Applied Biochemistry and Biotechnology (v.173, #1)

Plant Extract: A Promising Biomatrix for Ecofriendly, Controlled Synthesis of Silver Nanoparticles by Hemant P. Borase; Bipinchandra K. Salunke; Rahul B. Salunkhe; Chandrashekhar D. Patil; John E. Hallsworth; Beom S. Kim; Satish V. Patil (1-29).
Uses of plants extracts are found to be more advantageous over chemical, physical and microbial (bacterial, fungal, algal) methods for silver nanoparticles (AgNPs) synthesis. In phytonanosynthesis, biochemical diversity of plant extract, non-pathogenicity, low cost and flexibility in reaction parameters are accounted for high rate of AgNPs production with different shape, size and applications. At the same time, care has to be taken to select suitable phytofactory for AgNPs synthesis based on certain parameters such as easy availability, large-scale nanosynthesis potential and non-toxic nature of plant extract. This review focuses on synthesis of AgNPs with particular emphasis on biological synthesis using plant extracts. Some points have been given on selection of plant extract for AgNPs synthesis and case studies on AgNPs synthesis using different plant extracts. Reaction parameters contributing to higher yield of nanoparticles are presented here. Synthesis mechanisms and overview of present and future applications of plant-extract-synthesized AgNPs are also discussed here. Limitations associated with use of AgNPs are summarised in the present review.
Keywords: Plant extract; AgNPs; Antimicrobial; Biosensor; Toxicity

LC-MS/MS Based Identification of Piperine Production by Endophytic Mycosphaerella sp. PF13 from Piper nigrum by S. Chithra; B. Jasim; C. Anisha; Jyothis Mathew; E. K. Radhakrishnan (30-35).
Piper nigrum is very remarkable for its medicinal properties due to the presence of metabolites like piperine. Emerging understanding on the biosynthetic potential of endophytic fungi suggests the possibility to have piperine producing fungi in P. nigrum. In the current study, endophytic fungi isolated from P. nigrum were screened for the presence of piperine by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This resulted in the identification of a Mycosphaerella sp. with the ability to produce piperine extracellularly. The biosynthesis of piperine (C17H19NO3) by the endophytic fungal isolate was confirmed by the presence of m/z 286.1 (M + H+) in the LC-MS/MS analysis using positive mode ionization. This was further supported by the presence of specific fragment ions with masses 135, 143, 171 and 201 formed due to the fragmentation of piperine present in the fungal extract.
Keywords: Piperine; Piper nigrum ; Endophytic fungi; Mycosphaerella sp; Liquid chromatography-tandem mass spectrometry

Biocompatible APTES–PEG Modified Magnetite Nanoparticles: Effective Carriers of Antineoplastic Agents to Ovarian Cancer by Amaneh Javid; Shahin Ahmadian; Ali Akbar Saboury; Seyed Mehdi Kalantar; Saeed Rezaei-Zarchi; Sughra Shahzad (36-54).
Magnetite nanoparticles are particularly attractive for drug delivery applications because of their size-dependent superparamagnetism, low toxicity, and biocompatibility with cells and tissues. Surface modification of iron oxide nanoparticles with biocompatible polymers is potentially beneficial to prepare biodegradable nanocomposite-based drug delivery agents for in vivo and in vitro applications. In the present study, the bare (10 nm) and polyethylene glycol (PEG)–(3-aminopropyl)triethoxysilane (APTES) (PA) modified (17 nm) superparamagnetic iron oxide nanoparticles (SPIO NPs) were synthesized by coprecipitation method. The anticancer drugs, doxorubicin (DOX) and paclitaxel (PTX), were separately encapsulated into the synthesized polymeric nanocomposites for localized targeting of human ovarian cancer in vitro. Surface morphology analysis by scanning electron microscopy showed a slight increase in particle size (27 ± 0.7 and 30 ± 0.45 nm) with drug loading capacities of 70 and 61.5 % and release capabilities of 90 and 93 % for the DOX- and PTX-AP-SPIO NPs, respectively (p < 0.001). Ten milligrams/milliliter DOX- and PTX-loaded AP-SPIO NPs caused a significant amount of cytotoxicity and downregulation of antiapoptotic proteins, as compared with same amounts of free drugs (p < 0.001). In vivo antiproliferative effect of present formulation on immunodeficient female Balb/c mice showed ovarian tumor shrinkage from 2,920 to 143 mm3 after 40 days. The present formulation of APTES–PEG-SPIO-based nanocomposite system of targeted drug delivery proved to be effective enough in order to treat deadly solid tumor of ovarian cancer in vitro and in vivo.
Keywords: Salination; Ovarian cancer; Tumor volume; Cytotoxicity; Encapsulation efficiency; PEGylation

