Assessment of genetic fidelity of <Emphasis Type="Italic">Dioscorea bulbifera</Emphasis> L. and <Emphasis Type="Italic">Dioscorea hirtiflora</Emphasis> Benth. and medicinal bioactivity produced from the induced tuberous roots

Wild tubers of Dioscorea bulbifera (Db) and Dioscorea hirtiflora (Dh) mainly used as sources of famine food and in herbal preparations are often indiscriminately collected in Africa and Asia. Therefore, there is the need to complement wild sourcing of the tubers to promote their conservation. The present study reports in vitro tuberous induction (80%) for the first time from Dh cultured on MS?+?NAA (2.5 mg/L) with IC₅₀ of 472.5?±?1.77 µg/mL using DPPH, whereas tuberous root (60%) from Db on MS?+?Kn (2.5 mg/L)?+?NAA (0.25 mg/L) had IC₅₀ of 26.97?±?1.00 µg/mL. Genetic fidelity assessment of in vitro plants compared to the wild plants revealed similar amplicon size of amplified DNA using trnH–psbA and rbcL. Similarly, micromorphological diagnostic features like oil gland, crystals (raphides), trichome and stomata type were obtained from the epidermal peels of the wild and in vitro plants. The ethyl acetate (EtOAc) extract of the flesh of Dh (wild) had the highest catechin content (108.3?±?0.69 µg/g DW). Protocatechuic acid was highest in the methanol (MeOH) extract of the flesh of Dh (0.42?±?0.02 µg/g DW), while it was detected in trace amount in the in vitro tuberous roots of MeOH extracts of Dh treated with NAA. The in vitro protocol developed in this study could be employed to multiply Dioscorea bulbifera L. and Dioscorea hirtiflora Benth. to offer genetically stable clones for the optimization of bioactive compounds and germplasms conservation.     

Characterization of chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) lectin for biological activity

Lectins are proteins that are subject of intense investigations. Information on lectin from chickpea (Cicer arietinum L.) with respect to its biological activities are very limited. In this study, we purified lectin from the seeds of chickpea employing DEAE-cellulose and SP-Sephadex ion exchange chromatography and identified its molecular subunit mass as 35 kDa. The free radical scavenging activity of lectin measured by the DPPH assay has IC₅₀ of 0.88 µg/mL. Lectin exerted antifungal activity against Candida krusei, Fusarium oxysporium oxysporium, Saccharomyces cerevisiae and Candida albicans, while antibacterial activity against E. coli, B. subtilis, S. marcescens and P. aeruginosa. The minimum inhibitory concentrations were 200, 240, 160 and 140 µg for C. krusei, F. oxysporium, S. cerevisiae and C. albicans respectively. Lectin was further examined for its antiproliferative potential against cancerous cell line. The cell viability assay indicated a high inhibition activity on Ishikawa, HepG2, MCF-7 and MDA-MB-231 with IC₅₀ value of 46.67, 44.20, 53.58 and 37.46?µg/mL respectively. These results can provide a background for future research into the benefits of chickpea lectin to pharmacological perspective.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Melothria maderaspatana</Emphasis> via indirect organogenesis

An effective protocol was developed for in vitro regeneration of the Melothria maderaspatana via indirect organogenesis in liquid and solid culture systems. Organogenesis was achieved from liquid culture calluses derived from leaf and petiole explants of mature plants. Organogenic calluses (98.2?±?0.36 and 94.8?±?0.71%) were induced from both leaf and petiole explants on Murashige and Skoog (MS) liquid medium containing 6.0 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 µM thidiazuron (TDZ); and 6.0 µM 2,4-D and 1.0 µM benzyladenine (BA) combinations, respectively. Adventitious shoot regeneration (68.2?±?0.06 shoots per explant) was achieved on MS medium supplemented with 2.0 µM BA, 4.0 µM TDZ, 10% v/v coconut water and 0.06 mM glutamine from leaf-derived calluses. Petiole-derived calluses produced adventitious shoots (45.4?±?0.09 shoots per explant) on MS medium fortified with 2.0 µM BA, 4.0 µM TDZ, 10% v/v coconut water, and 0.08 mM glutamine. Elongation of shoots occurred in MS medium with 2.0 µM gibberellic acid (GA₃). Regenerated shoots (2–3 cm in length) rooted (74.2?±?0.38%) and hardened (85?±?1.24%) when they were transferred to 1/2-MS medium supplemented with 3.0 µM indole-3-butyric acid (IBA) followed by garden soil, vermiculate, and sand (2:1:1 ratio) mixture. The elongated shoots (4–5 cm in length) were exposed simultaneously for rooting as well as hardening (100%) in moistened [(1/8-MS basal salt solution with 5 µM IBA and 100 mg l^?1 Bavistin® (BVN)] garden soil, vermiculate, and sand (2:1:1 ratio) mixture. Subsequently, the plants were successfully established in the field. The survival percentage differed with seasonal variations.     

