Applied Biochemistry and Biotechnology (v.172, #4)

Genetic variation in three populations of Parkia timoriana (DC.) Merr. grown in the Manipur state of northeast India was analysed using inter-simple sequence repeat (ISSR) markers. A total of 30 individual trees representing three populations were sampled and studied using 22 University of British Columbia (UBC set no. 9) primers in the present study. Of the total 22 primers, 19 primers produced distinct, reproducible and well-resolved fragments. Overall, a total number of 111 fragments were generated by the 19 primers and of which, 51 were polymorphic (45.94 %). The average number of loci and polymorphic loci generated per primer were 5.84 and 2.68, respectively. The genetic variation generated by ISSR markers within the three populations studied ranges from 33.33 to 18.92 %. The overall genetic differentiation (Gst) among populations was estimated to be 0.29, and the number of gene flow (Nm) was estimated to be 1.23 per generation between populations. Of the total genetic variance, 70.04 % was attributed to within-population diversity while 4.72 % differences to the among-populations. The genetic similarity across the individuals belonging to the three populations was represented by the dendrogram showing the grouping of the individuals into three major groups which is also supported by the principle component analysis. The present finding asserts the effectiveness of ISSR procedure for assessing genetic variations of P. timoriana populations and provides valuable genetic information that can be utilized for breeding and conservation strategies.
Keywords: Parkia timoriana ; Population; Northeast India; Genetic variation; ISSR

In Vitro and In Vivo Characterization of Plant Growth Promoting Bacillus Strains Isolated from Extreme Environments of Eastern Algeria by Asma Ait-Kaki; Noreddine Kacem-Chaouche; Marc Ongena; Mounira Kara-Ali; Laid Dehimat; Karima Kahlat; Philippe Thonart (1735-1746).
This report is to our knowledge the first to study plant growth promotion and biocontrol characteristics of Bacillus isolates from extreme environments of Eastern Algeria. Seven isolates of 14 (50 %) were screened for their ability to inhibit growth of some phytopathogenic fungi on PDA and some roots exudates. The bacteria identification based on 16S r-RNA and gyrase-A gene sequence analysis showed that 71 % of the screened isolates belonged to Bacillus amyloliquefaciens and the rest were closely related to B. atrophaeus and B. mojavensis. Most of them had high spore yields (22 × 108–27 × 108 spores/ml). They produced protease and cellulase cell wall-degrading enzymes while the chitinase activity was only observed in the B. atrophaeus (6SEL). A wide variety of lipopeptides homologous was detected by liquid chromatography–electrospray ionization–mass spectrometry analysis. Interestingly, some additional peaks with new masses were characterized, which may correspond to new fengycin classes. The isolates produced siderophores and indole-3- acetic acid phytohormone. The greenhouse experiment using a naturally infested soil with Sclerotonia sclerotiorum showed that the B. atrophaeus (6SEL) significantly increased the size of the chickpea plants and reduced the stem rot disease (P < 0.05). These results suggest that these isolates may be used further as bio-inoculants to improve crop systems.
Keywords: Extreme environments; Bacillus ; Biocontrol; Plant growth promotion; Lipopeptides; IAA; Siderophores

Purification and Characterisation of a β-1,4-Xylanase from Remersonia thermophila CBS 540.69 and Its Application in Bread Making by Katrina McPhillips; Deborah M. Waters; Cécile Parlet; Daniel J. Walsh; Elke K. Arendt; Patrick G. Murray (1747-1762).
This work reports the first investigation of Remersonia thermophila hemicellulosic hydrolytic enzyme production, with subsequent purification of an extracellular endo-β-1,4-xylanase (RtXyl) and its application in bread making. The research describes RtXyl purification from sorghum-induced submerged liquid cultures of this moderately thermophilic, aerobic, ascomycete fungus. The purified enzyme is a single subunit protein with a molecular mass of 42 kDa and exhibits glycosyl hydrolase family-10-like activity over a broad pH and temperature range. Optimal activity was measured at pH 6.0 and 65 °C respectively, which is suitable for bread making applications. Substrate specificity studies revealed that RtXyl is purely xylanolytic with no side-activities against other plant polysaccharides. The RtXyl catalytic efficiency (K cat/K m) was highest with oats spelt xylan (810.90 mg mL−1 s−1), wheat arabinoxylan (809.52 mg mL−1 s−1) and beechwood xylan (417.40 mg mL−1 s−1) with less efficiency towards insoluble oats spelt xylan (236.40 mg mL−1 s−1). Hydrolysis products analysed by thin layer chromatography yielded a range of xylosaccharides, predominantly xylotriose and xylobiose. RtXyl application in a basic wheat bread recipe at low dosages (0.297 XU/g) showed its suitability to increase loaf volume by 8.0 % compared with the control bread. RtXyl increased loaf softness by 19.6 % while reducing bread staling by 20.4 % up to 4 days of storage.
Keywords: Xylanase; Remersonia thermophila ; Enzyme purification; Hemicellulose; Bread volume; Bread softness

Transfer and Targeted Overexpression of γ-Tocopherol Methyltransferase (γ-TMT) Gene Using Seed-Specific Promoter Improves Tocopherol Composition in Indian Soybean Cultivars by Muthukrishnan Arun; Kondeti Subramanyam; Jeevaraj Theboral; Ganeshan Sivanandhan; Manoharan Rajesh; Gnanajothi Kapil Dev; Balusamy Jaganath; Markandan Manickavasagam; Shanmugam Girija; Andy Ganapathi (1763-1776).
Soybean oil contains high levels of tocopherols which are an important source of vitamin E in human diet. The conversion of γ- to α-tocopherol catalyzed by γ-tocopherol methyltransferase (γ-TMT) is found to be the rate limiting factor in soybean which influences the tocopherol composition. Using Agrobacterium-mediated transformation, we overexpressed the γ-TMT gene of Perilla frutescens under the control of the seed-specific promoter vicillin in cultivar Pusa 16. Transgene integration and expression was confirmed in five independently transformed GUS positive soybean plants by polymerase chain reaction (PCR), Southern hybridization, and reverse transcriptase-PCR (RT-PCR). High-performance liquid chromatography (HPLC) analysis showed that overexpression of Pf-γ-TMT resulted in efficient conversion of γ-tocopherol to α-tocopherol and concomitant increase in seed α-tocopherol content in RT-PCR positive plants. The protocol was successfully applied to three more cultivars PK 416, Gujarat soybean 1, and VL soya 1 in which seeds of transformed plants showed elevated level of α-tocopherol than wild-type seeds.
Keywords: Agrobacterium tumefaciens EHA 105; α-tocopherol; γ-tocopherol; HPLC; Pf-γ-TMT

Evaluation and Functional Characterization of a Biosurfactant Produced by Lactobacillus plantarum CFR 2194 by Arenahalli Ningegowda Madhu; Siddalingaiya Gurudutt Prapulla (1777-1789).
The study details the investigations on the ability of Lactobacillus plantarum CFR 2194, an isolate from kanjika, a rice-based ayurvedic fermented product, to produce biosurfactant. Surfactant production, as a function of fermentation time, indicates that the maximum production occurred at 72 h under stationary conditions. Isolation, partial purification, and characterization of the biosurfactant produced have been carried out, and Fourier transform infrared spectroscopy (FTIR) spectra demonstrated that biosurfactants were constituted by protein and polysaccharide fractions, i.e., possessed the structure typical of glycoprotein, which is affected by the medium composition and the phase of growth of the biosurfactant-synthesizing strain. Critical micelle concentration (cmc) of the biosurfactant was found to be 6 g l−1. The emulsification index (EI), emulsification activity (EA), and emulsion stability (ES) values of the biosurfactant have confirmed its emulsification property. Aqueous fractions of the produced biosurfactant exhibited a significant antimicrobial activity against the food-borne pathogenic species: Escherichia coli ATCC 31705, E. coli MTCC 108, Salmonella typhi, Yersinia enterocolitica MTCC 859, and Staphylococcus aureus F 722. More importantly, the biosurfactant from L. plantarum showed antiadhesive property against above food-borne pathogens. The results thus indicate the potential for developing strategies to prevent microbial colonization of food contact surfaces and health-care prosthesis using these biosurfactants.
Keywords: Biosurfactants; Probiotics; Lactobacillus plantarum ; Glycoproteins

