scholarly journals Improving on nature’s shortcomings: evolving a lipase for increased lipolytic activity, expression and thermostability

2019 ◽  
Vol 32 (1) ◽  
pp. 13-24 ◽  
Author(s):  
Ana L Alfaro-Chávez ◽  
Jian-Wei Liu ◽  
Joanne L Porter ◽  
Adrian Goldman ◽  
David L Ollis
Keyword(s):  
Escherichia Coli ◽  
Thermal Stability ◽  
Drosophila Melanogaster ◽  
Lipolytic Activity ◽  
Industrial Applications ◽  
Wild Type ◽  
Expression In Escherichia Coli ◽  
A Value ◽  
Substrate Binding Domain ◽  
Final Round

Abstract An enzyme must be soluble, stable, active and easy to produce to be useful in industrial applications. Not all enzymes possess these attributes. We set out to determine how many changes are required to convert an enzyme with poor properties into one that has useful properties. Lipase Lip3 from Drosophila melanogaster had been previously optimised for expression in Escherichia coli. The expression levels were good, but Lip3 was mainly insoluble with poor activity. Directed evolution was used to identify variants with enhanced activity along with improved solubility. Five variants and the wild-type (wt) enzyme were purified and characterised. The yield of the wt enzyme was just 2.2 mg/L of culture, while a variant, produced under the same conditions, gave 351 mg. The improvement of activity of the best variant was 200 times higher than that of the wt when the crude lysates were analysed using pNP-C8, but with purified protein, the improvement observed was 1.5 times higher. This means that most of the increase of activity is due to increase in solubility and stability. All the purified variants showed increased thermal stability compared with the wt enzyme that had a T1/2 of 37°C, while the mutant with P291L of 42.2°C and the mutant R7_47D with five mutations had a value of 52.9°C, corresponding to an improvement of 16°C. The improved variants had between five and nine changes compared with the wt enzyme. There were four changes that were found in all 30 final round variants for which sequences were obtained; three of these changes were found in the substrate-binding domain.

scholarly journals Expression in Escherichia coli and characterization of a reconstituted recombinant 7Fe ferredoxin from Desulfovibrio africanus

1996 ◽  
Vol 314 (1) ◽  
pp. 63-71 ◽  
Author(s):  
Johanneke L. H. BUSCH ◽  
Jacques L. J. BRETON ◽  
Barry M. BARTLETT ◽  
Richard JAMES ◽  
E. Claude HATCHIKIAN ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Magnetic Circular Dichroism ◽  
Wild Type ◽  
E Coli ◽  
Excess Iron ◽  
Expression In Escherichia Coli ◽  
Type D

Desulfovibrio africanus ferredoxin III is a monomeric protein (molecular mass of 6585 Da) that contains one [3Fe-4S]1+/0 and one [4Fe-4S]2+/1+ cluster when isolated aerobically. The amino acid sequence consists of 61 amino acids, including seven cysteine residues that are all involved in co-ordination to the clusters. In order to isolate larger quantities of D. africanus ferredoxin III, we have overexpressed it in Escherichia coli by constructing a synthetic gene based on the amino acid sequence of the native protein. The recombinant ferredoxin was expressed in E. coli as an apoprotein. We have reconstituted the holoprotein by incubating the apoprotein with excess iron and sulphide in the presence of a reducing agent. The reconstituted recombinant ferredoxin appeared to have a lower stability than that of wild-type D. africanus ferredoxin III. We have shown by low-temperature magnetic circular dichroism and EPR spectroscopy that the recombinant ferredoxin contains a [3Fe-4S]1+/0 and a [4Fe-4S]2+/1+ cluster similar to those found in native D. africanus ferredoxin III. These results indicate that the two clusters have been correctly inserted into the recombinant ferredoxin.

scholarly journals Kinetics and thermodynamics of thermal inactivation for recombinant Escherichia coli cellulases, cel12B, cel8C, and polygalacturonase, peh28; biocatalysts for biofuel precursor production

