the antibacterial activity of β-chitosan was observed, but not of α-chitosan. Adding Co2+ and Ni2+ significantly improved the antibacterial activity of chitosan, while adding K+, Na+, and Li+ significantly weakened the antibacterial activity of some β- and α-chitosan samples (P < 0.05), and different Mw and forms of chitosan showed different metal ion absorption capacities. Results indicate that chitosan might insert into the groove of bacterial DNA double helix structure to induce DNA degradation and permeate through bacteria cell membranes and combine with genomic DNA to induce its dysfunction, providing evidences for the antibacterial mechanisms of chitosan.
Keywords: α- and β-Chitosan; Antibacterial activity; Agar well diffusion assay; Spectrophotometric and electrophoretic analysis; Confocal fluorescence microscopy

High Cell Density Cultivation of Recombinant Escherichia coli Strains Expressing 2-O-Sulfotransferase and C5-Epimerase for the Production of Bioengineered Heparin by Jianhua Zhang; Matt Suflita; Guoyun Li; Weihong Zhong; Lingyun Li; Jonathan S. Dordick; Robert J. Linhardt; Fuming Zhang (2986-2995).
Bioengineered heparin is being investigated as a potential substitute for the animal-sourced anticoagulant drug. One step in the current process to prepare bioengineered heparin involves the conversion of N-sulfo heparosan, rich in → 4)GlcNS(1 → 4) GlcA(1 → sequences (where S is sulfo, GlcN is α-d-glucosamine, and GlcA is β-d-glucuronic acid), to a critical intermediate, rich in → 4)GlcNS(1 → 4) IdoA2S(1 → sequences (where S is sulfo and IdoA is α-l-iduronic acid), using 2-O-sulfotransferase (2-OST) and C5 epimerase (C5-epi). Until now, these heparan sulfate biosynthetic enzymes have been expressed in Escherichia coli grown in shake flask culture as fusion proteins. The current study is focused on the high cell density fed-batch cultivation of recombinant E. coli strains expressing both enzymes. We report the high productivity expression of active 2-OST and C5-epi enzymes of 6.0 and 2.2 mg/g dry cell weight, respectively.
Keywords: Fermentation; Heparin; Sulfotransferase; Epimerase; Recombinant enzymes

In order to compare the effectiveness of two molecular marker systems, a set of six RAPD and nine SSR markers were used to study the genetic diversity in a population of 46 sour orange accessions, a common rootstock used in almost all citrus orchards in Tunisia. Genetic diversity parameters [average and effective number of alleles, percentage of polymorphism, polymorphic information content (PIC), effective marker index (EMI), and marker index (MI) parameters] for RAPD, SSR, and RAPD + SSR were determined in order to assess the efficiency of the two marker systems. The results revealed that these parameters were significantly higher when using RAPD markers. Similarly, cluster analysis using the results of RAPD was practically the same as that obtained when combining data from the two marker systems (RAPD + SSR) demonstrating the efficiency of RAPD in discriminating between sour orange accessions. Therefore, the use of SSR markers, known to be more efficient and discriminatory, does not bring significant supplementary information in this work. Indeed, results would have been obtained using only the RAPD markers. Accordingly, this work highlights the efficiency and advantages of RAPD, as an easy and efficient technique, in studying citrus rootstock’s genetic diversity, and establishing genetic relationships among citrus accessions.
Keywords: Genotyping; Sour orange (Citrus aurantium L.); RAPD; SSR