In vitro and in silico characterization of angiogenic inhibitors from <Emphasis Type="Italic">Sophora interrupta</Emphasis>

Sophora interrupta Bedd, (Fabaceae) is used in Indian folk medicine to treat cancer. Angiogenesis is one of the crucial characteristics of cancer metastasis and is regulated by vascular endothelial growth factor (VEGF). In this study, we examined the antiangiogenic properties of the root ethyl acetate extract of Sophora interrupta by various methods. In vitro antioxidant activity (100–600 μg/ml) of S. interrupta ethyl acetate (SEA) extract was evaluated by DPPH and ABTS, anti-inflammatory activity (50, 100 and 150 μg/ml) by estimating nitric oxide (NO) levels, anti-angiogenic activity (200 and 500 μg/ml) was validated by chorio allantoic membrane (CAM) assay and in silico molecular dynamic (MD) simulations analyses (25 ns) were performed to identify the anti-angiogenic compounds extracted from root extract. The antioxidative activity of SEA extract at IC₅₀ (200?±?0.6 μg/mL) is equal to that of ascorbic acid at IC₅₀ (50?±?0.6 μg/mL), and the anti-inflammatory activity of SEA extract at IC₅₀ (150?±?0.2 μg/mL) was inhibited significantly by nitric oxide (NO) production. The SEA extract significantly reduced the sprouting of new blood vessels at ID₅₀ 500?±?0.13 μg/mL in the CAM assay. Gas chromatography–mass spectrometry analysis of the SEA extract detected 34 secondary metabolites, of which 6a,12a-dihydro-6H-(1,3)dioxolo(5,6)benzofuro(3,2-c)chromen-3-ol (maackiain) and funiculosin formed strong hydrogen bond interactions with Lys 920, Thr 916 and Cys 919 (2H), as well as Glu 917 of VEGFR2, and these interactions were similar to those of the anti-angiogenic compound axitinib. Significant findings in all the assays performed indicate that SEA extract has potential anti-angiogenic compounds that may interfere with VEGF-induced cancer malignancy.     

In vitro anticancer properties of selected <Emphasis Type="Italic">Eucalyptus</Emphasis> species

In spite of the recent advancements in oncology, the overall survival rate for pancreatic cancer has not improved over the last five decades. Eucalypts have been linked with cytotoxic and anticancer properties in various studies; however, there is very little scientific evidence that supports the direct role of eucalypts in the treatment of pancreatic cancer. This study assessed the anticancer properties of aqueous and ethanolic extracts of four Eucalyptus species using an MTT assay. The most promising extracts were further evaluated using a CCK-8 assay. Apoptotic studies were performed using a caspase 3/7 assay in MIA PaCa-2 cells. The aqueous extract of Eucalyptus microcorys leaf and the ethanolic extract of Eucalyptus microcorys fruit inhibited the growth of glioblastoma, neuroblastoma, lung and pancreatic cancer cells by more than 80% at 100 μg/mL. The E. microcorys and Eucalyptus saligna extracts showed lower GI₅₀ values than the ethanolic Eucalyptus robusta extract in MIA PaCa-2 cells. Aqueous E. microcorys leaf and fruit extracts at 100 μg/mL exerted significantly higher cell growth inhibition in MIA PaCa-2 cells than other extracts (p < 0.05). Statistically similar IC₅₀ values (p > 0.05) were observed in aqueous E. microcorys leaf (86.05 ± 4.75 μg/mL) and fruit (64.66 ± 15.97 μg/mL) and ethanolic E. microcorys leaf (79.30 ± 29.45 μg/mL) extracts in MIA PaCa-2 cells using the CCK-8 assay. Caspase 3/7-mediated apoptosis and morphological changes of cells were also witnessed in MIA PaCa-2 cells after 24 h of treatment with the extracts. This study highlighted the significance of E. microcorys as an important source of phytochemicals with efficacy against pancreatic cancer cells. Further studies are warranted to purify and structurally identify individual compounds and elucidate their mechanisms of action for the development of more potent and specific chemotherapeutic agents for pancreatic cancer.     

