Apparent Melting Temperature Research Articles

Probing protein stability and proteolytic resistance by loop scanning: A comprehensive mutational analysis

Improvement in protein thermostability was often found to be associated with increase in its proteolytic resistance as revealed by comparative studies of homologous proteins from extremophiles or mutational studies. Structural elements of protein responsible for this association are not firmly established although loops are implicated indirectly due to their structural role in protein stability. To get a better insight, a detailed study of protein wide mutants and their influence on stability and proteolytic resistance would be helpful. To generate such a data set, a model protein, Bacillus subtilis lipase was subjected to loop scanning site-saturation mutagenesis on 86 positions spanning all loops including termini. Upon screening of ~16,000 clones, 17 single mutants with improved thermostability were identified with increment in apparent melting temperature (Tm(app) ) by 1-6°C resulting in an increase in free energy of unfolding (ΔG(unf) ) by 0.04-1.16 kcal/mol. Proteolytic resistance of all single mutants upon incubation with nonspecific protease, Subtilisin A, was determined. Upon comparison, post-proteolysis residual activities as well as kinetics of proteolysis of mutants showed excellent correlation with ΔG(unf) , (r > 0.9), suggesting that proteolysis was strongly correlated with the global stability of this protein. This significant correlation in this set, with least possible sequence changes (single aa substitution), while covering >60% of protein surface strongly argues for the covariance of these two variables. Compared to studies from extremophiles, with large sequence heterogeneity, the observed correlation in such a narrow sequence space (ΔΔG(unf) = 1.57 kcal⁻¹) justifies the robustness of this relation.

Miscibility of Polymer Blends Comprising Poly (Ethylene Oxide) - Epoxidized Natural Rubber

Miscibilty of thermoplastic-elastomer blends comprising poly(ethylene oxide) (PEO) and epoxidized natural rubber with 25 mol% epoxide level (ENR-25) were studied by differential scanning calorimetry (DSC) and polarized optical microscope (POM), respectively. Two glass transition temperatures (Tg), corresponding to those of neat PEO and ENR-25 are observed over the entire composition range. It reflects the immiscibility of the two constituents in the molten state. Hoffman-Weeks method was employed to determine the equilibrium melting temperatures (Tm o ) for the PEO/ENR-25 blends. No systematic variations of Tm o and equivalently the apparent melting temperature (Tm) with ascending ENR-25 content for the blends are detected. However, quantities Tm increase with ascending isothermal crystallization temperature (Tc). There is no melting transition of PEO in PEO/ENR-25 blends when the ENR-25 content in the blend > 50 wt%. Reciprocal half time (t1/2 -1 ) is applied to characterize the isothermal crystallization kinetics of the phase-separated blends. The rate of crystallization and equivalently the spherulite growth rate (G) of PEO decreases with increasing Tc but exhibit no systematic variations with ascending ENR-25 content. No crystallization is observed for the blends when the PEO content is ≤ 50 and  40 wt% when analyzed using DSC and POM, respectively. The birefringence Maltese cross structure of PEO appears deformed with irregular border and coarse texture when the amount of ENR-25 in the blend  40 wt%.

A thermal stability assay can help to estimate the crystallization likelihood of biological samples

The identification of crystallization conditions for biological molecules largely relies on a trial-and-error process in which a number of parameters are explored in large screening experiments. Currently, construct design and sample formulation are recognized as critical variables in this process and often a number of protein variants are assayed for crystallization either sequentially or in parallel, which adds complexity to the screening process. Significant effort is dedicated to sample characterization and quality-control experiments in order to identify at an early stage and prioritize those samples which would be more likely to crystallize. However, large-scale studies relating crystallization success to sample properties are generally lacking. In this study, the thermal stability of 657 samples was estimated using a simplified Thermofluor assay. These samples were also subjected to automated vapour-diffusion crystallization screening under a constant protocol. Analysis of the data shows that samples with an apparent melting temperature (T(m)) of 318 K or higher crystallized in 49% of cases, while the crystallization success rate decreased rapidly for samples with lower T(m). Only 23% of samples with a T(m) below 316 K produced crystals. Based on this analysis, a simple method for estimation of the crystallization likelihood of biological samples is proposed. This method is easy, rapid and consumes very small amounts of sample. The results of this assay can be used to determine optimal incubation temperatures for crystallization experiments or to prioritize certain constructs. More generally, this work provides an objective test that can contribute to making decisions in both focused and structural genomics crystallography projects.