Highly Potential Antifungal Activity of Quantum-Sized Silver Nanoparticles Against Candida albicans by Malathi Selvaraj; Prabhu Pandurangan; Nishanthi Ramasami; Suresh Babu Rajendran; Sriman Narayanan Sangilimuthu; Palani Perumal (55-66).
The antifungal activity of polyvinylpyrrolidone (PVP)-stabilized quantum-sized silver nanoparticles (SNPs) against the growth of Candida albicans has been demonstrated in the present study. C. albicans is a known opportunistic human pathogen causing superficial and systemic infections. Research data carried out on C. albicans so far have shown unequivocally that it develops resistance against conventional antifungal drugs and that the infections it causes are difficult to cure with conventional antifungal agents. Hence, it is urgent to find newer materials for the treatment of infections caused by C. albicans that must be safe for the host. PVP-capped SNPs were synthesized, and its surface plasmon band was observed at 410 nm. The growth of C. albicans was markedly inhibited when the cells were incubated with SNP. The minimum inhibitory concentration (MIC) of SNP was determined as 70 ng/ml, and this value is relatively lower when compared with the conventionally used antifungal drugs such as amphotericin B (0.5 μg/ml), fluconazole (0.5 μg/ml), and ketoconazole (8 μg/ml). The viability of SNP-treated cells was checked by measuring the metabolic activity using XTT assay. Field emission scanning electron microscopic (FE-SEM) and transmission electron microscopic (TEM) analyses of the cells treated with SNP have lost the structural integrity to a greater extent.
Keywords: Quantum-sized silver nanoparticles; Candida albicans ; Antifungal activity; Minimum inhibitory concentration; Polyvinylpyrrolidone

Improved Oxidation of Naringenin to Carthamidin and Isocarthamidin by Rhodotorula marina by Anna Madej; Jarosław Popłoński; Ewa Huszcza (67-73).
A novel single-step microbial transformation process for the efficient production of carthamidin and isocarthamidin from naringenin by yeast Rhodotorula marina in an aerated bioreactor was described. The biotransformation led to the total product concentration of 233 mg/l. The highest conversion efficiency observed for carthamidin was 0.31 mg/mg of naringenin and for isocarthamidin 0.47 mg/mg of naringenin.
Keywords: Naringenin; Carthamidin; Isocarthamidin; Biotransformation Rhodotorula marina

The objective of the present study was to evaluate the nutrient removal efficiency and the physiological responses in terms of growth, biochemical composition and photosynthetic activity of the autochthonous freshwater algal strain Desmodesmus communis. Microalgae were grown in a primary municipal effluent under different hydraulic retention times (HRTs) and in a two-phases process using both primary and secondary wastewater effluents. Semi-continuous cultures were operated for 7 day at 5-, 3- and 1.5-day HRT and the different dilution rate showed a greater influence on the biomass composition and nutrient removal efficiency. Removal of N-NH3 and P-PO4 was over 99 % and the highest accumulation of polysaccharides (57.2 wt.%) was obtained at high HRT (5 day); the maximum content of proteins (26.9 wt.%) was achieved at 1.5-day HRT, even if, under this condition, a clear inefficiency in terms of ammonia removal was observed. Moreover the accumulation of N-NH3 occurring at 1.5-day HRT caused the decrease of the photosynthetic response in terms of efficiency of light capture (α) and relative electron transport rate (rETR), both parameters extracted from the rapid light curves (RLC) measurements. No significant differences were observed for the total fatty acids (TFAs), with a content of 2–3.5 wt.% for each HRT condition. On the other hand, in the two-phases process, when a nutrient deprivation condition was induced by diluting the culture with the secondary wastewater effluent, the algal cells accumulated TFAs, achieving a maximum content of 9.7 wt.% and a great increment in terms of biomass (1.64 ± 0.02 g L−1) due to the ability of this algal strain to accumulate intracellular N. The wide and accurate investigation of the different aspects related to the whole process represents a relevant point of novelty in this research field and suggests the operational conditions for the start-up of an open pond system for wastewater treatment and biomass production for further applications.
Keywords: Desmodesmus communis ; Municipal wastewater; Hydraulic retention times (HRTs); Nutrient deprivation; Biomass composition; RLC (rapid light curve)

