Expression and non-chromatographic purification of 1,3-propanediol oxidoreductase in Escherichia coli

The gene dhaT from Klebsiella pneumoniae encodes 1,3-propanediol oxidoreductase (PDOR). Thermally responsive elastin-like polypeptides (ELPs) was used as a fusion tag to purify the proteins (PDOR). The ELP gene was attached to dhaT and ligated into the pET-22b vector. Different NaCl concentrations were employed to decrease the transition temperature (Tt) which was diminished as salt concentration increased. The optimal final concentration of NaCl was 1 M and the corresponding Tt was 39.5\ubaC. Enzymatic assays were determined via every step for purification of fusion PDOR. PDOR showed good stability during the purification process, the specific activity in the first and second round of inverse transition cycling (ITC) was 276.1 \ub1 13.3 and 213.3 \ub1 10.8 U/mg, respectively. The ELPs fusion PDOR was superior to histidine tagged PDOR in both yield and activity after the purification

Biosynthetic bifunctional enzyme complex with high-efficiency luciferin-recycling to enhance the bioluminescence imaging

Firefly luciferase is a prominent reporter on molecular imaging with the advantage of longer wavelength on light emission and the ATP linear correlation, which makes it useful in most of current bioluminescence imaging model. However, the utility of this biomaterial was limited by the signal intensity and stability which are respectively affected by enzyme activity and substrate consumption. This study demonstrated a series of novel synthetic bifunctional enzyme complex of Firefly luciferase (Fluc) and Luciferin-regenerating enzyme (LRE). A peptide linker library was constructed for the fusion strategy on biosynthesis. The findings of both experimental data and structural simulation demonstrated that the intervention of fused LRE remarkably improve the stability of in vitro bioluminescence signal through luciferin recycling; and revealed the competitive relationship of Fluc and LRE on luciferin binding: Fluc performed higher activity with one copy number of rigid linker (EAAAK) at the C terminal while LRE acted more efficiently with two copy numbers of flexible linker (GGGGS) at the N terminal. With the advantage of signal intensity and stability, this fused bifunctional enzyme complex may expand the application of firefly luciferase to in vitro bioluminescence imaging

Codon optimization of 1,3-propanediol oxidoreductase expression in Escherichia coli and enzymatic properties

The gene dhaT from Klebsiella pneumoniae encoding 1,3-propanediol oxidoreductase (PDOR) was de novo synthesized by splicing overlap extension polymerase chain reaction (SOE-PCR) primarily according to Escherichia coli's codon usage, as well as mRNA secondary structure. After optimization, Codon Adaptation Index (CAI) value was improved from 0.75 to 0.83, meanwhile energy of mRNA secondary structure was increased from -400.1 to -86.8 kcal/mol. This synthetic DNA was under control by phage T7 promoter in the expression vector pET-15b and transformed into the E. coli BL21 (DE3) strain. Inducers such as isopropyl \u3b2-D-thiogalactoside (IPTG) and lactose were compared by activity at different inducing time. The activity of PDOR after codon optimized was 385.4 \ub1 3.6 U/mL, which was almost 5-fold higher than wild type (82.3 \ub1 1.5 U/ml) under the flask culture at 25\ub0C for 10 hrs. Then his-tagged enzyme was separated by using Ni-IDA column. The favorite environment for enzyme activity was at 5\ub0C and pH 10.0, PDOR showed a certainly stability in potassium carbonate buffer for 2 hrs at diverse temperatures, enzyme activity was significantly improved by Mn2+

Application of a two-stage temperature control strategy to enhance 1,3-propanediol productivity by Clostridium butyricum

863 Project [2006AA020103]; NNSFC (National Natural Science Foundation of China) [21076172]; University of science and technology in Fujian province [2010H6023]BACKGROUND: The purpose of the present work was to enhance 1,3-propanediol productivity during the batch cultivation on a type of raw glycerol by application of a two-stage temperature control strategy. RESULTS: First, the effect of the raw glycerol on microbial growth and 1,3-propanediol production was investigated. The highest 1,3-propanediol productivity, 1.93 g L1 h1, was achieved when the initial raw glycerol concentration was 6% (v/v). Second, the effect of temperature on microbial growth and 1,3-propanediol production was investigated and kinetic analysis was carried out. The results indicated that 37 degrees C favored microbial growth while 35 degrees C was best for 1,3-propanediol production. Finally, a two-stage temperature control strategy was applied in 1,3-propanediol production. The incubation temperature was kept at 37 degrees C from inoculation to 2 h and then switched to 35 degrees C. Compared with batch cultivations at 35 and 37 degrees C, the fermentation time was shortened from 10 to 9.2 h, resulting in an increase in 1,3-propanediol productivity of around 11%. CONCLUSION: 1,3-propanediol productivity was enhanced effectively by application of a two-stage temperature control strategy. (c) 2012 Society of Chemical Industr

Codon-Optimized NADH Oxidase Gene Expression and Gene Fusion with Glycerol Dehydrogenase for Bienzyme System with Cofactor Regeneration.