Thermal protein unfolding in photo-activated adenylate cyclase nano-clusters from the amoeboflagellate Naegleria gruberi NEG-M strain

The photo-activated adenylate cyclase (nPAC) protein from the amoeboflagellate Naegleria gruberi NEG-M strain consists of a BLUF domain (sensor of blue light using flavin) and a cyclase homology domain (CHD). The nPAC thermal stability is determined by its protein unfolding behavior which is quantified by the protein melting temperature and protein melting time. The protein unfolding in nPAC nano-clusters in aqueous solution at pH 7.5 is studied by light attenuation and fluorescence measurements. The temporal behavior of protein unfolding (denaturation) is monitored by observation of spectral changes of the first absorption band of the flavin cofactor. The nPAC unfolding occurs irreversible in a bi-exponential manner (different melting time constants for proteins at nano-cluster surface and in nano-cluster interior). The nPAC apparent melting temperature (there half of the proteins are unfolded) is determined by light attenuation measurement (light scattering increases due to coalescing of unfolded protein nano-clusters) in the non-absorbing spectral region of the protein. A measurement standard is developed employing a staircase temperature heating and cooling profile. High thermal stability of nPAC nano-clusters in pH 7.5 aqueous solution was found with an apparent melting temperature of 60 °C.

Improvement in the Thermostability of d -Psicose 3-Epimerase from Agrobacterium tumefaciens by Random and Site-Directed Mutagenesis

The S213C, I33L, and I33L S213C variants of D-psicose 3-epimerase from Agrobacterium tumefaciens, which were obtained by random and site-directed mutagenesis, displayed increases of 2.5, 5, and 7.5°C in the temperature for maximal enzyme activity, increases of 3.3-, 7.2-, and 29.9-fold in the half-life at 50°C, and increases of 3.1, 4.3, and 7.6°C in apparent melting temperature, respectively, compared with the wild-type enzyme. Molecular modeling suggests that the improvement in thermostability in these variants may have resulted from increased putative hydrogen bonds and formation of new aromatic stacking interactions. The immobilized wild-type enzyme with and without borate maintained activity for 8 days at a conversion yield of 70% (350 g/liter psicose) and for 16 days at a conversion yield of 30% (150 g/liter psicose), respectively. After 8 or 16 days, the enzyme activity gradually decreased, and the conversion yields with and without borate were reduced to 22 and 9.6%, respectively, at 30 days. In contrast, the activities of the immobilized I33L S213C variant with and without borate did not decrease during the operation time of 30 days. These results suggest that the I33L S213C variant may be useful as an industrial producer of D-psicose.

Improving the Resistance of a Eukaryotic β-Barrel Protein to Thermal and Chemical Perturbations

β-Barrel membrane proteins have regular structures with extensive hydrogen-bond networks between their transmembrane (TM) β-strands, which stabilize their protein fold. Nevertheless, weakly stable TM regions, which are important for the protein function and interaction with other proteins, exist. Here, we report on the apparent stability of human Tom40A, a member of the “mitochondrial porin family” and main constituent of the mitochondrial protein-conducting channel TOM ( translocase of the outer membrane). Using a physical interaction model, TmSIP, for β-barrel membrane proteins, we have identified three unfavorable β-strands in the TM domain of the protein. Substitution of key residues inside these strands with hydrophobic amino acids results in a decreased sensitivity of the protein to chemical and/or thermal denaturation. The apparent melting temperature observed when denatured at a rate of 1 °C per minute is shifted from 73 to 84 °C. Moreover, the sensitivity of the protein to denaturant agents is significantly lowered. Further, we find a reduced tendency for the mutated protein to form dimers. We propose that the identified weakly stable β-strands 1, 2 and 9 of human Tom40A play an important role in quaternary protein–protein interactions within the mammalian TOM machinery. Our results show that the use of empirical energy functions to model the apparent stability of β-barrel membrane proteins may be a useful tool in the field of nanopore bioengineering.