An efficient method for cloning Syzygium cumini (above 40 years old) through mature nodal segments has been successfully developed and that could be exploited for large-scale production of this valuable multipurpose tree. Nodal segments from mature tree were taken as explants and cultured on MS basal medium with different cytokinins (BA, Kin, AdS). The application of BA proved to be the best responsive cytokinin for the induction of shoot buds and shoots, but the proliferated shoots exhibited slower and stunted growth accompanied with abscission of leaves and shoot tip necrosis (STN). The problem of leaf abscission and STN was considerably reduced by the application of an adjuvant, adenine sulphate (AdS) in the optimal medium which led to the production of a maximum of 14 shoots. Further improvement in shoot bud regeneration and improved growth pattern of the regenerating tissue was obtained on the media comprised of MS + BA (10 μM) + GA3 (2.5 μM). A total number of 15 shoots with mean shoot length of 5.9 cm was obtained. The healthy elongated shoots were then rooted on MS basal augmented with NAA (5 μM). The plantlets obtained were healthy and were successfully acclimatized and transferred under field condition with 70 % survival rate.
Keywords: Clonal propagation; Nodal segment; Black plum; Leaf abscission; Myrtaceae; Timber

S-layer is a self-assemble regularly crystalline surface that covers major cell wall component of many bacteria and archaea and exhibits a high metal-binding capacity. We have studied the effect of the calcium ions and type of solid support (glass or mica) on the structure of the S-layers from Bacillus coagulans HN-68 and Bacillus thuringiensis MH14 upon simple methods based on light microscopy and AFM. Furthermore, the Fourier transform infrared spectroscopy (FTIR) study is indicated that the calcium–S-layer interaction occurred mainly through the carboxylate groups of the side chains of aspartic acid (Asp) and glutamic acid (Glu) and nitrogen atoms of Lys, Asn, and histidine (His) amino acids and N–H groups of the peptide backbone. Studied FTIR revealed that inner faces of S-layer are mainly negative, and outer faces of S-layer are mainly positive. Probably, calcium ions with positive charges bound to the carboxyl groups of Glu and Asp. Accordingly, calcium ions are anchored in the space between the inner faces of S-layer with negative charge and the surface of mica with negative charge. This leads to regular arrangement of the S-layer subunits.
Keywords: S-layer; Bacillus coagulans ; Bacillus thuringiensis ; XRD–light microscopy; FTIR

The in vitro synergistic antibacterial activity of six proline-based cyclic dipeptides [cyclo(d-Pro-l-Leu), cyclo(l-Pro-l-Met), cyclo(d-Pro-l-Phe), cyclo(l-Pro-l-Phe), cyclo(l-Pro-l-Tyr), and cyclo(l-Pro-d-Tyr)] in combination imipenem and ceftazidime was investigated in the present manuscript. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the cyclic dipeptides were compared with those of the standard antibiotics (imipenem and ceftazidime). The synergistic antibacterial activities of cyclic dipeptides with imipenem or ceftazidime were assessed using the checkerboard and time-kill methods. The results of the present study showed that the combined effect of six cyclic dipeptides with imipenem predominantly recorded synergistic interaction (FIC index <0.5), whereas combination of certain cyclic dipeptides with ceftazidime recorded additive. The time-kill curve showed that the growth of the test bacteria was completely attenuated after 12–24 h of treatment with a 50:50 ratio of proline-based cyclic dipeptides and antibiotics. These synergistic effects have a potential role in delaying the development of resistance as the antibacterial activity is achieved with the very low concentrations of cyclic dipeptides and antibiotics. The cytotoxicity of cyclic dipeptides was tested against VERO cell line (African green monkey kidney cell line), and no cytotoxicity was recorded for cyclic dipeptides up to 100 μg/mL. These findings suggest that combination of cyclic dipeptides and antibiotics might be a good strategy for the individualization of novel templates for the development of new antimicrobial agents or combinations of drugs for antimicrobial chemotherapy. Moreover, these combinations may lead to the development of a new and vital antimicrobial combination against the infections caused by pathogenic bacteria. The in vitro synergistic activity of cyclic dipeptides with antibiotics against medically important bacteria is reported here for the first time.
Keywords: Diketopiperazines; Antibiotics; Synergism; Bacteria

This study aimed at optimizing the production of hyaluronic acid by Lactobacillus acidophilus FTDC 1231 using response surface methodology and evaluating the effects of divalent metal ions along the production pathway using molecular docking. Among different divalent metal ions that were screened, only iron (II) sulphate and copper (II) sulphate significantly (P < 0.05) affected the production of hyaluronic acid. Subsequent optimization yielded hyaluronic acid at concentration of 0.6152 mg/mL in the presence of 1.24 mol L−1 iron (II) sulphate and 0.16 mol L−1 of copper (II) sulphate (103 % increase compared to absence of divalent metal ions). Data from molecular docking showed Fe2+ improved the binding affinity of UDP-pyrophophorylase towards glucose-1-phosphate, while Cu2+ contributed towards the interaction between UDP-glucose dehydrogenase and UDP-glucose. We have demonstrated that lactobacilli could produce hyaluronic acid at increased concentration upon facilitation by specific divalent metal ions, via specific targets of enzymes and substrates along pentose phosphate pathway.
Keywords: Hyaluronic acid; Lactobacilli; Divalent metal ions; Molecular docking; Optimization

