Production and Characterization of Cellulase-Free Xylanase from <Emphasis Type="Italic">Trichoderma inhamatum</Emphasis>

The production of extracellular cellulase-free xylanase from Trichoderma inhamatum was evaluated in liquid Vogel medium with different carbon sources as natural substrates and agricultural or agro-industrial wastes. Optimal production of 244.02 U/mL was obtained with xylan as carbon source, pH 6.0 at 25 degrees C, 120 rpm, and 60-h time culture. Optimal conditions for enzyme activity were 50 degrees C and pH 5.5. Thermal stability of T. inhamatum xylanolytic complex expressed as T1/2 was 2.2 h at 40 degrees C and 2 min at 50 degrees C. The pH stability was high from 4.0 to 11.0. These results indicate possible employment of such enzymatic complex in some industrial processes which require activity in acid pH, wide-ranging pH stability, and cellulase activity absence.     

Purification and Biochemical Characterization of a Highly Thermostable Xylanase from <Emphasis Type="Italic">Actinomadura</Emphasis> sp. Strain Cpt20 Isolated from Poultry Compost

An extracellular thermostable xylanase from a newly isolated thermophilic Actinomadura sp. strain Cpt20 was purified and characterized. Based on matrix-assisted laser desorption–ionization time-of-flight mass spectrometry analysis, the purified enzyme is a monomer with a molecular mass of 20,110.13 Da. The 19 residue N-terminal sequence of the enzyme showed 84% homology with those of actinomycete endoxylanases. The optimum pH and temperature values for xylanase activity were pH 10 and 80 °C, respectively. This xylanase was stable within a pH range of 5–10 and up to a temperature of 90 °C. It showed high thermostability at 60 °C for 5 days and half-life times at 90 °C and 100 °C were 2 and 1 h, respectively. The xylanase was specific for xylans, showing higher specific activity on soluble oat-spelt xylan followed by beechwood xylan. This enzyme obeyed the Michaelis–Menten kinetics, with the K _m and k _cat values being 1.55 mg soluble oat-spelt xylan/ml and 388 min⁻¹, respectively. While the xylanase from Actinomadura sp. Cpt20 was activated by Mn²⁺, Ca²⁺, and Cu²⁺, it was, strongly inhibited by Hg²⁺, Zn²⁺, and Ba²⁺. These properties make this enzyme a potential candidate for future use in biotechnological applications particularly in the pulp and paper industry.     

Purification and Characterization of Thermostable Chitinase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> SK-1

Chitinase was purified from the culture medium of Bacillus licheniformis SK-1 by colloidal chitin affinity adsorption followed by diethylamino ethanol-cellulose column chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular size and pI of chitinase 72 (Chi72) were 72 kDa and 4.62 (Chi72) kDa, respectively. The purified chitinase revealed two activity optima at pH 6 and 8 when colloidal chitin was used as substrate. The enzyme exhibited activity in broad temperature range, from 40 to 70°C, with optimum at 55°C. It was stable for 2 h at temperatures below 60°C and stable over a broad pH range of 4.0–9.0 for 24 h. The apparent K _m and V _max of Chi72 for colloidal chitin were 0.23 mg ml⁻¹ and 7.03 U/mg, respectively. The chitinase activity was high on colloidal chitin, regenerated chitin, partially N-acetylated chitin, and chitosan. N-bromosuccinamide completely inhibited the enzyme activity. This enzyme should be a good candidate for applications in the recycling of chitin waste.     

<Emphasis Type="Italic">Z</Emphasis>-and <Emphasis Type="Italic">E</Emphasis>-conformers of <Emphasis Type="Italic">N</Emphasis>-chloroacetylcytisine

N-Chloroacetylcytisine was synthesized by acylation of (–)-cytisine. Stable Z- and E-conformers with respect to rotational isomerism around the N-12–CO bond were found in PMR spectra at room temperature. The point at which PMR resonances of the Z- and E-conformers coalesced upon heating was measured. The transition barrier between the conformers was estimated.     