Bacillus anthracis is a well-known bioweapon pathogen, which coordinates the expression of its virulence factors in response to a specific environmental signal by its protein architecture. Absences of sortase signal functioning may fail to assemble the surface linked proteins and so B. anthracis cannot sustain an infection with host cells. Targeting the signaling mechanism of B. anthracis can be achieved by inhibition of SrtA enzyme through λphage-derived plyG. The lysin enzyme plyG is experimentally proven as bacteriolytic agent, specifically kill's B. anthracis by inhibiting the SrtA. Here, we have screened the peptides from λphage lysin, and these peptides are having the ability as LPXTG competitive inhibitors. In comparison to the activator peptide LPXTG binding motif, λphage lysin based inhibitor peptides are having much supremacy towards binding of SrtA. Finally, peptide structures extracted from PlyG are free from toxic, allergic abilities and also have the ability to terminate the signal transduction mechanism in B. anthracis.
Keywords: Activator; LPXTG; Peptide; PlyG; Peptidomimetic; SrtA

A Stable and Efficient Agrobacterium tumefaciens-Mediated Genetic Transformation of the Medicinal Plant Digitalis purpurea L. by Ying Li; Zhenrui Gao; Chunlan Piao; Kaiwen Lu; Zhiping Wang; Min-Long Cui (1807-1817).
In this study, we developed a rapid and efficient method for in vitro propagation and Agrobacterium tumefaciens-mediated transformation of Digitalis purpurea L. (syn. foxglove), an important medicinal plant. Mature leaf explants of D. purpurea were used for 100 % adventitious shoot regeneration on Murashige and Skoog (MS) medium supplemented with 1 mg L−1 thidiazuron (TDZ) (a cytokine) and 0.1 mg L−1 1-naphthaleneacetic acid (NAA) (an auxin). Transformation was achieved by inoculating leaf explants with the A. tumefaciens strains GV2260/pBI121 or GV3101/pBI121. The binary vector pBI121 contained the reporter β-glucuronidase gene (GUS) and kanamycin selection marker nptII. Kanamycin-resistant shoots were regenerated directly on the selection medium 4–6 weeks after co-cultivation. Approximately, 52.2 and 60 % of kanamycin-resistant shoots transformed with Agrobacterium strains GV2260 and GV3101, respectively, showed strong GUS staining by histochemical assay. Furthermore, PCR and Southern blot analysis confirmed the presence of nptII and GUS on the chromosome of the transformed D. purpurea plants, and stable GUS expression was detected in the transformants by RT-PCR analysis. This efficient method of shoot regeneration and genetic transformation of D. purpurea will provide a powerful tool to increase and produce valuable components such as digitoxin, digoxin, and digoxigenin in D. purpurea through improved secondary metabolic pathways via a biotechnological approach.
Keywords: Digitalis purpurea L; Common medicinal plant; Mature leaf explants; High-efficiency shoot regeneration; Agrobacterium tumefaciens-mediated transformation; Stable GUS expression

In this work, a new approach for acetone–butanol–ethanol (ABE) production has been proposed. Direct fermentation of native starches (uncooked process) was investigated by using granular starch hydrolyzing enzyme (GSHE) and Clostridium saccharoperbutylacetonicum N1-4. Even the process was carried out under suboptimal condition for activity of GSHE, the production of ABE was similar with that observed in conventional process or cooked process in terms of final solvent concentration (21.3 ± 0.4 to 22.4 ± 0.4 g/L), butanol concentration (17.5 ± 0.4 to 17.8 ± 0.3 g/L) and butanol yield (0.33 to 0.37 g/g). The production of solvents was significantly dependent on the source of starches. Among investigated starches, corn starch was more susceptible to GSHE while cassava starch was the most resistant to this enzyme. Fermentation using native corn starch resulted in the solvent productivity of 0.47 g/L h, which was about 15 % higher than that achieved in cooked process. On the contrary, uncooked process using cassava and wheat starch resulted in the solvent productivity of 0.30 and 0.37 g/L h, which were respectively about 30 % lower than those obtained in cooked process. No contamination was observed during all trials even fermentation media were prepared without sterilization. During the fermentation using native starches, no formation of foam is observed. This uncooked process does not require cooking starchy material; therefore, the thermal energy consumption for solvent production would remarkably be reduced in comparison with cooked process.
Keywords: Granular starch hydrolyzing enzyme; Native starch; Acetone; Butanol; Ethanol fermentation; Clostridium saccharoperbutylacetonicum N1-4; Uncooked process

Integrated Use of Residues from Olive Mill and Winery for Lipase Production by Solid State Fermentation with Aspergillus sp. by José Manuel Salgado; Luís Abrunhosa; Armando Venâncio; José Manuel Domínguez; Isabel Belo (1832-1845).
Two-phase olive mill waste (TPOMW) is presently the major waste produced by the olive mill industry. This waste has potential to be used as substrate for solid state fermentation (SSF) despite of its high concentration of phenolic compounds and low nitrogen content. In this work, it is demonstrated that mixtures of TPOMW with winery wastes support the production of lipase by Aspergillus spp. By agar plate screening, Aspergillus niger MUM 03.58, Aspergillus ibericus MUM 03.49, and Aspergillus uvarum MUM 08.01 were chosen for lipase production by SSF. Plackett–Burman experimental design was employed to evaluate the effect of substrate composition and time on lipase production. The highest amounts of lipase were produced by A. ibericus on a mixture of TPOMW, urea, and exhausted grape mark (EGM). Urea was found to be the most influent factor for the lipase production. Further optimization of lipase production by A. ibericus using a full factorial design (32) conducted to optimal conditions of substrate composition (0.073 g urea/g and 25 % of EGM) achieve 18.67 U/g of lipolytic activity.
Keywords: Olive mill wastes; Winery wastes; Plackett–Burman; Solid state fermentation; Aspergillus sp.

