Cloning and characterization of dichloromethane dehalogenase from Methylobacterium rhodesianum for dichloromethane degradation

The gene encoding dichloromethane dehalogenase from Methylobacterium rhodesianum was cloned. Bioinformatic analysis showed that dichloromethane dehalogenase gene sequence from M. rhodesianum is almost identical to the one from Methylobacterium extorquens, with only one base difference. Dichloromethane dehalogenase was subsequently expressed in Escherichia coli BL21 (DE3) and purified. It was found that enzyme activity in recombinant cells was 3 times higher than that in the wild-type M. rhodesianum. Further investigation showed that recombinant dichloromethane dehalogenase was most active at 40°C at pH 7–8, and its K_M was 10.96 mM when treated with dichloromethane as substrate. The fitted curve of dichloromethane degradation gave a V_max of 0.43 mM/h of in 0.01 M phosphate buffer. Degradation efficiency of dichloromethane reached 86.11% within 20 h. In addition, it was found that degradation efficiency of dichloromethane was highly associated with glutathione concentration, supporting the reports that glutathione functions as coenzyme of dichloromethane dehalogenase for dichloromethane degradation.     

Thermophilic and halophilic β-agarase from a halophilic archaeon Halococcus sp. 197A

An agar-degrading archaeon Halococcus sp. 197A was isolated from a solar salt sample. The agarase was purified by hydrophobic column chromatography using a column of TOYOPEARL Phenyl-650 M. The molecular mass of the purified enzyme, designated as Aga-HC, was ~55 kDa on both SDS-PAGE and gel-filtration chromatography. Aga-HC released degradation products in the order of neoagarohexose, neoagarotetraose and small quantity of neoagarobiose, indicating that Aga-HC was a β-type agarase. Aga-HC showed a salt requirement for both stability and activity, being active from 0.3 M NaCl, with maximal activity at 3.5 M NaCl. KCl supported similar activities as NaCl up to 3.5 M, and LiCl up to 2.5 M. These monovalent salts could not be substituted by 3.5 M divalent cations, CaCl₂ or MgCl₂. The optimal pH was 6.0. Aga-HC was thermophilic, with optimum temperature of 70 °C. Aga-HC retained approximately 90 % of the initial activity after incubation for 1 hour at 65–80 °C, and retained 50 % activity after 1 hour at 95 °C. In the presence of additional 10 mM CaCl₂, approximately 17 % remaining activity was detected after 30 min at 100 °C. This is the first report on agarase purified from Archaea.     

蛇足石杉中的新内生真菌及其代谢产物的分离与鉴定

从蛇足石杉内生菌的次级代谢产物中寻找活性成分,为进一步开发利用蛇足石杉药用植物资源提供了新途径,但至今其内生菌代谢产物的系统性研究较为少见。种类丰富的内生真菌普遍存在于各种植物中,但蕨类植物中内生真菌的研究较少。为了寻找蛇足石杉内生菌中的细胞毒活性成分,该研究从蛇足石杉根部分离得到一株球毛壳属(Chaetomium sp.)真菌M336,对其化学成分进行了研究。对蛇足石杉内生真菌M336采用PDA固体培养基扩大发酵,发酵物经提取及乙酸乙酯萃取后,通过正相硅胶柱色谱法、Sephadex LH-20凝胶柱色谱法、薄层制备、高效液相色谱等色谱手段对其发酵物中的化学成分进行分离纯化,利用理化性质、质谱、核磁等波谱分析技术,并结合相关文献数据鉴定化合物的结构。结果表明：从内生真菌M336发酵提取物的乙酸乙酯萃取部分分离并鉴定出8个化合物,分别为chaetoviridines F、chaetoviridines E、5′-epichaetoviridin A、5′-epichaetoviridin A、xanthoquinodins Al、xanthoquinodins A2、xanthoquinodins B1和毛壳菌素。从M336中分离得到8个化合物,化合物3有一定的抑菌作用,其余化合物有一定的细胞毒活性。该研究结果丰富了蛇足石杉内生真菌球毛壳属中的天然细胞毒活性的化合物。     

