Thermal Half-life Research Articles

Correction to "Arylazopyrazoles: Azoheteroarene Photoswitches Offering Quantitative Isomerization and Long Thermal Half-Lives.

ADVERTISEMENT RETURN TO ISSUEPREVAddition/CorrectionNEXTORIGINAL ARTICLEThis notice is a correctionCorrection to “Arylazopyrazoles: Azoheteroarene Photoswitches Offering Quantitative Isomerization and Long Thermal Half-LivesClaire E. Weston, Robert D. Richardson, Peter R. Haycock, Andrew J. P. White, and Matthew J. Fuchter*Cite this: J. Am. Chem. Soc. 2016, 138, 33, 10716Publication Date (Web):August 16, 2016Publication History Published online16 August 2016Published inissue 24 August 2016https://doi.org/10.1021/jacs.6b08265Copyright © 2016 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views1814Altmetric-Citations2LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (682 KB) Get e-Alerts Get e-Alerts

Purification and biochemical characterization of novel acidic chitinase from Paenicibacillus barengoltzii

The novel chitinase (PbChi67) from the marine bacterium Paenicibacillus barengoltzii CAU904 was purified and biochemically characterized. PbChi67 was purified to apparent homogeneity with 10.2 fold purification and 8.0% recovery yield. The molecular mass of the enzyme was 67.0kDa by SDS-PAGE and 67.9kDa by gel filtration, respectively. PbChi67 was most active at pH 3.5 and was stable within pH 3.0–9.0. The optimal temperature of PbChi67 was 60°C and it was stable up to 55°C with a thermal denaturing half-life of 43min at 65°C. The enzyme exhibited strict substrate specificity towards colloidal chitin and glycol chitin but showed no or trace activities towards other tested substrates. The Km and Vmax values of PbChi67 for colloidal chitin and glycol chitin were 3.35mg/mL and 17.1μmol/min/mg, and 2.66mg/mL and 15.0μmol/min/mg, respectively. PbChi67 hydrolyzed colloidal chitin to yield N-acetyl chitooligosaccharides (COSs) with degree of polymerization (DP) of 2–4 at the initial hydrolysis stage, indicating that it is an endo-type chitinase. These properties make the enzyme as a good candidate for recycling of chitin materials.

Acylhydrazones as Widely Tunable Photoswitches.

Molecular photoswitches have attracted much attention in biological and materials contexts. Despite the fact that existing classes of these highly interesting functional molecules have been heavily investigated and optimized, distinct obstacles and inherent limitations remain. Considerable synthetic efforts and complex structure-property relationships render the development and exploitation of new photoswitch families difficult. Here, we focus our attention on acylhydrazones: a novel, yet underexploited class of photochromic molecules based on the imine structural motif. We optimized the synthesis of these potent photoswitches and prepared a library of over 40 compounds, bearing different substituents in all four crucial positions of the backbone fragment, and conducted a systematic study of their photochromic properties as a function of structural variation. This modular family of organic photoswitches offers a unique combination of properties and the compounds are easily prepared on large scales within hours, through an atom-economic synthesis, from commercially available starting materials. During our thorough spectroscopic investigations, we identified photoswitches covering a wide range of thermal half-lives of their (Z)-isomers, from short-lived T-type to thermally stable P-type derivatives. By proper substitution, excellent band separation between the absorbance maxima of (E)- and (Z)-isomers in the UV or visible region could be achieved. Our library furthermore includes notable examples of rare negative photochromic systems, and we show that acylhydrazones are highly fatigue resistant and exhibit good quantum yields.

Thermal stability of foam during steam drive

A fine-long tube model was established for carrying out the thermal degradation experiments of steam foam to determine the foam stability in steam chamber. The real core sand from the Yulou oil layer of Block Jin45 of Liaohe Oilfield was packed in the model, and the experiment lasting for twenty days was conducted under the typical reservoir pressure and saturated steam temperature. The analysis on the experimental results and theoretical calculation results indicate that the thermal degradation reaction model of the added foam is the first-order reaction, and the degradation rate constants at the temperatures of 200 °C, 250 °C, and 300 °C are 9.35×10−8 s−1, 17.80×10−8 s−1, and 24.53×10−8 s−1 respectively. The foam thermal degradation rate kinetic model was deduced, which can be used to calculate the foam thermal degradation rate and half-life at any temperature condition. The half-lives of the added foam are 370 d, 160 d, 83 d, 49 d and 31 d at the temperatures of 100 °C, 150 °C, 200 °C, 250 °C and 300 °C respectively, which indicates that the production performance of most heavy oil reservoirs developed by steam drive in China can be improved by injecting foam agent. But in terms of the deep and high-pressure heavy oil reservoirs, the foam stability is poor when the saturated steam temperature is over 300 °C.

