Thermostability Of Luciferase Research Articles

Rational Side-Chain Amino Acid Substitution in Firefly Luciferase for Improved Thermostability

Improving the thermostability of the luciferase from firefly (Photinus pyralis) needs to be solved to broaden its industrial applications. In this paper, molecular dynamic (MD) simulations were used to identify 4 amino acid substitutions (P183V, E325K, Q338V, and E354K) which might have a significant influence on the thermostability of luciferase. Root-mean-square deviation values were calculated to further evaluate the effect of these mutations on thermostability of the enzyme and demonstrated that the thermostability of the corresponding protein variants was in the order E354K > E325K > WT > P183V > Q338V. Following the MD simulation, the enzyme variants were expressed in a recombinant host, and the results showed that the t1/2, T50, and Tm of mutant E354K were increased 2.32-fold, and 4.5 and 3.3°C more compared with the wild type, respectively. MD simulations, as well as circular dichroism and fluorescence spectroscopy were further applied to elucidate the conformational differences between the wild-type and E354K luciferases. The results indicated that a possible explanation for the improved thermostability of E354K enzyme lies in the formation of a salt bridge between Lys354 and Glu311 and alteration of protein conformation.

The role of proline substitutions within flexible regions on thermostability of luciferase

Improving the stability of firefly luciferase has been a critical issue for its wider industrial applications. Studies about hyperthermophile proteins show that flexibility could be an effective indicator to find out weak spots to engineering thermostability of proteins. However, the relationship among flexibility, activity and stability in most of proteins is unclear. Proline is the most rigid residue and can be introduced to rigidify flexible regions to enhance thermostability of proteins. We firstly apply three different methods, molecular dynamics (MD) simulation, B-FITTER and framework rigidity optimized dynamics algorithm (FRODA) to determine the flexible regions of Photinus pyralis luciferase: Fragment 197–207; Fragment 471–481 and Fragment 487–495. Then, introduction of proline is used to rigidify these flexible regions. Two mutants D476P and H489P within most flexible regions are finally designed. In the results, H489P mutant shows improved thermostability while maintaining its catalytic efficiency compared to that of wild type luciferase. Flexibility analysis confirms that the overall rigidity and local rigidity of H489P mutant are greatly strengthened. D476P mutant shows decreased thermosatbility and the reason for this is elucidated at the molecular level. S307P mutation is randomly chosen outside the flexible regions as a control. Thermostability analysis shows that S307P mutation has decreased kinetic stability and enhanced thermodynamic stability.

LuxCDABE—Transformed Constitutively Bioluminescent Escherichia coli for Toxicity Screening: Comparison with Naturally Luminous Vibrio fischeri

We show that in vitro toxicity assay based on inhibition of the bioluminescence of recombinant Escherichia coli encoding thermostable luciferase from Photorhabdus luminescens is a versatile alternative to Vibrio fischeri Microtox™ test. Performance of two luxCDABE-transformed E. coli MC1061 constructs (pDNlux) and (pSLlux) otherwise identical, but having 100-fold different background luminescence was compared with the performance of V. fischeri. The microplate luminometer and a kinetic Flash-Assay test format was used that differently from Microtox test is also applicable for high throughput analysis. Toxic effects (30-s till 30-min EC50) of four heavy metals (Zn, Cd, Hg, Cu) and three organic chemicals (aniline, 3,5-dichloroaniline and 3,5-dichlorophenol) were studied. Both E. coli strains had comparable sensitivity and the respective 30-min EC50 values highly correlated (log-log R2 = 0.99; p < 0.01) showing that the sensitivity of the recombinant bacteria towards chemicals analyzed did not depend on the bioluminescence level of the recombinant cells. The most toxic chemical for all used bacterial strains (E. coli, V. fischeri) was mercury whereas the lowest EC50 values for Hg (0.04–0.05 mg/L) and highest EC50 values for aniline (1,300–1,700 mg/L) were observed for E. coli strains. Despite of that, toxicity results obtained with both E. coli strains (pSLlux and pDNlux) significantly correlated with V. fischeri results (log-log R2 = 0.70/0.75; p < 0.05/0.01). The use of amino acids (0.25%) and glucose (0.05%)-supplemented M9 medium instead of leucine-supplemented saline significantly (p < 0.05) reduced the apparent toxicity of heavy metals to both E. coli strains up to three orders of magnitude, but had little or no complexing effect on organic compounds. Thus, P. luminescens luxCDABE-transformed E. coli strains can be successfully used for the acute toxicity screening of various types of organic chemicals and heavy metals and can replace V. fischeri in certain cases where the thermostability of luciferase >30 °C is crucial. The kinetic Flash Assay test format of the bioluminescence inhibition assay facilitates high throughput analysis. The assay medium, especially in case of testing heavy metals should be a compromise: optimal for the viability/luminescence of the recombinant test strain and of minimum complexing potential.

Thermostabilization of protein A-luciferase fusion protein by single amino acid mutation

A gene expression plasmid, pMALU7, for coding a fusion protein between protein A (SpA) and mutated firefly luciferase (Luc), was constructed. The fused gene was expressed in Escherichia coli and the resulting protein (SpA-LucTS) was purified with affinity chromatography. By changing a single amino acid, from Glu to Lys at the position 354 in the luciferase moiety, the thermostability of luciferase was improved.

Thermostabilization of firefly luciferase by a single amino acid substitution at position 217.

Random mutagenesis of the luciferase cDNA from "Genji" firefly, Luciola cruciata, was induced by hydroxylamine in an attempt to isolate thermostable mutants. Three mutants were isolated, and the cDNAs encoding these proteins were sequenced. All mutant cDNAs carried the same C to T transition mutation that conferred an amino acid substitution of Thr by Ile at position 217. The wild-type luciferase and the thermostable variant (Thr217Ile) were purified to homogeneity, and their enzymatic properties were determined. Thr217Ile was superior to wild-type in thermal and pH stability. Furthermore, the specific activity of the Thr217Ile mutant was increased to 130% of that of the wild-type. In order to examine the effect of amino acid residue substitution at position 217 on the thermostability of luciferase, we replaced the Thr residue at position 217 with all of the rest of the possible amino acid residues by site-directed mutagenesis. The thermostability of these substitution mutants seemed to correlate with the hydrophobicity of the substituted amino acid residue.