Purification and Characterization of Pepsin-Solubilized Collagen from Skin of Sea Cucumber Holothuria parva by Nasim Adibzadeh; Saeed Aminzadeh; Shahla Jamili; Ali Asghar Karkhane; Naser Farrokhi (143-154).
Pepsin-solubilized collagen (PSC) was extracted from the skin of sea cucumber Holothuria parva and was fractionally characterized. The PSC from H. parva skin consisted of three α1 chains (α1)3, in contrast to calf skin collagen type I with two α1 and one α2 chains (α1)2α2 with approximately 130 kDa each. The maximum transition (Tm) and denaturation temperature (Td) of PSC were determined to be 46.94 and 32.5 °C, respectively. The amino acid composition analysis revealed that glycine, proline, alanine, and hydroxyproline were the abundant amino acids available in extracted PSC. The results showed that the isolated collagen from H. parva has some similar characteristics to previously reported collagens used in food and pharmaceutical industries.
Keywords: Sea cucumber; Pepsin-solubilized collagen; Isolation and characterization

In the present investigation, we have investigated the effect of zinc oxide nanoparticles (ZnONP) on the production of β-glucosidase (BGL) in Saccharomyces cerevisiae under various conditions. ZnONP was synthesized chemically and characterized using various standard techniques. The results revealed that yeast culture administered with 5 mM ZnONP enhanced the intracellular BGL activity up to 28 % compared to control with simultaneous growth of cells. However, at a higher dose of ZnONP (10 and 15 mM), both the activity of the enzyme and yeast growth was dropped. When yeast cells were grown in alcoholic medium (2, 5, and 10 % ethanol), the growth was found inhibited with substantial reduction of intracellular BGL activity. Interestingly, the administration of ZnONP further inhibited the cell growth, however, suppressed the alcoholic effect on enzyme activity. Moreover, under the same condition, ZnONP enhanced the biological activity of the enzyme in cells, indicated a higher yield of BGL production. When the mechanism of ZnONP-mediated cell growth inhibition was investigated, N-acetylcysteine (NAC)-based cell growth study proved that reactive oxygen species (ROS) was not the sole cell death mechanism induced by ZnONP, indicating a second mechanism of cell death. Our findings provide a new insight on the potential application of ZnONP as an external supplement to enhance the active production of BGL like important industrial enzyme in S. cerevisiae in both normal and alcohol stressed condition as well as to produce baker’s yeast in higher amount.
Keywords: ZnO nanoparticles; β-Glucosidase; Saccharomyces cerevisiae ; Alcohol; Reactive oxygen species

Trypsin Inhibitor from Edible Mushroom Pleurotus floridanus Active against Proteases of Microbial Origin by P. P. Manzur Ali; K. Sapna; K. R. Rekha Mol; Sarita G Bhat; M. Chandrasekaran; K. K Elyas (167-178).
Protease inhibitors can be versatile tools mainly in the fields of medicine, agriculture and food preservative applications. Fungi have been recognized as sources of protease inhibitors, although there are only few such reports on mushrooms. This work reports the purification and characterization of a trypsin inhibitor from the fruiting body of edible mushroom Pleurotus floridanus (PfTI) and its effect on the activity of microbial proteases. The protease inhibitor was purified up to 35-fold by DEAE-Sepharose ion exchange column, trypsin-Sepharose column and Sephadex G100 column. The isoelectric point of the inhibitor was 4.4, and its molecular mass was calculated as 37 kDa by SDS-PAGE and 38.3 kDa by MALDI-TOF. Inhibitory activity confirmation was by dot-blot analysis and zymographic activity staining. The specificity of the inhibitor toward trypsin was with Ki of 1.043 × 10−10 M. The inhibitor was thermostable up to 90 °C with maximal stability at 30 °C, active over a pH range of 4–10 against proteases from Aspergillus oryzae, Bacillus licheniformis, Bacillus sp. and Bacillus amyloliquefaciens. Results indicate the possibility of utilization of protease inhibitor from P. floridanus against serine proteases.
Keywords: Trypsin inhibitor; Pleurotus floridanus ; Purification; Ki; Thermostable