Purification,Characterization, and Crystallization of Crocodylus siamensis Hemoglobin

Crocodylus siamensis hemoglobin was purified by a size exclusion chromatography, Sephacryl S-100 with buffer containing dithiothreitol. The purified Hb was dissociated to be two forms (α chain and β chain) which observed by SDS-PAGE, indicated that the C. siamensis Hb was an unpolymerized form. The unpolymerized Hb (composed of two α chains and two β chains) showed high oxygen affinity at 3.13 mmHg (P₅₀) and 1.96 (n value), and a small Bohr effect (δH⁺ = ?0.29) at a pH of 6.9–8.4. Adenosine triphosphate did not affect the oxygenation properties, whereas bicarbonate ions strongly depressed oxygen affinity. Crude C. siamensis Hb solutions were showed high O₂ affinity at P₅₀ of 2.5 mmHg which may assure efficient utilization of the lung O₂ reserve during breath holding and diving. The purified Hbs were changed to cyanmethemoglobin forms prior crystallization. Rod- and plate-shaped crystals were obtained by the sitting-drop vapor-diffusion method at 5 °C using equal volumes of protein solution (37 mg/ml) and reservoir [10–13 % (w/v) PEG 4000, with 0.1 M Tris buffer in present of 0.2 M MgCl₂·6H₂O] solution at a pH of 7.0–8.5.     

Purification and characterization of an extracellular haloalkaline serine protease from the moderately halophilic bacterium, <Emphasis Type="Italic">Bacillus iranensis (</Emphasis>X5B)

This study reports the purification and characterization of an extracellular haloalkaline serine protease from the moderately halophilic bacterium, Bacillus iranensis, strain X5B. The enzyme was purified to homogeneity by acetone precipitation, ultrafiltration and carboxymethyl (CM) cation exchange chromatography, respectively. The purified protease was a monomeric enzyme with a relative molecular mass of 48–50 kDa and it was inhibited by PMSF indicating that it is a serine-protease. The optimum pH, temperature and NaCl concentration were 9.5, 35 °C and 0.98 M, respectively. The enzyme showed a significant tolerance to salt and alkaline pH. It retained approximately 50 % of activity at 2.5 M NaCl and about 70 % of activity at highly alkaline pH of 11.0; therefore, it was a moderately halophilic and also can be activated by metals, especially by Ca²⁺. The specific activity of the purified protease was measured to be 425.23 μmol of tyrosine/min per mg of protein using casein as a substrate. The apparent K _m and V _max values were 0.126 mM and 0.523 mM/min, respectively and the accurate value of k _cat was obtained as 3.284 × 10^?2 s^?1. These special and important characteristics make this serine protease as valuable tool for industrial applications.     

Synthesis and in vitro cytotoxic evaluation of new 1H-benzo[d]imidazole derivatives of dehydroabietic acid

A series of new 1H-benzo[d]imidazole derivatives of dehydroabietic acid were designed and synthesized as potent antitumor agents. Structures of the target molecules were characterized using MS, IR, ¹H NMR, ¹³C NMR and elemental analyses. In the in vitro cytotoxic assay, most compounds showed significant cytotoxic activities against two hepatocarcinoma cells (SMMC-7721 and HepG2) and reduced cytotoxicity against noncancerous human hepatocyte (LO2). Among them, compound 7b exhibited the best cytotoxicity against SMMC-7721 cells (IC₅₀: 0.36 ± 0.13 μM), while 7e was most potent to HepG2 cells (IC₅₀: 0.12 ± 0.03 μM). The cell cycle analysis indicated that compound 7b caused cell cycle arrest of SMMC-7721 cells at G2/M phase. Further, compound 7b also induced the apoptosis of SMMC-7721 cells in Annexin V-APC/7-AAD binding assay.     