Contribution of Disulfide Bridges to the Thermostability of a Type A Feruloyl Esterase from Aspergillus usamii.

The contribution of disulfide bridges to the thermostability of a type A feruloyl esterase (AuFaeA) from Aspergillus usamii E001 was studied by introducing an extra disulfide bridge or eliminating a native one from the enzyme. MODIP and DbD, two computational tools that can predict the possible disulfide bridges in proteins for thermostability improvement, and molecular dynamics (MD) simulations were used to design the extra disulfide bridge. One residue pair A126-N152 was chosen, and the respective amino acid residues were mutated to cysteine. The wild-type AuFaeA and its variants were expressed in Pichia pastoris GS115. The temperature optimum of the recombinant (re-) AuFaeAA126C-N152C was increased by 6°C compared to that of re-AuFaeA. The thermal inactivation half-lives of re-AuFaeAA126C-N152C at 55 and 60°C were 188 and 40 min, which were 12.5- and 10-folds longer than those of re-AuFaeA. The catalytic efficiency (k cat/K m) of re-AuFaeAA126C-N152C was similar to that of re-AuFaeA. Additionally, after elimination of each native disulfide bridge in AuFaeA, a great decrease in expression level and at least 10°C decrease in thermal stability of recombinant AuEaeA variants were also observed.

Engineering a thermostable fungal GH10 xylanase, importance of N-terminal amino acids.

Xylanases are used in many industrial processes including pulp bleaching, baking, detergent, and the hydrolysis of plant cell wall in biofuels production. In this work we have evolved a single domain GH10 xylanase, Xyn10A_ASPNG, from Aspergillus niger to improve its thermostability. We introduced a rational approach involving as the first step a computational analysis to guide the design of a mutagenesis library in targeted regions which identified thermal important residues that were subsequently randomly mutagenized through rounds of iterative saturation mutagenesis (ISM). Focusing on five residues, four rounds of ISM had generated a quintuple mutant 4S1 (R25W/V29A/I31L/L43F/T58I) which exhibited thermal inactivation half-life (t1/2 ) at 60°C that was prolonged by 30 folds in comparison with wild-type enzyme. Whereas the wild-type enzyme retained 0.2% of its initial activity after a heat treatment of 10 min at 60°C and was completely inactivated after 2 min at 65°C, 4S1 mutant retained 30% of its initial activity after 15 min heating at 65°C. Furthermore, the mutant melting temperature (Tm ) increased by 17.4°C compared to the wild type. Each of the five mutations in 4S1 was found to contribute to thermoresistance, but the dramatic improvement of enzyme thermoresistance of 4S1 was attributed to the synergistic effects of the five mutations. Comparison of biochemical data and model structure between 4S1 and the wild-type enzyme suggested that the N-terminal coil of the enzyme is important in stabilizing GH10 xylanase structure. Based on model structure analyses, we propose that enforced hydrophobic interactions within N-terminal elements and between N- and C-terminal ends are responsible for the improved thermostability of Xyn10A_ASPNG.

Highly thermostable carbonic anhydrase from Persephonella marina EX-H1: Its expression and characterization for CO2-sequestration applications

The codon-optimized carbonic anhydrase gene of Persephonella marina EX-H1 (PMCA) was expressed and characterized. The gene with the signal peptide removed, PMCA(sp−), resulted in the production of approximately five times more purified protein than from the intact gene PMCA using an Escherichia coli expression system. PMCA(sp−) is formed as homo-dimer complex. PMCA(sp−) has a wide pH tolerance (optimum pH 7.5) and a high thermostability even at 100°C (88min of thermal deactivation half-life). The melting temperature for PMCA(sp−) was 84.5°C. The apparent kcat and Km values for CO2 hydration were 3.2×105s−1 and 10.8mM. The activity of the PMCA(sp−) enzyme was enhanced by Zn2+, Co2+, and Mg2+, but was strongly inhibited by Cu2+, Fe3+, Al3+, Pb2+, Ag+, and Hg2+. PMCA(sp−) readily catalyzed the hydration of CO2, precipitating CaCO3 as calcite in the presence of Ca2+.