Thermodynamic characterization of muscle tropomyosins from marine invertebrates

Structural properties of invertebrate muscle tropomyosin (TM) have not been characterized in detail to date. TMs were thus purified from the mantle muscle of Japanese common squid Todarodes pacificus, the foot muscle of tokobushi abalone Haliotis diversicolor and the tail muscle of kuruma prawn Marsupenaeus japonicus, and investigated for their thermodynamic properties by circular dichroism (CD) spectrometry and differential scanning calorimetry (DSC). From the CD spectrometry data, the apparent melting temperature and the apparent free energy of unfolding at 20°C were calculated to be 43.5°C and 14.5 kJ/mol for the squid TM, 43.0°C and 23.9 kJ/mol for the abalone TM, and 47.3°C and 50.2 kJ/mol for the prawn TM, respectively. From the DSC data, the total free energy of unfolding at 20°C was calculated to be 129 kJ/mol, 253 kJ/mol, and 271 kJ/mol for the squid, abalone, and prawn TMs, respectively. These results suggest that the thermal stability was in the order of prawn TM>abalone TM>squid TM.

Structural Basis of Poly(ADP-ribose) Recognition by the Multizinc Binding Domain of Checkpoint with Forkhead-associated and RING Domains (CHFR)

Cellular stress in early mitosis activates the antephase checkpoint, resulting in the decondensation of chromosomes and delayed mitotic progression. Checkpoint with forkhead-associated and RING domains (CHFR) is central to this checkpoint, and its activity is ablated in many tumors and cancer cell lines through promoter hypermethylation or mutation. The interaction between the PAR-binding zinc finger (PBZ) of CHFR and poly(ADP-ribose) (PAR) is crucial for a functional antephase checkpoint. We determined the crystal structure of the cysteine-rich region of human CHFR (amino acids 425-664) to 1.9 Å resolution, which revealed a multizinc binding domain of elaborate topology within which the PBZ is embedded. The PBZ of CHFR closely resembles the analogous motifs from aprataxin-like factor and CG1218-PA, which lie within unstructured regions of their respective proteins. Based on co-crystal structures of CHFR bound to several different PAR-like ligands (adenosine 5'-diphosphoribose, adenosine monophosphate, and P(1)P(2)-diadenosine 5'-pyrophosphate), we made a model of the CHFR-PAR interaction, which we validated using site-specific mutagenesis and surface plasmon resonance. The PBZ motif of CHFR recognizes two adenine-containing subunits of PAR and the phosphate backbone that connects them. More generally, PBZ motifs may recognize different numbers of PAR subunits as required to carry out their functions.

The RNA polymerase subunits E/F from the Antarctic archaeon Methanococcoides burtonii bind to specific species of mRNA

RNA polymerase in Archaea is composed of 11 or 12 subunits - 9 or 10 that form the core, and a heterodimer formed from subunits E and F that associates with the core and can interact with general transcription factors and facilitate transcription. While the ability of the heterodimer to bind RNA has been demonstrated, it has not been determined whether it can recognize specific RNA targets. In this study we used a recombinant archaeal MbRpoE/F to capture cellular mRNA in vitro and a microarray to determine which transcripts it specifically binds. Only transcripts for 117 genes (4% of the total) representing 48 regions of the genome were bound by MbRpoE/F. The transcripts represented important genes in a number of functional classes: methanogenesis, cofactor biosynthesis, nucleotide metabolism, transcription, translation, import/export. The arrangement and characteristics (e.g. codon and amino acid usage) of genes relative to the putative origin of replication indicate that MbRpoE/F preferentially binds to mRNA of genes whose expression may be important for cellular fitness. We also compared the biophysical properties of RpoE/F from M. burtonii and Methanocaldococcus jannaschii, demonstrating a 50°C difference in their apparent melting temperatures. By using MbRpoE/F to capture and characterize cellular RNA we have identified a previously unknown functional property of the MbRpoE/F heterodimer.

Characterization of cytochrome P450 monooxygenase CYP154H1 from the thermophilic soil bacterium Thermobifida fusca

Cytochrome P450 monooxygenases are valuable biocatalysts due to their ability to hydroxylate unactivated carbon atoms using molecular oxygen. We have cloned the gene for a new cytochrome P450 monooxygenase, named CYP154H1, from the moderately thermophilic soil bacterium Thermobifida fusca. The enzyme was overexpressed in Escherichia coli at up to 14% of total soluble protein and purified to homogeneity in three steps. CYP154H1 activity was reconstituted using putidaredoxin reductase and putidaredoxin from Pseudomonas putida DSM 50198 as surrogate electron transfer partners. In biocatalytic reactions with different aliphatic and aromatic substrates of varying size, the enzyme converted small aromatic and arylaliphatic compounds like ethylbenzene, styrene, and indole. Furthermore, CYP154H1 also accepted different arylaliphatic sulfides as substrates chemoselectively forming the corresponding sulfoxides and sulfones. The enzyme is moderately thermostable with an apparent melting temperature of 67°C and exhibited still 90% of initial activity after incubation at 50°C.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-010-2965-9) contains supplementary material, which is available to authorized users.

