A gene (pueA, polyurethane esterase A) encoding an extracellular polyurethanase (PueA) was cloned from Pseudomonas chlororaphis into Escherichia coli. The enzyme secreted from E. coli showed esterase activity when assayed with p-nitrophenyl acetate. Subcloning of a 3. 2-kb SalI-EcoRI fragment into a T7 RNA polymerase expression vector (pT7-6) produced a (35)S-labeled protein of 65 kDa. Nucleoti...

Here we report the construction of a histidine-tagged T4 RNA ligase expression plasmid (pRHT4). The construct, when overexpressed in BL21 (DE3) cells, allows the preparation of large quantities of T4 RNA ligase in high purity using only a single purification column. The histidine affinity tag does not inhibit enzyme function, and we were able to purify 1–3 mg pure protein/g cell pellet. A simpl...

Leptospira interrogans serovar autumnalis, a causative agent of leptospirosis in Thailand, was isolated from a patient for DNA extraction and amplification of LipL32 gene by polymerase chain reaction (PCR). The 782 bp PCR product was obtained, which was inserted into pAE plasmid with polyhistidine (His6 tag) to construct pAE-LipL32. This recombinant plasmid was transfected into E. coli BL21 (DE...

To isolate novel and useful microbial enzymes from uncultured gastrointestinal microorganisms, a fecal microbial metagenomic library of the pygmy loris was constructed. The library was screened for amylolytic activity, and 8 of 50,000 recombinant clones showed amylolytic activity. Subcloning and sequence analysis of a positive clone led to the identification a novel gene (amyPL) coding for α-am...

The amnE gene from Pseudomonas sp. AP-3 has been verified as encoding a deaminase with 142 amino-acid residues. In order to change the substrate specificity via structure-based protein engineering, the amnE gene, after gene-code optimization, was chemically synthesized and cloned into the expression vector pET-28a. The protein was expressed in Escherichia coli BL21 (DE3) and purified by Ni(2+)-...

D-Amino acid oxidase is a key enzyme in the production of semisynthetic cephalosporins. The Trigonopsis variabilis D-amino acid oxidase with 12amino-acid peptide at N-terminus was expressed in Escherichia coli BL21(DE3), in a series of short fed-batch cultivations. The strategy of gradual induction and gradual feeding with D-methionine during expression resulted in active enzyme production twic...

© 2015 Lin and Yan. Erratum to: Microbial Cell Factories 2012, 11:42 DOI 10.1186/1475‐2859‐11‐42 Recently, we have noticed an accidental error existing in this article [1]. In the section Materials and Methods “Construction of Plasmids”, we mistakenly described that “the gene cluster hpaBC was amplified from E. coli MG1655 genome”. This information should be corrected as “the gene cluster hpaBC...

Here we report the construction of a histidine-tagged T4 RNA ligase expression plasmid (pRHT4). The construct, when overexpressed in BL21 (DE3) cells, allows the preparation of large quantities of T4 RNA ligase in high purity using only a single purification column. The histidine affinity tag does not inhibit enzyme function, and we were able to purify 1-3 mg pure protein/g cell pellet. A simpl...