High power (60mW) single frequency erbium:ytterbium codoped fiber laser

The characteristics of a high power Er3+:Yb3+ single frequency fiber laser pumped at 980nm are reported. The device gives 60mW output power with RIN 10MHz and linewidth 500kHz. At low output powers (< 30mW) the slope efficiency is as high as 25%, falling to 12% at higher powers, the saturation behaviour is related to a bottleneck effect due to the finite Yb-Er transfer rate. Improved performance can be obtained using new fibers with an increased rare-earth concentration which show negligible signs of erbium clustering

Efficient single frequency fibre lasers using novel photosensitive Er/Yb optical fibres

Boron- and germanium-doped highly photosensitive cladding is used in a novel design to achieve photosensitive Er/Yb-doped fibers, permitting short, strong gratings (length ~1cm, reflectivity >99%) to be written without hydrogenation. The high absorption at 980nm in Er/Yb fibers permits efficient pump absorption over a short device length, which is ideal for achieving highly efficient single-frequency fiber lasers. Both single-frequency Bragg-grating reflector and distributed-feedback lasers with slope efficiencies of 25% with respect to launched pump power have been realized in such fibers

Mutational analysis of feedback inhibition and catalytic sites of prephenate dehydratase from Corynebacterium glutamicum

Prephenate dehydratase is a key regulatory enzyme in the phenylalanine-specific pathway of Corynebacterium glutamicum. PCR-based random mutagenesis and functional complementation were used to screen for m-fluorophenylalanine (mFP)-resistant mutants. Comparison of the amino acid sequence of the mutant prephenate dehydratases indicated that Ser-99 plays a role in the feedback regulation of the enzyme. When Ser-99 of the wild-type enzyme was replaced by Met, the specific activity of the mutant enzyme was 30% lower than that of the wild-type. The Ser99Met mutant was active in the presence of 50 muM phenylalanine, whereas the wild-type enzyme was not. The functional roles of the eight conserved residues of prephenate dehydratase were investigated by site-directed mutagenesis. Glu64Asp substitution reduced enzyme activity by 15%, with a 4.5- and 1.7-fold increase in K-m and k(cat) values, respectively. Replacement of Thr-183 by either Ala or Tyr resulted in a complete loss of enzyme activity. Substitution of Arg-184 with Leu resulted in a 50% decrease of enzyme activity. The specific activity for Phe185Tyr was more than 96% lower than that of the wild-type, and the K-m value was 26-fold higher. Alterations in the conserved Asp-76, Glu-89, His-115, and Arg-236 residues did not cause a significant change in the K-m and k(cat) values. These results indicated that Glu-64, Thr-183, Arg-184, and Phe-185 residues might be involved in substrate binding and/or catalytic activity

Expression of Pseudomonas amyloderamosa isoamylase gene in Saccharomyces cerevisiae

Isoamylase gene (iso) of Pseudomonas amyloderamosa was amplified by polymerase chain reaction and cloned into Saccharomyces cerevisiae vectors under the control of alcohol dehydrogenase gene and glyceraldehyde-3-phosphate dehydrogenase gene promoters. The signal sequence of iso gene was also replaced with that of Schwanniomyces occidentalis alpha-amylase gene. The extracellular isoamylase activity of transformed Sacc. cerevisiae could reach 86 U ml(-1) after a 4-days cultivation

Phylogenetic analysis and biochemical characterization of a thermostable dihydropyrimidinase from alkaliphilic Bacillus sp TS-23

Two degenerate primers established from the alignment of highly conserved amino acid sequences of bacterial dihydropyrimidinases (DHPs) were used to amplify a 330-bp gene fragment from the genomic DNA of Bacillus sp. TS-23 and the amplified DNA was successfully used as a probe to clone a dhp gene from the strain. The open reading frame of the gene consisted of 1422 bp and was deduced to contain 472 amino acids with a molecular mass of 52 kDa. The deduced amino acid sequence exhibited greater than 45% identity with that of prokaryotic D-hydantoinases and eukaryotic DHPs. Phylogenetic analysis showed that Bacillus sp. TS-23 DHP is grouped together with Bacillus stearothermophilus D-hydantoinase and related to dihydroorotases and allantoinases from various organisms. His(6)-tagged DHP was over-expressed in Escherichia coli and purified by immobilized metal affinity chromatography to a specific activity of 3.46 U mg(-1) protein. The optimal pH and temperature for the purified enzyme were 8.0 and 60 degrees C, respectively. The half-life of His(6)-tagged DHP was 25 days at 50 degrees C. The enzyme activity was stimulated by Co2+ and Mn2+ ions. His(6)-tagged DHP was most active toward dihydrouracil followed by hydantoin derivatives. The catalytic efficiencies (k(cat)/K-m) of the enzyme for dihydrouracil and hydantoin were 2.58 and 0.61 s(-1) mM(-1), respectively

Expression, crystallization and preliminary X-ray diffraction studies of N-carbamyl-D-amino-acid amidohydrolase from Agrobacterium radiobacter

