Title: Impact of Salt Concentration and pH on Surface Charged Residues: Controlling Protein Association Pathways in Carboxylesterase EstGtA2 Moisan JK, Meddeb-Mouelhi F, Charbonneau DM, Beauregard M Ref: Protein Pept Lett, 24:561, 2017 : PubMed
BACKGROUND: Understanding the relationship between enzymatic stability and the amino acid sequence encoding carboxylesterases is of utmost importance. OBJECTIVES: Here we thoroughly characterized the behavior of the carboxylesterase EstGtA2 from Geobacillus thermodenitrificans during thermal denaturation at different pH with various salt concentrations. METHOD: EstGtA2 was characterized by circular dichroism regarding conformation and thermal stability, by dynamic light scattering for detection of association/aggregation, by enzymatic assays for activity and by monitoring the impact of heat treatments on activity. RESULTS: Our investigation revealed a particular dependence between aggregation/association and preservation of secondary structures upon heating in EstGtA2. At pH 7, 8 and 9, depending on salt concentration, a folded but non-native associated state characterised by an apparent particle size of 300 nm resisted secondary structure unfolding up to 95 degrees C. CONCLUSION: The paths leading to various aggregative states were found to be controlled by pH (depending on proximity to pI) and to a lesser extent, ionic strength, suggesting that ionic interactions at the surface of the protein are responsible for behavior of EstGtA2. The various paths available to EstGtA2 could be important for protection of Geobacillus termodenitrificans when exposed to heat stress. The understanding and/or control of these paths would allow for optimal use of EstGtA2 in industrial processes.
Title: Characterization of a Novel Alkalophilic Lipase From Aneurinibacillus thermoaerophilus: Lid Heterogeneity and Assignment to Family I.5 Zottig X, Meddeb-Mouelhi F, Charbonneau DM, Beauregard M Ref: Protein J, 36:478, 2017 : PubMed
Recent investigations of Aneurinibacillus thermoaerophilus strains have allowed identification of a unique solvent tolerant lipase, distinct from known lipases. This work reports the expression and purification of this lipase (LipAT) and the first characterization of its structure and temperature and pH-dependent behaviour. LipAT has a secondary structural content compatible with the canonical lipase alpha/beta hydrolase fold, and is dimeric at neutral pH. The protein was folded from pH 5 to 10, and association into folded aggregates at pH 7 and 8 likely protected its secondary structures from thermal unfolding. The enzyme was active from 25 to 65 degrees C under neutral pH, but its maximal activity was detected at pH 10 and 45 degrees C. The ability of LipAT to recover from high temperature was investigated. Heating at 70 degrees C and pH 10 followed by cooling prevented the restoration of activity, while similar treatments performed at pH 8 (where folded aggregates may form) allowed recovery of 50% of the initial activity. In silico analyses revealed a high conservation (85% or more) for the main lipase signature sequences in LipAT despite an overall low residue identity (60% identity compared to family I.5 lipases). In contrast, the active site lid region in LipAT is very distinct showing only 25% amino acid sequence identity to other homologous lipases in this region. Comparison of lids among lipases from the I.5 family members and LipAT reveals that this region should be a primary target for elucidation, optimisation and prediction of structure-function relationships in lipases.
Title: Role of key salt bridges in thermostability of G. thermodenitrificans EstGtA2: distinctive patterns within the new bacterial lipolytic enzyme family XV Charbonneau DM, Beauregard M Ref: PLoS ONE, 8:e76675, 2013 : PubMed
Bacterial lipolytic enzymes were originally classified into eight different families defined by Arpigny and Jaeger (families I-VIII). Recently, the discovery of new lipolytic enzymes allowed for extending the original classification to fourteen families (I-XIV). We previously reported that G. thermodenitrificans EstGtA2 (access no. AEN92268) belonged to a novel group of bacterial lipolytic enzymes. Here we propose a 15(th) family (family XV) and suggest criteria for the assignation of protein sequences to the N' subfamily. Five selected salt bridges, hallmarks of the N' subfamily (E3/R54, E12/R37, E66/R140, D124/K178 and D205/R220) were disrupted in EstGtA2 using a combinatorial alanine-scanning approach. A set of 14 (R/K-->A) mutants was produced, including five single, three double, three triple and three quadruple mutants. Despite a high tolerance to non-conservative mutations for folding, all the alanine substitutions were destabilizing (decreasing T m by 5 to 14 degrees C). A particular combination of four substitutions exceeded this tolerance and prevents the correct folding of EstGtA2, leading to enzyme inactivation. Although other mutants remain active at low temperatures, the accumulation of more than two mutations had a dramatic impact on EstGtA2 activity at high temperatures suggesting an important role of these conserved salt bridge-forming residues in thermostability of lipolytic enzymes from the N' subfamily. We also identified a particular interloop salt bridge in EstGtA2 (D194/H222), located at position i -2 and i -4 residues from the catalytic Asp and His respectively which is conserved in other related bacterial lipolytic enzymes (families IV and XIII) with high tolerance to mutations and charge reversal. We investigated the role of residue identity at position 222 in controlling stability-pH dependence in EstGtA2. The introduction of a His to Arg mutation led to increase thermostability under alkaline pH. Our results suggest primary targets for optimization of EstGtA2 for specific biotechnological purposes.