Improving the thermostability of a mesophilic family 10 xylanase, AuXyn10A, from Aspergillus usamii by in silico design.

To improve the thermostability of a mesophilic GH family 10 xylanase, AuXyn10A, from Aspergillus usamii E001, its modification was performed by in silico design. Based on the comparison of B-factor values, a mutant xylanase ATXyn10 was predicted by substituting a segment YP from Tyr(25) to Pro(34) of AuXyn10A with the corresponding one from Asn(24) to Ala(32) of TaXyn10, a thermophilic GH family 10 xylanase from Thermoascus aurantiacus. Analysis of a TaXyn10 crystal structure indicated that there is a close interaction between segments YP and FP. For that reason, another mutant xylanase ATXyn10(M) was designed by mutating Ser(286) and His(288) of ATXyn10 into the corresponding Gly(285) and Phe(287) in the FP of TaXyn10. Then, two ATXyn10- and ATXyn10(M)-encoding genes, ATxyn10 and ATxyn10 (M), were expressed in Pichia pas toris GS115. The temperature optimum of recombinant (re) ATXyn10(M) was 60 °C, 10 °C higher than that of reAuXyn10A. Its thermal inactivation half-life (t(1/2)) at 55 °C was 10.4-fold longer than that of reAuXyn10A. As compared with reAuXyn10A, reATXyn10(M) displayed a slight decrease in K(m) value and a significant increase in V(max) value from 6,267 to 8,870 U/mg.

Photoresponsive switches at surfaces based on supramolecular functionalization with azobenzene-oligoglycerol conjugates.

The synthesis, supramolecular complexation, and switching of new bifunctional azobenzene-oligoglycerol conjugates in different environments is reported. Through the formation of host-guest complexes with surface immobilized β-cyclodextrin receptors, the bifunctional switches were coupled to gold surfaces. The isomerization of the amphiphilic azobenzene derivatives was examined in solution, on gold nanoparticles, and on planar gold surfaces. The wettability of functionalized gold surfaces can be reversibly switched under light-illumination with two different wavelengths. Besides the photoisomerization processes and concomitant effects on functionality, the thermal cis to trans isomerization of the conjugates and their complexes was monitored. Thermal half-lives of the cis isomers were calculated for different environments. Surprisingly, the half-lives on gold nanoparticles were significantly smaller compared to planar gold surfaces.

Exploration of Disulfide Bridge and N-Glycosylation Contributing to High Thermostability of a Hybrid Xylanase

A comparison between three-dimensional structures of a wild-type xylanase AoXyn11A and a hybrid xylanase AEx11A revealed that a disulfide bridge (Cys(5)-Cys(32)) and an N-glycosylation site (Asn(42)) were imported into AEx11A by N-terminal substitution of AoXyn11A with EvXyn11(TS). Two mutant genes AEx11A(C5T) and AEx11A(N42Q) were constructed by mutating Cys(5)- and Asn(42)-encoding codons of AEx11A into Thr(5)- and Gln(42)-encoding ones, and heterologously expressed in Pichia pastoris GS115, respectively. The temperature optimum of the recombinant AEx11A(C5T) (reAEx11A(C5T)) was decreased to 60°C from 80°C of reAEx11A, while its thermal inactivation half-lives at 70 and 80°C shortened to 3 and 1 min from 197 and 25 min of reAEx11A, respectively. However, there was no obvious alteration between reAEx11A and reAEx11A(C5T) in pH characteristics and kinetic parameters. Furthermore, both reAEx11A(N42Q) and reAEx11A displayed no significant difference in all enzymatic properties tested, except for the apparent molecular weight. We concluded based on this study that the disulfide bridge of AEx11A was vital to its high thermostability, but the N-glycosylation had no effect on.

Shaping Crystals with Light: Crystal-to-Crystal Isomerization and Photomechanical Effect in Fluorinated Azobenzenes

Unusually long thermal half-lives of perhalogenated cis-azobenzenes enabled their structural characterization and the first evidence of a crystal-to-crystal cis → trans azobenzene isomerization. Irradiation with visible light transforms a perhalogenated cis-azobenzene single crystal into a polycrystalline aggregate of its trans-isomer in a photomechanical transformation that involves a significant, controllable, and thermally irreversible change of crystal shape. This is the first demonstration of permanent photomechanical modification of crystal shape in an azobenzene.