Enhanced Production of 2,3-Butanediol from Sugarcane Molasses by Jian-Ying Dai; Pan Zhao; Xiao-Long Cheng; Zhi-Long Xiu (3014-3024).
2,3-Butanediol has been known as a platform green chemical, and the production cost is the key problem for its large-scale production in which the carbon source occupies a major part. Sugarcane molasses is a by-product of sugar industry and considered as a cheap carbon source for biorefinery. In this paper, the fermentation of 2,3-butanediol with sugarcane molasses was studied by reducing the medium ingredients and operation steps. The fermentation medium was optimized by response surface methodology, and 2,3-butanediol production was explored under the deficiency of sterilization, molasses acidification, and organic nitrogen source. Based on these experiments, the fermentation medium with sugarcane molasses as carbon source was simplified to five ingredients, and the steps of molasses acidification and medium sterilization were reduced; thus, the cost was reduced and the production of 2,3-butanediol was enhanced. Under fed-batch fermentation, 99.5 g/L of 2,3-butanediol and acetoin was obtained at 60 h with a yield of 0.39 g/g sugar.
Keywords: 2,3-Butanediol; Sugarcane molasses; Nonsterile fermentation; Medium simplification; Diol

Production of Cell-Penetrating Peptides in Escherichia coli Using an Intein-Mediated System by Vida Rodríguez; Jorge Lascani; Juan A. Asenjo; Barbara A. Andrews (3025-3037).
Cell-penetrating peptides are molecules with the ability to cross membranes and enter cells. Attention has been put on these peptides as a tool for drug delivery research, as they are able to serve as delivery vectors for large molecules. Intracellular delivery of bioactive peptides is a very promising research area for clinical applications, since peptides are able to simulate protein regions and thus modulate key intracellular protein-protein interactions. Therefore, evaluation of different strategies for production of these peptides is necessary. In this work, an intein-mediated system was used to evaluate Escherichia coli recombinant production of p53pAnt and PNC27 anticancer cell-penetrating peptides. It was demonstrated that the pTXB1 and the pTYB11 vector systems are suitable for production of this kind of peptides. The production process involves a low-temperature induction process and an efficient on-column intein-mediated cleavage, which allowed an effective peptide recovery using a single chromatographic step.
Keywords: Cell-penetrating peptides; Intein cleavage; Recombinant peptide production; E. coli

Continuous Removal of Nonylphenol by Versatile Peroxidase in a Two-Stage Membrane Bioreactor by J. E. Méndez-Hernández; G. Eibes; A. Arca-Ramos; T. A. Lú-Chau; G. Feijoo; M. T. Moreira; J. M. Lema (3038-3047).
The ligninolytic enzymes versatile peroxidase (VP) and manganese peroxidase (MnP) have been previously described as efficient oxidizers of the endocrine disrupting chemical (EDC) nonylphenol at high concentrations of the pollutant. Envisaging the application of an enzymatic technology as a tertiary treatment in wastewater treatment plants, it is important to design a continuous reactor that performs the efficient removal of nonylphenol under environmental conditions. In the present research, a two-stage membrane bioreactor based on the production and use of Mn3+-malonate (chemical oxidant) was applied. The bioreactor consisted of an enzymatic reactor (R1) for the production of Mn3+-malonate by VP, coupled to an oxidation reactor (R2), where the oxidation of nonylphenol by Mn3+-malonate took place. The production of Mn3+-malonate in R1 was maintained constant: 500–700 μM with minimal deactivation of the enzyme. The oxidation reactor attained nearly complete removal of nonylphenol, even at a hydraulic retention time (HRT) shorter than 20 min. The operation with real wastewater containing nonylphenol at environmental concentrations (454 nM) was also successful, with a nonylphenol removal of 99.5 % at a rate of 0.73 μM h−1. Moreover, when the HRT of R2 was sharply reduced to 6.8 and 3.6 min, the removal of nonylphenol was maintained beyond 99 %, which proves the feasibility of the system to remove the target compound present in a real effluent, even at very short HRTs.
Keywords: Nonylphenol; Versatile peroxidase (VP); Two-stage reactor; Mn3+ complex