Thermostability of striated and smooth adductor muscle tropomyosins from Yesso scallop Mizuhopecten yessoensis

The striated and smooth adductor muscle tropomyosins (TMs) of Yesso scallop Mizuhopecten yessoensis have been known to express different isoforms, but have not been characterized in detail to date. In the present study, TMs from both muscles of Yesso scallop were purified and their stabilities were compared by circular dichroism (CD) spectrometry and differential scanning calorimetry (DSC). From the CD data, the apparent melting temperature and the apparent free energy of folding at 20 degrees C were calculated to be 30.5 degrees C and -13.4 kJ/mol, and 36.0 degrees C and -31.9 kJ/mol for the striated and smooth muscle TMs, respectively. From the DSC data, DeltaH values were calculated to be 1.87 x 10(3) and 2.19 x 10(3) kJ/mol for striated and smooth muscle TMs. These results suggest that smooth muscle TM has higher thermostability than striated muscle TM. The amino acid residues responsible for such stability difference were considered to be the six amino acid substitutions in the middle region of the TM molecules.

Dependence of melting, roughness and contact resistances on Ge and Ni content in alloyed AuGe/Ni/Au-type electrical contacts to GaAs/AlGaAs multilayer structures

Annealed AuGe/Ni/Au film structures on GaAs/AlGaAs multilayers have been examined for contact resistance, roughness, magnetization and melting as functions of anneal temperature, Ni-layer thickness and three AuGe compositions. Magnetization data indicate that a solid state, solubility-limited dissolution of Ni into AuGe takes place even for low-temperature anneals and that this dissolution is complete when alloying occurs at ∼400 °C. An apparent melting temperature, detected in differential scanning calorimetry, increases with increasing Ni-layer thickness and decreasing Ge content in the AuGe alloy. Electrical contact formation and roughening of the surface occur in the range of melting temperatures of the structure. The eutectic alloy with a Ni-layer thickness of ∼25–30 nm gives the optimum contact resistance. The contact resistance can be traded off for the reduction in roughness by either increasing the Ni-layer thickness or reducing the Ge content, with the latter being the better choice of the two. The temperature dependence (4–300 K) of the contact resistance shows indications of both thermionic and tunneling behaviors. The barrier height for the current conduction increases with the increase of the Ni-layer thickness and a decrease of the Ge content in the AuGe layer, relative to that of the structure with optimum contact resistance.

Generic Approach for the Generation of Stable Humanized Single-chain Fv Fragments from Rabbit Monoclonal Antibodies

Despite their favorable pharmacokinetic properties, single-chain Fv antibody fragments (scFvs) are not commonly used as therapeutics, mainly due to generally low stabilities and poor production yields. In this work, we describe the identification and optimization of a human scFv scaffold, termed FW1.4, which is suitable for humanization and stabilization of a broad variety of rabbit antibody variable domains. A motif consisting of five structurally relevant framework residues that are highly conserved in rabbit variable domains was introduced into FW1.4 to generate a generically applicable scFv scaffold, termed FW1.4gen. Grafting of complementarity determining regions (CDRs) from 15 different rabbit monoclonal antibodies onto FW1.4 and their derivatives resulted in humanized scFvs with binding affinities in the range from 4.7 x 10(-9) to 1.5 x 10(-11) m. Interestingly, minimalistic grafting of CDRs onto FW1.4gen, without any substitutions in the framework regions, resulted in affinities ranging from 5.7 x 10(-10) to <1.8 x 10(-12) m. When compared with progenitor rabbit scFvs, affinities of most humanized scFvs were similar. Moreover, in contrast to progenitor scFvs, which were difficult to produce, biophysical properties of the humanized scFvs were significantly improved, as exemplified by generally good production yields in a generic refolding process and by apparent melting temperatures between 53 and 86 degrees C. Thus, minimalistic grafting of rabbit CDRs on the FW1.4gen scaffold presents a simple and reproducible approach to humanize and stabilize rabbit variable domains.