Purification and Characterization of a Novel Thermostable Mycelial Lectin from <Emphasis Type="Italic">Aspergillus terricola</Emphasis>

Lectin has been isolated from mycelia of Aspergillus terricola by single step purification on porcine stomach mucin-Sepharose 4B affinity column. Lectin could be effectively purified with 75% recovery and 4.47-fold increase in specific activity. Lectin migrated as a single band on SDS-PAGE with an apparent molecular mass of 32.5 kDa. Sugar inhibition assay revealed that the lectin did not strongly interact with most carbohydrates and their derivatives tested while strong binding affinity to d-glucose, d-sucrose, N-acetyl-d-galactosamine, asialofetuin, porcine stomach mucin, and bovine submaxillary mucin was indicated. Neuraminidase and protease treatment to erythrocytes enhanced lectin titre. Lectin activity was stable within the pH range of 7.0–10.5. A. terricola lectin displayed remarkable thermostability and remained unaffected upon incubation at 70 °C for 2.5 h. Lectin did not require metal ions for its activity. Incubation with denaturants (urea, thiourea, and guanidine–HCl) substantially reduced lectin activity. Carbohydrate analysis revealed that it is a glycoprotein with 9.76% total sugars.     

Potential Application of Alkaline Pectinase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> SS in Pulp and Paper Industry

Pectinase production from Bacillus subtilis SS was optimized under solid-state fermentation (5,943 U/g of dry bacterial bran). The pectinase produced was stable in neutral to alkaline pH range at 70 degrees C; therefore, the suitability of this pectinase in pulp and paper industry was investigated. The enzyme pretreatment process was optimized, and a pectinase dose of 5 IU/g of oven-dried pulp (10% consistency) at pH 9.5 temperature 70 degrees C after 150 min of treatment gave the best pretreatment to the pulp. An increase of 4.3% in brightness along with an increase of 14.8 and 65.3% in whiteness and fluorescence, respectively, whereas a 15% decrease in the yellowness of the pretreated pulp were observed. There was a 5.85% reduction in kappa number and 6.1% reduction in permanganate number along with a reduction in the chemical oxygen demand value. Significant characteristics showed by pectinase open new possibilities of application of this cellulase-free enzyme in the pulp and paper industry by reducing the negative environmental impact of chemicals apart from improving the properties of paper.     

Isolation,Purification and Characterization of a Surfactants-, Laundry Detergents- and Organic Solvents-Resistant Alkaline Protease from <Emphasis Type="Italic">Bacillus</Emphasis> sp. HR-08

Bacillus sp. HR-08 screened from soil samples of Iran, is capable of producing proteolytic enzymes. 16S rDNA analysis showed that this strain is closely related to Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus mojavensis, and Bacillus atrophaeus. The zymogram analysis of the crude extract revealed the presence of five extracellular proteases. One of the proteases was purified in three steps procedure involving ammonium sulfate precipitation, DEAE-Sepharose ionic exchange and Sephacryl S-200 gel filtration chromatography. The molecular mass of the enzyme on SDS-PAGE was estimated to be 29 kDa. The protease exhibited maximum activity at pH 10.0 and 60 °C and was inhibited by PMSF but it was not affected by cysteine inhibitors, suggesting that the enzyme is a serine alkaline protease. Irreversible thermoinactivation of enzyme was examined at 50, 60, and 70 °C in the presence of 10 mM CaCl₂. Results showed that the protease activity retains more than 80% and 50% of its initial activity after incubation for 30 min at 60 and 70 °C, respectively. This enzyme had good stability in the presence of H₂O₂, nonionic surfactant, and local detergents and its activity was enhanced in the presence of 20% of dimethyl sulfoxide (DMSO), dimethyl formamide (DMF) and isopropanol. The enzyme retained more than 90% of its initial activity after pre-incubation 1 h at room temperature in the presence of 20% of these solvents. Also, activation can be seen for the enzyme at high concentration (50%, v/v) of DMF and DMSO.     

