Screening carbonyl reductases with the ability to catalyze the reduction of complex carbonyl compounds is of great significance for the biosynthesis of R-tolvaptan(R-TVP). In this study, the target carbonyl reductase in the crude enzyme extract of rabbit liver was separated, purified, and identified by ammonium sulfate precipitation, gel-filtration chromatography, ion exchange chromatography, affinity chromatography, and protein mass spectrometry. With the rabbit liver genome as the template, the gene encoding the carbonyl reductase rlsr5 was amplified by PCR and the recombinant strain was successfully constructed. After RLSR5 was purified by affinity chromatography, its enzymatic properties were characterized. The results indicated that the gene sequence of rlsr5 was 972 bp, encoding a protein with a molecular weight of 40 kDa. RLSR5 was a dimeric protein, and each monomer was composed of a (α/β)₈-barrel structure. RLSR5 could asymmetrically reduce 7-chloro-1-[2-methyl-4-[(2- methylbenzoyl)amino]benzoyl]-5-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine (prochiral ketone, PK) to synthesize R-TVP. The specific activity of the enzyme was 36.64 U/mg, and the optical purity of the product was 99%. This enzyme showcased the optimal performance at pH 6.0 and 30 °C. It was independent of metal ions, with the activity enhanced by Mn²⁺. This study lays a foundation for the biosynthesis of tolvaptan of optical grade.
Animals ; Rabbits ; Alcohol Oxidoreductases/biosynthesis* ; Recombinant Proteins/metabolism* ; Escherichia coli/metabolism* ; Liver/enzymology*

L-phosphinothricin (L-PPT) is an efficient broad-spectrum herbicide. To realize the multi-enzyme catalytic preparation of L-PPT, we constructed an engineered strain Escherichia coli YM-1 for efficient expression of D-amino acid transaminase, which could catalyze the generation of the intermediate 2-oxo-4-[(hydroxymethylphosphonyl)] butyric acid (PPO) from D-phosphinothricin (D-PPT). In addition, E. coli pLS was constructed to co-express glutamate dehydrogenase and glucose dehydrogenase, which not only catalyzed the generation of L-PPT from PPO but also regenerated the coenzyme nicotinamide adenine dinucleotide phosphate (NADPH). A fed-batch fermentation process was then established for E. coli YM-1 and pLS, and the apparent activities of D-amino acid transaminase and glutamate dehydrogenase were increased by 22.68% and 100.82%, respectively, compared with those in shake flasks. The process parameters were optimized for the catalytic preparation of L-PPT by whole-cell cascade of E. coli YM-1 and pLS with D, L-PPT as the substrate. After reaction for 8 h, 91.36% conversion of D-PPT was achieved, and the enantiomeric excess of L-PPT reached 90.22%. The findings underpin the industrial production of L-PPT.
Escherichia coli/enzymology* ; Aminobutyrates/metabolism* ; Glutamate Dehydrogenase/biosynthesis* ; Glucose 1-Dehydrogenase/biosynthesis* ; Herbicides/metabolism* ; Multienzyme Complexes/metabolism* ; Transaminases/metabolism* ; Phosphinic Acids/metabolism*

Endo-β-N-acetylglucosaminidase is used widely in the glycobiology studies and industries. In this study, a new endo-β-N-acetylglucosaminidase, designated as Endo SA, was cloned from Streptomyces alfalfae ACCC 40021 and expressed in Escherichia coli BL21 (DE3). The purified recombinant Endo SA exhibited the maximum activity at 35 ºC and pH 6.0, good thermo/pH stability and high specific activity (1.0×10⁶ U/mg). It displayed deglycosylation activity towards different protein substrates. These good properties make EndoSA a potential tool enzyme and industrial biocatalyst.
Cloning, Molecular ; Enzyme Stability ; Escherichia coli ; genetics ; Gene Expression ; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Streptomyces ; enzymology ; genetics

L-2-aminobutyric acid (L-ABA) is an important chemical raw material and chiral pharmaceutical intermediate. The aim of this study was to develop an efficient method for L-ABA production from L-threonine using a trienzyme cascade route with Threonine deaminase (TD) from Escherichia. coli, Leucine dehydrogenase (LDH) from Bacillus thuringiensis and Formate dehydrogenase (FDH) from Candida boidinii. In order to simplify the production process, the activity ratio of TD, LDH and FDH was 1:1:0.2 after combining different activity ratios in the system in vitro. The above ratio was achieved in the recombinant strain E. coli 3FT+L. Moreover, the transformation conditions were optimized. Finally, we achieved L-ABA production of 68.5 g/L with a conversion rate of 99.0% for 12 h in a 30-L bioreactor by whole-cell catalyst. The environmentally safe and efficient process route represents a promising strategy for large-scale L-ABA production in the future.
Aminobutyrates ; chemical synthesis ; Bacillus thuringiensis ; enzymology ; Candida ; enzymology ; Escherichia coli ; enzymology ; Formate Dehydrogenases ; metabolism ; Leucine Dehydrogenase ; metabolism ; Threonine ; metabolism ; Threonine Dehydratase ; metabolism