Square Planar Platinum(II) Complexes with N,S-Donor Ligands: Synthesis, Characterisation, DNA Interaction and Cytotoxic Activity by Mohan N. Patel; Chintan R. Patel; Hardik N. Joshi; Pankaj A. Vekariya (1846-1858).
The platinum(II) complexes with N,S-donor ligand have been synthesised and characterised by physiological techniques like elemental, electronic, Fourier transform infrared, hydrogen-1 nuclear magnetic resonance (1H NMR) and liquid chromatography–mass spectrometry spectra. The synthesised complexes have been checked for their DNA binding ability by absorption titration and viscosity measurement, and the results show that the complexes binds to herring sperm DNA (HS DNA) via covalent mode of binding. The DNA cleavage activity of synthesised complexes has been carried out by gel electrophoresis experiment using supercoiled form of pUC19 DNA, showing the unwinding of the negatively charged supercoiled DNA. Brine shrimp (Artemia cysts) lethality bioassay technique has been applied for the determination of toxic property of synthesised complexes in terms of micromolars.
Keywords: N,S-donor ligand; Platinum(II) complexes; DNA interaction; Unwinding; Cytotoxicity

This work presents a new chemiluminescent (CL) probe array assay. The new type CL probe array is based on enzyme mimics of Co3O4–SiO2 mesoporous nanocomposite material, which not only have an excellent catalytic effect on the luminol–H2O2 CL reaction in an alkaline medium but also can be used for the immobilization of enzymes. The linear range of the lactose concentration is 3.0 × 10−7 to 1.0 × 10−5 g mL−1 and the detection limit is 6.9 × 10−8 g mL−1. β-Galactosidase and glucose oxidase were selected as a model for enzyme assays to demonstrate the applicability of Co3O4–SiO2 mesoporous nanocomposite material in multienzyme immobilization. The novel bifunctional CL probe array has been successfully applied to the determination of lactose in milk.
Keywords: Chemiluminescent probe array; Co3O4 − SiO2 mesoporous nanocomposite material; Immobilization; Lactose

The increasing evidence on the differential biochemical effects of eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) raises the need of n-3 highly unsaturated fatty acid concentrates with different amounts of these fatty acids. In the present work, physicochemical and enzymatic techniques were combined to obtain acylglycerols, mainly triacylglycerols (TAG), rich in n-3 fatty acids. Sardine oil was obtained by washing sardine (Sardina pilchardus) mince with a NaHCO3 solution, hydrolyzed in a KOH–ethanol solution, and concentrated with urea. The esterification reaction was performed in the stoichiometric proportion of substrates for re-esterification to TAG, with 10 % level of Rhizomucor miehei lipase based on the weight of substrates, without any solvent, during 48 h. This procedure led to approximately 88 % of acylglycerols, where more than 66 % were TAG and the concentration of n-3 fatty acids was higher than 60 %, the EPA and DHA ratio (EPA/DHA) was 4:1. The content of DHA in the unesterifed fraction (free fatty acids) increased from 20 to 54 %, while the EPA level in the same fraction decreased from 33 to 12.5 % (EPA/DHA ratio ≈1:4). Computational methods (density functional theory calculations) have been carried out at the B3LYP/6-31G(d,p) level to explain some of the experimental results.
Keywords: n-3 HUFA; EPA/DHA ratios; Triacylglycerols; Rhizomucor miehei lipase; Sardine oil; Ab initio geometry optimization

Identification of Novel Rab27a/Melanophilin Blockers by Pharmacophore-Based Virtual Screening by Jong Young Joung; Ha Yeon Lee; Jongil Park; Jee-Young Lee; Byung Ha Chang; Kyoung Tai No; Ky-Youb Nam; Jae Sung Hwang (1882-1897).
Melanocytes are unique cells that produce specific melanin-containing intracellular organelles called melanosomes. Melanosomes are transported from the perinuclear area of melanocytes toward the plasma membrane as they become more melanized in order to increase skin pigmentation. In this vesicular trafficking of melanosomes, Rab27a, melanophilin, and myosin Va play crucial roles in linking melanosomes to actin-based motors. To identify novel compounds to inhibit binding interface between Rab27a and melanophilin, a pharmacophore model was built based on a modeled 3D structure of the protein complex that describes the essential binding residues in the intermolecular interaction. A pharmacophore model was employed to screen a chemical library database. Finally, 25 virtual hits were selected for biological evaluations. The biological activities of 11 analogues were evaluated in a second assay. Two compounds were identified as having concentration-dependent inhibitory activity. By analyzing structure–activity relationships of derivatives of BMD-20, two hydroxyl functional groups were found to be critical for blocking the intermolecular binding between Rab27a and melanophilin.
Keywords: Rab27a/melanophilin; Pharmacophore-based virtual screening; Skin pigmentation; Mekanosome tansport; Molecular docking

In recent years, a significant interest has been generated in discovering and developing exopolysaccharides (EPS) produced by microorganisms, especially fungi due to their multifaceted industrial and pharmacological applications. A number of filamentous and cellular fungi have been explored for this; however, according to the existing literature, the work on exopolysaccharide production by indigenous culture on this aspect is still very less and requires a serious attention. The present work is an attempt in this regard and aims to optimize the submerged culture conditions to produce the exopolysaccharides from an indigenous yeast Aureobasidium pullulans RYLF-10 with respect to several operating parameters in shake flask fermentation. The yeast A. pullulans RYLF-10 was identified by 18s RNA sequencing and detailed study on its nutritional requirements, and environmental conditions for submerged culture have been optimized. The optimal temperature and pH for both the vegetative growth and EPS production were found to be 28 ± 1 °C and 5.0, respectively, while the agitation speed and inoculum size were reported to be 150 rpm and 1 % (v/v), respectively. Sucrose (50 g/l) and yeast extract (1 g/l) were found to be the most suitable carbon and nitrogen sources which worked best in the ratio of 60:1 and resulted in the maximum EPS yield. Similarly, the other variables like growth regulator (riboflavin) and minerals (NaCl + K2HPO4 + MgSO4) altogether resulted in a noteworthy EPS yield of 45.24 g/l which is the maximum yield from this indigenous isolate of A. pullulans RYLF-10.
Keywords: Exopolysaccharides; Indigenous; Aureobasidium pullulans ; Hyperproduction; Fermentation

A Review on Optimization Production and Upgrading Biogas Through CO2 Removal Using Various Techniques by Dian Andriani; Arini Wresta; Tinton Dwi Atmaja; Aep Saepudin (1909-1928).
Biogas from anaerobic digestion of organic materials is a renewable energy resource that consists mainly of CH4 and CO2. Trace components that are often present in biogas are water vapor, hydrogen sulfide, siloxanes, hydrocarbons, ammonia, oxygen, carbon monoxide, and nitrogen. Considering the biogas is a clean and renewable form of energy that could well substitute the conventional source of energy (fossil fuels), the optimization of this type of energy becomes substantial. Various optimization techniques in biogas production process had been developed, including pretreatment, biotechnological approaches, co-digestion as well as the use of serial digester. For some application, the certain purity degree of biogas is needed. The presence of CO2 and other trace components in biogas could affect engine performance adversely. Reducing CO2 content will significantly upgrade the quality of biogas and enhancing the calorific value. Upgrading is generally performed in order to meet the standards for use as vehicle fuel or for injection in the natural gas grid. Different methods for biogas upgrading are used. They differ in functioning, the necessary quality conditions of the incoming gas, and the efficiency. Biogas can be purified from CO2 using pressure swing adsorption, membrane separation, physical or chemical CO2 absorption. This paper reviews the various techniques, which could be used to optimize the biogas production as well as to upgrade the biogas quality.
Keywords: Biogas; CO2 ; Production; Optimization; Upgrading

Bioremediation of Cd by Microbially Induced Calcite Precipitation by Chang-Ho Kang; Sang-Hyun Han; YuJin Shin; Soo Ji Oh; Jae-Seong So (1929-1937).
Contamination by Cd is a significant environmental problem. Therefore, we examined Cd removal from an environmental perspective. Ureolysis-driven calcium carbonate precipitation has been proposed for use in geotechnical engineering for soil remediation applications. In this study, 55 calcite-forming bacterial strains were newly isolated from various environments. Biomineralization of Cd by calcite-forming bacteria was investigated in laboratory-scale experiments. A simple method was developed to determine the effectiveness of microbially induced calcite precipitation (MICP). Using this method, we determined the effectiveness of biomineralization for retarding the flow of crystal violet through a 25-mL column. When the selected bacteria were analyzed using an inductively coupled plasma optical emission spectrometer, high removal rates (99.95 %) of Cd were observed following incubation for 48 h. Samples of solids that formed in the reaction vessels were examined using a scanning electron microscope. The CdCO3 compounds primarily showed a spherical shape. The results of this study demonstrate that MICP-based sequestration of soluble heavy metals via coprecipitation with calcite may be useful for toxic heavy metal bioremediation.
Keywords: Cd; MICP; Calcite; Urease; Bioremediation