Molecular properties of the copolymers of <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-diallyl-<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-dimethylammonium chloride and maleic acid

The hydrodynamic and conformational properties of molecules of poly(N,N-diallyl-N,N-dimethylammonium chloride) and N,N-diallyl-N,N-dimethylammonium chloride-maleic acid copolymers of different compositions in solutions with various ionic-strength and pH values, as well as of the polyelectrolyte complex based on the copolymer with dodecyl sulfate anions in chloroform, are studied. For poly(N,N-diallyl-N,N-dimethylammonium chloride) molecules in a 1 M NaCl solution, the Kuhn segment length and the hydrodynamic diameter of the chain are estimated as A = 3.9 nm and d = 0.48 nm, respectively. In acidic solutions with pH 3.5, the copolymers demonstrate behavior typical for polyelectrolytes. In an alkaline solution with pH 13, when 1 M NaCl is added to the solution of the copolymer containing 29 mol % maleic acid units, there is an antipolyelectrolyte effect that manifests itself as an increase in the intrinsic viscosity of the copolymer and in the hydrodynamic radius of its molecules. It is found that an increase in the fraction of maleic acid units in the copolymer from 12 to 42 mol % brings about a reduction in the equilibrium rigidity of its macromolecules from 4.1 to 2.2 nm. The equilibrium rigidity of polyelectrolyte-complex molecules is higher than that of initial copolymer molecules owing to steric interactions arising between the aliphatic chains of dodecyl sulfate anions. In an electric field, the molecules of the complex are oriented owing to the induced dipole moment resulting from the displacement of dodecyl sulfate anions along the chain contour.     

Purification and Properties of a Psychrotrophic <Emphasis Type="Italic">Trichoderma</Emphasis> sp. Xylanase and its Gene Sequence

A psychrotrophic fungus identified as Trichoderma sp. SC9 produced 36.7 U/ml of xylanase when grown on a medium containing corncob xylan at 20 °C for 6 days. The xylanase was purified 37-fold with a recovery yield of 8.2%. The purified xylanase appeared as a single protein band on SDS-PAGE with a molecular mass of approximately 20.5 kDa. The enzyme had an optimal pH of 6.0, and was stable over pH 3.5–9.0. The optimal temperature of the xylanase was 42.5 °C and it was stable up to 35 °C at pH 6.0 for 30 min. The xylanase was thermolabile with a half-life of 23.9 min at 45 °C. The apparent K _m values of the xylanase for birchwood, beechwood, and oat-spelt xylans were found to be 3, 2.1, and 16 mg/ml respectively. The xylanase hydrolyzed beechwood xylan and birchwood xylan to yield mainly xylobiose as end products. The enzyme-hydrolysed xylotriose, xylotetraose, and xylopentose to produce xylobiose, but it hardly hydrolysed xylobiose. A xylanase gene (xynA) with an open reading frame of 669 nucleotide base pairs (bp), encoding 222 amino acids, from the strain was cloned and sequenced. The deduced amino acid sequence of XynA showed 85% homology with Xyn2 from a mesophilic strain of Trichoderma viride.     

Synthesis of 6-methyl-8<Emphasis Type="Italic">H</Emphasis>-dibenzo[<Emphasis Type="Italic">a</Emphasis>,<Emphasis Type="Italic">g</Emphasis>]quinolizin-8-imines <Emphasis Type="Italic">via Reissert</Emphasis> compounds

Alkylation of Reissert compounds derived from 3-methylisoquinolines with several 2-cyanobenzylbromides followed by hydrolytic cleavage provided the corresponding 1-benzyl-3-methylisoquinolines. Treatment of the latter with methylmagnesiumiodide caused cyclization to the title compounds rather than formation of 2-acetylbenzylisoquinolines.     

Analysis of <Emphasis Type="Italic">Escherichia coli</Emphasis> nicotinate mononucleotide adenylyltransferase mutants <Emphasis Type="Italic">in vivo</Emphasis> and <Emphasis Type="Italic">in vitro</Emphasis>

Background  

Adenylation of nicotinate mononucleotide to nicotinate adenine dinucleotide is the penultimate step in NAD⁺ synthesis. In Escherichia coli, the enzyme nicotinate mononucleotide adenylyltransferase is encoded by the nadD gene. We have earlier made an initial characterization in vivo of two mutant enzymes, NadD72 and NadD74. Strains with either mutation have decreased intracellular levels of NAD⁺, especially for one of the alleles, nadD72.     