A short terpene synthase gene was obtained by screening the transcriptome data of Senecio scandens. The phylogenetic tree and sequence alignment putatively identified this gene as a nerolidol synthase gene, named SsNES(GenBank MH518312). Protein homology modeling indicated that SsNES contained a complete conserved domain and folded correctly. SsNES was cloned and successfully expressed in Escherichia coli as soluble protein. The biochemical function of SsNES was characterized by E. coli metabolic engineering, which showed that SsNES catalyzed formation of trans-nerolidol with(E, E)-farnesyl diphosphate as the substrate. Nerolidol was also detected in stems and leaves of S. scandens, indicating that SsNES might act as the nerolidol synthase in plant. RT-PCR analysis indicated that SsNES was mainly expressed in stem, flowers and leaves, and no expression was observed in roots. After the treatment of SA, MeJA or Ala, SsNES was induced significantly at 6 h, indicating involvement in the defense response of S. scandens. The identification of SsNES not only clarified biosynthesis of nerolidol in S. scandens, but also provided diversity of sesquiterpene synthase, as well as theoretical basis for disease and pest defense mediated by the terpene metabolites.
Escherichia coli ; Genes, Plant ; Phylogeny ; Senecio ; enzymology ; Sesquiterpenes ; metabolism

BACKGROUND/AIMS: The number of urinary tract infections (UTIs) caused by extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-EC) is increasing. In an outpatient setting, there are limited therapeutic options to treat ESBL-producing pathogens. We evaluated the outcomes of amikacin outpatient parenteral antibiotic therapy (OPAT) for UTIs caused by ESBL-EC in patients not pre-treated with carbapenem. METHODS: We retrospectively evaluated the outcomes of amikacin OPAT for UTIs caused by ESBL-EC. RESULTS: From November 2011 to October 2012, eight females, who could not be hospitalized for carbapenem treatment, were treated with amikacin OPAT for nine episodes of non-bacteremic ESBL-EC UTIs. Seven of the eight patients had one or more comorbidities. Of the nine UTI cases, three had symptomatic lower UTIs and six had non-bacteremic upper UTIs. In all of the cases, symptomatic and laboratory improvements were observed following amikacin OPAT. One patient showed a delayed relapse with bilateral microabscesses 3 weeks after treatment cessation; however, a clinical and microbiological cure was eventually reached. All of the patients were able to tolerate amikacin OPAT without any significant nephrotoxicity or ototoxicity. CONCLUSIONS: Amikacin OPAT represents a feasible therapeutic option for non-bacteremic UTIs caused by ESBL-EC in settings with limited resources.
Adult ; Aged ; Aged, 80 and over ; Ambulatory Care ; Amikacin/administration & dosage/adverse effects/*therapeutic use ; Drug Administration Schedule ; Escherichia coli/*drug effects/enzymology/isolation & purification ; Escherichia coli Infections/diagnosis/*drug therapy/microbiology/urine ; Humans ; Microbial Sensitivity Tests ; Middle Aged ; Recurrence ; Remission Induction ; Retrospective Studies ; Time Factors ; Treatment Outcome ; Urinalysis ; Urinary Tract Infections/diagnosis/*drug therapy/microbiology/urine ; Urine/microbiology ; beta-Lactamase Inhibitors/administration & dosage/adverse effects/*therapeutic use ; beta-Lactamases/*metabolism

The observation statistics suggested that the haemolymph melanization speed of larvae became fast and the growth inhibition of Escherichia coli was strong as the quantities of feeding on mulberry leaves increased. The RT-PCR result showed that the mRNA expressions of melanin biosynthesis enzyme BmTan, BmPo-1, BmYellow-f and BmDdc were high in the haemolyph of 5 L 3 d larvae. The qPCR analysis showed Bmtan, Bmddc, Bmyellow, Bmebony and Bmblack, especially Bmddc expression were significantly higher in black disease larvae than in normal larvae. Compared with control, Ddc inhibitors drastically inhibited the lipopolysaccharide-induced haemolymph melanization. In addition, the content of Dopa and Dopamine markedly rose after E. coli injection. These indicated that haemolymph melanization was linked to immune defenses and Bmddc may play a role in melanization response of haemolymph immune in silkworm.
Animals ; Bombyx ; enzymology ; genetics ; microbiology ; Escherichia coli ; Genes, Insect ; Hemolymph ; chemistry ; Larva ; Melanins ; biosynthesis