Effects of Different Biomass Drying and Lipid Extraction Methods on Algal Lipid Yield, Fatty Acid Profile, and Biodiesel Quality by Javid Hussain; Yan Liu; Wilson A. Lopes; Janice I. Druzian; Carolina O. Souza; Gilson C. Carvalho; Iracema A. Nascimento; Wei Liao (3048-3057).
Three lipid extraction methods of hexane Soxhlet (Sox-Hex), Halim (HIP), and Bligh and Dyer (BD) were applied on freeze-dried (FD) and oven-dried (OD) Chlorella vulgaris biomass to evaluate their effects on lipid yield, fatty acid profile, and algal biodiesel quality. Among these three methods, HIP was the preferred one for C. vulgaris lipid recovery considering both extraction efficiency and solvent toxicity. It had the highest lipid yields of 20.0 and 22.0 % on FD and OD biomass, respectively, with corresponding neutral lipid yields of 14.8 and 12.7 %. The lipid profiling analysis showed that palmitic, oleic, linoleic, and α-linolenic acids were the major fatty acids in the algal lipids, and there were no significant differences on the amount of these acids between different drying and extraction methods. Correlative models applied to the fatty acid profiles concluded that high contents of palmitic and oleic acids in algal lipids contributed to balancing the ratio of saturated and unsaturated fatty acids and led to a high-quality algal biodiesel.
Keywords: Biodiesel; Chlorella vulgaris ; Lipid extraction; Fatty acid profile; Correlative models

Isolation and Molecular Characterization of Thermostable Phytase from Bacillus subtilis (BSPhyARRMK33) by Chinreddy Subramanyam Reddy; V. Mohan Murali Achary; Mrinalini Manna; Jitender Singh; Tanushri Kaul; Malireddy K. Reddy (3058-3067).
The thermostable phytase gene was isolated from Bacillus subtilis ARRMK33 (BsPhyARRMK33). The gene has an ORF of 1152 bp and that encodes a protein of 383 amino acids. Sequence analysis showed high homology with Bacillus sp. phytase proteins, but no similarity was found with other phytases. SDS-PAGE analysis exhibited a predicted molecular mass of 42 kDa. Homology modeling of BsPhyARRMK33 protein based on Bacillus amyloliquefaciens crystal structure disclosed its β-propeller structure. BsPhyARRMK33 recombinant plasmid in pET-28a(+) was expressed in Rosetta gami B DE3 cells and the maximum phytase activity 15.3 U mg−1 obtained. The enzyme exhibits high thermostability at various temperatures and broad pH ranges. The recombinant protein retained 74 % of its original activity after incubation at 95 °C for 10 min. In the presence of Ca2+, the recombinant phytase activity was maximal where as it was inhibited by EDTA. The optimal pH and temperature for the recombinant phytase activity is achieved at 7.0 and 55 °C, respectively. Thermostable nature and wide range of pH are promising features of recombinant BsPhyARRMK33 protein that may be employed as an efficient alternative to commercially known phytases and thereby alleviate environmental eutrophication.
Keywords: Bacillus subtilis ; Phytase; Thermostable; Feed additive; Phytate

High Levan Production by Bacillus licheniformis NS032 Using Ammonium Chloride as the Sole Nitrogen Source by B. D. Kekez; G. D. Gojgic-Cvijovic; D. M. Jakovljevic; J. R. Stefanovic Kojic; M. D. Markovic; V. P. Beskoski; M. M. Vrvic (3068-3083).
In this study, levan production by Bacillus licheniformis NS032 isolated from a petroleum sludge sample was investigated. High levan yield was obtained in a wide range of sucrose concentrations (up to 400 g/L) and, contrary to most levan-producing strains, using ammonium chloride as the sole N source. Interaction between sucrose, ammonium chloride, and initial pH of the medium in a low sucrose (60–200 g/L) and a high sucrose (300–400 g/L) system was analyzed by response surface methodology. According to the calculated model in the low sucrose system, maximum predicted levan yield was 47.8 g/L (sucrose 196.8 g/L, ammonium chloride 2.4 g/L, pH 7.0), while in the high sucrose system, levan yield was 99.2 g/L (sucrose 397.6 g/L, ammonium chloride 4.6 g/L, pH 7.4). In addition, protective effect of microbial levan against copper toxicity to Daphnia magna is observed for the first time. The acute toxicity (48 h EC50) of copper decreased from 0.14 to 0.44 mg/L by levan in concentration of 50 ppm.
Keywords: Levan; Box–Behnken design; pH; Bacillus licheniformis ; Response surface methodology; Copper; Daphnia magna