Medium Optimization for Improved Production of Dihydrolipohyl Dehydrogenase from <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> PAD-91 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Dihydrolipohyl dehydrogenase (DLD) is a FAD-dependent enzyme that catalyzes the reversible oxidation of dihydrolipoamide. Herein, we report medium optimization for the production of a recombinant DLD with NADH-dependent diaphorase activity from a strain of Bacillus sphaericus PAD-91. The DLD gene that consisted of 1413 bp was expressed in Escherichia coli BL21 (DE3), and its enzymatic properties were studied. The composition of production medium was optimized using one-variable-at-a-time method followed by response surface methodology (RSM). B. sphaericus DLD catalyzed the reduction of lipoamide by NAD⁺ and exhibited diaphorase activity. The molecular weight of enzyme was about 50 kDa and determined to be a monomeric protein. Recombinant diaphorase showed its optimal activity at temperature of 30 °C and pH 8.5. K _m and V _max values with NADH were estimated to be 0.025 mM and 275.8 U/mL, respectively. Recombinant enzyme was optimally produced in fermentation medium containing 10 g/L sucrose, 25 g/L yeast extract, 5 g/L NaCl and 0.25 g/L MgSO₄. At these concentrations, the actual diaphorase activity was calculated to be 345.0 ± 4.1 U/mL. By scaling up fermentation from flask to bioreactor, enzyme activity was increased to 486.3 ± 5.5 U/mL. Briefly, a DLD with diaphorase activity from a newly isolated B. sphaericus PAD-91 was characterized and the production of recombinant enzyme was optimized using RSM technique.     

Antifungal activity of magnoflorine against <Emphasis Type="Italic">Candida</Emphasis> strains

Candida albicans is a major invasive pathogen, and the development of strains resistant to conventional antifungal agents has been reported in recent years. We evaluated the antifungal activity of 44 compounds against Candida strains. Magnoflorine showed the highest growth inhibitory activity of the tested Candida strains, with a minimum inhibitory concentration (MIC) of 50 μg/mL based on microdilution antifungal susceptibility testing. Disk diffusion assay confirmed the antifungal activity of magnoflorine and revealed that this activity was stable over 3 days compared to those of berberine and cinnamaldehyde. Cytotoxicity testing showed that magnoflorine could potentially be used in a clinical setting because it didn’t have any toxicity to HaCaT cells even in 200 μg/mL of treatment. Magnoflorine at 50 μg/mL inhibited 55.91?±?7.17% of alpha-glucosidase activity which is required for normal cell wall composition and virulence of Candida albicans. Magnoflorine also reduced the formation of C. albicans’ biofilm. Combined treatment with magnoflorine and miconazole decreased the amount of miconazole required to kill various Candida albicans. Therefore, magnoflorine is a good candidate lead compound for novel antifungal agents.     

Novel aromatic polyketides from soil <Emphasis Type="Italic">Streptomyces</Emphasis> spp.: purification,characterization and bioactivity studies