Insight into the structural requirements of aminopyrimidine derivatives for good potency against both purified enzyme and whole cells of M. tuberculosis: combination of HQSAR,CoMSIA, and MD simulation studies

The Mycobacterium tuberculosis protein kinase B (PknB) is critical for growth and survival of M. tuberculosis within the host. The series of aminopyrimidine derivatives show impressive activity against PknB (IC₅₀ < .5 μM). However, most of them show weak or no cellular activity against M. tuberculosis (MIC > 63 μM). Consequently, the key structural features related to activity against of both PknB and M. tuberculosis need to be investigated. Here, two- and three-dimensional quantitative structure–activity relationship (2D and 3D QSAR) analyses combined with molecular dynamics (MD) simulations were employed with the aim to evaluate these key structural features of aminopyrimidine derivatives. Hologram quantitative structure–activity relationship (HQSAR) and CoMSIA models constructed from IC₅₀ and MIC values of aminopyrimidine compounds could establish the structural requirements for better activity against of both PknB and M. tuberculosis. The NH linker and the R₁ substituent of the template compound are not only crucial for the biological activity against PknB but also for the biological activity against M. tuberculosis. Moreover, the results obtained from MD simulations show that these moieties are the key fragments for binding of aminopyrimidine compounds in PknB. The combination of QSAR analysis and MD simulations helps us to provide a structural concept that could guide future design of PknB inhibitors with improved potency against both the purified enzyme and whole M. tuberculosis cells.     

Partial Purification and Characterization of A Novel Histidine Decarboxylase from Enterobacter aerogenes DL-1

Histidine decarboxylase (HDC) from Enterobacter aerogenes DL-1 was purified in a three-step procedure involving ammonium sulfate precipitation, Sephadex G-100, and DEAE-Sepharose column chromatography. The partially purified enzyme showed a single protein band of 52.4 kD on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH for HDC activity was 6.5, and the enzyme was stable between pH 4 and 8. Enterobacter aerogenes HDC had optimal activity at 40°C and retained most of its activity between 4 and 50°C. HDC activity was reduced in the presence of numerous tested compounds. Particularly with SDS, it significantly (p < 0.01) inhibited enzyme activity. Conversely, Ca²⁺ and Mn²⁺ showed prominent activation effects (p < 0.01) with activity increasing to 117.20% and 123.42%, respectively. The Lineweaver–Burk plot showed that K _m and V _max values of the enzyme for L-histidine were 0.21 mM and 71.39 µmol/min, respectively. In comparison with most HDCs from other microorganisms and animals, HDC from E. aerogenes DL-1 displayed higher affinity and greater reaction velocity toward L-histidine.     

Design,synthesis, in silico and in vitro evaluation of thiophene derivatives: A potent tyrosine phosphatase 1B inhibitor and anticancer activity

A series of novel methyl 4-(4-amidoaryl)-3-methoxythiophene-2-carboxylate derivatives were designed against the active site of protein tyrosine phosphatise 1B (PTP1B) enzyme using MOE.2008.10. These molecules are also subjected for in silico toxicity prediction studies and considering their corresponding drug scores, it implied that, the molecules are promising as anticancer agents. The designed compounds were synthesized by using suitable methods and characterized. They were subjected to inhibitory activity against PTP1B and in vitro anticancer activity by MTT assay. Most of the tested compounds showed potent inhibitory activity against PTP1B, among the compounds tested, compound 5b exhibited the highest activity (IC₅₀ = 5.25 µM) and remarkable cytotoxic activity at 0.09 µM of IC₅₀ against the MCF-7 cell line. In addition to this, compound 5c also showed potential anticancer activity at 2.22 µM of IC₅₀ against MCF-7 and 0.72 µM against HepG2 cell lines as well as PTP1B inhibitory activity at IC₅₀ of 6.37 µM.     