To improve the yield of sucrose isomerase from Pantoea dispersa UQ68J, we studied the effect of different signal peptides and fermentation conditions on sucrose isomerase expression in Escherichia coli. The gene of sucrose isomerase was optimized and expressed in E. coli BL21 (DE3) with native signal peptide which was named as ORI strain. The total and extracellular enzyme activity was 85 and 65 U/mL in the flask, respectively. The mature protein, which started from the 22th amino acid, was connected with the PelB and OmpA signal peptide to construct P22 and O22 strain, respectively. The total activity of P22 reached 138 U/mL, which was 1.6 times of ORI strain. The total activity of O22 strain was similar to that of ORI strain. Induced by 3.0 g/L lactose, the total activity of P22 strain increased to 168 U/mL. In 3 L fermentor, the effects of glycine concentration and induction time were studied. Induction when the DCW reached 18 g/L (OD₆₀₀=30), with 0.5% glycine, the extracellular enzyme activity reached 1 981 U/mL, and the total enzyme activity reached 2 640 U/mL, which is the highest activity of sucrose isomerase that was expressed in recombinant E. coli.
Bacterial Proteins ; biosynthesis ; Bioreactors ; Escherichia coli ; metabolism ; Fermentation ; Gene Expression ; Glucosyltransferases ; biosynthesis ; Lactose ; Pantoea ; enzymology ; Protein Sorting Signals ; Recombinant Proteins ; biosynthesis

Arabinose-5-phosphate isomerase (KdsD) is the first key limiting enzyme in the biosynthesis of 3-deoxy-D-manno-octulosonate (KDO). KdsD gene was cloned into prokaryotic expression vector pET-HTT by seamless DNA cloning method and the amount of soluble recombinant protein was expressed in a soluble form in E. coli BL21 (DE3) after induction of Isopropyl β-D-1-thiogalactopyranoside (IPTG). The target protein was separated and purified by Ni-NTA affinity chromatography and size exclusion chromatography, and its purity was more than 85%. Size exclusion chromatography showed that KdsD protein existed in three forms: polymers, dimmers, and monomers in water solution, different from microbial KdsD enzyme with the four polymers in water solution. Further, the purified protein was identified through Western blotting and MALDI-TOF MASS technology. The results of activity assay showed that the optimum pH and temperature of AtKdsD isomerase activities were 8.0 and 37 ℃, respectively. The enzyme was activated by metal protease inhibitor EDTA (5 mmol/L) and inhibited by some metal ions at lower concentration, especially with Co²⁺ and Cd²⁺ metal ion. Furthermore, when D-arabinose-5-phosphate (A5P) was used as substrate, Km and Vmax of AtKdsD values were 0.16 mmol/L, 0.18 mmol/L·min. The affinity of AtKdsD was higher than KdsD in E. coli combined with substrate. Above results have laid a foundation for the KdsD protein structure and function for its potential industrial application.
Aldose-Ketose Isomerases ; biosynthesis ; Arabidopsis ; enzymology ; Arabidopsis Proteins ; biosynthesis ; Cloning, Molecular ; Escherichia coli ; metabolism ; Metals ; Pentosephosphates ; Recombinant Proteins ; biosynthesis

Glutathione S-transferase (GST) is important in plants to resist various stresses. In this study, two Phi GST genes (SmGSTF1 and SmGSTF2) were cloned from Selaginella moellendorffii. SmGSTF1 and SmGSTF2 genes encode proteins of 215 amino acid residues. Gene expression analysis showed that the two genes were expressed in roots, stems and leaves. The recombinant SmGSTF1 and SmGSTF2 proteins were overexpressed in Escherichia coli, and purified by Ni-affinity chromatography. SmGSTF1 and SmGSTF2 had the catalytic activity towards 1-Chloro-2,4-Dieitrobenzene, 4-Chloro-7-nitro-1,2,3-benzoxadiazole (NBD-Cl), and 4-Nitrobenzyl chloride substrates. SmGSTF1 also had the activity towards Fluorodifen and Cumyl hydroperoxide (Cum-OOH), whereas SmGSTF2 not. The enzyme kinetics analysis showed that SmGSTF1 and SmGSTF2 had high affinity towards glutathione, and low affinity towards 1-Chloro-2, 4-Dieitrobenzene. The enzymatic activity of SmGSTF1 and SmGSTF2 had high catalytic activity between pH 7 and 8.5, and between 45 and 55 °C. SmGSTF1 and SmGSTF2 may have an important role in the resistance of Selaginella moellendorfii against stress.
Amino Acid Sequence ; Cloning, Molecular ; Escherichia coli ; Glutathione Transferase ; genetics ; metabolism ; Plant Proteins ; genetics ; metabolism ; Selaginellaceae ; enzymology