Aromatic polyketides are important therapeutic compounds which include front line antibiotics and anticancer drugs. Since most of the aromatic polyketides are known to be produced by soil dwelling Streptomyces, 54 Streptomyces strains were isolated from the soil samples. Five isolates, R1, B1, R3, R5 and Y8 were found to be potent aromatic polyketide producers and were identified by 16S rRNA gene sequencing as Streptomyces spectabilis, Streptomyces olivaceus, Streptomyces purpurascens, Streptomyces coeruleorubidus and Streptomyces lavendofoliae respectively. Their sequences have been deposited in the GenBank under the accession numbers KF468818, KF681280, KF395224, KF527511 and KF681281 respectively. The Streptomyces strains were cultivated in the media following critically optimised culture conditions. The resulting broth extracts were fractionated on a silica gel column and preparative TLC to obtain pure compounds. The pure compounds were tested for bioactivity and the most potent compound from each isolate was identified by UV–Vis, IR and NMR spectroscopic methods. Isolated S. spectabilis (R1), yielded one potent compound identified as dihydrodaunomycin with an MIC of 4 µg/ml against Bacillus cereus and an IC₅₀ value of 24 µM against HeLa. S. olivaceus (B1), yielded a comparatively less potent compound, elloramycin. S. purpurascens (R3) yielded three compounds, rhodomycin, epelmycin and obelmycin. The most potent compound was rhodomycin with an MIC of 2 µg/ml against B. cereus and IC₅₀ of 15 µM against HeLa. S. coeruleorubidus (R5), yielded daunomycin showing an IC₅₀ of 10 µM and also exhibiting antimetastatic properties against HeLa. S. lavendofoliae (Y8), yielded a novel aclacinomycin analogue with IC₅₀ value of 2.9 µM and potent antimetastatic properties at 1 µM concentration against HeLa. The study focuses on the characterization of aromatic polyketides from soil Streptomyces spp., which can serve as potential candidates for development of chemotherapeutic drugs in future.     

<Emphasis Type="Italic">In Vitro</Emphasis>-<Emphasis Type="Italic">In Vivo</Emphasis> Evaluation of Novel Co-spray Dried Rifampicin Phospholipid Lipospheres for Oral Delivery

The objective of this study comprises of developing novel co-spray dried rifampicin phospholipid lipospheres (SDRPL) to investigate its influence on rifampicin solubility and oral bioavailability. Solid-state techniques were employed to characterize the liposphere formulation. SDRPL solubility was determined in distilled water. BACTEC 460TB System was employed to evaluate SDRPL antimycobacterial activity. The oral bioavailability of the lipospheres was evaluated in Sprague Dawley rats. Lipospheres exhibited amorphous, smooth spherical morphology with a significant increase (p?<?0.001) in solubility of SDRPL (2:1), 350.9?±?23 versus 105.1?±?12 μg/ml and SDRPL (1:1) 306.4?±?20 versus 105.1?±?12 μg/ml in comparison to rifampicin (RMP). SDRPL exhibited enhanced activity against Mycobacterium tuberculosis, H37Rv strain, with over twofolds less minimum inhibitory concentration (MIC) than the free drug. Lipospheres exhibited higher peak plasma concentration (109.92?±?25 versus 54.31?±?18 μg/ml), faster T _max (two versus four hours), and enhanced area under the curve (AUC_0–∞) (406.92?±?18 versus 147.72?±?15 μg h/L) in comparison to pure RMP. Thus, SDRPL represents a promising carrier system exhibiting enhanced antimycobacterial activity and oral bioavailability of rifampicin.     

Growth kinetics and scale-up of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Production of recombinant proteins in plants through Agrobacterium-mediated transient expression is a promising method of producing human therapeutic proteins, vaccines, and commercial enzymes. This process has been shown to be viable at a large scale and involves growing large quantities of wild-type plants and infiltrating the leaf tissue with a suspension of Agrobacterium tumefaciens bearing the genes of interest. This study examined one of the steps in this process that had not yet been optimized: the scale-up of Agrobacterium production to sufficient volumes for large-scale plant infiltration. Production of Agrobacterium strain C58C1 pTFS40 was scaled up from shake flasks (50–100 mL) to benchtop (5 L) scale with three types of media: Lysogeny broth (LB), yeast extract peptone (YEP) media, and a sucrose-based defined media. The maximum specific growth rate (μ _max) of the strain in the three types of media was 0.46 ± 0.04 h^?1 in LB media, 0.43 ± 0.03 h^?1 in YEP media, and 0.27 ± 0.01 h^?1 in defined media. The maximum biomass concentration reached at this scale was 2.0 ± 0.1, 2.8 ± 0.1, and 2.6 ± 0.1 g dry cell weight (DCW)/L for the three media types. Production was successfully scaled up to a 100-L working volume reactor with YEP media, using k _L a as the scale-up parameter.     