Renewable and carbon neutral, marine algal biomass could be an attractive alternative substrate for the production of biofuel and various biorefinery products. Thus, the feasibility of brown seaweed (Laminaria japonica) hydrolysate as a carbon source was investigated here for l-lactate production. This work reports the homofermentative route for l-lactate production by introducing Streptococcus bovis/equinus l-lactate dehydrogenase in an engineered Escherichia coli strain where synthesis of the competing by-product was blocked. The engineered strain utilized both glucose and mannitol present in the hydrolysate under microaerobic condition and produced 37.7 g/L of high optical purity l-lactate at 80 % of the maximum theoretical value. The result shown in this study implies that algal biomass would be as competitive with lignocellulosic biomass in terms of lactic acid production and that brown seaweed can be used as a feedstock for the industrial production of other chemicals.
Keywords: l-Lactate; Mannitol; Seaweed; Metabolic engineering; Escherichia coli

Electroanalysis may be used in the Vanillin Biotechnological Production by William Giraud; Marie Mirabel; Maurice Comtat (1953-1963).
This study shows that electroanalysis may be used in vanillin biotechnological production. As a matter of fact, vanillin and some molecules implicated in the process like eugenol, ferulic acid, and vanillic acid may be oxidized on electrodes made of different materials (gold, platinum, glassy carbon). By a judicious choice of the electrochemical method and the experimental conditions the current intensity is directly proportional to the molecule concentrations in a range suitable for the biotechnological process. So, it is possible to imagine some analytical strategies to control some steps in the vanillin biotechnological production: by sampling in the batch reactor during the process, it is possible to determine out of line the concentration of vanillin, eugenol, ferulic acid, and vanillic acid with a gold rotating disk electrode, and low concentration of vanillin with addition of hydrazine at an amalgamated electrode. Two other possibilities consist in the introduction of electrodes directly in the batch during the process; the first one with a gold rotating disk electrode using linear sweep voltammetry and the second one requires three gold rotating disk electrodes held at different potentials for chronoamperometry. The last proposal is the use of ultramicroelectrodes in the case when stirring is not possible.
Keywords: Vanillin; Eugenol; Gold; Amalgamated electrode; Sensor

A membrane based affinity purification system was developed for the purification of the DNA specific nuclease, DNase I. Single stranded DNA was bound to unmodified polyvinylidene fluoride (PVDF) membranes which were used to purify DNase I from a solution of bovine serum albumin. Using coated membranes, a 6-fold increase in specific activity was achieved with 80 % enzyme recovery. This method provides a simple yet effective way to purify DNase I and can be very useful for the purification of other DNA specific enzymes.
Keywords: DNase I; Affinity purification; PVDF membranes; DNase purification; Membrane purification

Bile Enhances Cell Surface Hydrophobicity and Biofilm Formation of Bifidobacteria by Padma Ambalam; Kanthi Kiran Kondepudi; Ingrid Nilsson; Torkel Wadström; Åsa Ljungh (1970-1981).
Twenty-four human bifidobacterial strains were analysed for cell surface hydrophobicity (CSH) using a salt aggregation test (SAT) and a Congo red binding (CRB) assay. Three strains were selected for a systematic study on the CSH and biofilm formation: Bifidobacterium breve 46, Bifidobacterium animalis ssp. lactis 8:8 and a reference strain B. animalis ssp. lactis JCM 10602. CRB of the B. breve 46 and B. animalis ssp. lactis JCM 10602 was significantly enhanced (P < 0.05) when grown in deMan–Rogosa–Sharpe cysteine (MRSC) broth supplemented with taurocholic acid (TA) or native porcine bile (PB). An enhanced CSH of the strains grown with PB and gastric mucin correlated with an increased mucin binding and an enhanced biofilm formation in prebiotic oligosaccharide-supplemented cultures. The three strains showed late bile-induced biofilm (72 h) under an anaerobic growth condition, and both B. animalis ssp. lactis strains showed a late bile-induced biofilm formation under aerobic conditions shown by crystal violet staining. These two strains were thus considered to be oxygen tolerant and more robust. Furthermore, enhanced biofilm formation of these robust bifidobacterial strains in the presence of prebiotics may allow for strong colonisation in the gastrointestinal tract when administered to in vivo models as a “synbiotic supplement”.
Keywords: Bifidobacteria; Cell surface hydrophobicity; Congo red binding; Bile stress; Biofilm; Mucus binding; Prebiotics

Sugarcane (Saccharum spp.) is a dynamic C4 polyploidy grass used as a major source of sucrose and an alternative for ethanol, food, and energy. Despite growing scientific interest, various sucrose metabolism regulatory aspects have been limited. Biochemical and gene expression studies were conducted on developmental stages, 240–420 days of planting (DAP) in mature leaves of three high and three low sucrose sugarcane cultivars. Sucrose synthase (SS) and sucrose phosphate synthase (SPS) activities were found to be remarkably higher at 240–360 DAP but decrease at 420 DAP. Twofold increases of SS activity was estimated at 240–360 DAP while SPS activity trend was found to be lower than the SS activity. In comparing SS and SPS activities with the brix of respective DAP, results show that these activities are significant and positively correlated with ‘r’ values of 0.69 and 0.68 for SS and SPS, respectively. However, the soluble acid invertase (SAI) and neutral invertase (NI) activities were found to decrease significantly with the maturity of cultivars, negatively correlating with brix at ‘r’ values 0.83 and 0.89 for SAI and NI, respectively. The antioxidant enzyme activity was modulated similar to the invertases activity. Of the six genes, ESAS 11 and 23 associated with sucrose accumulation and ESTS 34 and 41 associated with sugar transport in sugarcane were differentially expressed among the selected high and low sugarcane cultivars. Hence, these findings reinforce the selection of diverse sugarcane cultivars for gene expression studies targeting to quantitative traits and candidate marker determination.
Keywords: Sugarcane; Sucrose accumulating enzymes and genes; Antioxidant enzymes; Sugar; Transporter; Brix; Gene expression

Proteases are some of the most important industrial enzymes, and one of their main applications is for the production of cheese in the dairy industry. Due to a shortage of animal rennet, microbial coagulant proteases are being sought. In this work, the production of microbial rennet from Thermomucor indicae-seudaticae N31 was studied in submerged fermentation. The best enzyme production was obtained in a fermentation medium containing 4 % wheat bran as the substrate in 0.3 % saline solution, incubated for 72 h at 45 °C and 150 rpm. The value of the milk clotting activity (MCA) was 60.5 U/mL, and the ratio to proteolytic activity (MCA/PA) was 510. The crude enzyme showed optimum pH at 5.5 and two peaks of optimum temperature (MCA at 65 °C and PA at 60 °C). The MCA was stable in the pH range 4.0–4.5 for 24 h and up to 55 °C for 1 h. It was stable during storage at different temperatures (−20 to 25 °C) for 10 weeks. Based on these results, we conclude that microbial rennet from T. indicae-seudaticae N31 produced by submerged fermentation showed good prospects of replacing traditional rennet.
Keywords: Microbial coagulant; Wheat bran; Thermophilic fungus; Rennet; Fermentation