Effect of the structure of dietary epoxylignan on its cytotoxic activity: relationship between the structure and the activity of 7,7′-epoxylignan and the introduction of apoptosis by caspase 3/7

We compared the cytotoxic activities of dietary epoxylignans and their stereoisomers and found (?)-verrucosin, which is (7S,7′R,8R,8′R)-7,7′-epoxylignan, to be the most cytotoxic epoxylignan against HeLa cells (IC₅₀ = 6.6 μM). On the other hand, the activity was about a factor of 10 less against HL-60. In this research on the relationship between the structure and cytotoxic activity of (?)-verrucosin 13, the 7-(4-methoxyphenyl)-7′-(3,4-dimethoxyphenyl) derivative 60, for which the activity (IC₅₀ = 2.4 μM) is three times greater than (?)-verrucosin 13, was discovered. The induction of apoptosis by caspase 3/7 was observed upon treatment with the (?)-verrucosin derivative.     

Stachybotrychromenes A–C: novel cytotoxic meroterpenoids from <Emphasis Type="Italic">Stachybotrys</Emphasis> sp.

In the course of gaining new insights into the secondary metabolite profile of various Stachybotrys strains, in particular concerning triprenyl phenol-like compounds, so far, unknown metabolites with analogous structural features were discovered. Three novel meroterpenoids containing a chromene ring moiety, namely stachybotrychromenes A–C, were isolated from solid culture of the filamentous fungus Stachybotrys chartarum DSMZ 12880 (chemotype S). Their structures were elucidated by means of comprehensive spectroscopic analysis (1D and 2D NMR, ESI-HRMS, and CD) as well as by comparison with spectroscopic data of structural analogues described in literature. Stachybotrychromenes A and B exhibited moderate cytotoxic effects on HepG2 cells after 24 h with corresponding IC₅₀ values of 73.7 and 28.2 μM, respectively. Stachybotrychromene C showed no significant cytotoxic activity up to 100 μM. Moreover, it is noteworthy that stachybotrychromenes A–C are produced not only by S. chartarum chemotype S but also S. chartarum chemotype A and Stachybotrys chlorohalonata.     

Effects of alginate oligomer on the expression of cell cycle- and stress-related genes in Chlamydomonas reinhardtii

Enzymatically prepared alginate oligomer (AO) promoted the growth of Chlamydomonas reinhardtii in a concentration-dependent manner. AO at 2.5 mg/mL induced increase in expression levels of cyclin A, cyclin B, and cyclin D in C. reinhardtii. CuSO₄ at 100 μM suppressed the growth of C. reinhardtiin, and AO at 2.5 mg/mL significantly alleviated the toxicity of CuSO₄. Increased intracellular reactive oxygen species level in C. reinhardtii induced by CuSO₄ was reduced by AO. After cultivation with CuSO₄ at 100 μM, expression levels of ascorbate peroxidase and superoxide dismutase in C. reinhardtii were increased, and AO reduced the increased levels of these enzymes. These results suggest that AO exhibits beneficial effects on C. reinhardtii through influencing the expression of various genes not only at normal growth condition but also under CuSO₄ stress.     

Purification and characterization of salicylate 5-hydroxylase, a three-component monooxygenase from Ralstonia sp. strain U2

Salicylate is an important intermediate in the bacterial degradation of polycyclic aromatic hydrocarbons and salicylate hydroxylases play essential roles in linking the peripheral and ring-cleavage catabolic pathways. Unlike the well-characterized salicylate 1-hydroxylases, the rarely occurred salicylate 5-hydroxylase (S5H) has not been characterized in detail. In this study, the three-component Fe-S protein complex (NagAaGHAb) of S5H from Ralstonia sp. strain U2 was purified, and its biochemical and catalytic properties were characterized. The oxygenase component NagGH exhibited an α₃β₃ heterohexameric structure and contained one Rieske-type [2Fe-2S] cluster and one mononuclear iron per α subunit. NagAa is the ferredoxin-NADP⁺ reductase component containing flavin and plant type [2Fe–2S] cluster. The ferredoxin component NagAb was characterized as a [2Fe-2S] dimer which remains remarkably stable in denaturing gel electrophoresis after being heated at 100 °C for 1 h. Purified NagAa and NagAb, NagGH catalyzed the hydroxylation of salicylate to gentisate with a specific activity of 107.12?±?14.38 U/g and showed an apparent K _m for salicylate of 102.79?±?27.20 μM and a similar K _m value for both NADH and NADPH (59.76?±?7.81 μM versus 56.41?±?12.76 μM). The hydroxylase exhibited different affinities for two hydroxysalicylates (2,4-dihydroxybenzoate K _m of 93.54?±?18.50 μM versus 2,6-dihydroxybenzoate K _m of 939.80?±?199.46 μM). Interestingly, this S5H also showed catalytic activity to the pollutant 2-nitrophenol and exhibited steady-state kinetic data of the same order of magnitude as those for salicylate. This study will allow further comparative studies of structure–function relationships of the ring hydroxylating mono- and di-oxygenase systems.     