Isolation and Purification of Condensed Tannins from Flamboyant Tree and Their Antioxidant and Antityrosinase Activities by Hui-Ling Feng; Ling Tian; Wei-Ming Chai; Xiao-Xin Chen; Yan Shi; Yu-Sen Gao; Chong-Ling Yan; Qing-Xi Chen (179-192).
Flamboyant tree, a kind of medicinal plant, was studied as a source of condensed tannins. The antioxidant activities of the condensed tannins from the leaf, fruit, and stem bark of flamboyant tree were screened by ABTS radical and hydroxyl radical scavenging activity methods. The results indicated that these compounds possessed potent antioxidant activity. Their structures were then characterized by high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) after thiolytic degradation. The results showed that the leaf condensed tannins were composed of afzelechin/epiafzelechin, catechin/epicatechin, and gallocatechin/epigallocatechin, while the fruit and stem bark condensed tannins had afzelechin/epiafzelechin and catechin/epicatechin. In addition, the condensed tannins were evaluated for their antityrosinase ability. They were found to have significant antityrosinase activity. The IC50 values were 35 ± 2.0 and 40 ± 0.5 μg/ml for the condensed tannins of fruit and stem bark, respectively. Further, fluorescence quenching and copper interacting techniques were utilized to unravel the molecular mechanisms of the inhibition. The results showed that the hydroxyl group of the condensed tannins could chelate the dicopper center of the enzyme and interact with tryptophan residues. Our studies revealed that condensed tannins might be suitable for use in food, agriculture, cosmetic, nutraceutical, and pharmaceutical applications.
Keywords: Flamboyant tree; Condensed tannins; ABTS; Hydroxyl radical scavenging activity; HPLC-ESI-MS; Antityrosinase activity

Microbial Leaching of Waste Solder for Recovery of Metal by H. Hocheng; T. Hong; U. Jadhav (193-204).
This study proposes an environment-friendly bioleaching process for recovery of metals from solders. Tin-copper (Sn-Cu), tin-copper-silver (Sn-Cu-Ag), and tin-lead (Sn-Pb) solders were used in the current study. The culture supernatant of Aspergillus niger removed metals faster than the culture supernatant of Acidithiobacillus ferrooxidans. Also, the metal removal by A. niger culture supernatant is faster for Sn-Cu-Ag solder as compared to other solder types. The effect of various process parameters such as shaking speed, temperature, volume of culture supernatant, and increased solder weight on bioleaching of metals was studied. About 99 (±1.75) % metal dissolution was achieved in 60 h, at 200-rpm shaking speed, 30 °C temperature, and by using 100-ml A. niger culture supernatant. An optimum solder weight for bioleaching was found to be 5 g/l. Addition of sodium hydroxide (NaOH) and sodium chloride (NaCl) in the bioleached solution from Sn-Cu-Ag precipitated tin (85 ± 0.35 %) and silver (80 ± 0.08 %), respectively. Passing of hydrogen sulfide (H2S) gas at pH 8.1 selectively precipitated lead (57.18 ± 0.13 %) from the Sn-Pb bioleached solution. The proposed innovative bioleaching process provides an alternative technology for recycling waste solders to conserve resources and protect environment.
Keywords: Solders; Bioleaching; Aspergillus niger ; Acidithiobacillus ferrooxidans ; Culture supernatant

Effective wastewater treatment and electricity generation using dual-chamber microbial fuel cell (MFC) will require a better understanding of how operational parameters affect system performance. Therefore, the main aim of this study is to investigate the bioelectricity production in a dual-chambered MFC-operated batch mode under different operational conditions. Initially, platinum (Pt) and mixed metal oxide titanium (Ti-TiO2) electrodes were used to investigate the influence of the electrode materials on the power generation at initial dissolved organic carbon (DOC) concentration of 400 mg/L and cycle time of 15 days. MFC equipped with Ti-TiO2 electrode performed better and was used to examine the effect of influent DOC concentration and cycle time on MFC performance. Increasing influent DOC concentration resulted in improving electricity generation, corresponding to a 1.65-fold increase in power density. However, decrease in cycle time from 15 to 5 days adversely affected reactor performance. Maximum DOC removal was 90 ± 3 %, which was produced at 15-day cycle time with an initial DOC of 3,600 mg/L, corresponding to maximum power generation of about 7,205 mW/m2.
Keywords: Cycle time; Substrate; MFC; Pt electrode; Ti-TiO2 electrode