Cloning of Thermostable Cellulase Genes of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> and Their Secretive Expression in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Screening for the powerful cellulase genes with improved activities remains a challenge for the biorefinery research. In this study, five cellobiohydrolase genes and one endoglucanase gene sourced from Clostridium thermocellum DSM 1237, cbhA, celK, celO, cel48Y, cel48S, and celA were cloned into a newly established tool vector pP43JM2 and expressed in two Bacillus subtilis strains, B. subtilis WB600 and B. subtilis WB800, respectively. Most of the cellulases produced in the B. subtilis recombinants were efficiently secreted into the culture medium. These secreted soluble proteins showed distinct cellulase activities using phosphoric acid swollen cellulose (PASC) as the substrate and they also demonstrated strong synergistic effects for PASC, Avicel cellulose, and the dilute acid pretreated corn stover. The current work provided a quick secretive cloning method for screening cellulase genes and may provide a host strain for constructing a consolidated bioprocessing platform with the capacity of secretive expression of multiple cellulases.     

<Emphasis Type="Italic">seco</Emphasis>-Kaurane skeleton diterpenoids from <Emphasis Type="Italic">Croton oblongifolius</Emphasis>

A new seco-kaurane type diterpenoid, ent-3,4-seco-17-oxo-kaur-4(19),15(16)-dien-3-oic acid, and a known compound, ent-3,4-seco-kaur-4(19),16(17)-dien-3-oic acid, were isolated from the stem bark of Croton oblongifolius. The structures of these compounds were established on the basis of spectroscopic data.     

Characterization of <Emphasis Type="Italic">Pf</Emphasis>TrxR inhibitors using antimalarial assays and <Emphasis Type="Italic">in silico</Emphasis>techniques

Background

The compounds 1,4-napthoquinone (1,4-NQ), bis-(2,4-dinitrophenyl)sulfide (2,4-DNPS), 4-nitrobenzothiadiazole (4-NBT), 3-dimethylaminopropiophenone (3-DAP) and menadione (MD) were tested for antimalarial activity against both chloroquine (CQ)-sensitive (D6) and chloroquine (CQ)-resistant (W2) strains of Plasmodium falciparum through an in vitro assay and also for analysis of non-covalent interactions with P. falciparum thioredoxin reductase (PfTrxR) through in silico docking studies.

Results

The inhibitors of PfTrxR namely, 1,4-NQ, 4-NBT and MD displayed significant antimalarial activity with IC₅₀ values of?<?20 μM and toxicity against 3T3 cell line. 2,4-DNPS was only moderately active. In silico docking analysis of these compounds with PfTrxR revealed that 2,4-DNPS, 4-NBT and MD interact non-covalently with the intersubunit region of the enzyme.

Conclusions

In this study, tools for the identification of PfTrxR inhibitors using phenotyphic screening and docking studies have been validated for their potential use for antimalarial drug discovery project.

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Extracellular Production and Characterization of Two <Emphasis Type="Italic">Streptomyces</Emphasis><Emphasis Type="SmallCaps">l</Emphasis>-Asparaginases

l-Asparaginase (ASNase) has proved its use in medical and food industries. Sequence-based screening showed the thermophilic Streptomyces strain Streptomyces thermoluteus subsp. fuscus NBRC 14270 (14270 ASNase) to positive against predicted ASNase primary sequences. The 14270 ASNase gene and four l-asparaginase genes from Streptomyces coelicolor, Streptomyces avermitilis, and Streptomyces griseus (SGR ASNase) were expressed in Streptomyces lividans using a hyperexpression vector: pTONA5a. Among those genes, only 14270 ASNase and SGR ASNase were successful for overexpression and detected in culture supernatants without an artificial signal peptide. Comparison of the two Streptomyces enzymes described above demonstrated that 14270 ASNase was superior to SGR ASNase in terms of optimum temperature, thermal stability, and pH stability.     

Purification and Characterization of Two Extracellular Xylanases from <Emphasis Type="Italic">Penicillium sclerotiorum</Emphasis>: A Novel Acidophilic Xylanase