<Emphasis Type="Italic">Nibribacter flagellatus</Emphasis> sp. nov., isolated from rhizosphere of <Emphasis Type="Italic">Hibiscus syriacus</Emphasis> and emended description of the genus <Emphasis Type="Italic">Nibribacter</Emphasis>

A Gram-stain negative, aerobic, motile by flagella, rod-shaped strain (THG-T16^T) was isolated from rhizosphere of Hibiscus syriacus. Growth occurred at 10–40 °C (optimum 28–30 °C), at pH 6.0–8.0 (optimum 7.0) and at 0–1.0% NaCl (optimum 0%). Based on 16S rRNA gene sequence analysis, the near phylogenetic neighbours of strain THG-T16^T were identified as Nibribacter koreensis KACC 16450^T (98.6%), Rufibacter roseus KCTC 42217^T (94.7%), Rufibacter immobilis CCTCC AB 2013351^T (94.5%) and Rufibacter tibetensis CCTCC AB 208084^T (94.4%). The DNA G+C content of strain THG-T16^T was determined to be 46.7 mol%. DNA–DNA hybridization values between strain THG-T16^T and N. koreensis KACC 16450^T, R. roseus KCTC 42217^T, R. immobilis CCTCC AB 2013351^T, R.tibetensis CCTCC AB 208084^T were 33.5?±?0.5% (31.7?±?0.7% reciprocal analysis), 28.1?±?0.2% (25.2?±?0.2%), 17.1?±?0.9% (10.2?±?0.6%) and 8.1?±?0.3% (5.2?±?0.1%). The polar lipids were identified as phosphatidylethanolamine, two unidentified aminophospholipids, an unidentified aminolipid and three unidentified lipids. The quinone was identified as MK-7 and the polyamine as sym-homospermidine. The major fatty acids were identified as C_16:1 ω5c, C_17:1 ω6c, iso-C_15:0, summed feature 3 (C_16:1 ω7c and/or C_16:1 ω6c) and summed feature 4 (iso-C_17:1 I and/or anteiso-C_17:1 B). On the basis of the phylogenetic analysis, chemotaxonomic data, physiological characteristics, and DNA–DNA hybridization data, strain THG-T16^T represents a novel species of the genus Nibribacter, for which the name Nibribacter flagellatus sp. nov. is proposed. The type strain is THG-T16^T(=?KACC 19188^T?=?CCTCC AB 2016246^T).     

Assessing biodegradation of furfuryl alcohol using <Emphasis Type="Italic">Pseudomonas putida</Emphasis> MTCC 1194 and <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> MTCC 1034 and its kinetics

The biodegradation of furfuryl alcohol (FA) in shake flask experiments using a pure culture of Pseudomonas putida (MTCC 1194) and Pseudomonas aeruginosa (MTCC 1034) was studied at 30 °C and pH 7.0. Experiments were performed at different FA concentrations ranging from 50 to 500 mg/l. Before carrying out the biodegradation studies, the bacterial strains were acclimatized to the concentration of 500 mg/l of FA by gradually raising 100 mg/l of FA in each step. The well acclimatized culture of P. putida and P. aeruginosa degraded about 80 and 66% of 50 mg/l FA, respectively. At higher concentration of FA, the percentage of FA degradation decreased. The purpose of this study was to determine the kinetics of biodegradation of FA by measuring biomass growth rates and concentration of FA as a function of time. Substrate inhibition was calculated from experimental growth parameters using the Haldane equation. Data for P. putida were determined as µ _max ?=?0.23 h^?1, K _s ?=?23.93 mg/l and K _i ?=?217.1 mg/l and for P. aeruginosa were determined as µ _max ?=?0.13 h^?1, K _s ?=?21.3 mg/l and K _i ?=?284.9 mg/l. The experimental data were fitted in Haldane, Aiba and Edwards inhibition models.     