O-Fluoroazobenzenes as Readily Synthesized Photoswitches Offering Nearly Quantitative Two-Way Isomerization with Visible Light

Azobenzene functionalized with ortho-fluorine atoms has a lower energy of the n-orbital of the Z-isomer, resulting in a separation of the E and Z isomers' n→π* absorption bands. Introducing para-substituents allows for further tuning of the absorption spectra of o-fluoroazobenzenes. In particular, electron-withdrawing ester groups give rise to a 50 nm separation of the n→π* transitions. Green and blue light can therefore be used to induce E→Z and Z→E isomerizations, respectively. The o-fluoroazobenzene scaffold is readily synthesized and can be inserted into larger structures via its aryl termini. These new azobenzene derivatives can be switched in both ways with high photoconversions, and their Z-isomers display a remarkably long thermal half-life.

The fusion Vibrio campbellii luciferase as a eukaryotic gene reporter

Bacterial luciferase from Vibrio campbellii is a thermostable enzyme with an in vitro thermal inactivation half-life of ∼1020min at 37°C. The enzyme also binds tightly to reduced FMN. In this study, a V. campbellii fusion luciferase construct in which the α and β subunits are linked with a decapeptide was made and characterized. In general, the overall enzymatic properties of the two enzymes are similar. Expression of the enzymes in Escherichia coli demonstrated that the V. campbellii fusion luciferase emits less light than the native luciferase, but still emits a much greater amount of light than native luciferase from Vibrio harveyi and Photobacterium leiognathi TH1. The intensity of light emitted by the V. campbellii fusion luciferase was more than 80-fold greater than that from the V. harveyi native luciferase when expressed at 37°C. Biochemical characterization has shown that the V. campbellii fusion luciferase also retains a high binding affinity for reduced flavin mononucleotide and high thermostability. The levels of bioluminescence emitted by the V. campbellii fusion luciferase expressed in HEK293T cells reached ∼1×106 Relative Light Units/mg total protein. These findings suggest that the V. campbellii fusion luciferase is a promising candidate for further development as a luciferase-based reporter for eukaryotic systems.

Domain-swapping of mesophilic xylanase with hyper-thermophilic glucanase

BackgroundDomain fusion is limited at enzyme one terminus. The issue was explored by swapping a mesophilic Aspergillus niger GH11 xylanase (Xyn) with a hyper-thermophilic Thermotoga maritima glucanase (Glu) to construct two chimeras, Xyn-Glu and Glu-Xyn, with an intention to create thermostable xylanase containing glucanase activity.ResultsWhen expressed in E. coli BL21(DE3), the two chimeras exhibited bi-functional activities of xylanase and glucanase. The Xyn-Glu Xyn moiety had optimal reaction temperature (Topt) at 50 °C and thermal in-activation half-life (t1/2) at 50 °C for 47.6 min, compared to 47 °C and 17.6 min for the Xyn. The Glu-Xyn Xyn moiety had equivalent Topt to and shorter t1/2 (5.2 min) than the Xyn. Both chimera Glu moieties were more thermostable than the Glu, and the three enzyme Topt values were higher than 96 °C. The Glu-Xyn Glu moiety optimal pH was 5.8, compared to 3.8 for the Xyn-Glu Glu moiety and the Glu. Both chimera two moieties cooperated with each other in degrading substrates.ConclusionsDomain-swapping created different effects on each moiety properties. Fusing the Glu domain at C-terminus increased the xylanase thermostability, but fusing the Glu domain at N-terminus decreased the xylanase thermostability. Fusing the Xyn domain at either terminus increased the glucanase thermostability, and fusing the Xyn domain at C-terminus shifted the glucanase pH property 2 units higher towards alkaline environments. Fusing a domain at C-terminus contributes more to enzyme catalytic activity; whereas, fusing a bigger domain at N-terminus disturbs enzyme substrate binding affinity.

