scholarly journals Production of d-Tagatose by Whole-Cell Conversion of Recombinant Bacillus subtilis in the Absence of Antibiotics

Biology ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1343
Author(s):  
Xian Zhang ◽  
Ruiqi Lu ◽  
Qiang Wang ◽  
Mengkai Hu ◽  
Zhiyue Li ◽  
...  
Keyword(s):  
Heterologous Gene Expression ◽  
Maximum Yield ◽  
Alanine Racemase ◽  
Lacz Gene ◽  
Whole Cell ◽  
Cell Conversion ◽  
One Step ◽  
Overlapping Extension Pcr ◽  
Recombinant Bacillus Subtilis ◽  
Arabinose Isomerase

d-tagatose is a popular functional monosaccharide produced from lactose by β-galactosidase and arabinose isomerase. In this study, two d-alanine-deficient heterologous gene expression systems were constructed, B. subtilis 168 D1 and B. subtilis 168 D2, using overlapping extension PCR and the CRE/loxP system. The lacZ gene for β-galactosidase was integrated into a specific locus of the chassis B. subtilis 168 D2. A mutually complementary plasmid pMA5 with the alanine racemase gene alrA attached to it was constructed and used to assemble recombinant plasmids overexpressing β-galactosidase and arabinose isomerase. Afterward, an integrated recombinant was constructed by the plasmid expressing the arabinose isomerase gene araA of E. coli transform-competent B. subtilis 168 D2 cells. The co-expressing plasmids were introduced into alanine racemase knockout B. subtilis 168 D1. Whole-cell bioconversion was performed using the integrated recombinant with a maximum yield of 96.8 g/L d-tagatose from 500 g/L lactose, and the highest molar conversions were 57.2%. B. subtilis 168 D1/pMA5-alrA-araA-lacZ is capable of single-cell one-step production of d-tagatose. This study provides a new approach to the production of functional sugars.

scholarly journals Optimized recombinant Bacillus Subtilis 168 whole-cell catalyzes one-step biosynthesis of high fructose syrup

2021 ◽  
Vol 6 (2) ◽  
pp. 067-075
Author(s):  
Ildephonse Habimana ◽  
Qiao Zhina ◽  
Aqeel Sahibzada Muhammad ◽  
Jean Damascene Harindintwali ◽  
Al-Adeeb Abdulquader ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Whole Cell ◽  
Bacillus Subtilis 168 ◽  
High Fructose ◽  
One Step ◽  
High Fructose Syrup ◽  
Recombinant Bacillus Subtilis

Microbiological transformations 35: Enantioselective one-step preparative scale synthesis of 1,3-dithiane-1-oxide via whole-cell bacterial oxidation

1996 ◽  
Vol 37 (34) ◽  
pp. 6117-6120 ◽  
Author(s):  
Véronique Alphand ◽  
Nicoletta Gaggero ◽  
Stefano Colonna ◽  
Roland Furstoss
Keyword(s):  
Bacterial Oxidation ◽  
Preparative Scale ◽  
Whole Cell ◽  
One Step

Synthesis of the Carboxyl and Pyridine Functional Ionic Liquids

2014 ◽  
Vol 513-517 ◽  
pp. 246-250 ◽  
Author(s):  
Ying Xiong ◽  
Min Yang
Keyword(s):  
Ionic Liquids ◽  
Reaction Time ◽  
Anion Exchange ◽  
Room Temperature ◽  
Exchange Resin ◽  
Anion Exchange Resin ◽  
Maximum Yield ◽  
Step Method ◽  
Strong Base ◽  
One Step

The effects of different solvents on synthesis of base functional ionic liquid, butyl pyridinium hydroxide ([bPy]OH), from butyl pyridinium bromine ([bPy]Br) were investigated systematically using KOH/NaOH as the base agent and strong base anion exchange resin. The results showed that the yield of [bPy]OH achieved 35% with the molar ratio of 1:1.1 ([bPy]Br to NaOH) using dichloromethane under room temperature. With isopropanol and 8 h of the reaction time, the yield could reach 88% with byproducts. The yield of 97% without byproduct was achieved by using strong base anion exchange resin in column chromatography static reaction for 0.25 h. The yield of carboxyl and pyridine functional ionic liquids based on neutralization method, exchange method and one-step method were compared and the results showed that the one-step method possessed the maximum yield of 88% with 3 h of the reaction time at room temperature.