NADH oxidases (NOXs) play an important role in maintaining balance of NAD+/NADH by catalyzing cofactors regeneration. The expression of nox gene from Lactobacillus brevis in Escherichia coli BL21 (BL21 (DE3)) was studied. Two strategies, the high AT-content in the region adjacent to the initiation codon and codon usage of the whole gene sequence consistent with the host, obtained the NOX activity of 59.9 U/mg and 73.3 U/mg (crude enzyme), with enhanced expression level of 2.0 and 2.5-folds, respectively. Purified NOX activity was 213.8 U/mg. Gene fusion of glycerol dehydrogenase (GDH) and NOX formed bifuctional multi-enzymes for bioconversion of glycerol coupled with coenzyme regeneration. Kinetic parameters of the GDH-NOX for each substrate, glycerol and NADH, were calculated as Vmax(Glycerol) 20 μM/min, Km(Glycerol) 19.4 mM, Vmax (NADH) 12.5 μM/min and Km (NADH) 51.3 μM, respectively, which indicated the potential application of GDH-NOX for quick glycerol analysis and dioxyacetone biosynthesis

Mechanistic study of manganese-substituted glycerol dehydrogenase using a kinetic and thermodynamic analysis.

Mechanistic insights regarding the activity enhancement of dehydrogenase by metal ion substitution were investigated by a simple method using a kinetic and thermodynamic analysis. By profiling the binding energy of both the substrate and product, the metal ion's role in catalysis enhancement was revealed. Glycerol dehydrogenase (GDH) from Klebsiella pneumoniae sp., which demonstrated an improvement in activity by the substitution of a zinc ion with a manganese ion, was used as a model for the mechanistic study of metal ion substitution. A kinetic model based on an ordered Bi-Bi mechanism was proposed considering the noncompetitive product inhibition of dihydroxyacetone (DHA) and the competitive product inhibition of NADH. By obtaining preliminary kinetic parameters of substrate and product inhibition, the number of estimated parameters was reduced from 10 to 4 for a nonlinear regression-based kinetic parameter estimation. The simulated values of time-concentration curves fit the experimental values well, with an average relative error of 11.5% and 12.7% for Mn-GDH and GDH, respectively. A comparison of the binding energy of enzyme ternary complex for Mn-GDH and GDH derived from kinetic parameters indicated that metal ion substitution accelerated the release of dioxyacetone. The metal ion's role in catalysis enhancement was explicated

Pretreatment of raw glycerol with activated carbon for 1,3-propanediol production by Clostridium butyricum

National "863" Project [2006AA020103]; National Natural Science Foundation of China [21076172]; University of Science and Technology in Fujian province [2010H6023]Raw glycerol contains inhibiting impurities that render its bioconversion difficult. In this work, raw glycerol used in 1,3-propanediol fermentation by Clostridium butyricum was pretreated with activated carbon. Compared with pure glycerol, the untreated raw glycerol inhibited fermentation. Therefore, the raw glycerol was treated using dusty activated carbon as an adsorbent in batch fermentation, resulting in a shortening of the fermentation time from 18.5 to 13.3 h. The effects of pH, dusty activated carbon amount, and adsorption time on the pretreatment results were investigated using uniform design. The pH had a much lower impact and the optimal pretreatment conditions were achieved: pH 3.0, 0.5 g dusty activated carbon/150 g raw glycerol, adsorption time 3 h. Batch cultures on pretreated raw glycerol were comparable to those on pure glycerol. The 1,3-propanediol concentration in fed-batch cultivation was 49.3 g/L on pretreated raw glycerol while it was 40.8 g/L on untreated raw glycerol. The FTIR spectra of dusty activated carbon before and after adsorption indicated that the inhibiting impurities were probably aromatic compounds. This work indicates that pretreatment is necessary to ferment raw glycerol to 1,3-propanediol and that pretreatment with dusty activated carbon is an efficient, economical, and simple method

Expression and non-chromatographic purification of 1,3-propanediol oxidoreductase in Escherichia coli

The gene dhaT from Klebsiella pneumoniae encodes 1,3-propanediol oxidoreductase (PDOR). Thermally responsive elastin-like polypeptides (ELPs) was used as a fusion tag to purify the proteins (PDOR). The ELP gene was attached to dhaT and ligated into the pET-22b vector. Different NaCl concentrations were employed to decrease the transition temperature (Tt) which was diminished as salt concentration increased. The optimal final concentration of NaCl was 1 M and the corresponding Tt was 39.5ºC. Enzymatic assays were determined via every step for purification of fusion PDOR. PDOR showed good stability during the purification process, the specific activity in the first and second round of inverse transition cycling (ITC) was 276.1 ± 13.3 and 213.3 ± 10.8 U/mg, respectively. The ELPs fusion PDOR was superior to histidine tagged PDOR in both yield and activity after the purification

Artificially constructed quorum-sensing circuits are used for subtle control of bacterial population density.

Vibrio fischeri is a typical quorum-sensing bacterium for which lux box, luxR, and luxI have been identified as the key elements involved in quorum sensing. To decode the quorum-sensing mechanism, an artificially constructed cell-cell communication system has been built. In brief, the system expresses several programmed cell-death BioBricks and quorum-sensing genes driven by the promoters lux pR and PlacO-1 in Escherichia coli cells. Their transformation and expression was confirmed by gel electrophoresis and sequencing. To evaluate its performance, viable cell numbers at various time periods were investigated. Our results showed that bacteria expressing killer proteins corresponding to ribosome binding site efficiency of 0.07, 0.3, 0.6, or 1.0 successfully sensed each other in a population-dependent manner and communicated with each other to subtly control their population density. This was also validated using a proposed simple mathematical model