Metabolic Engineering of Escherichia coli for Production of 2-phenylethanol from Renewable Glucose by Zhen Kang; Chuanzhi Zhang; Guocheng Du; Jian Chen (2012-2021).
2-Phenylethanol (2-PE) is an important aromatic alcohol with a rose-like odor and has wide applications. The present work aims to construct a synthetic pathway for 2-PE synthesis from glucose in Escherichia coli. First, the genes adh1 (encoding alcohol dehydrogenase) and kdc (encoding phenylpyruvate decarboxylase) from Saccharomyces cerevisiae S288c and Pichia pastoris GS115 were investigated in E. coli, respectively, and single overexpression of adh1 or kdc significantly increased 2-PE accumulation. When co-overexpressing adh1 and kdc, 2-PE was increased up to 130 from 57 mg/L. Furthermore, by optimizing coordinated expression of the four committed genes aroF, pheA, adh1 and kdc, 2-PE was improved to 285 mg/L which was the highest production of 2-PE by the recombinant E. coli system. In addition, our results also demonstrated that the tyrB gene, which encodes aromatic-amino-acid transaminase, plays an important role on 2-PE synthesis.
Keywords: Metabolic engineering; 2-Phenylethanol; Gene expression control; Escherichia coli ; l-phenylalanine

Endo-β-1,4-xylanase derived from Trichoderma reesei was covalently immobilized on poly (ethylene glycol) methyl ether 5000 (mPEG5000), and the resulting immobilized enzyme had a residual activity of 72.4 % with 82.9 % of PEGylated amino groups. Compared with the free enzyme, the immobilized xylanase was stable at pH values in the range of 4.0–6.0 and temperatures in the range of 50–65 °C. A self-extractive bioconversion system composed of immobilized xylanase, mPEG5000, and sodium citrate was used to produce xylo-oligosaccharides and provided a better distribution of the xylo-oligosaccharides than the free enzyme. Furthermore, the immobilized xylanase could be effectively recovered in situ following the hydrolysis reaction.
Keywords: Endo-β-1,4-xylanase; Immobilization; PEGylation; Self-extractive bioconversion; Xylo-oligosaccharides

Acetaldehyde Detoxification Using Resting Cells of Recombinant Escherichia coli Overexpressing Acetaldehyde Dehydrogenase by Zhengying Yao; Chong Zhang; Junfeng Zhao; Fengxia Lu; Xiaomei Bie; Zhaoxin Lu (2030-2040).
Acetaldehyde dehydrogenase (E.C. plays a key role in the acetaldehyde detoxification. The recombinant Escherichia coli cells producing acetaldehyde dehydrogenase (ist-ALDH) were applied as whole-cell biocatalysts for biodegradation of acetaldehyde. Response surface methodology (RSM) was employed to enhance the production of recombinant ist-ALDH. Under the optimum culture conditions containing 20.68 h post-induction time, 126.75 mL medium volume and 3 % (v/v) inoculum level, the maximum ist-ALDH activity reached 496.65 ± 0.81 U/mL, resulting in 12.5-fold increment after optimization. Furthermore, the optimum temperature and pH for the catalytic activity of wet cells were 40 °C and pH 9.5, respectively. The biocatalytic activity was improved 80 % by permeabilizing the recombinant cells with 0.075 % (v/v) Triton X-100. When using 2 mmol/L NAD+ as coenzyme, the permeabilized cells could catalyze 98 % of acetaldehyde within 15 min. The results indicated that the recombinant E. coli with high productivity of ist-ALDH might be highly efficient and easy-to-make biocatalysts for acetaldehyde detoxification.
Keywords: Acetaldehyde dehydrogenase; Recombinant E. coli ; Response surface methodology; Whole-cell biocatalysts

The present study is concerned with the microbiological transformation of L-tyrosine to L-dopa by a newly isolated turkey tail mushroom Coriolus versicolor DOB-4. As tyrosinase (catechol oxidase, EC is an extracellular enzyme, therefore biomass was used as an enzyme source in the reaction mixture. Biomass particles were pretreated with methanol and oven dried at 105 °C for 2 h. The optimal L-dopa production was achieved when 1.5 mg/ml L-tyrosine was used as the basal substrate. Thin layer chromatography and high-performance liquid chromatography analysis depicted that citric acid supports higher substrate conversion and product formation rates. A noticeable enhancement was observed when process parameters viz. L-tyrosine concentration (1.5 mg/ml), citric acid (1.5 mg/ml), time of incubation (50 min), and reaction temperature (60 °C) were optimized using Plackett–Burman design. The maximum production of L-dopa was found to be 0.872 mg/ml with L-tyrosine consumption of 1.002 mg/ml. The model terms were found highly significant (HS, p ≤ 0.05), suggesting the potential commercial utility of the culture (df = 3, LSD = 0.342).
Keywords: Coriolus versicolor ; L-Dopa; Microbiological transformation; Mushroom biomass; Plackett–Burman design

miRNAs: A New Method for Erythroid Differentiation of Hematopoietic Stem Cells Without the Presence of Growth Factors by Fatemeh Kouhkan; Maryam Hafizi; Naser Mobarra; Majid Mossahebi-Mohammadi; Shahin Mohammadi; Mehrdad Behmanesh; Mina Soufi Zomorrod; Shaban Alizadeh; Reyhaneh Lahmy; Morteza Daliri; Masoud Soleimani (2055-2069).
Micro RNAs (miRNAs) are a novel class of non-coding regulatory RNA molecules that contribute to post-transcriptional gene regulation. Recent studies have demonstrated that specific miRNAs such as miR-150, miR-154, and miR-451 have key roles in erythropoiesis. To date, stimulatory cytokines are considered as unique effectors for in vitro differentiation of HSCs to erythropoietic lineage. However, the use of these factors is not cost-effective for clinical applications and therapeutic strategies. Here, we present a novel and cost-effective strategy in which miRNAs expression modulation promotes erythroid differentiation in HSCs in the absence of any extrinsic factors. Thus, CD133+ hematopoietic stem cells purified from human umbilical cord blood were treated with pre-miR-451 containing lentiviruses, anti-miR-150 and anti-miR-154 in the absence of growth factors and cytokines. Obtained results indicated that miR-451 upregulation and miR-150 downregulation have positive effect on GATA-1, FOG-1, and EKLF, CD71 and CD235a genes expression and induce hemoglobinization efficiently. However, downregulation of miR-154 had no effect on erythropoiesis indexes compared to that observed in the control group. In conclusion, the data presented here for the first time demonstrate that expression modulation of miR-451 and miR-150 could be an efficient alternative to stimulatory cytokines for CD133+ differentiation into erythroid lineage. Modulation of erythropoiesis in stem cells via miRNA holds promising potential for vascular tissue engineering and regenerative medicine applications.
Keywords: microRNAs; miR-451; miR-150; CD133+ hematopoietic stem cells; Erythroid differentiation; Stimulatory cytokines

Aptamer-based Biosensor for Detection of Phenylalanine at Physiological pH by Eskandar Omidinia; Nasrin Shadjou; Mohammad Hasanzadeh (2070-2080).
A simple, sensitive aptamer-based biosensor for the detection of phenylalanine is developed using the electrochemical transduction method. For this proposed aptasensor, a 5-thiol-terminated aptamer is covalently attached onto a gold electrode. At the first time, the electrode was evaluated as an electrochemical aptasensor for determination of phenylalanine in aqueous solutions. This sensor was tested in a Tris–HCl buffer with physiological pH = 7.4 by cyclic voltammetry and differential pulse voltammetry. The detection limit and sensitivity of the modified electrode toward phenylalanine were estimated to be 1 nM (S/N = 3) and 0.367 μA nM−1, respectively. The linear range of the signal was observed between 1 and 10 nM of phenylalanine with 0.9914 correlation factor. The herein-described approach is expected to promote the exploitation of aptamer-based biosensors for protein assays in biochemical and biomedical studies.
Keywords: Aptamer; Electrochemical biosensor; Phenylalanine; Covalently attachment