Biocidal effect of (<Emphasis Type="Italic">E</Emphasis>)-anethole on the cyanobacterium <Emphasis Type="Italic">Aphanizomenon gracile</Emphasis> Lemmermann

Biocidal natural substances of botanical origin offer a promising ecofriendly option for controlling toxic cyanobacteria. Herein, we study 11 essential oils and some of their major components for their activity on Aphanizomenon gracile. On the basis of our results we support that Origanum vulgare and O. dictamnus, Ocimum basilicum, Eucalyptus meliodora, Melissa officinalis, and Pimpinella anisum exhibited the strongest activities, and the IC_50/1d values of the extracts were calculated to be between 168.43 and 241.97 μg mL^?1. When the major components of the biocidal essential oils were tested individually, (E)-anethole was found active, exhibiting an IC_50/1d value of 71.35 μg mL^?1. On the other hand, the half-life (t _1/2 ) of (E)-anethole was calculated at 1 h. A preliminary attempt of (E)-anethole microencapsulation was conducted, in order to slowly release this biocidal agent, increasing the residual life under open air conditions and thus the biological activity. Results were promising since the microencapsulated product exhibited better activity than did the non-formulated (E)-anethole. This is a first report on the biocidal activity of EOs and (E)-anethole on A. gracile and a preliminary indication of the microencapsulated (E)-anethole potential use as a natural biocidal in fresh waters.

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Graphical abstract Filaments of Aphanizomenon gracile capable of bloom forming (strain SAG 31.79, in a batch culture) and (E)-anethole

     

Simultaneous production of intracellular triacylglycerols and extracellular polyol esters of fatty acids by <Emphasis Type="Italic">Rhodotorula babjevae</Emphasis> and <Emphasis Type="Italic">Rhodotorula</Emphasis> aff. <Emphasis Type="Italic">paludigena</Emphasis>

Microbial oils have been analyzed as alternatives to petroleum. However, just a handful of microbes have been successfully adapted to produce chemicals that can compete with their petroleum counterparts. One of the reasons behind the low success rate is the overall economic inefficiency of valorizing a single product. This study presents a lab-scale analysis of two yeast species that simultaneously produce multiple high-value bioproducts: intracellular triacylglycerols (TG) and extracellular polyol esters of fatty acids (PEFA), two lipid classes with immediate applications in the biofuels and surfactant industries. At harvest, the yeast strain Rhodotorula aff. paludigena UCDFST 81-84 secreted 20.9 ± 0.2 g L^?1 PEFA and produced 8.8 ± 1.0 g L^?1 TG, while the yeast strain Rhodotorula babjevae UCDFST 04-877 secreted 11.2 ± 1.6 g L^?1 PEFA and 18.5 ± 1.7 g L^?1 TG. The overall glucose conversion was 0.24 and 0.22 g_{(total lipid)} g _(glucose) ^?1 , respectively. The results present a stable and scalable microbial growth platform yielding multiple co-products.     

Enhancement of Nisin Production by <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis>

Lactococcus lactis subsp lactis BSA (L. lactis BSA) was isolated from a commercial fermented product (BSA Food Ingredients, Montreal, Canada) containing mixed bacteria that are used as starter for food fermentation. In order to increase the bacteriocin production by L. lactis BSA, different fermentation conditions were conducted. They included different volumetric combinations of two culture media (the Man, Rogosa and Sharpe (MRS) broth and skim milk), agitation level (0 and 100 rpm) and concentration of commercial nisin (0, 0.15, and 0.30 µg/ml) added into culture media as stimulant agent for nisin production. During fermentation, samples were collected and used for antibacterial evaluation against Lactobacillus sakei using agar diffusion assay. Results showed that medium containing 50 % MRS broth and 50 % skim milk gave better antibacterial activity as compared to other medium formulations. Agitation (100 rpm) did not improve nisin production by L. lactis BSA. Adding 0.15 µg/ml of nisin into the medium-containing 50 % MRS broth and 50 % skim milk caused the highest nisin activity of 18,820 AU/ml as compared to other medium formulations. This activity was 4 and ~3 times higher than medium containing 100 % MRS broth without added nisin (~4700 AU/ml) and 100 % MRS broth with 0.15 µg/ml of added nisin (~6650 AU/ml), respectively.     