Activity and thermostability increase of xylanase following transplantation with modules sub-divided from hyper-thermophilic CBM9_1-2

Transplantation is useful for elucidating the functions of structural modules and for engineering enzyme properties. Unexpectedly, transplanting a hyper-thermophilic carbohydrate-binding module, CBM9_1-2, into the mesophilic Aspergillus niger GH11 xylanase (Xyn) slightly decreased the thermal inactivation half-life of Xyn. This effect was further investigated by dividing the CBM9_1-2 module into two smaller parts, C1 and C2, which were transplanted into Xyn to create the chimeras Xyn-C1 and Xyn-C2. Both chimeras exhibited higher catalytic activities on xylan than native Xyn. Xyn-C2 exhibited higher binding affinities for both oat spelt and birch wood xylans, and its thermal inactivation half-life (69.3min) was 4 or 5 times longer than that of Xyn (17.6min), Xyn-C1 (13.4min), and the original chimera containing CBM9_1-2 (13.8min). In contrast, Xyn-C1 exhibited higher binding affinity for oat spelt xylan, but not for birch wood xylan. Through this rational engineering of the fungal xylanase, the C2 sub-module was shown to have a different thermostabilizing effect than the C1 sub-module. The different functions of the smaller parts of a large module can play pivotal roles in transplantation.

Lentivirus Immobilization to Nanoparticles for Enhanced and Localized Delivery From Hydrogels

Hydrogels can provide a controllable cell microenvironment for numerous applications in regenerative medicine, and delivery of gene therapy vectors can be employed to enhance their bioactivity. We investigated the delivery of lentiviral vectors from hydrogels, and employed the immobilization of lentivirus to hydroxylapatite (HA) nanoparticles as a means to retain and stabilize vectors within hydrogels, and thereby increase delivery efficiency. Entrapment of the vector alone within the hydrogel maintained the activity of the virus more effectively compared to the absence of a hydrogel, and release was slowed with an increasing solid content of the hydrogel. Association of the lentivirus with HA increased the activity of the complexes, with HA increasing the virus activity for 72 hours. Cells seeded onto lentivirus-HA-loaded hydrogels had a decreased number of infected cells outside of the hydrogel relative to the absence of HA. In vivo studies with collagen hydrogels loaded with lentivirus and HA-lentivirus demonstrated sustained and localized transgene expression for at least 4 weeks, with increased expression using the lentivirus-HA complex. This strategy of nanoparticle immobilization to stabilize and retain vectors is broadly applicable to hydrogels, and may provide a versatile tool to combine gene therapy and biomaterials for applications in regenerative medicine.

Production, Purification, and Characterization of a Potential Thermostable Galactosidase for Milk Lactose Hydrolysis from Bacillus stearothermophilus

β-Galactosidase, commonly named lactase, is one of the most important enzymes used in dairy processing; it catalyzes the hydrolysis of lactose to its constituent monosaccharides glucose and galactose. Here, a thermostable β-galactosidase gene bgaB from Bacillus stearothermophilus was cloned and expressed in B. sub-tilis WB600. The recombinant enzyme was purified by a combination of heat treatment, ammonium sulfate fractionation, ion exchange, and gel filtration chromatography techniques. The purified β-galactosidase appeared as a single protein band in sodium dodecyl sulfate-PAGE gel with a molecular mass of approximately 70 kDa. Its isoelectric point, determined by polyacryl-amide gel isoelectric focusing, was close to 5.1. The optimum temperature and pH for this β-galactosidase activity were 70°C and pH 7.0, respectively. Kinetics of thermal inactivation and half-life times for this thermostable enzyme at 65 and 70°C were 50 and 9h, respectively, and the Km and Vmax values were 2.96mM and 6.62μmol/min per mg. Metal cations and EDTA could not activate this thermostable enzyme, and some divalent metal ions, namely, Fe2+, Zn2+, Cu2+, Pb2+, and Sn2+, inhibited its activity. Thiol reagents had no effect on the enzyme activity, and sulfhydryl group blocking reagents inactivated the enzyme. This enzyme possessed a high level of transgalactosylation activity in hydrolysis of lactose in milk. The results suggest that this recombinant thermostable enzyme may be suitable for both the hydrolysis of lactose and the production of galactooligosaccharides in milk processing.