In vitro salt and thermal tolerance of fungal endophytes of Nicotiana spp. growing in arid regions of north-western Australia

Abstract

Endophytic fungal strains isolated from indigenous Nicotiana plants naturally growing in dry and hot regions of north-western Australia were characterised based on their tolerance to salinity and temperature. Sixty-eight fungal isolates were tested on eight levels (0.5 M, 1.0 M, 1.5 M, 2.0 M, 2.5 M, 3.0 M, 3.5 M and 4.0 M) of five different of salts solutions NaCl, KCl, MgCl₂, CaCl₂ and MgSO₄ and at various temperatures (25–50?°C). The salt adaptation test indicated that the fungal strains namely Aspergillus niger (E-202), A. ochraceous-A (E-134), Aurantiporus sp. (E-135), Cladosporium halotolerance (E-128), Pleurostomophora richardsiae (E-13) and Trichoderma sp. (E-185.1) were tolerant to higher concentrations of various salts. The most growth-limiting salt turned out to be MgCl₂ followed by the chaotrope CaCl₂. Responses to temperature tolerance revealed that most fungi tested could grow at 30?°C. About 50% all the fungi did not show any growth when the temperature was raised above 30?°C. When the temperature was raised up to 50?°C all the fungi failed to grow but the fungus Rasamsonia piperina (E-172). Endophyte strains identified could be promising candidates for future research in investigating the fungus–plant interactions and their roles in plant adaptation to inhospitable environments.     

Purification and partial characterization of the extradiol dioxygenase, 2′-carboxy-2,3-dihydroxybiphenyl 1,2-dioxygenase,in the fluorene degradation pathway from Rhodococcus sp. strain DFA3

Type II extradiol dioxygenase, 2′-carboxy-2,3-dihydroxybiphenyl 1,2-dioxygenase (FlnD1D2) involved in the fluorene degradation pathway of Rhodococcus sp. DFA3 was purified to homogeneity from a heterologously expressing Escherichia coli. Gel filtration chromatography and SDS-PAGE suggested that FlnD1D2 is an α₄β₄ heterooctamer and that the molecular masses of these subunits are 30 and 9.9 kDa, respectively. The optimum pH and temperature for enzyme activity were 8.0 and 30 °C, respectively. Assessment of metal ion effects suggested that exogenously supplied Fe²⁺ increases enzyme activity 3.2-fold. FlnD1D2 catalyzed meta-cleavage of 2′-carboxy-2,3-dihydroxybiphenyl homologous compounds, but not single-ring catecholic compounds. The K_m and k_cat/K_m values of FlnD1D2 for 2,3-dihidroxybiphenyl were 97.2 μM and 1.5 × 10^?2 μM^?1sec^?1, and for 2,2′,3-trihydroxybiphenyl, they were 168.0 μM and 0.5 × 10^?2 μM^?1sec^?1, respectively. A phylogenetic tree of the large and small subunits of type II extradiol dioxygenases suggested that FlnD1D2 constitutes a novel subgroup among heterooligomeric type II extradiol dioxygenases.     

