Multiplex polymerase chain reaction detection of Curvularia lunata, Bipolaris maydis, and Aureobasidium zeae in infected maize leaf tissues.

Multiplex polymerase chain reaction (PCR) is a method for simultaneous identification and detection of multiple pathogenic fungi, however, its complexity limits its application. To simplify the protocol and to improve the effectiveness, three-level designs for six factors (three primers, Taq DNA polymerase, dNTP, Mg2+ ) were constructed to optimize the multiplex PCR system by using the orthogonal design method and the annealing temperature of the PCR reactions was also optimized. Finally, a multiplex PCR system for the simultaneous detection of these three pathogens of maize was successfully established. The reaction volume was 25 µl and the annealing temperature was 57℃. The optimal conditions for multiplex PCR reaction contained 0.48 µmol/L Cl-1/Cl-2, 0.72 µmol/L Bm-1/Bm-2, 0.24 µmol/L Az-1/Az-2, 1.5 U polymerase, 0.35 mmol/L dNTP, and 1.25 mmol/L MgCl2 . The multiplex PCR system can detect Curvularia lunata, Bipolaris maydis, and Aureobasidium zeae in infected plant tissues rapidly with the sensitivity at 10 pg DNA/µl.

Complete Genome Sequence of Exiguobacterium sp. Strain MH3, Isolated from Rhizosphere of Lemna minor.

We report the complete genome sequence of Exiguobacterium sp. strain MH3, isolated from the rhizosphere of duckweed. The genome assembly is 3.16 Mb, with a G+C content of 47.24%, and it may provide useful information about plant-microbe interactions and the genetic basis for the tolerance of the strain to various environmental stresses.

Overexpression of a recombinant amidase in a complex auto-inducing culture: purification, biochemical characterization, and regio- and stereoselectivity.

Rhodococcus erythropolis AJ270 metabolizes a wide range of nitriles via the two-step nitrile hydratase/amidase pathway. In this study, an amidase gene from R. erythropolis AJ270 was cloned and expressed in Escherichia coli BL21 (DE3). The activity reached the highest level of 22.04 U/ml in a complex auto-inducing medium using a simplified process of fermentation operation. The recombinant amidase was purified to more than 95% from the crude lysate using Ni-NTA affinity chromatography and Superose S10-300 gel filtration. The V(max) and K(m) values of the purified enzyme with acetamide (50 mM) were 6.89 µmol/min/mg protein and 4.12 mM, respectively, which are similar to those of the enzyme from the wild-type cell. The enzyme converted racemic α-substituted amides, O-benzylated ß-hydroxy amides, and N-benzylated ß-amino amides to the corresponding (S)-acids with remarkably high enantioselectivity. The ionic liquid [BMIm][PF6] (1-butyl-3-methylimidazolium hexafluorophosphate) enhanced the activity by 1.5-fold compared with water. The adequate expression of the enzyme and excellent enantioselectivity of the recombinant amidase to a broad spectrum of amides suggest that the enzyme has prospective industrial-scale practical applications in pharmaceutical chemistry.

Purification and characterization of four keratinases produced by Streptomyces sp. strain 16 in native human foot skin medium.

Four extracellular keratinases (designated KI, KII, KIII, and KIV) were produced during submerged aerobic cultivation in a medium containing native human foot skin (NHFS) for enzyme synthesis. The molecular weights, determined by SDS-PAGE, were 25, 50, 34, and 19 kDa, respectively. Gel filtration of the four purified enzymes in native conditions indicated that active keratinase KI is a novel homo-octamer, KII a homo-dimer, and KIII and KIV monomers. All four keratinases exhibited high activities at pH 8.0-10.0 with an optimal pH of 9.0. The optimal temperature for keratinolytic activity of KI, KII, and KIII was approximately 50, and 60 degrees C for KIV. One millimolar of PMSF completely inhibited the keratinolytic activities of the four enzymes. The N-terminal sequences of KI, KII, and KIII showed that they were different from previously described enzymes, whereas KIV shared an identical N-terminal sequence with two other peptidases from Streptomyces.

Stereoselective epoxidation of cis-propenylphosphonic acid to fosfomycin by a newly isolated bacterium Bacillus simplex strain S101.

In industry, fosfomycin is mainly prepared via chemical epoxidation of cis-propenylphosphonic acid (cPPA). The conversion yield of fosfomycin is less than 50% in the whole process and a large quantity of waste is produced. Biotransformation by microorganisms is an alternative method of preparation. This kind of conversion is more delicate, environmentally friendly, and the conversion yield of fosfomycin would be higher. In this work, an aerobic bacterium capable of transforming cPPA to fosfomycin was isolated. The organism, designated as strain S101, was identified as Bacillus simplex by morphological and physiological characteristics as well as by analysis of the gene encoding the 16S rRNA. Fosfomycin was assayed by two means, bioassay and gas chromatography (GC). Glycerol was a good carbon source for growth and cPPA conversion of strain S101. When cPPA was used as the sole carbon source, neither growth nor conversion to fosfomycin occurred. The optimum cPPA concentration in the conversion medium was 2,000 microg ml(-1). After 6 days of incubation, the concentration of fosfomycin reached its maximum level (1,838.2 microg ml(-1)), with a conversion ratio of 81.3%. Air was indispensable for the growth but not for the conversion to fosfomycin. Furthermore, vanadium ions were found to be essential for the conversion. High concentrations of cPPA had fewer inhibitory effects on the growth of strain S101.

Molecular evolution of Fome lignosus laccase by ethyl methane sulfonate-based random mutagenesis in vitro.

In order to improve the laccase activity, mutant libraries are constructed through ethyl methane sulfonate-based (EMS) random mutagenesis. Mutagenesis improved expression 3.7-fold to 144 mgl(-1) laccase in yeast, together with a 1.4-fold increase in K(cat). Thus, the total activity is enhanced 5-fold for 2,2'-azino-bis 3-ethylbenzothiaoline-6-sulfonic acid (ABTS). In the presence of 0.6mM copper, the highest activity value reached 30 Uml(-1) after a 3-day cultivation at a temperature of 30 degrees C(.) In comparison with the wild type, the best mutant enzymatic properties (K(m) for ABTS and guaiacol, thermo- and pH stability, optimal pH) are not changed. Moreover, amino acid sequence analysis indicates that there are four substitutions in the best mutant laccase (Gly160Asp, Ala167Thr, Gly174Asp, and Glu234Gly). The best mutant laccase model showed that the Gly160 and Ala167 are to be found near the water channel; especially the distance of Ala167 to the Cu3a is 14.46 A. This implies that it is likely involved in the formation of water channel and that it helps facilitate the easy incoming and outgoing of water.

High resolution X-ray molecular structure of the nitrile hydratase from Rhodococcus erythropolis AJ270 reveals posttranslational oxidation of two cysteines into sulfinic acids and a novel biocatalytic nitrile hydration mechanism.

The crystal structure of Fe-type nitrile hydratase from Rhodococcus erythropolis AJ270 was determined at 1.3A resolution. The two cysteine residues (alphaCys(112) and alphaCys(114)) equatorially coordinated to the ferric ion were post-translationally modified to cysteine sulfinic acids. A glutamine residue (alphaGln(90)) in the active center gave double conformations. Based on the interactions among the enzyme, substrate and water molecules, a new mechanism of biocatalysis of nitrile hydratase was proposed, in which the water molecule activated by the glutamine residue performed as the nucleophile to attack on the nitrile which was simultaneously interacted by another water molecule coordinated to the ferric ion.