Terpenoid bioactive compound from <Emphasis Type="Italic">Streptomyces rochei</Emphasis> (M32): taxonomy,fermentation and biological activities

The present study emphasized the production of biologically active terpenoid compound from Streptomyces rochei M32, which was isolated from Western Ghats ecosystem, South India. The presence of resistant genes like mecA, vanA of Staphylococcus aureus and bla _SHV, bla _TEM of Pseudomonas aeruginosa was confirmed by molecular studies. The isolated compound from Streptomyces rochei M32 inhibited wide range of standard and clinical drug resistant pathogens and enteric pathogens. The rice bran supplemented basal medium influenced the active compound production on 8th day of fermentation and yielded 1875 mg of crude extract from 10 g of rice bran substrate. Purification and characterization of crude ethyl acetate extract was achieved by preparative thin layer chromatography. The active fraction was identified as terpenoid class compound by chemical screening. Based on the results of spectral studies (NMR, LC–MS, FTIR, etc.), the active compound was tentatively identified as 1, 19-bis (3-hydroxyazetidin-1-yl) nonadeca-5, 14-diene-1, 8, 12, 19-tetraone with molecular weight 462.41 g/mol. Minimum inhibitory concentration value ranges between 7.6 and 31.2 µg/mL against test organisms was observed. The cytotoxicity results on cervical cancer (HeLa) cell line showed IC₅₀ value of 2.034 µg/mL. The corresponding compound is not previously reported from any microbial resources.     

Production of <Emphasis Type="Italic">trans</Emphasis>-10,<Emphasis Type="Italic">cis</Emphasis>-12-conjugated linoleic acid using permeabilized whole-cell biocatalyst of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Objective

To improve the production of trans-10,cis-12-conjugated linoleic acid (t10,c12-CLA) from linoleic acid in recombinant Yarrowia lipolytica.

Results

Cells of the yeast were permeabilized by freeze/thawing. The optimal conditions for t10,c12-CLA production by the permeabilized cells were at 28 °C, pH 7, 200 rpm with 1.5 g sodium acetate l^?1, 100 g wet cells l^?1, and 25 g LA l^?1. Under these conditions, the permeabilized cells produced 15.6 g t10,c12-CLA l^?1 after 40 h, with a conversion yield of 62 %. The permeabilized cells could be used repeatedly for three cycles, with the t10,c12-CLA extracellular production remaining above 10 g l^?1.

Conclusion

Synthesis of t10,c12-CLA was achieved using a novel method, and the production reported in this work is the highest value reported to date.

     

Single point mutations enhance activity of <Emphasis Type="Italic">cis</Emphasis>-epoxysuccinate hydrolase

Objectives

To enhance activity of cis-epoxysuccinate hydrolase from Klebsiella sp. BK-58 for converting cis-epoxysuccinate to tartrate.

Results

By semi-saturation mutagenesis, all the mutants of the six important conserved residues almost completely lost activity. Then random mutation by error-prone PCR and high throughput screening were further performed to screen higher activity enzyme. We obtained a positive mutant F10D after screening 6000 mutations. Saturation mutagenesis on residues Phe10 showed that most of mutants exhibited higher activity than the wild-type, and the highest mutant was F10Q with activity of 812 U mg^?1 (k _cat /K _m , 9.8 ± 0.1 mM^?1 s^?1), which was 230 % higher than that of wild-type enzyme 355 U mg^?1 (k _cat /K _m , 5.3 ± 0.1 mM^?1 s^?1). However, the thermostability of the mutant F10Q slightly decreased.

Conclusions

The catalytic activity of a cis-epoxysuccinate hydrolase was efficient improved by a single mutation F10Q and Phe10 might play an important role in the catalysis.