Hypocrea jecorina CEL6A protein engineering.

The complex technology of converting lignocellulose to fuels such as ethanol has advanced rapidly over the past few years, and enzymes are a critical component of this technology. The production of effective enzyme systems at cost structures that facilitate commercial processes has been the focus of research for many years. Towards this end, the H. jecorina cellobiohydrolases, CEL7A and CEL6A, have been the subject of protein engineering at Genencor. Our first rounds of cellobiohydrolase engineering were directed towards improving the thermostability of both of these enzymes and produced variants of CEL7A and CEL6A with apparent melting temperatures above 70°C, placing their stability on par with that of H. jecorina CEL5A (EG2) and CEL3A (BGL1). We have now moved towards improving CEL6A- and CEL7A-specific performance in the context of a complete enzyme system under industrially relevant conditions. Achievement of these goals required development of new screening strategies and tools. We discuss these advances along with some results, focusing mainly on engineering of CEL6A.

A Second Look at Mini-Protein Stability: Analysis of FSD-1 Using Circular Dichroism, Differential Scanning Calorimetry, and Simulations

Mini-proteins that contain <50 amino acids often serve as model systems for studying protein folding because their small size makes long timescale simulations possible. However, not all mini-proteins are created equal. The stability and structure of FSD-1, a 28-residue mini-protein that adopted the ββα zinc-finger motif independent of zinc binding, was investigated using circular dichroism, differential scanning calorimetry, and replica-exchange molecular dynamics. The broad melting transition of FSD-1, similar to that of a helix-to-coil transition, was observed by using circular dichroism, differential scanning calorimetry, and replica-exchange molecular dynamics. The N-terminal β-hairpin was found to be flexible. The FSD-1 apparent melting temperature of 41°C may be a reflection of the melting of its α-helical segment instead of the entire protein. Thus, despite its attractiveness due to small size and purposefully designed helix, sheet, and turn structures, the status of FSD-1 as a model system for studying protein folding should be reconsidered.

Adaptive Evolution of p53 Thermodynamic Stability

The thermodynamic stability of a protein plays an important role during evolution and adaptation in order to maintain a folded and active conformation. p53 is a tumour suppressor involved in the regulation of numerous genes. Human p53 has an unusually low thermodynamic stability and is frequently inactivated by oncogenic missense mutations. Here, we examined the thermodynamic and kinetic stability of p53 DNA binding domains from selected invertebrate and vertebrate species by differential scanning calorimetry and equilibrium urea denaturation. There is a correlation in the apparent melting temperature of p53 with the body temperature of homeotherm vertebrates. We found that p53 from these organisms has a half-life for spontaneous unfolding at organismal body temperature of 10–20 min. We also found that p53 from invertebrates has higher stability, bearing more resemblance towards p63 and p73 from humans. Using structure-guided mutagenesis on the human p53 scaffold, we demonstrated that the amino acid changes on the protein surface and in the protein interior lead to the elevated stability of p53 orthologs. We propose a model in which the p53 DNA binding domain has been shaped by the complex interplay of different selective pressures and underwent adaptive evolution leading to pronounced effects on its stability. p53 from vertebrates has evolved to have a low thermodynamic stability and similarly short spontaneous half-life at organismal body temperature, which is related to function.

Gene cloning, expression and characterization of a new cold-active and salt-tolerant endo-β-1,4-xylanase from marine Glaciecola mesophila KMM 241

Although a lot of xylanases are studied, only a few xylanases from marine microorganisms have been reported. A new xylanase gene, xynA, was cloned from marine bacterium Glaciecola mesophila KMM 241. Gene xynA contains 1,272 bp and encodes a 423-amino acid xylanase precursor. The recombinant xylanase, XynA, expressed in Escherichia coli BL21 is a monomer with a molecular mass of 43 kDa. Among the characterized xylanases, XynA shares the highest identity (46%) to the xylanase from Flavobacterium sp. strain MSY2. The optimum pH and temperature for XynA is 7.0 and 30 degrees C. XynA retains 23% activity and 27% catalytic efficiency at 4 degrees C. XynA has low thermostability, remaining 20% activity after 60-min incubation at 30 degrees C. Its apparent melting temperature (T (m)) is 44.5 degrees C. These results indicate that XynA is a cold-active xylanase. XynA shows a high level of salt-tolerance, with the highest activity at 0.5 M NaCl and retaining 90% activity in 2.5 M NaCl. It may be the first salt-tolerant xylanase reported. XynA is a strict endo-beta-1,4-xylanase with a demand of at least four sugar moieties for effective cleavage. It efficiently hydrolyzes xylo-oligosaccharides and xylan into xylobiose and xylotriose without producing xylose, suggesting its potential in xylo-oligosaccharides production.