Replacement P212H Altered the pH–Temperature Profile of Phytase from Aspergillus niger NII 08121 by Mrudula Vasudevan Ushasree; Jalaja Vidya; Ashok Pandey (3084-3092).
Microbial phytase, a widely used animal feed enzyme, needs to be active and stable in the acidic milieu for better performance in the monogastric gut. Aspergillus niger phytases exhibit an activity dip in the pH range from 3.0 to 3.5. Replacement of amino acids, which changed the pKa of catalytic residues H82 and D362, resulted in alteration of the pH profile of a thermostable phytase from A. niger NII 08121. Substitution P212H in the protein loop at 14 Å distance to the active site amended the pH optimum from 2.5 to pH 3.2 nevertheless with a decrease in thermostability than the wild enzyme. This study described the utility of amino acid replacements based on pKa shifts of catalytic acid/base to modulate the pH profile of phytases.
Keywords: Aspergillus niger ; Phytase; pH stability; Thermostability; Site-directed mutagenesis

Biosensor Technology for Pesticides—A review by Neelam Verma; Atul Bhardwaj (3093-3119).
Pesticides, due to their lucrative outcomes, are majorly implicated in agricultural fields for crop production enhancement. Due to their pest removal properties, pesticides of various classes have been designed to persist in the environment over a longer duration after their application to achieve maximum effectiveness. Apart from their recalcitrant structure and agricultural benefits, pesticides also impose acute toxicological effects onto the other various life forms. Their accumulation in the living system may prove to be detrimental if established in higher concentrations. Thus, their prompt and accurate analysis is a crucial matter of concern. Conventional techniques like chromatographic techniques (HPLC, GC, etc.) used for pesticides detection are associated with various limitations like stumpy sensitivity and efficiency, time consumption, laboriousity, requirement of expensive equipments and highly trained technicians, and many more. So there is a need to recruit the methods which can detect these neurotoxic compounds sensitively, selectively, rapidly, and easily in the field. Present work is a brief review of the pesticide effects, their current usage scenario, permissible limits in various food stuffs and 21st century advancements of biosensor technology for pesticide detection. Due to their exceptional performance capabilities, easiness in operation and on-site working, numerous biosensors have been developed for bio-monitoring of various environmental samples for pesticide evaluation immensely throughout the globe. Till date, based on sensing element (enzyme based, antibody based, etc.) and type of detection method used (Electrochemical, optical, and piezoelectric, etc.), a number of biosensors have been developed for pesticide detection. In present communication, authors have summarized 21st century’s approaches of biosensor technology for pesticide detection such as enzyme-based biosensors, immunosensors, aptamers, molecularly imprinted polymers, and biochips technology. Also, the major technological advancements of nanotechnology in the field of biosensor technology are discussed. Various biosensors mentioned in manuscript are found to exhibit storage stability of biocomponent ranging from 30–60 days, detection limit of 10−6 − 10−16 M, response time of 1–20 min and applications of developed biosensors in environmental samples (water, food, vegetables, milk, and juice samples, etc.) are also discussed. Researchers all over the globe are working towards the development of different biosensing techniques based on contrast approaches for the detection of pesticides in various environmental samples.
Keywords: Pesticides; Biosensors; Acetylcholinesterase (AChE); Oragnophosphate (OP); Aptamers; Transducers