Biochemical and Kinetic Study of Laccase from Ganoderma cupreum AG-1 in Hydrogels by Mayur Gahlout; Shilpa Gupte; Akshaya Gupte (215-227).
In the present study, three different types of hydrogels i.e., (poly (−acrylamide)/alginate (P (AAm)/Alg), poly (acrylamide-N-isopropylacrylamide) (P (AAm-NIPA)), and poly (acrylamide-N-isopropylacrylamide)/alginate (P (AAm-NIPA)/Alg)) were synthesized by acrylamide, alginate, and N-isopropylacrylamide for the entrapment of laccase. The hydrogel-entrapped and free laccase showed optimum temperature of 50 °C for the oxidation of ABTS, but the entrapped laccase showed high temperature, pH, and storage stability as compared to the free enzyme. The K m values of free laccase, (P (AAm)/Alg)-L, (P (AAm-NIPA))-L, and (P (AAm-NIPA)/Alg)-L were found to be 0.13, 0.28, 0.33, and 0.50 mM, respectively. The V max values of free laccase, (P (AAm)/Alg)-L, (P (AAm-NIPA))-L, and (P (AAm-NIPA)/Alg)-L were found to be 22.22 × 102, 5.55 × 102, 5.0 × 102, and 4.54 × 102 mM/min, respectively. The entrapped laccase hydrogels were used for the decolorization of Reactive Violet 1 dye, with 39 to 45 % decolorization efficiency till the 10th cycle.
Keywords: Laccase; Hydrogel; Acrylamide; Alginate and N-isopropylacrylamide

mga Genosensor for Early Detection of Human Rheumatic Heart Disease by Swati Singh; Ankur Kaushal; Shashi Khare; Ashok Kumar (228-238).
The 5′ amino-labeled DNA probe complementary to mga gene of Streptococcus pyogenes was immobilized on carboxylated multiwall carbon nanotubes electrode and hybridized with 0.1–100 ng/6 μl single-stranded genomic DNA (ssG-DNA) of S. pyogenes from throat swab of suspected rheumatic heart disease (RHD) patients. Electrochemical response was measured by cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance (EI). The sensitivity of the sensor was 106.03 (μA/cm2)/ng and limit of detection (LOD) was found 0.014 ng/6 μl with regression coefficient (R 2) of 0.921 using DPV. The genosensor was characterized by FTIR and SEM, and electrode was found stable for 6 months on storage at 4 °C with 5–6 % loss in initial DPV current. mga genosensor is the first report on RHD sensor which can save life of several suspected patients by early diagnosis in 30 min.
Keywords: Genosensor; mga genosensor; Rheumatic heart disease; Streptococcus pyogenes

Expression of the Key Genes Involved in ABA Biosynthesis in Rice Implanted by Ion Beam by Q. F. Chen; H. Y. Ya; Y. R. Feng; Z. Jiao (239-247).
The plant hormone abscisic acid (ABA) plays a central role in adaptive stress responses to abiotic environments, but little information exists about its responses to implantation with low-energy ion beams. The genes related to ABA synthesis including zeaxanthin epoxidase(ZEP), 9-cis-epoxycarotenoid dioxygenase (NCED), abscisic aldehyde oxidase (AAO), short-chain dehydrogenase/reductase-like(SDR), and cytochrome P450 in rice seedlings germinating from seeds implanted by ion beam for 72, 96, and 120 h after imbibitions (HAI) were determined by real-time PCR. Moreover, we also explored the changes of endogenous ABA content in rice seedlings after 48, 72, 96, 120, and 144 h after imbibitions using enzyme-linked immunosorbent assay (ELISA). The results showed that ion beam implantation could enhance the genes’ transcription activity which was involved in ABA biosynthesis. However, the response of each gene is not consistent with the underlying differences in ion flux. The obviously up-regulated expression of ZEP, AAO2, SDR, and P450-2 were observed underlying the behaviour at an ion flux of 1 × 1017 N+/cm2. However, the expression of NCED, AAO1, and SDR2 can be enhanced by 5 × 1017 N+/cm2. The expression of AAO3, SDR1, and P450-1 can be elevated underlying the both ion flux of 1 × 1017 N+/cm2 and 5 × 1017 N+/cm2. The expression of SDR3 can be enhanced in every ion flux. The results of ELISA showed that endogenous ABA level in rice seedlings increased at treatment with vacuum, 1 × 1017 and 5 × 1017 N+/cm2. Collectively, ion beam irritation can enhance the expression of genes involved in ABA biosynthesis, resulting in increasing content of endogenous ABA in rice. Our findings suggest that ABA pathway was involved in the adaption to irradiation with ion beam in plants.
Keywords: Ion beam implantation; ABA biosynthesis; Gene expression