Stabilization of the Human β 2-Adrenergic Receptor TM4–TM3–TM5 Helix Interface by Mutagenesis of Glu122 3.41, A Critical Residue in GPCR Structure

G protein-coupled receptor (GPCR) instability represents one of the most profound obstacles in the structural study of GPCRs that bind diffusible ligands. The introduction of targeted mutations at nonconserved residues that lie proximal to helix interfaces has the potential to enhance the fold stability of the receptor helix bundle while maintaining wild-type receptor function. To test this hypothesis, we studied the effect of amino acid substitutions at Glu122 3.41 in the well-studied β 2-adrenergic receptor (β 2AR), which was predicted from sequence conservation to lie at a position equivalent to a tryptophan residue in rhodopsin at the 3,4,5 helix interface among transmembrane (TM) domains 3, 4, and 5. Replacement of Glu122 3.41 with bulky hydrophobic residues, such as tryptophan, tyrosine, and phenylalanine, increases the yield of functionally folded β 2AR by as much as 5-fold. Receptor stability in detergent solution was studied by isothermal denaturation, and it was found that the E122W and E122Y mutations enhanced the β 2AR thermal half-life by 9.3- and 6.7-fold, respectively, at 37 °C. The β 1AR was also stabilized by the introduction of tryptophan at Glu147 3.41, and the effect on protein behavior was similar to the rescue of the unstable wild-type receptor by the antagonist propranolol. Molecular modeling of the E122W and E122Y mutants revealed that the tryptophan ring edge and tyrosine hydroxyl are positioned proximal to the helical break in TM5 introduced by the conserved Pro211 5.50 and may stabilize the helix by interacting favorably with the unpaired carbonyl oxygen of Val206 5.45. Conformational flexibility of TM5 is likely to be a general property of class A GPCRs; therefore, engineering of the TM4–TM3–TM5 interface at the 3.41 position may provide a general strategy for the stabilization of other receptors.

Photochemically Induced Disturbance of the Alkyl Chain Packing in Vesicular Membranes

In previous reports, we presented the synthesis and properties of double-tailed azobenzene-substituted phosphate amphiphiles (Kuiper et al. Synthesis 2003, 695 and Kuiper et al. Langmuir 2004, 20, 1152). We also reported that an ion channel can be regulated by trans-cis isomerization of these amphiphiles, which were incorporated in the membrane (Folgering et al. Langmuir 2004, 20, 6985). In the present study, the effect of trans-cis isomerization of both single- and double-tailed azobenzene-substituted amphiphiles on the aggregation and packing behavior has been studied. The phase transition temperature of a membrane and the thermal half-life times of the cis azobenzene-substituted amphiphiles in membranes have been measured. Furthermore, the synthesis and properties of single-tailed azobenzene-substituted phosphate amphiphiles are described and compared with those of the double-tailed analogues. The single-tailed azobenzene-substituted phosphates have a low solubility in water and form micelles, sheets, and crystals. In all cases the trans-cis isomerization leads to a disturbance of the chain packing. Both single- and double-tailed cis azobenzene-substituted phosphates lowered the main phase transition temperature of bilayer membranes. The effect increased when the azobenzene moiety was situated closer to the head group. Accordingly, the half-life times of the cis azobenzene group was shorter when the azobenzene group was positioned closer to the head group for both the single- and double-tailed amphiphiles. Interestingly, the thermal cis-trans isomerization of the single-tailed azobenzene-substituted phosphates was faster in a DOPC membrane than that for the free monomer in aqueous solution.

Expression of the melA gene from Rhizobium etli CFN42 in Escherichia coli and characterization of the encoded tyrosinase

The gene melA from the nitrogen-fixing bacterium Rhizobium etli CFN42 was amplified using PCR, cloned in the expression vector pTtrc99A to obtain plasmid pTrc melA, and transformed into E. coli strain W3110. The resulting recombinant strain W3110/pTrc melA synthesized a dark pigment when growing in solid or liquid media containing l-tyrosine and copper. This pigment was identified as melanin by comparing it with analytical grade melanin using a spectrophotometric assay. Melanin was synthesized when recombinant E. coli cells were incubated at 30 °C; however, at 37 °C significantly less polymer was produced. The recombinant tyrosinase expressed intracellularly in E. coli was purified 40-fold with a 25% yield from a cell extract by ammonium sulfate precipitation and ion exchange chromatography. With the partially purified tyrosinase, the K m for l-dopa and l-tyrosine were determined as 2.44 and 0.19 mM, respectively. Temperature and pH for maximum activity were 50 °C and 6.5–7.5, respectively. Activation energy for thermal inactivation (50.77 kJ/mol; using l-dopa as substrate at pH 7) and half-life values indicate a higher thermal stability of R. etli tyrosinase in comparison with mushroom tyrosinase. Interestingly, for a bacterial tyrosinase, MelA showed an unusually higher activity for l-tyrosine than for l-dopa.