Formulation and optimisation of various nuclear polyhedrosis virus isolates and assessment of their insecticidal activity against Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) larvae

Helicoverpa armigera (Hubner) is a pest that poses serious threat to the tomato crop in India. Larvae of this species are susceptible to the nucleopolyhedrovirus (NPV), which has attracted interest as a potential biocontrol agent. Rearing of larvae in natural diet resulted in less number of pupae, while in artificial diet more pupae and healthy adults were obtained. The NPV was tested for its insecticidal action against H. armigera in natural and artificial diets. The bioassay results of inoculated H. armigera nucleopolyhedrosis virus (HaNPV) with different concentrations indicate that the 4.0 g/l dosage caused maximum mortality (70.3% and 60.54%), and minimum mortality 46.83% and 44.08% was recorded in the 0.5 g/l dosage under laboratory and pot culture conditions, respectively. Polyhedral inclusion bodies (PIBs) were estimated using a standard haemocytometer. According to the estimate, purified PIBs were found at a concentration of 1 × 10⁹/ml. The different formulation of NPV, i.e. wettable fine powder with a hue of white, had a particle size of 100 μm with a concentration of 1 × 10⁸/ml. The encapsulated product was found to be in the form of beads and was red in colour with a concentration of 1 × 10⁸ PIBs/ml. The effectiveness of NPV against H. armigera larvae was noted at maximum LC₅₀ values at a concentration of 1 × 10⁹/ml. Qualitative analysis estimates showed that protein polyhedrin and enzyme chitinase had a molecular weight of 33 and 45 kDa, respectively. Viral DNA was isolated from NPV-infected larvae and was then separated using SDS-PAGE. The estimated polyhedrin with a molecular weight of 33 kDa, which is responsible for the increased mortality percent of larvae, was confirmed by the bioassay. Hence it was concluded that NPV is ecofriendly in the management of pests on tomato and other crops.     

Characterization and preliminary mutation analysis of a thermostable alanine racemase from Thermoanaerobacter tengcongensis MB4

A thermostable alanine racemase from Thermoanaerobacter tengcongensis MB4 was successfully expressed in Escherichia coli and characterized. The full-length gene MBalr2 (1164 bp) encodes 388 amino acid residues including 6 out of 8 highly conserved amino acid residues at the entryway to the active site of alanine racemase. Recombinant MBAlr2 and three mutants (S171A, H359Y and double mutation S171A/H359Y) of MBAlr2 were purified by His₆-tag affinity column and gel filtration chromatography. The purified protein MBAlr2 was a dimeric PLP-dependent enzyme with broad substrate specificity. The optimal racemization temperature and pH were 70–75 °C and 11.0, respectively. The kinetic parameters K _m and V _max of MBAlr2 at 70 °C, determined by HPLC, were 20.16 mM and 1414 μmol min^?1 for l-alanine, and 9.95 mM and 702.6 μmol min^?1 for d-alanine, respectively. Enzymatic assays showed that the activity of both mutants (S171A and H359Y) was lost, but the activity of mutant S171A/H359Y was recovered to 69.8 % of wild type, which suggested that residues Ser171 and His359 might be the important residues for catalytic mechanisms of MBAlr2.     

Plant defense approach of Bacillus subtilis (BERA 71) against Macrophomina phaseolina (Tassi) Goid in mung bean

This study was aimed to elucidate the mitigation mechanism of an endophytic bacterium, Bacillus subtilis (BERA 71) against Macrophomina phaseolina (Tassi) Goid disease in mung bean. M. phaseolina reduced the plant growth by inducing disease, hydrogen peroxide (H₂O₂) and lipid peroxidation. The inoculation of B. subtilis to diseased plants increased chlorophyll, ascorbic acids, and superoxide dismutase, catalase, peroxidase, ascorbate peroxidase and glutathione reductase activities, and while inhibited H₂O₂ and lipid peroxidation for enhancing plant growth. In addition, B. subtilis association in plants mitigated the M. phaseolina infection due to increase of indole acetic acids and indole butyric acid, and also a decrease of abscisic acid. However, the nutrients (N, K, Ca, Mg, Zn, Cu, Mn and Fe) were increased, except Na in M. phaseolina diseased plants treated with B. subtilis. The result of this study suggests that B. subtilis interaction with plants can modulate the metabolism of pigments, hormones, antioxidants and nutrients against M. phaseolina to induce disease resistance in mung bean.