Activity of Lactobacillus brevis Alcohol Dehydrogenase on Primary and Secondary Alcohol Biofuel Precursors
Abstract
:1. Introduction
2. Experimental Section
2.1. Chemicals
2.2. Microorganisms and Media
2.3. Plasmid Construction
Strain | Genotype or Description | Source |
---|---|---|
E. coli NEB10-beta | araD139∆(ara-leu)7697 fhuA lacX74 galK (ϕ80 ∆(lacZ)M15) mcrA galU recA1 endA1 nupG rpsL (StrR) ∆(mrr-hsdRMS-mcrBC) | New England Biolabs |
E. coli BW25113 | F’ λ− ∆(araD-araB)567, ∆lacZ4787(::rrnB-3), lambda−, rph-1, ∆(rhaD-rhaB)568, hsdR514 | CGSC |
E. coli BW25113(DE3) | λDE3 lysogen of BW25113λ | This study |
L. brevis LB19 | Genetic source of LbADH | CIRM-BIA |
Plasmid | Features | Source |
pACYCDuet-1 | Expression vector, Cmr, PT7, pACYC184 Ori | Novagen |
pACYC- LbADH | LbADH from L. brevis LB19 inserted between NdeI and XhoI sites of pACYCDuet-1 | This study |
pACYC- LbADH-His | LbADH from L. brevis LB19 inserted between BamHI and EcoRI sites of pACYCDuet-1 | This study |
2.4. Whole Cell Conversion of 2-Butanone to 2-Butanol by E. coli Growing Cells
2.5. Whole Cell Conversion of 2-Butanone to 2-Butanol by E. coli Resting Cells
2.6. Metabolite Analyses
2.7. In Vitro Enzyme Assays
2.8. Modeling Enzyme Kinetics
3. Results and Discussion
3.1. Confirming the In Vivo Function of Recombinant LbADH for 2-Butanol Production
3.2. Characterizing the In Vitro Activity of Recombinant LbADH on Short-Chain 2-Alkanones and Aldehydes
Product Alcohol | Substrate | kcat (s−1) | KM (mM) | KI (mM) | kcat/KM (mM−1s−1) |
---|---|---|---|---|---|
2° | Acetone | 1.52 ± 0.01 | 0.88 ± 0.16 | 30.7 ± 6.8 | 1.73 ± 0.05 |
2-Butanone | 0.11 ± 0.01 | 0.10 ± 0.02 | 1.34 ± 0.26 | 1.12 ± 0.28 | |
2-Pentanone | 0.11 ± 0.01 | 0.04 ± 0.01 | 0.68 ± 0.17 | 3.03 ± 1.05 | |
1° | Propionaldehyde | 3.36 ± 0.19 | 3.1 ± 0.7 | N.D. | 1.09 ± 0.25 |
Butyraldehyde | 4.42 ± 0.11 | 0.17 ± 0.02 | N.D. | 25.5 ± 3.5 | |
Valeraldehyde | 0.18 ± 0.01 | 0.12 ± 0.03 | N.D. | 1.57 ± 0.41 |
3.3. Comparing LbADH with Other Bacterial ADHs
Organism Enzyme(s) | Substrate(s) | KM (mM) | kcat (s-1) | kcat/KM (mM−1s−1) | Relative Activity | Reference |
---|---|---|---|---|---|---|
L. brevis LB19 LbADH | 2-butanone | 0.096 | 0.107 | 1.12 | - | This Study |
Clostridium beijerinckii NRRL B593 ADH | acetone 2-butanone | 0.98 1.5 | 139 64.2 | 142 43.3 | - - | [26] |
Rhodococcus sp. GK1 SADH | acetone 2-octanone | 65 2.1 | - - | - - | - - | [21] |
C. acetobutylicum ATCC 824 BDH I BDH II | Butyraldehyde butyraldehyde | 3.6 14 | - - | - - | - - | [25] |
Burkholderia sp. AIU652 ADH | acetone 2-butanone 2-pentanone | 0.065 0.040 - | - - - | - - - | 100% 83% 44% | [19] |
Pseudomonas sp. PED ADH | 2-butanone 2-pentanone | - - | - - | - - | 100% 6% | [20] |
4. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
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Halloum, I.; Thompson, B.; Pugh, S.; Nielsen, D.R. Activity of Lactobacillus brevis Alcohol Dehydrogenase on Primary and Secondary Alcohol Biofuel Precursors. Fermentation 2015, 1, 24-37. https://doi.org/10.3390/fermentation1010024
Halloum I, Thompson B, Pugh S, Nielsen DR. Activity of Lactobacillus brevis Alcohol Dehydrogenase on Primary and Secondary Alcohol Biofuel Precursors. Fermentation. 2015; 1(1):24-37. https://doi.org/10.3390/fermentation1010024
Chicago/Turabian StyleHalloum, Ibrahim, Brian Thompson, Shawn Pugh, and David R. Nielsen. 2015. "Activity of Lactobacillus brevis Alcohol Dehydrogenase on Primary and Secondary Alcohol Biofuel Precursors" Fermentation 1, no. 1: 24-37. https://doi.org/10.3390/fermentation1010024
APA StyleHalloum, I., Thompson, B., Pugh, S., & Nielsen, D. R. (2015). Activity of Lactobacillus brevis Alcohol Dehydrogenase on Primary and Secondary Alcohol Biofuel Precursors. Fermentation, 1(1), 24-37. https://doi.org/10.3390/fermentation1010024