BACKGROUND:
Biodiesels are methyl esters of fatty acids that are usually produced by base catalyzed transesterification of triacylglyerol with methanol. Some lipase enzymes are effective catalysts for biodiesel synthesis and have many potential advantages over traditional base or acid catalyzed trasesterification. Natural lipases are often rapidly inactivated by the high methanol concentrations used for biodiesel synthesis, however, limiting their practical use. The lipase from Proteus mirabilis is a particularly promising catalyst for biodiesel synthesis as it produces high yields of methyl esters even in the presence of large amounts of water and expresses very well in Escherichia coli. However, since the Proteus mirabilis lipase is only moderately stable and methanol tolerant, these properties need to be improved before the enzyme can be used industrially.
RESULTS:
We employed directed evolution, resulting in a Proteus mirabilis lipase variant with 13 mutations, which we call Dieselzyme 4. Dieselzyme 4 has greatly improved thermal stability, with a 30-fold increase in the half-inactivation time at 50[degree sign]C relative to the wild-type enzyme. The evolved enzyme also has dramatically increased methanol tolerance, showing a 50-fold longer half-inactivation time in 50% aqueous methanol. The immobilized Dieselzyme 4 enzyme retains the ability to synthesize biodiesel and has improved longevity over wild-type or the industrially used Brukholderia cepacia lipase during many cycles of biodiesel synthesis. A crystal structure of Dieselzyme 4 reveals additional hydrogen bonds and salt bridges in Dieselzyme 4 compared to the wild-type enzyme, suggesting that polar interactions may become particularly stabilizing in the reduced dielectric environment of the oil and methanol mixture used for biodiesel synthesis.
CONCLUSIONS:
Directed evolution was used to produce a stable lipase, Dieselzyme 4, which could be immobilized and re-used for biodiesel synthesis. Dieselzyme 4 outperforms the industrially used lipase from Burkholderia cepacia and provides a platform for still further evolution of desirable biodiesel production properties.
Ten thermophilic bacterial strains were isolated from manure compost. Phylogenetic analysis based on 16S rRNA genes and biochemical characterization allowed identification of four different species belonging to four genera: Geobacillus thermodenitrificans, Bacillus smithii, Ureibacillus suwonensis and Aneurinibacillus thermoaerophilus. PCR-RFLP profiles of the 16S-ITS-23S rRNA region allowed us to distinguish two subgroups among the G. thermodenitrificans isolates. Isolates were screened for thermotolerant hydrolytic activities (60-65 degrees C). Thermotolerant lipolytic activities were detected for G. thermodenitrificans, A. thermoaerophilus and B. smithii. Thermotolerant protease, alpha-amylase and xylanase activities were also observed in the G. thermodenitrificans group. These species represent a source of potential novel thermostable enzymes for industrial applications.
Title: N-terminal purification tag alters thermal stability of the carboxylesterase EstGtA2 from G. thermodenitrificans by impairing reversibility of thermal unfolding Charbonneau DM, Meddeb-Mouelhi F, Beauregard M Ref: Protein Pept Lett, 19:264, 2012 : PubMed
The novel thermostable carboxylesterase EstGtA2 from G. thermodenitrificans (accession no. AEN92268) was functionally expressed and purified using an N-terminal fusion tag peptide. We recently reported general properties of the recombinant enzyme. Here we report preliminary data on thermal stability of EstGtA2 and of its tagged form. Conformational stability was investigated using circular dichroism and correlated with residual activity measurements using a colorimetric assay. The tag peptide had no considerable impact on the apparent melting temperature: T(m) value = 64.8 degrees C (tagged) and 65.7 degrees C (cleaved) at pH 8. After thermal unfolding, the tag-free enzyme rapidly recovered initial activity at 25 degrees C (1.2 Umg(-1)), which was corroborated by substantial refolding (83%) as determined by far-UV CD transitions. However, after thermal unfolding, the purification tag drastically decreased specific activity at 25 degrees C (0.07 Umg(-1)). This was corroborated by the absence of refolding transition. Although the purification tag has no undesirable impact on activity before thermal unfolding as well as on Tm, it drastically hinders EstGtA2 refolding resulting in a major loss of thermal stability.
Title: A novel thermostable carboxylesterase from Geobacillus thermodenitrificans: evidence for a new carboxylesterase family Charbonneau DM, Meddeb-Mouelhi F, Beauregard M Ref: J Biochem, 148:299, 2010 : PubMed
A novel gene encoding an esterase from Geobacillus thermodenitrificans strain CMB-A2 was cloned, sequenced and functionally expressed in Escherichia coli M15. Sequence analysis revealed an open reading frame of 747 bp corresponding to a polypeptide of 249 amino acid residues (named EstGtA2). After purification, a specific activity of 2.58 U mg(-1) was detected using p-NP caprylate (C8) at 50 degrees C and pH 8.0 (optimal conditions). The enzyme catalyses the hydrolysis of triglycerides (tributyrin) and a variety of p-nitrophenyl esters with different fatty acyl chain length (C4-C16). The enzyme has potential for various industrial applications since it is characterized by its activity under a wide range of pH, from 25 to 65 degrees C. Using Geobacillus stearothermophilus Est30 esterase structure as template, a model of EstGtA2 was built using ESyPred3D. Analysis of this structural model allowed identifying putative sequence features that control EstGtA2 enzymatic properties. Based on sequence properties, multiple sequence comparisons and phylogenetic analyses, this enzyme appears to belong to a new family of carboxylesterases.