Two xylanases from the crude culture filtrate of Penicillium sclerotiorum were purified to homogeneity by a rapid and efficient procedure, using ion-exchange and molecular exclusion chromatography. Molecular masses estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 23.9 and 33.1 kDa for xylanase I and II, respectively. The native enzymes’ molecular masses of 23.8 and 30.8 kDa were estimated for xylanase I and II, respectively, by molecular exclusion chromatography. Both enzymes are glycoproteins with optimum temperature and pH of 50 °C and pH 2.5 for xylanase I and 55 °C and pH 4.5 for xylanase II. The reducing agents β-mercaptoethanol and dithio-treitol enhanced xylanase activities, while the ions Hg²⁺ and Cu²⁺ as well the detergent SDS were strong inhibitors of both enzymes, but xylanase II was stimulated when incubated with Mn²⁺. The K _m value of xylanase I for birchwood xylan and for oat spelt xylan were 6.5 and 2.6 mg mL⁻¹, respectively, whereas the K _m values of xylanase II for these substrates were 26.61 and 23.45 mg mL⁻¹. The hydrolysis of oat spelt xylan by xylanase I released xylobiose and larger xylooligosaccharides while xylooligosaccharides with a decreasing polymerization degree up to xylotriose were observed by the action of xylanase II. The present study is among the first works to examine and describe an extracellular, highly acidophilic xylanase, with an unusual optimum pH at 2.5. Previously, only one work described a xylanase with optimum pH 2.0. This novel xylanase showed interesting characteristics for biotechnological process such as feed and food industries.     

Purification and Characterization of a Naringinase from <Emphasis Type="Italic">Cryptococcus albidus</Emphasis>

Naringinase which was extracted from the fermented broth of Cryptococcus albidus was purified about 42-folds with yield 0.7% by sulfate fractionation and chromatography on Toyopearl HW-60, Fractogel DEAE-650-s, and Sepharose 6B columns. Molecular weight of protein determined by gel filtration and SDS-PAGE was 50 kDa. Naringinase of C. albidus includes high content of the dicarbonic and hydrophobic amino acids. Enzyme contains also carbohydrate component, represented by mannose, galactose, rhamnose, ribose, arabinose, xylose, and glucose. The enzyme was optimally active at pH 5.0 and 60 °C. Naringinase was found to exhibit specificity towards p-nitrophenyl-α-L-rhamnose, p-nitrophenyl-β-D-glucose, naringin, and neohesperidin. Its K _m towards naringin was 0.77 mM and the V _max was 36 U/mg. Naringinase was inhibited by high concentrations of reaction product—L-rhamnose. Enzyme revealed stability to 20% ethanol and 500 mM glucose in the reaction mixture that makes it possible to forecast its practical use in the food industry in the production of juices and wines.     

Stability of <Emphasis Type="Italic">s</Emphasis>-, <Emphasis Type="Italic">p</Emphasis>-, and <Emphasis Type="Italic">d</Emphasis>-element diphosphates in water

The mean atomic Gibbs energies of formation of (Δ _f ? _at ⁰ ) of s-, p-, and d-element diphosphates have been calculated using ion increments of the Gibbs energy (Δ _f G ⁰). The diphosphate hydrolysis kinetics is considered, and a correlation between the Δ _f ? _at ⁰ values and the hydrolysis rate constants is presented.     

Isolation and characterization of the cellulose synthase genes <Emphasis Type="Italic">PpCesA6</Emphasis> and <Emphasis Type="Italic">PpCesA7</Emphasis> in <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Analysis of cellulose biosynthesis using molecular approaches has been successful in identifying genes in many cellulose-producing organisms, yet the mechanism of cellulose biosynthesis still remains to be understood. We are interested in developing the moss Physcomitrella patens as a useful system for the study of cellulose biosynthesis. This moss affords a number of advantages including a haploid dominated gametophyte and a very high efficiency of homologous recombination in its nuclear DNA for constructing gene knockouts. In addition, P. patens has only a primary cell wall unlike Arabidopsis thaliana, which has both a primary and a secondary cell wall. We identified two full-length cellulose synthase (CesA) genes of P. patens, PpCesA6 and PpCesA7 from an EST database and have analyzed the genomic sequences. PpCesA6 and PpCesA7 show high similarity to each other, both at the cDNA and genomic DNA levels. Single and double knockouts of PpCesA6 and PpCesA7 were generated and screened for phenotypic changes. While the PpCesA6 and PpCesA7 single knockouts did not show any obvious phenotypic differences from the wild-type, the double knockout had significantly reduced stem length. These results suggest that PpCesA6 and PpCesA7 probably have a very similar role in cellulose biosynthesis and their functions may be redundant. Additionally, their roles may overlap with the other P. patens CesAs as observed for CesAs involved in primary cell wall biosynthesis in A. thaliana.