The Agrobacterium radiobacter CCRC 14924 N-carbamyl-D-amino-acid amidohydrolase, the enzyme used for production of D-amino acids, was overexpressed in Escherichia coli JM109. The expressed protein was crystallized by vapour diffusion using lithium sulfate as precipitant. It crystallizes in space group P2(1) with unit-cell parameters cr = 69.8, b = 67.9 and c = 137.8 Angstrom and beta = 96.4 degrees. There are four molecules per asymmetric unit. Crystals diffract to 2.8 Angstrom resolution using a rotating-anode source at cryogenic (113 K) temperatures

High-level expression of Trigonopsis variabilis D-amino acid oxidase in Escherichia coli using lactose as inducer

The use of lactose as inducer for the expression of Trigonopsis variabilis D-amino acid oxidase gene (daao) was investigated in Escherichia coli regulated by T7 or T5 promoter. The daao gene was prepared by reverse transcriptase-polymerase chain reaction and cloned into pET21b and pQE-30 to yield pET-DAAO and pQE-DAAO, respectively. The His(6)-tagged DAAO was expressed in E. coli and had a M-r value of approximately 39.3 kDa. In lactose-induced E. coli BL21 (DE3) (pET-DAAO), the expressed DAAO could comprise up to 15% of total soluble proteins and a productivity of 23.4 U ml(-1) was obtained

Substitution of the critical methionine residues in Trigonopsis variabilis D-amino acid oxidase with leucine enhances its resistance to hydrogen peroxide

Each of the six oxidative-sensitive methionine residues in Trigonopsis variabilis D-amino acid oxidase (DAAO) was changed to leucine by site-directed mutagenesis. The wild-type and mutant enzymes with an apparent molecular mass of about 39.3 kDa were expressed in Escherichia coli. The specific activity of four mutant DAAOs (Met(104)Leu, Met(226)Leu, Met(245)Leu, and Met(339)Leu) was decreased by more than 96% while Met(156)Leu and Met(209)Leu showed about 23% and 96% higher activity, respectively, than the wild-type enzyme. The kinetic parameters of the two mure active enzymes were determined and a 2.2-fold increase in K(m) was observed for Met(209)Leu. Comparison of Met(156)Leu and wild-type DAAO revealed a 95% increase in k(cat)/K(m). Met(156)Leu, Met(209)Leu, and Met(226)Leu were resistant to inactivation by 50 mM H(2)O(2) The other three mutant DAAOs were also slightly more resistant than the wild-type enzyme to chemical oxidation. These observations indicate that the oxidative stability in T. variabilis DAAO can be improved by substitution of methionine residues with leucine. (C) 2000 Published by Elsevier Science B.V. All rights reserved

Screening of compactin-resistant microorganisms capable of converting compactin to pravastatin

A simple method of using compactin for effective screening of microbial strains with high hydroxylation activity at the 6 beta position of compactin was developed. Agar plates containing different carbon sources and 500 mu g compactin mL(-1) were used to screen the microorganisms that can convert compactin to pravastatin. About 100 compactin-resistant strains were isolated from the Basal agar containing 7% (w/v) mannitol as a carbon source, in which two bacteria, Pseudomocardia autotrophica BCRC 12444 and Streptomyces griseolus BCRC 13677, capable of converting compactin to pravastatin with the yield of 20 and 32% (w/w), respectively, were found. High-performance liquid chromatography using C-18 column and two sequential mobile phases, 30% and 50% (v/v) acetonitrile, was also established to simultaneously determine the concentration of compactin and pravastatin in the culture broth. As such, about 2% of target microorganisms could be obtained from the screening program

Overexpression, one-step purification, and biochemical characterization of a recombinant gamma-glutamyltranspeptidase from Bacillus licheniformis

A truncated gene from Bacillus lichenifromis ATCC 27811 encoding a recombinant gamma-glutamyltranspeptidase (BLrGGT) was cloned into pQE-30 to generate pQE-BLGGT, and the overexpressed enzyme was purified from the crude extract of IPTG-induced E. coli M15 (pQE-BLGGT) to homogeneity by nickel-chelate chromatography. This protocol yielded over 25 mg of purified BLrGGT per liter of growth culture under optimum conditions. The molecular masses of the subunits of the purified enzyme were determined to be 41 and 22 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pH and temperature for the recombinant enzyme were 6-8 and 40 degrees C, respectively. The chloride salt of metal ions Mg2+, K+, and Na+ can activate BLrGGT, whereas that of Pb2+ dramatically inhibited it. The substrate specificity study showed that L-gamma-glutamyl-p-nitroanilide (L-gamma-Glu-p-NA) is a preference for the enzyme. Steady-state kinetic study revealed that BLrGGT has a k (cat) of 105 s(-1) and a K (m) of 21 mu M when using L-gamma-Glu-p-NA as the substrate. With this overexpression and purification system, BLrGGT can now be obtained in quantities necessary for structural characterization and synthesis of commercially important gamma-glutamyl compounds