2020 ◽  
Author(s):  
Eman Ibrahim ◽  
Ahmed Mahmoud ◽  
Kim D Jones ◽  
Keith E Taylor ◽  
Ebtesam N Hosseney ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Thermal Stability ◽  
Lignocellulosic Biomass ◽  
Thermal Inactivation ◽  
Biomass Conversion ◽  
Industrial Applications ◽  
Recombinant Escherichia Coli ◽  
Thermal Stabilities ◽  
Kinetics And Thermodynamics ◽  
Lignocellulosic Biomass Conversion

Abstract Lignocellulosic biomass conversion using cellulases/polygalacturonases is a process that can be progressively influenced by several determinants involved in cellulose microfibril degradation. This article focuses on the kinetics and thermodynamics of thermal inactivation of recombinant Escherichia coli cellulases, cel12B, cel8C and a polygalacturonase, peh 28, derived from Pectobacterium carotovorum sub sp. carotovorum. Several consensus motifs conferring the enzymes’ thermal stability in both cel12B and peh28 model structures have been detailed earlier, which were confirmed for the three enzymes through the current study of their thermal inactivation profiles over the 20–80°C range using the respective activities on carboxymethylcellulose and polygalacturonic acid. Kinetic constants and half-lives of thermal inactivation, inactivation energy, plus inactivation entropies, enthalpies and Gibbs free energies, revealed high stability, less conformational change and protein unfolding for cel12B and peh28 due to thermal denaturation compared to cel8C. The apparent thermal stability of peh28 and cel12B, along with their hydrolytic efficiency on a lignocellulosic biomass conversion as reported previously, makes these enzymes candidates for various industrial applications. Analysis of the Gibbs free energy values suggests that the thermal stabilities of cel12B and peh28 are entropy-controlled over the tested temperature range.

scholarly journals The Periplasmic Protein MppA Requires an Additional Mutated Locus To Repress marA Expression in Escherichia coli

2003 ◽  
Vol 185 (4) ◽  
pp. 1465-1469 ◽  
Author(s):  
Xiaowen Bina ◽  
Vincent Perreten ◽  
Stuart B. Levy
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Antibiotic Susceptibility ◽  
Type Strain ◽  
Wild Type Strain ◽  
Binding Protein ◽  
Periplasmic Protein ◽  
Multiple Antibiotic Resistance ◽  
Wild Type ◽  
Expression In Escherichia Coli

ABSTRACT Escherichia coli strain TP985, which has an insertional mutation in the gene for the periplasmic murein tripeptide binding protein MppA, was previously reported to overproduce MarA and exhibit a multiple-antibiotic resistance (Mar) phenotype (H. Li and J. T. Park, J. Bacteriol. 181:4842-4847, 1999). We found that TP985 contained a previously unrecognized marR mutation which was responsible for the Mar phenotype. Transduction of the mppA mutation from TP985 to another wild-type strain did not affect antibiotic susceptibility. Overproduction of MppA repressed marA transcription in TP985 but not in other mppA or marR mutants. Therefore, TP985 contains an additional unknown mutation(s) that facilitates the repression of marA expression by MppA.

Wild-type and mutant bacterioopsins D85N, D96N, and R82Q: high-level expression in Escherichia coli

Biochemistry ◽  
1991 ◽  
Vol 30 (12) ◽  
pp. 3082-3088 ◽  
Author(s):  
Richard F. Shand ◽  
Larry J. W. Miercke ◽  
Alok K. Mitra ◽  
Susan K. Fong ◽  
Robert M. Stroud ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Wild Type ◽  
High Level Expression ◽  
Expression In Escherichia Coli ◽  
High Level

scholarly journals Improving the Thermostability of a Fungal GH11 Xylanase via Fusion of a Submodule (C2) from Hyperthermophilic CBM9_1-2

2021 ◽  
Vol 23 (1) ◽  
pp. 463
Author(s):  
Huabiao Miao ◽  
Yu Ma ◽  
Yuanyuan Zhe ◽  
Xianghua Tang ◽  
Qian Wu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Md Simulation ◽  
Industrial Applications ◽  
Enzymatic Property ◽  
Optimal Temperature ◽  
Wild Type ◽  
Hybrid Proteins ◽  
Neocallimastix Patriciarum ◽  
Thermal Stability Of ◽  
Additional Hydrogen