Optical fiber bioluminescent whole-cell microbial biosensors to genotoxicants

2000 ◽  
Vol 42 (1-2) ◽  
pp. 305-311 ◽  
Author(s):  
B. Polyak ◽  
E. Bassis ◽  
A. Novodvorets ◽  
S. Belkin ◽  
R.S. Marks
Keyword(s):  
Optical Fiber ◽  
Optical Fiber Sensor ◽  
Photon Counting ◽  
Genetically Engineered ◽  
Whole Cell ◽  
Light Production ◽  
Dna Damaging Agents ◽  
Sensor Module ◽  
One Step ◽  
Dose Dependent

Conservation of water resources calls for ever stricter regulatory measures and better monitoring systems. Whole-cell bacterial sensors have been genetically engineered to react to target toxicants by the induction of a selected promoter and the subsequent production of bioluminescent light through a recombinant lux reporter. In order to create a one-step assay, we have designed a new, self-contained, disposable optical fiber sensor module and a customized photodetector system that integrates these microorganisms. A photon-counting photomultiplier tube-based instrument was constructed. Optical fiber tip cores were covered with adlayer films consisting of calcium alginate containing bioluminescent bacterial sensors of genotoxicants. Multiplying these steps thickened the adlayer in increments, increasing the number of bacterial reporters attached to the optical fiber transducer. These whole cell optrodes are responsive to external traces of DNA damaging agents such as mitomycin C. Light production was shown to be dose-dependent and proportional to the number of bacterial layers.

scholarly journals Differential Selectivity of the Escherichia coli Cell Membrane Shifts the Equilibrium for the Enzyme-Catalyzed Isomerization of Galactose to Tagatose

2008 ◽  
Vol 74 (8) ◽  
pp. 2307-2313 ◽  
Author(s):  
Jin-Ha Kim ◽  
Byung-Chul Lim ◽  
Soo-Jin Yeom ◽  
Yeong-Su Kim ◽  
Hye-Jung Kim ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Membrane ◽  
Gene Knockout ◽  
Equilibrium Constants ◽  
Isomerization Reaction ◽  
Whole Cells ◽  
Cell Conversion ◽  
Equilibrium Shift ◽  
Differential Selectivity ◽  
Arabinose Isomerase

ABSTRACT An Escherichia coli galactose kinase gene knockout (ΔgalK) strain, which contains the l-arabinose isomerase gene (araA) to isomerize d-galactose to d-tagatose, showed a high conversion yield of tagatose compared with the original galK strain because galactose was not metabolized by endogenous galactose kinase. In whole cells of the ΔgalK strain, the isomerase-catalyzed reaction exhibited an equilibrium shift toward tagatose, producing a tagatose fraction of 68% at 37°C, whereas the purified l-arabinose isomerase gave a tagatose equilibrium fraction of 36%. These equilibrium fractions are close to those predicted from the measured equilibrium constants of the isomerization reaction catalyzed in whole cells and by the purified enzyme. The equilibrium shift in these cells resulted from the higher uptake and lower release rates for galactose, which is a common sugar substrate, than for tagatose, which is a rare sugar product. A ΔmglB mutant had decreased uptake rates for galactose and tagatose, indicating that a methylgalactoside transport system, MglABC, is the primary contributing transporter for the sugars. In the present study, whole-cell conversion using differential selectivity of the cell membrane was proposed as a method for shifting the equilibrium in sugar isomerization reactions.

scholarly journals Characterization and Heterologous Gene Expression of a Novel Esterase from Lactobacillus casei CL96

2004 ◽  
Vol 70 (6) ◽  
pp. 3213-3221 ◽  
Author(s):  
Young J. Choi ◽  
Carlos B. Miguez ◽  
Byong H. Lee
Keyword(s):  
Lactobacillus Casei ◽  
Heterologous Gene Expression ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Catalytic Triad ◽  
Methylotrophic Bacterium ◽  
Methylobacterium Extorquens ◽  
Reading Frame ◽  
One Step ◽  
Novel Esterase

ABSTRACT A novel esterase gene (estI) of Lactobacillus casei CL96 was localized on a 3.3-kb BamHI DNA fragment containing an open reading frame (ORF) of 1,800 bp. The ORF of estI was isolated by PCR and expressed in Escherichia coli, the methylotrophic bacterium Methylobacterium extorquens, and the methylotrophic yeast Pichia pastoris under the control of T7, methanol dehydrogenase (PmxaF ), and alcohol oxidase (AOX1) promoters, respectively. The amino acid sequence of EstI indicated that the esterase is a novel member of the GHSMG family of lipolytic enzymes and that the enzyme contains a lipase-like catalytic triad, consisting of Ser325, Asp516, and His558. E. coli BL21(DE3)/pLysS containing estI expressed a novel 67.5-kDa protein corresponding to EstI in an N-terminal fusion with the S � tag peptide. The recombinant L. casei CL96 EstI protein was purified to electrophoretic homogeneity in a one-step affinity chromatography procedure on S-protein agarose. The optimum pH and temperature of the purified enzyme were 7.0 and 37�C, respectively. Among the pNP (p-nitrophenyl) esters tested, the most selective substrate was pNP-caprylate (C8), with Km and k cat values of 14 � 1.08 μM and 1,245 � 42.3 S−1, respectively.

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