Yangia sp. ND199, a moderate halophile isolated from mangrove soil sample in Vietnam, was found to accumulate poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from unrelated carbon sources in a medium with 4.5 % (w/v) NaCl. Cultivation with glycerol as carbon source and yeast extract as nitrogen source resulted in maximum cell dry weight of 5.7 g/l and PHBV content of 52.8 wt% (containing 2.9 mol% of 3HV) after 40 h. The 3HV content of the PHBV was the highest during initial stages of copolymer production and decreased with increase in the copolymer amount with time, but was not affected by changing the pH of the culture medium. Only homopolymer poly(3-hydroxybutyrate) was synthesized when monosodium glutamate was used as the nitrogen source. Fed-batch cultivation of Yangia sp. ND199 with glycerol and yeast extract gave PHBV content and productivity of 53.2 wt% and 0.44 g/l/h, respectively, which were reduced to 40.6 wt% and 0.25 g/l/h, respectively, with crude glycerol as carbon source. Both the copolymer content and productivity were improved to 56 wt% and 0.61 g/l/h, respectively, by using 1:1 mixture of crude glycerol and high fructose corn syrup. This is the first report of PHBV production by a wild-type halophilic bacterium using glycerol as carbon source.
Keywords: Yangia sp. ND199; Poly(3-hydroxybutyrate-co-3-hydroxyvalerate); Glycerol; Carbon source; 3HV content

Systemic Mechanism of Taste, Flavour and Palatability in Brain by Muthuraman Pandurangan; Inho Hwang (3133-3147).
Taste is considered as one of the five traditional senses and has the ability to detect the flavour of food and certain minerals. Information of taste is transferred to the cortical gustatory area for identification and discrimination of taste quality. Animals have memory recognition power to maintain the familiar foods which are already encountered. Animal shows neophobic response when it encounters novel taste and shows no hesitation when the food is known to be safe. Palatability is the hedonic reward provided by foods and fluids. Palatability is closely related to neurochemicals, and this chemical influences the consumption of food and fluid. Even though, the food is palatable that can become aversive and avoided as a consequence of postingestional unpleasant experience such as malaise. This review presents the overall view on brain mechanisms of taste, flavour and palatability.
Keywords: Taste; Flavour; Palatability; Brain; Amygdala

Purification and Characterization of a Thermostable Hypothetical Xylanase from Aspergillus oryzae HML366 by Haiyan He; Yongling Qin; Nan Li; Guiguang Chen; Zhiqun Liang (3148-3161).
In the current study, fermentation broth of Aspergillus oryzae HML366 in sugar cane bagasse was subjected to ultrafiltration and ion exchange chromatography, and two xylanases, XynH1 and XynH2, were purified. Time-of-flight mass spectrometry coupled with SDS-PAGE analysis revealed that XynH1 is identical to the hypothetical A. oryzae RIB40 protein XP_001826985.1, with a molecular weight of 33.671 kDa. Likewise, XynH2 was identified as xylanase XynF1 with a molecular weight of 35.402 kDa. Sequence analysis indicated that XynH1 belongs to glycosyl hydrolases family 10. The specific activity of XynH1 was measured at 476.9 U/mg. Optimal xylanase activity was observed at pH 6.0, and enzyme remained active within pH 4.0–10.0 and at a temperature below 70 °C. Mg2+, Mn2+, Ca2+, and K+ enhanced the XynH1 xylanase activity to 146, 122, 114, and 108 %, respectively. XynH1 hydrolyzed Birchwood xylan and Larchwood xylan effectively. The K m and V max of XynH1 values determined were 1.16 mM and 336 μmol/min/mg with Birchwood xylan as the substrate. A. oryzae HML366 xylanase XynH1 showed superior heat and pH tolerance, therefore may have significant applications in paper and biofuel industries. These studies constitute the first investigation of the xylanase activities of the hypothetical protein XP_001826985.1 form A. oryzae.
Keywords: Extracellular xylanases; Hypothetical protein; Aspergillus oryzae ; Time-of-flight mass spectrometry; Glycosyl hydrolases family 10