Xylanases have been applied in many industrial fields. To improve the activity and thermostability of the xylanase CDBFV from Neocallimastix patriciarum (GenBank accession no. KP691331), submodule C2 from hyperthermophilic CBM9_1-2 was inserted into the N- and/or C-terminal regions of the CDBFV protein (producing C2-CDBFV, CDBFV-C2, and C2-CDBFV-C2) by genetic engineering. CDBFV and the hybrid proteins were successfully expressed in Escherichia coli BL21 (DE3). Enzymatic property analysis indicates that the C2 submodule had a significant effect on enhancing the thermostability of the CDBFV. At the optimal temperature (60.0 °C), the half-lives of the three chimeras C2-CDBFV, CDBFV-C2, and C2-CDBFV-C2 are 1.5 times (37.5 min), 4.9 times (122.2 min), and 3.8 times (93.1 min) longer than that of wild-type CDBFV (24.8 min), respectively. More importantly, structural analysis and molecular dynamics (MD) simulation revealed that the improved thermal stability of the chimera CDBFV-C2 was on account of the formation of four relatively stable additional hydrogen bonds (S42-S462, T59-E277, S41-K463, and S44-G371), which increased the protein structure’s stability. The thermostability characteristics of CDBFV-C2 make it a viable enzyme for industrial applications.

scholarly journals Kinetic analysis of an inhibitor-resistant variant of the OHIO-1 β-lactamase, an SHV-family class A enzyme

1998 ◽  
Vol 333 (2) ◽  
pp. 395-400 ◽  
Author(s):  
Shan LIN ◽  
Mary THOMAS ◽  
David M. SHLAES ◽  
Susan D. RUDIN ◽  
James R. KNOX ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Activation Energy ◽  
Carboxylic Acid ◽  
Active Site ◽  
Wild Type ◽  
Small Energy ◽  
Resistant Variant ◽  
Expression In Escherichia Coli ◽  
Class A ◽  
The Difference

The Met69 → Ile mutant of the OHIO-1 β-lactamase, an SHV-family enzyme, is resistant to inactivation by β-lactamase inhibitors. Analysis of purified Met69 → Ile enzyme reveals that its isoelectric point (pI 7.0) and CD spectrum are identical with those of the OHIO-1 enzyme. Levels of β-lactamase expression in Escherichia coli as determined by immunoblotting are similar for OHIO-1 and Met69 → Ile β-lactamase. The kinetic constants of the Met69 → Ile enzyme compared with OHIO-1 are smaller for benzylpenicillin (Km = 6 µM compared with 17 µM; kcat = 234 s-1 compared with 345 s-1 respectively) and carbenicillin (Km = 3 µM compared with 17 µM; kcat = 131 s-1 compared with 320 s-1 respectively). For the cephalosporins cephaloridine and 7 - (thienyl - 2 - acetamido) - 3 - [2 - (4 - N,N - dimethylaminophenylazo)pyridinium-methyl]-3-cephem-4-carboxylic acid (PADAC), a similar pattern is also seen (Km = 38 µM compared with 96 µM and 6 µM compared with 75 µM respectively; kcat = 235 s-1 compared with 1023 s-1 and 9 s-1 compared with 50 s-1 respectively). Consistent with minimum inhibitory concentrations that show resistance to β-lactam β-lactamase inhibitors, the apparent Ki values, turnover numbers and partition ratios (kcat/kinact) for the mechanism-based inactivators clavulanate, sulbactam and tazobactam are increased. The inactivation rate constants (kinact) are decreased. The difference in activation energy, a measurement of altered affinity for the wild-type and mutant enzymes leading to acylation of the active site, reveals small energy differences of less than 8.4 kJ/mol. In total, these results suggest that the Met → Ile substitution at position 69 in the OHIO-1 β-lactamase alters the active site, primarily affecting the interactions with β-lactamase inhibitors.

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