     

The nitrite reductase activity of horse heart carboxymethylated-cytochrome <Emphasis Type="Italic">c</Emphasis> is modulated by cardiolipin

Horse heart carboxymethylated cytc (CM-cytc) displays myoglobin-like properties. Here, the effect of cardiolipin (CL) liposomes on the nitrite reductase activity of ferrous CM-cytc [CM-cytc-Fe(II)], in the presence of sodium dithionite, is reported between pH 5.5 and 7.6, at 20.0 °C. Cytc-Fe(II) displays a very low value of the apparent second-order rate constant for the NO₂ ^?-mediated conversion of cytc-Fe(II) to cytc-Fe(II)-NO [k _on = (7.3 ± 0.7) × 10^?2 M^?1 s^?1; at pH 7.4], whereas the value of k _on for NO₂ ^? reduction by CM-cytc-Fe(II) is 1.1 ± 0.2 M^?1 s^?1 (at pH 7.4). CL facilitates the NO₂ ^?-mediated nitrosylation of CM-cytc-Fe(II) in a dose-dependent manner, the value of k _on for the NO₂ ^?-mediated conversion of CL–CM-cytc-Fe(II) to CL–CM-cytc-Fe(II)-NO (5.6 ± 0.6 M^?1 s^?1; at pH 7.4) being slightly higher than that for the NO₂ ^?-mediated conversion of CL–cytc-Fe(II) to CL–cytc-Fe(II)-NO (2.6 ± 0.3 M^?1 s^?1; at pH 7.4). The apparent affinity of CL for CM-cytc-Fe(II) is essentially pH independent, the average value of B being (1.3 ± 0.3) × 10^?6 M. In the absence and presence of CL liposomes, the nitrite reductase activity of CM-cytc-Fe(II) increases linearly on lowering pH and the values of the slope of the linear fittings of Log k _on versus pH are ?1.05 ± 0.07 and ?1.03 ± 0.03, respectively, reflecting the involvement of one proton for the formation of the transient ferric form, NO, and OH^?. These results indicate that Met80 carboxymethylation and CL binding cooperate in the stabilization of the highly reactive heme-Fe atom of CL–CM-cytc.     

Analysis of the effect of plant growth regulators and organic elicitors on antibacterial activity of <Emphasis Type="Italic">Eucomis autumnalis</Emphasis> and <Emphasis Type="Italic">Drimia robusta</Emphasis> ex vitro-grown biomass

The effects of plant growth regulators (PGRs) and organic elicitors (OEs) on in vitro propagation of Eucomis autumnalis was established. Three-year-old ex vitro grown plants from organogenesis of E. autumnalis and somatic embryogenesis (previously reported protocol) of Drimia robusta were investigated for antibacterial activity. In vitro propagation from leaf explants of E. autumnalis was established using different PGRs and OE treatments for mass propagation, biomass production and bioactivity analysis to supplement the use of wild plant material. Prolific shoots (16.0?±?0.94 shoots per explant) were obtained with MS (Murashige and Skoog in Physiol Plant 15:473–497, 1962) medium containing 100 mg l^?1 haemoglobin (HB), 10 µM benzyladenine (BA) and 2 µM naphthaleneacetic acid (NAA). The shoots were rooted effectively with a combination of 2.5 µM indole-3-acetic acid and 5.0 µM indole-3-butyric acid. The plantlets were successfully acclimatized in a vermiculite-soil mixture (1:1 v/v) in the greenhouse. Three-year-old ex vitro-grown E. autumnalis and D. robusta plants derived via organogenesis and somatic embryogenesis respectively exhibited antibacterial activity and varied with PGR and OE treatments, plant parts and bacteria. The leaves of E. autumnalis ex vitro-derived from a combination of HB, BA and NAA followed by the individual treatments of BA and HB gave the best antibacterial activities (<?1 mg ml^?1: minimum inhibitory concentration from 0.098 to 0.78 mg ml^?1) against all tested pathogenic bacteria (Bacillus subtilis, Enterococcus faecalis, Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa). The bulbs of D. robusta ex vitro-derived from solid culture with 10 µM picloram, 1 µM thidiazuron and 20 µM glutamine exhibited good antibacterial activity against E. faecalis, M. luteus and S. aureus when compared with other treatments and mother plants. The ex vitro-grown E. autumnalis and D. robusta biomass produced with PGRs along with OE treatments confirmed a good potent bioresource and can be used as antibacterial agents. The in vitro plant regeneration of E. autumnalis and D. robusta protocols and ex vitro plants could be used for conservation strategies, bioactivity and traditional medicinal use.