Effects of Redox Mediators on Anaerobic Degradation of Phenol by Shewanella sp. XB by Jing Wang; Yang Zhou; Peiliang Li; Hong Lu; Ruofei Jin; Guangfei Liu (3162-3172).
Up to now, no information is available on the role of redox mediators in anaerobic degradation of phenol without exogenous electron acceptors. The effects of mediators on anaerobic biodegradation of phenol by Shewanella sp. XB were investigated for the first time in this study. The results showed that the removal efficiencies of phenol were enhanced by 2.87-, 3.31-, and 2.89-fold under fermentation conditions in the presence of 0.20 mM anthraquinone-2-sulfonate (AQS), 0.05 mM anthraquinone-2,6-disulfonate (AQDS), and 0.05 mM riboflavin, respectively, in 3-day incubation. Further analysis found that phenol was fermented via volatile fatty acids (VFAs) as intermediates, particularly acetate, and further converted to CH4. Under denitrification conditions, phenol removal efficiencies were enhanced by 3.92-, 4.58-, and 3.57-fold when AQS, AQDS, and riboflavin, respectively, were added into reaction systems. During this process, phenol was preferentially degraded to CO2 and H2O rather than CH4. These results indicate that the three mediators not only increased significantly phenol biodegradation rates under fermentation conditions, but also considerably accelerated nitrate bioreduction-coupled phenol degradation. These findings are beneficial to develop efficient strategies of phenol biodegradation and to better understand phenol fate in natural environments.
Keywords: Redox mediators; Phenol; Fermentation; Denitrification; Shewanella

Utilization of lignocellulose materials to replace the pure glucose for preparation of the fermenting yeast seeds could reduce the cost of ethanol fermentation, because a large quantity of glucose is saved in the large-scale seed fermentor series. In this study, Saccharomyces cerevisiae DQ1 was cultured using the freshly pretreated corn stover material as the carbon source, and then the culture broth was used as the inoculation seeds after a series of seed transfer and inoculated into the ethanol production fermentor. The results show that the yeast cell growth and ethanol fermentation performance have essentially no difference when the yeast seeds were cultured by glucose, the corn stover hydrolysate liquid, and the pretreated corn stover solids as carbon sources, respectively. Approximately 22 % of the yeast cell culture cost was saved, and the process flow sheet in industrial scale plants was simplified by using the pretreated corn stover for seed culture. The results provided a practical method for materials and operational cost reduction for cellulosic ethanol production.
Keywords: Seed culture; Corn stover (CS) solids; Carbon source; Saccharomyces cerevisiae DQ1; Simultaneous saccharification and ethanol fermentation (SSF)

Neutral phytase is used as a feed additive for degradation of anti-nutritional phytate in aquatic feed industry. Site-directed mutagenesis of Bacillus amyloliquefaciens DSM 1061 phytase was performed with an aim to increase its activity. Mutation residues were chosen based on multiple sequence alignments and structure analysis of neutral phytsaes from different microorganisms. The mutation sites on surface (D148E, S197E and N156E) and around the active site (D52E) of phytase were selected. Analysis of the phytase variants showed that the specific activities of mutants D148E and S197E remarkably increased by about 35 and 13 % over a temperature range of 40–75 °C at pH 7.0, respectively. The k cat of mutants D148E and S197E were 1.50 and 1.25 times than that of the wild-type phytase, respectively. Both D148E and S197E showed much higher thermostability than that of the wild-type phytase. However, mutants N156E and D52E led to significant loss of specific activity of the enzyme. Structural analysis revealed that these mutations may affect conformation of the active site of phytase. The present mutant phytases D148E and S197E with increased activities and thermostabilities have application potential as additives in aquaculture feed.
Keywords: Phytase; Bacillus amyloliquefaciens ; Site-directed mutagenesis; Activity