Cell suspension cultures of Arnebia euchroma were established from the friable callus on liquid Murashige and Skoog medium supplemented with 6-benzylaminopurine (10.0 μM) and indole-3-butyric acid (5.0 μM). Salicylic acid was used to study its effect on the enzymes which participate in shikonin biosynthesis with respect to metabolite (shikonin) content in the cell suspension culture of A. euchroma. In our study, phenylalanine ammonia lyase and PHB geranyltransferase were selected from the entire biosynthetic pathway. Results showed that phenylalanine ammonia lyase is responsible for growth and PHB geranyltransferase for metabolite production. Salicylic acid exhibited an inverse relationship with the metabolite content (shikonin); salicylic acid (100 μM) completely inhibited shikonin biosynthesis. The results presented in the current study can be successfully employed for the metabolic engineering of its biosynthetic pathway for the enhancement of shikonin, which will not only help in meeting its industrial demand but also lead to the conservation of species in its natural habitat.
Keywords: Arnebia euchroma ; Boraginaceae; Cell suspension culture; Shikonin derivatives; Secondary metabolites; PAL; PHB geranyltransferase

Probiotic Attributes of Autochthonous Lactobacillus rhamnosus Strains of Human Origin by Sheetal Pithva; Satyamitra Shekh; Jayantilal Dave; Bharatkumar Rajiv Manuel Vyas (259-277).
The study was aimed at evaluating the probiotic potential of indigenous autochthonous Lactobacillus rhamnosus strains isolated from infant feces and vaginal mucosa of healthy female. The survival of the selected strains and the two reference strains (L. rhamnosus GG and L. casei Actimel) was 67–81 % at pH 2 and 70–80 % after passage through the simulated gastrointestinal fluid. These strains are able to grow in the presence of 4 % bile salt, 10 % NaCl, and 0.6 % phenol. The cell surface of L. rhamnosus strains is hydrophilic in nature as revealed by bacterial adhesion to hydrocarbons (BATH) assay. Despite this, L. rhamnosus strains showed mucin adherence, autoaggregation and coaggregation properties that are strain-specific. In addition, they produce bile salt hydrolase (BSH) and β-galactosidase activities. L. rhamnosus strains exhibit antimicrobial activity against food spoilage organisms and gastrointestinal pathogens, as well as Candida and Aspergillus spp. L. rhamnosus strains have similar antibiotic susceptibility pattern, and resistance to certain antibiotics is intrinsic or innate. The strains are neither haemolytic nor producer of biogenic amines such as histamine, putrescine, cadaverine and tyramine. Lyophilized cells of L. rhamnosus Fb exhibited probiotic properties demonstrating potential of the strain for technological suitability and in the preparation of diverse probiotic food formulations.
Keywords: Antimicrobial activity; Autoaggregation; Coaggregation; Lactobacillus rhamnosus ; Probiotics

To maintain biological functions, thousands of different reactions take place in human body at physiological pH (7.0) and mild conditions, which is associated with health and disease. Therefore, to examine the catalytic function of the intrinsically occurring molecules, such as amino acids at neutral pH, is of fundamental interests. Natural basic α-amino acid of l-lysine, l-arginine, and l-histidine neutralized to physiological pH as salts were investigated for their ability to catalyze Knoevenagel condensation of benzaldehyde and ethyl cyanoacetate. Compared with their free base forms, although neutralized alkaline amino acid salts reduced the catalytic activity markedly, they were still capable to perform an efficient catalysis at physiological pH as porcine pancreatic lipase (PPL), one of the best enzymes that catalyze Knoevenagel condensation. In agreement with the fact that the three basic amino acids were well neutralized, stronger basic amino acid Arg and Lys showed more obvious variation in NH bend peak from the FTIR spectroscopy study. Study of ethanol/water system and quantitative kinetic analysis suggested that the microenvironment in the vicinity of amino acid salts and protonability/deprotonability of the amine moiety may determine their catalytic activity and mechanism. The kinetic study of best approximation suggested that the random binding might be the most probable catalytic mechanism for the neutralized alkaline amino acid salt-catalyzed Knoevenagel condensation.
Keywords: Knoevenagel condensation; Amino acid; Lipase; Neutralized alkaline amino acid salt; Physiological pH