Purification and Applications of a Lectin from the Mushroom Gymnopilus spectabilis by Silvana Alborés; Paola Mora; María José Bustamante; María Pía Cerdeiras; Laura Franco Fraguas (2081-2090).
A lectin was isolated from fruiting bodies of the mushroom Gymnopilus spectabilis (GSL) by ionic exchange chromatography. The lectin agglutinates mouse red cells exhibiting broad specificity towards several monosaccharides including the N-acetylneuraminic acid. Agglutination was also inhibited by the glycoproteins: fetuin, lactoferrin, and recombinant erythropoietin. GSL is a glycoprotein possessing 16 % of carbohydrates; the SDS-PAGE showed two bands with molecular mass of 52.1 and 64.4 kDa. Isoelectric focusing displayed microheterogeneity, with two bands at pIs 5.1 and 5.3. The lectin was stable between pH 2 and pH 8 while at pH 10, the agglutination decayed to 50 % of initial activity. Incubation at 40 and 80 °C led to 50 and 100 % loss in activity of the lectin, respectively. Synthesized GSL-Sepharose interacts with serum pregnant mare gonadotropin, and at least two subpopulations of this glycoprotein were separated. There was no interaction between transferrin and soluble GSL while a partial recognition was achieved with GSL-Sepharose. The terminal sialic acid seems to play an active role in modifying the interaction with GSL, depending if the lectin is in a soluble or immobilized form. The purified lectin inhibited in vitro the growth of Staphylococcus aureus and Aspergillus niger.
Keywords: Lectin; Gymnopilus spectabilis ; Basidiomycete; Fruiting body; Glycoproteins

Proteomic Identification of Syzygium cumini Seed Extracts by MALDI-TOF/MS by Kumari Binita; Sanjay Kumar; Vinay Kumar Sharma; Veena Sharma; Savita Yadav (2091-2105).
Syzygium cumini is traditionally used medicinal plant. The different part of the plant such as bark, leaves, seed and fruits are widely used as an alternative medicine in various diseases. Although the scientific community has a strong interest on S. cumini seed biochemistry focusing on metabolite composition, proteins have not yet been investigated. In the present study, we have applied a proteomic approach to study the proteome of the S. cumini seed using phenol extraction method for protein isolation, which were never analysed before. Fifteen brightly silver stained protein spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry after resolving on two-dimensional gel electrophoresis. These proteins have been found to involve in various functions such as antifungal, sulphur metabolism, carbohydrate metabolism, fruit ripening and softening, dormancy breaking and seed germination, hormone signalling, secondary metabolite transport, defence and stress response, nitrogen metabolism, synthesis and stabilization. Amongst the identified protein, lactoferrin was a mammalian origin protein with high nutritious and pharmaceutical value, which was purified by different types of chromatographic techniques and confirmed by western blotting. The antibacterial activity of lactoferrin was assessed by disc diffusion assay. We suggest that the protein constituents of S. cumini may have role in various functions required for plant physiology and its dietary values.
Keywords: Syzygium cumini seeds; Proteomics; Glycoprotein; Lactoferrin; Antibacterial property

Addition of additives has been confirmed to increase cellulase performance in the hydrolysis of lignocellulosic materials. In the hydrolysis of xylan-containing lignocellulosic biomass, xylanase can synergistically enhance the performance of cellulase. However, the role of additives in xylan hydrolysis by xylanase is not yet clear. In this work, with the presence of additives (bovine serum albumin, poly(ethylene glycol), and Tween), the hydrolysis of isolated xylan and the xylan in corn stover increased to different extents. Additives increased free xylanase in supernatants in the hydrolysis with xylanase, indicating the reduction of the adsorption of xylanase on corn stover and insoluble xylan. Enhanced hydrolysis of Avicel and corn stover by additives suggested that besides the prevention of unproductive binding of xylanase to lignin by additives, reducing the adsorption of xylanase on substrates was also contributed to enzymatic hydrolysis. The increment of xylanase activity by additives suggests that the additives were activators of xylanase. The results of this work indicate that the supplementation of additives could improve xylanase performance, synergistically enhanced the cellulose hydrolysis, and beneficial for the recycling of xylanase.
Keywords: Additive; Xylanase; Xylan; Enzymatic hydrolysis; Adsorption

Cloning, Expression, and Characterization of a Milk-Clotting Aspartic Protease Gene (Po-Asp) from Pleurotus ostreatus by Chaomin Yin; Liesheng Zheng; Liguo Chen; Qi Tan; Xiaodong Shang; Aimin Ma (2119-2131).
An aspartic protease gene from Pleurotus ostreatus (Po-Asp) had been cloned based on the 3′ portion of cDNA in our previous work. The Po-Asp cDNA contained 1,324 nucleotides with an open reading frame (ORF) of 1,212 bp encoding 403 amino acid residues. The putative amino acid sequence included a signal peptide, an activation peptide, two most possible N-glycosylation sites and two conserved catalytic active site. The mature polypeptide with 327 amino acid residues had a calculated molecular mass of 35.3 kDa and a theoretical isoelectric point of 4.57. Basic Local Alignment Search Tool analysis showed 68–80 % amino acid sequence identical to other basidiomycetous aspartic proteases. Sequence comparison and evolutionary analysis revealed that Po-Asp is a member of fungal aspartic protease family. The DNA sequence of Po-Asp is 1,525 bp in length without untranslated region, consisting of seven exons and six introns. The Po-Asp cDNA without signal sequence was expressed in Pichia pastoris and sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the molecular mass of recombinant Po-Asp was about 43 kDa. The crude recombinant aspartic protease had milk-clotting activity.
Keywords: Pleurotus ostreatus ; Aspartic protease (Po-Asp); Bioinformatics analysis; Pichia pastoris ; Expression; Milk clotting

Image Analysis Technique as a Tool to Identify Morphological Changes in Trametes versicolor Pellets According to Exopolysaccharide or Laccase Production by Ana P. M. Tavares; Rui P. Silva; António L. Amaral; Eugénio C. Ferreira; Ana M. R. B. Xavier (2132-2142).
Image analysis technique was applied to identify morphological changes of pellets from white-rot fungus Trametes versicolor on agitated submerged cultures during the production of exopolysaccharide (EPS) or ligninolytic enzymes. Batch tests with four different experimental conditions were carried out. Two different culture media were used, namely yeast medium or Trametes defined medium and the addition of lignolytic inducers as xylidine or pulp and paper industrial effluent were evaluated. Laccase activity, EPS production, and final biomass contents were determined for batch assays and the pellets morphology was assessed by image analysis techniques. The obtained data allowed establishing the choice of the metabolic pathways according to the experimental conditions, either for laccase enzymatic production in the Trametes defined medium, or for EPS production in the rich Yeast Medium experiments. Furthermore, the image processing and analysis methodology allowed for a better comprehension of the physiological phenomena with respect to the corresponding pellets morphological stages.
Keywords: Filamentous fungi; Pellets morphology; Image analysis; Enzyme activity; Exopolysaccharide production; Pulp and paper effluent

Enzymatic Trends of Fructooligosaccharides Production by Microorganisms by Mohd Anis Ganaie; Agbaje Lateef; Uma Shanker Gupta (2143-2159).
Fructooligosaccharides are influential prebiotics that affect various physiological functions in such a way that they promote positive impact to health. They occur naturally in many fruits and vegetables in trace amounts. However, they are mainly produced commercially by the reaction of microbial enzymes with di- or polysaccharides, such as sucrose or inulin as a substrate. For maximum production of fructooligosaccharides on an industrial level, development of more enzymes with high activity and stability is required. This has attracted the attention of biotechnologists and microbiologists worldwide. This study aims to discuss the new trends in the production of fructooligosaccharide and its effect on numerous health qualities through which it creates great demand in the sugar market.
Keywords: Prebiotic; Fructooligosaccharides; Inulin; Microorganisms; Fructosyltransferase; Immobilisation

