ABSTRACT
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.
Subject(s)
Aminobutyrates , Bacillus thuringiensis , Candida , Escherichia coli , Formate Dehydrogenases , Metabolism , Leucine Dehydrogenase , Metabolism , Threonine , Metabolism , Threonine Dehydratase , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To construct a rapid and high-throughput assay for identifying recombinant bacteria based on mass spectrometry.</p><p><b>METHODS</b>Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) techniques were used to identify 12 recombinant proteins (10 of Yersinia pestis, 1 of Campylobacter jejuni and 1 of Helicobacter pylori). A classification model for the various phase of recombinant bacteria was established, optimized and validated, using MALDI-TOF MS-ClinProTools system. The differences in the peptide mass spectra were analyzed by using Biotyper and FlexAnalysis softwares.</p><p><b>RESULTS</b>Models of GA, SNN, and QC were established. After optimizing the parameters, the GA recognition model showed good classification capabilities: RC=100%, mean CVA=98.7% (the CVA was 96.4% in phase 1, 100% in phase 2, 98.4% in phase 3, and 100% in phase 4, respectively) and PPV=95%. This model can be used to classify the bacteria and their recombinant, which only requires 3.7×103 cells for analysis. The total time needed is only 10 min from protein extraction to reporting the result for one sample. Furthermore, this assay can automatically detect and test 96 samples concurrently. A total of 48 specific peaks (9, 16, 9, and 14 for the four stages, respectively) was found in the various phase of recombinant bacteria.</p><p><b>CONCLUSION</b>MALDI-TOF MS can be used as a fast, accurate, and high-throughput method to identify recombinant bacteria, which provide a new ideas not only for recombinant bacteria but also for the identification of mutant strains and bioterrorism pathogens.</p>
Subject(s)
Bacterial Proteins , Cloning, Molecular , Escherichia coli , Mass Spectrometry , Organisms, Genetically Modified , Peptide Mapping , Recombinant ProteinsABSTRACT
Objective In order to research the best fermentation conditions of recombinant bacteria pThioHisA-B1/DH5? which contains gene of esterase B1.Methods Recombinant bacteria pThioHisA-B1/DH5? was induced in different culture media, induction temperatures, different inductor concentrations, different induction time in order to obtain the best expression effect.The expression products of different conditions were detected by SDS-PAGE and thin-layer gel scanning analysis.Results Based on the experiments, the optimized fermentation condition were that the culture medium was TB, inducing temperature was 28 ℃ and inducing time was 6 h,the concentration of IPTG was 0.4 mmol/L.The expression quantity of target protein attained 40.3% in total bacterial protein, and the soluble fusion protein accounted for 66.2% in total target proteins at the optimum conditions.Conclusion The optimized fermentation conditions of expressing fusion protein in recombinant bacteria have been found and in the conditions, soluble esterase B1 fusion protein of high yield can be obtained.