Transition Metals and Organic Ligands Influence Biodegradation of 1,4-Dioxane by Peerapong Pornwongthong; Anjali Mulchandani; Phillip B. Gedalanga; Shaily Mahendra (291-306).
1,4-Dioxane, a contaminant increasingly detected in water supplies, is a public health concern because it is classified as a possible human carcinogen. 1,4-Dioxane can be biodegraded by aerobic bacteria via monooxygenase-catalyzed reactions. While these metalloenzymes require trace metals as cofactors in their catalytic sites, these metals may be toxic at elevated concentrations. In this study, the effects of transition metals on 1,4-dioxane biodegradation by Pseudonocardia dioxanivorans CB1190, a monooxygenase-expressing bacterium, were investigated. Dose-dependent inhibition of 1,4-dioxane biodegradation by Cd(II), Cu(II), and Ni(II) was observed, whereas Zn(II) had no measurable effect on biodegradation rates. 1,4-Dioxane biodegradation in cultures exposed to 2 mg/L Cu(II) was restored in the presence of 0.005, 0.05, and 0.5 mM alginin, 0.05, and 0.5 mM cysteine, and 0.005 mM tannin. These results indicated that specific ligands bind with transition metals and alleviate bacterial toxicity. In parallel experiments, tannin and cysteine inhibited 1,4-dioxane biodegradation, but alginin, BSA, and SRNOM did not affect the biodegradation rates. Thus, monooxygenase-catalyzed biodegradation rates are subject to interactions among transition metals and natural organic ligands in the environment. Mechanistic insights and quantitative data obtained in this study will be useful for designing bioremediation strategies at sites simultaneously contaminated with metals and organic pollutants.
Keywords: Dioxane; Biotransformation; Co-contaminants; Oxygenase; Metal resistance; Metal ions; Trace metals; Natural organic matter; NOM; Metal–ligand complex; Bioavailable; Bioavailability

Stimulation of microbially induced calcium carbonate precipitation (MICCP) is likely to be influenced by calcium sources. In order to study such influences, we performed MICCP using Bacillus sp. CR2 in nutrient broth containing urea, supplemented with different calcium sources (calcium chloride, calcium oxide, calcium acetate and calcium nitrate). The experiment lasted 7 days, during which bacterial growth, urease activity, calcite production and pH were measured. Our results showed that calcium chloride is the better calcium source for MICCP process, since it provides higher urease activity and more calcite production. The influences of calcium sources on MICCP were further studied using Fourier transform-infrared (FTIR) spectroscopy, scanning electron microscopy (SEM) and X-ray diffraction (XRD) analyses. These analyses confirmed that the precipitate formed was CaCO3 and composed of predominantly calcite crystals with a little amount of aragonite and vaterite crystals. The maximum yield of calcite precipitation was achievable with calcium chloride followed by calcium nitrate as a calcium source. The results of present study may be applicable to media preparation during efficient MICCP process.
Keywords: Bacillus ; Calcite; Aragonite; Urease; Calcium chloride

Green Synthesized Iron Oxide Nanoparticles Effect on Fermentative Hydrogen Production by Clostridium acetobutylicum by Sundaresan Mohanraj; Shanmugam Kodhaiyolii; Mookan Rengasamy; Velan Pugalenthi (318-331).
A green synthesis of iron oxide nanoparticles (FeNPs) was developed using Murraya koenigii leaf extract as reducing and stabilizing agent. UV–vis spectra show that the absorption band centred at a wavelength of 277 nm which corresponds to the surface plasmon resonances of synthesized FeNPs. Fourier transform infrared spectroscopy spectrum exhibits that the characteristic band at 580 cm−1 is assigned to Fe–O of γ-Fe2O3. Transmission electron microscopy image confirms that the spherical with irregular shaped aggregates and average size of nanoparticles was found to be ∼59 nm. The effect of synthesized FeNPs on fermentative hydrogen production was evaluated from glucose by Clostridium acetobutylicum NCIM 2337. The hydrogen yield in control experiment was obtained as 1.74 ± 0.08 mol H2/mol glucose whereas the highest hydrogen yield in FeNPs supplemented experiment was achieved as 2.33 ± 0.09 mol H2/mol glucose at 175 mg/L of FeNPs. In addition, the hydrogen content and hydrogen production rate were also increased from 34 ± 0.8 to 52 ± 0.8 % and 23 to 25.3 mL/h, respectively. The effect of FeNPs was compared with supplementation of FeSO4 on fermentative process. The supplementation of FeNPs enhanced the hydrogen production in comparison with control and FeSO4. The supplementation of FeNPs led to the change of the metabolic pathway towards high hydrogen production due to the enhancement of ferredoxin activity. The fermentation type was shifted from butyrate to acetate/butyrate fermentation type at the addition of FeNPs.
Keywords: Green synthesis; Iron oxide nanoparticles; Murraya koenigii ; Hydrogen production; Clostridium acetobutylicum NCIM 2337