Screening Factors Influencing the Production of Astaxanthin from Freshwater and Marine Microalgae by Elda Nurafnie Binti Ibnu Rasid; Shaza Eva Mohamad; Haryati Jamaluddin; Madihah Md Salleh (2160-2174).
Astaxanthin, a carotenoid pigment found in several aquatic organisms, is responsible for the red colour of salmon, trout and crustaceans. In this study, astaxanthin production from freshwater microalga Chlorella sorokiniana and marine microalga Tetraselmis sp. was investigated. Cell growth and astaxanthin production were determined spectrophotometrically at 620 and 480 nm, respectively. Astaxanthin was extracted using acetone and measured subsequent to biomass removal. Aerated conditions favoured astaxanthin production in C. sorokiniana, whereas Tetraselmis sp. was best cultured under unaerated conditions. C. sorokiniana produced more astaxanthin with the highest yield reached at 7.83 mg/l in 6.0 mM in nitrate containing medium compared to Tetraselmis sp. which recorded the highest yield of only 1.96 mg/l in 1.5 mM nitrate containing medium. Production in C. sorokiniana started at the early exponential phase, indicating that astaxanthin may be a growth-associated product in this microalga. Further optimization of astaxanthin production was performed using C. sorokiniana through a 23 full factorial experimental design, and a yield of 8.39 mg/l was achieved. Overall, the study has shown that both microalgae are capable of producing astaxanthin. Additionally, this research has highlighted C. sorokiniana as a potential astaxanthin producer that could serve as a natural astaxanthin source in the current market.
Keywords: Astaxanthin production; Carotenoid; Chlorella sorokiniana ; Tetraselmis sp.; Two-factorial design; Growth-associated product; Marine microalgae; Freshwater microalgae

Development of a Biosensor for Copper Detection in Aqueous Solutions Using an Anemonia sulcata Recombinant GFP by Tiziana Masullo; Riccardo Puccio; Mariarosaria Di Pierro; Marcello Tagliavia; Paolo Censi; Valeria Vetri; Valeria Militello; Angela Cuttitta; Paolo Colombo (2175-2187).
Fluorescent proteins from marine organisms represent potential candidates for biosensor development. In this paper, we described the isolation of a native green fluorescent protein from Anemonia sulcata and the cloning and purification of its equivalent as a recombinant protein in Escherichia coli. Furthermore, the spectroscopic behaviours of the native and recombinant GFPs were investigated as a function of Cu2+, Cd2+, Pb2+ and Ni2+ concentration. Our results suggest the high selectivity of both proteins at copper than the other metals and, for the recombinant protein, a great sensitivity at a very low concentration (0.1–1 μM). Moreover, starting from these data, using the combination of molecular biology techniques and optical setup, we developed a device for the detection of Cu2+ in water solutions. The quenching effect detected with the device showed that the relative attenuation of the signal (0.46 ± 0.02 AU) was slightly larger than the data measured by fluorescence spectra (0.65 ± 0.03 AU). The good sensitivity in the span of two orders of the magnitude of Cu2+ concentration, the fact that the instrument is made up of low-cost and sturdy parts and the selective quenching of rAsGFP to copper ions make this setup suited as a low cost, on-the-field, copper ion-specific biosensor.
Keywords: Green fluorescent protein; Recombinant protein; Divalent metal ions; Copper; Quenching; Detector

The reaction of 5,5′-(pyridine-2,6-diyl)bis(4-amino-3-mercapto-1,2,4-triazole) with various aromatic aldehydes in acetic acid yielded the corresponding 5,5′-(pyridine-2,6-diyl)bis(4-arylideneamino-3-mercapto-1,2,4-triazole) derivatives. The structures of the synthesized compounds as well as their intermediates were confirmed by elemental analysis, infrared spectra, 1H NMR spectra and mass spectra studies. All the synthesized title compounds were screened for their antibacterial activities, and the preliminary results revealed that some of them showed good activities against Escherichia coli and Pseudomonas aeruginosa.
Keywords: Pyridine-2,6-dicarboxylic acid; Synthesis; 1,2,4-Triazole; Antibacterial activity

Bioconversion of Corncob Acid Hydrolysate into Microbial Oil by the Oleaginous Yeast Lipomyces starkeyi by Chao Huang; Xue-Fang Chen; Xiao-Yan Yang; Lian Xiong; Xiao-Qing Lin; Juan Yang; Bo Wang; Xin-De Chen (2197-2204).
For the first time, corncob acid hydrolysate was used for microbial oil production by the oleaginous yeast Lipomyces starkeyi. After hydrolysis by dilute sulfuric acid, corncob could turn into an acid hydrolysate with a sugar concentration of about 42.3 g/L. Detoxified by overliming and absorption with activated carbon, the corncob hydrolysate could be used by L. starkeyi efficiently that a total biomass of 17.2 g/L with a lipid content of 47.0 % (corresponding to a lipid yield of 8.1 g/L) and a lipid coefficient of 20.9 could be obtained after cultivation on the corncob hydrolysate for 8 days. Therefore, L. starkeyi is a promising strain for microbial oil production from lignocellulosic biomass. Glucose and xylose were used by L. starkeyi simultaneously during lipid fermentation while arabinose could not be utilized by it. Besides, the lipid composition of L. starkeyi was similar to that of vegetable oils; thus, it is a promising feedstock for biodiesel production.
Keywords: Lipomyces starkeyi ; Microbial oil; Corncob acid hydrolysate; Biodiesel

Empty fruit bunch (EFB), an underutilized waste product of oil palm processing, was studied as a substrate for the production of humic acids (HA) by a Trichoderma reesei strain by solid-state fermentation (SSF) in Raimbault columns. HA have attracted the attention of many investigators due to their applications in agriculture, industry, the environment, and biomedicine. Commercial HA are currently chemically extracted from peat and coal, which are nonrenewable carbon sources. Biotechnological processes are important for their sustainable and controlled production, with SSF being especially promising for mimicking the natural habitat of fungi. Trichoderma sporulation and HA production are related, and the results of this study showed that SSF stimulated fast sporulation. The productivity related to HA was much higher than that of the biomass, indicating an efficient utilization of EFB. These findings, added to the low cost of EFB, make SSF an attractive process for HA production.
Keywords: Humic acids; Trichoderma reesei ; Solid-state fermentation; Raimbault columns; Empty fruit bunch

Internal air circulation affects the temperature field distribution in a gas double-dynamic solid-state fermentation bioreactor (GDSFB). To enhance heat transfer through strengthening internal air circulation in a GDSFB, we put an air distribution plate (ADP) into the bioreactor and studied the effects of forced internal air circulation on airflow, heat transfer, and cellulase activity of Trichoderma viride L3. Results showed that ADP could help form a steady and uniform airflow distribution, and with gas-guide tubes, air reversal was formed inside the bioreactor, thus resulting in a smaller temperature difference between medium and air by enhancing convective heat transfer inside the bioreactor. Using an ADP of 5.35 % aperture ratio caused a 1 °C decrease in the average temperature difference during the solid-state fermentation process of T. viride L3. Meanwhile, the cellulase activity of T. viride L3 increased by 13.5 %. The best heat-transfer effect was attained when using an ADP of 5.35 % aperture ratio and setting the fan power to 125 V (4.81 W) in the gas double-dynamic solid-state fermentation (GDSF) process. An option of suitable aperture ratio and fan power may be conducive to ADPs’ industrial amplification.
Keywords: Forced internal air circulation; Air distribution plate; Solid-state fermentation; Heat transfer; Cellulase