Cold Adaptation of Zinc Metalloproteases in the Thermolysin Family from Deep Sea and Arctic Sea Ice Bacteria Revealed by Catalytic and Structural Properties and Molecular Dynamics

Increased conformational flexibility is the prevailing explanation for the high catalytic efficiency of cold-adapted enzymes at low temperatures. However, less is known about the structural determinants of flexibility. We reported two novel cold-adapted zinc metalloproteases in the thermolysin family, vibriolysin MCP-02 from a deep sea bacterium and vibriolysin E495 from an Arctic sea ice bacterium, and compared them with their mesophilic homolog, pseudolysin from a terrestrial bacterium. Their catalytic efficiencies, k(cat)/K(m) (10-40 degrees C), followed the order pseudolysin < MCP-02 < E495 with a ratio of approximately 1:2:4. MCP-02 and E495 have the same optimal temperature (T(opt), 57 degrees C, 5 degrees C lower than pseudolysin) and apparent melting temperature (T(m) = 64 degrees C, approximately 10 degrees C lower than pseudolysin). Structural analysis showed that the slightly lower stabilities resulted from a decrease in the number of salt bridges. Fluorescence quenching experiments and molecular dynamics simulations showed that the flexibilities of the proteins were pseudolysin < MCP-02 < E495, suggesting that optimization of flexibility is a strategy for cold adaptation. Molecular dynamics results showed that the ordinal increase in flexibility from pseudolysin to MCP-02 and E495, especially the increase from MCP-02 to E495, mainly resulted from the decrease of hydrogen-bond stability in the dynamic structure, which was due to the increase in asparagine, serine, and threonine residues. Finally, a model for the cold adaptation of MCP-02 and E495 was proposed. This is the first report of the optimization of hydrogen-bonding dynamics as a strategy for cold adaptation and provides new insights into the structural basis underlying conformational flexibility.

Mutating the Tight-Dimer Interface of Dihydrodipicolinate Synthase Disrupts the Enzyme Quaternary Structure: Toward a Monomeric Enzyme

Dihydrodipicolinate synthase (DHDPS) is a tetrameric enzyme that is the first enzyme unique to the ( S)-lysine biosynthetic pathway in plants and bacteria. Previous studies have looked at the important role of Tyr107, an amino acid residue located at the tight-dimer interface between two monomers, in participating in a catalytic triad of residues during catalysis. In this study, we examine the importance of this residue in determining the quaternary structure of the DHDPS enzyme. The Tyr107 residue was mutated to tryptophan, and structural, biophysical, and kinetic studies were carried out on the mutant enzyme. These revealed that while the solid-state structure of the mutant enzyme was largely unchanged, as judged by X-ray crystallography, it exists as a mixture of primarily monomer and tetramer in solution, as determined by analytical ultracentrifugation, size-exclusion chromatography, and mass spectrometry. The catalytic ability of the DHDPS enzyme was reduced by the mutation, which also allowed the adventitious binding of alpha-ketoglutarate to the active site. A reduction in the apparent melting temperature of the mutant enzyme was observed. Thus, the tetrameric quaternary structure of DHDPS is critical to controlling specificity, heat stability, and intrinsic activity.

Unscrambling thermal stability and temperature adaptation in evolved variants of a cold-active lipase

Directed evolution by error-prone PCR was applied to stabilize the cold-active lipase from Pseudomonas fragi (PFL). PFL displays high activity at 10 °C, but it is highly unstable even at moderate temperatures. After two rounds of evolution, a variant was generated with a 5-fold increase in half-life at 42 °C and a shift of 10 °C in the temperature optimum, nevertheless retaining cold-activity. The evolved lipase displayed specific activity higher than the wild type enzyme in the temperature range 29–42 °C. Biophysical measurements did not indicate any obvious difference between the improved variant and the wild type enzyme in terms of loss of secondary structure upon heat treatment, nor a shift in the apparent melting temperature.