The phospholipase A2 (PLA2) from Streptomyces violaceoruber was successfully expressed in the methylotrophic yeast Pichia pastoris GS115 under the control of AOX1 promoter for the first time. The maximum activity of the recombinant PLA2 (rPLA2) reached 34.7 ± 0.2 U/mL, and specific activity was 170 ± 4 U/mg after purification. On the sodium dodecyl sulfate polyacrylamide gel electrophoresis of the culture supernatants, three bands of 21, 18, and 14.3 kDa were observed. The peptide mass fingerprinting analysis showed that all of these three bands were rPLA2 from S. violaceoruber. By the treatment with Endo H and PNGase F, it indicated that the rPLA2 occurred N-glycosylation. The enzymatic properties of this enzyme were determined. The rPLA2 exhibited a lower optimum pH (pH = 6.0) compared to the wild-type enzyme, which was a desirable property in the application of oil degumming. In the enzymatic degumming process, the phosphorus content decreased from 261.77 ± 3.51 mg/kg to 20.74 ± 0.23 mg/kg, which is very promising for the industrial application.
Keywords: Streptomyces violaceoruber ; Phospholipase; Expression; Pichia pastoris ; Degumming

Functional Analysis of the GlcP Promoter in Streptomyces peucetius var. caesius by Alba Romero; Beatriz Ruiz; Jae Kyung Sohng; Niranjan Koirala; Romina Rodríguez-Sanoja; Sergio Sánchez (3207-3217).
In Streptomyces, carbon utilization is of significant importance for the expression of genes involved in morphological differentiation and antibiotic production. Glucose is mainly transported by GlcP, a membrane protein encoded by glcp. In Streptomyces coelicolor, this protein is encoded by sco5578. However, there is little information about the physiology of the GlcP promoter in Streptomyces. The aim of the present work was to clone and perform a functional analysis of the sp7066 promoter (ortholog of sco5578) from Streptomyces peucetius var. caesius. Hydrophobicity and cellular location analysis of the putative amino acid sequence of the cloned gene predicted SP7066 would be a membrane protein with a topology of six plus six transmembrane segments interrupted by a large cytoplasmic loop. In silico analysis of the upstream region of the sp7066 transcription initiation site predicted the sequences 5′-AGGAATAGT-3′ and 5′-TTGACT-3′ for regions -10 and -35 of sp7066 promoter. To reflect sp7066 expression, the promoter sequence was amplified, subcloned, and fused to the egfp reporter gene. Immunoblot analysis revealed that D-glucose and its analog 2-deoxyglucose were able to induce sp7066 expression. This effect was not modified by the presence of equimolar concentrations of D-galactose or N-acetylglucosamine. No expression of egfp was detected with the use of other carbon sources such as L-arabinose, D-fructose, and glycerol. Based on these analyses, we conclude that D-glucose is a preferred carbon source in S. peucetius var. caesius and that the sp7066 expression product, a putative non-PTS glucose permease, likely is a H+/symporter, localized to the membrane, and shows a strong specificity for D-glucose for inducing expression.
Keywords: Streptomyces ; Membrane protein; GlcP promoter; Permease; Ortholog; Reporter gene; Induction

Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) is one of the important strains for industrial cellulase production. An understanding of the control of cellulase gene expression in T. cellulolyticus is insufficient because only a few transcriptional factors related to cellulase gene expression have been identified. In the present study, we disrupted seven putative transcription regulator genes that showed similarity with cellulase or hemicellulase regulator genes in other filamentous fungi and investigated whether these genes are related to cellulase and xylanase production. Among the seven genes, five (tclA, tbgA, tlaA, tmcA, tclB2) had a smaller effect on cellulase and xylanase activities when culturing with cellulose. On the other hand, disruption of tacA and tctA, which are respectively homologues of ace1 (repressor of cellulase) and ctf1 (inducer of cutinase), led to a decrease in cellulase and hemicellulase production due to effects at both the enzymatic and transcriptional levels, indicating that tacA and tctA have positive roles in cellulase and xylanase production in T. cellulolyticus. These results suggest that cellulase and xylanase gene regulation in T. cellulolyticus differs from that in other filamentous fungi and imply that unknown transcriptional mechanisms function in T. cellulolyticus.
Keywords: Biomass; Cellulase; Xylanase; ace1 ; ctf1 ; Talaromyces cellulolyticus ; Acremonium cellulolyticus