This paper presents the stoichiometry section of a bioenergetics investigation into the biogas plasticization of wastewater sludge using the Anaerobic Pump (TAP). Three residue samples, an input substrate and two residual products, were collected from two side by side operated AD systems, a conventional continuous flow and stirred reactor, and TAP, and submitted for elemental and calorimetric analyses. The elemental compositions of the residues were fitted to a heterotrophic metabolism model [1] for both systems. To facilitate balanced stoichiometric models, a simple “cell” correction computation separates measured residual composites into “real” residual composition and cell growth (C5H7NO2) components. The elemental data and model results show that the TAP stage II residual composition (C1H0.065O0.0027N0.036) was nearly devoid of hydrogen and oxygen, leaving only fixed carbon and cells grown as the composition of the remaining mass. This quantitative evidence supports prior measurements of very high methane yields from TAP stage II reactor during steady-state experiments [2]. All performance parameters derived from the stoichiometric model(s) showed good agreement with measured steady-state averaged values. These findings are strong evidence that plasticization–disruption (TAP) cycle is the mechanism responsible for the observed increases in methane yield. The accuracy achieved by the stoichiometry models qualifies them for thermodynamic analysis to obtain potentials and bioconversion efficiencies. How applied pressure causes matrix conformation changes triggered by a functional consequence (plasticization and disruption) is this study’s essential focus.
Keywords: Advanced anaerobic bioconversion; Biogas; Biomass; Continuous flow; Pressure cycle; Plasticization; Renewable energy; Waste

Characterization and High Level Expression of Acidic Endoglucanase in Pichia pastoris by Ali Akbarzadeh; Seyed Omid Ranaei Siadat; Mostafa Motallebi; Mohammad Reza Zamani; Mohammad Barshan Tashnizi; Sakineh Moshtaghi (2253-2265).
Bioconversion of cellulosic material into glucose needs cellulase enzymes. One of the most important organisms that produces cellulases is Trichoderma reesei, whose cellulose enzymes are probably the most widely used in the industry. However, these enzymes are not stable enough at high pH and temperatures. The optimized synthetic endoglucanase II gene with Pichia pastoris codon preferences was secretary expressed in P. pastoris. Recombinant enzyme characterization showed maximum activity at pH 4.8 and temperature 75 °C, and it demonstrated increasing thermal stability in high temperature. The enzyme maintained its activity in a wide pH range from 3.5 to 6.5. The optimization of fermentation medium was carried out in shaking flasks. Recombinant protein expression at optimum conditions (pH 7, temperature 25 °C, and 1 % methanol induction) for 72 h demonstrated 2,358.8 U/ml endoglucanase activity units. To our knowledge, this is the highest acidic thermophilic endoglucanase activity that is reported in crude intracellular medium in P. pastoris. We conclude that P. pastoris is an appropriate host for high-level expression of optimized endoglucanase gene with improved thermal stability.
Keywords: Endoglucanse II; Codon optimization; Acidic thermophilic endoglucanase; Cellulase

Aqueous Extract of Crataegus azarolus Protects Against DNA Damage in Human Lymphoblast Cell K562 and Enhances Antioxidant Activity by Nadia Mustapha; Inès Bouhlel; Fadwa Chaabane; Imèn Mokdad Bzéouich; Kamel Ghedira; Thierry Hennebelle; Leila Chekir-Ghedira (2266-2275).
The present study was carried out to characterize the cellular antioxidant effect of the aqueous extract of Crataegus azarolus and its antigenotoxic potential using human myelogenous cells, K562. The antioxidant capacity of this extract was evaluated by determining its cellular antioxidant activity (CAA) in K562 cells. Also, preceding antigenotoxicity assessment, its eventual genotoxicity property was investigated by evaluating its capacity to induce the DNA degradation of treated cell nuclei. As no genotoxicity was detected at different exposure times, its ability to protect cell DNA against H2O2 oxidative effect was investigated, using the “comet assay.” It appears that 800 μg/mL of extract inhibited the genotoxicity induced by H2O2 with a rate of 41.30 %, after 4 h of incubation. In addition, this extract revealed a significant cellular antioxidant capacity against the reactive oxygen species in K562 cells.
Keywords: Crataegus azarolus ; Antigenotoxic activity; Comet assay; Cellular antioxidant activity; K562

DNA Damage and Effects on Antioxidative Enzymes in Earthworm (Eisenia fetida) Induced by Flumorph by Xiangyu Cao; Chao Yang; Jianli Liu; Xiujuan Hui; Wei Yang; Shuangshuang Li; Yanan Tian; Leiming Cai (2276-2285).
Flumorph is an Oomycete fungicide, which is used extensively as an effective fungicide in vegetables and fruits, but little is known about its effect on nontarget soil organisms. In the present study, biochemical responses including changes in the activity of antioxidative enzymes catalase (CAT), superoxide dismutase (SOD), glutathione-S-transferase (GST), malondialdehyde (MDA), and DNA damage induced by flumorph were investigated in earthworms (Eisenis fetida). The CAT concentrations were stimulated at 5.0 mg kg−1 over 28 days and inhibited at 10 and 20 mg kg−1, except 10 mg kg−1 on days 21 and 28 compared with the controls. The overall SOD activities were inhibited except 5 mg kg−1 on day 28 and 10 mg kg−1 on days 7 and 14. Meanwhile, the GST activities were stimulated on day 7 and decreased on the other days in summary. The MDA activities were increased notably at 5, 10, and 20 mg kg−1 after 14 days. Clear dose-dependent DNA damage to Eisenia fetida was observed by olive tail moments in comet assay compared with controls. The results demonstrate that flumorph induces oxidative stress and DNA damage to earthworms, and the effects may be the important mechanisms of its toxicity.
Keywords: Flumorph; Earthworm; Oxidative stress; Genotoxicity; Comet assay

The present study involved strategies for enhancement in in vitro azadirachtin (commercially used biopesticide) production by hairy root cultivation of Azadirachta indica. Improvement in the azadirachtin production via triggering its biosynthetic pathway in plant cells was carried out by the exogenous addition of precursors and elicitors in the growth medium. Among the different abiotic stress inducers (Ag+, Hg+2, Co+2, Cu+2) and signal molecules (methyl jasmonate and salicylic acid) tested, salicylic acid at 15 mg l−1 of concentration was found to enhance the azadirachtin yield in the hairy roots to the maximum (up to 4.95 mg g−1). Similarly, among the different biotic elicitors tested (filter-sterilized fungal culture filtrates of Phoma herbarium, Alternaria alternata, Myrothecium sp., Fusarium solani, Curvularia lunata, and Sclerotium rolfsii; yeast extract; and yeast extract carbohydrate fraction), addition of filter-sterilized fungal culture filtrate of C. lunata (1 % v/v) resulted in maximum azadirachtin yield enhancement in hairy root biomass (up to 7.1 mg g−1) with respect to the control (3.3 mg g−1). Among all the biosynthetic precursors studied (sodium acetate, cholesterol, squalene, isopentynyl pyrophosphate, mavalonic acid lactone, and geranyl pyrophosphate), the overall azadirachtin production (70.42 mg l−1 in 25 days) was found to be the highest with cholesterol (50 mg l−1) addition as an indirect precursor in the medium.
Keywords: Azadirachta indica ; Hairy roots; Azadirachtin; Yield; Elicitors; Precursors