Half-life Of Thermal Inactivation Research Articles

Engineering a Highly Thermostable High-Redox Potential Laccase

Fungal high-redox potential laccases (HRPLs) are considered to be among the most relevant biocatalysts for industrial and environmental applications, although the lack of thermostable variants is hindering their practical implementation. In this letter, we describe the design of a highly thermostable HRPL. By introducing 27 stabilizing mutations identified in previous evolutionary campaigns focused on the HRPL from basidiomycete PM1, we have designed a variant with a half-life of thermal inactivation at 75 °C of 225 min, 32-fold superior to that of the parental enzyme. In addition, this variant displays remarkable stability at both acidic and basic pHs. The synergetic effect of the set of stabilizing mutations introduced is responsible for the improvements in thermal and pH stability, while maintaining the high-redox potential of this robust biocatalyst.

Uncovering key residues responsible for the thermostability of a thermophilic 1,3(4)-β-d-glucanase from Nong flavor Daqu by rational design

Fungal 1,3(4)-β-D-glucanases were usually applied in brewing and feedstuff industries, however, the thermostability limits the most their application. The characterized 1,3(4)-β-D-glucanase (NFEg16A) from Chinese Nong-flavor (NF) Daqu showed the highest thermostability among GH16 fungal 1,3(4)-β-D-glucanases, with half-lives of thermal inactivation (t1/2) of 44.9 min at 90 °C, so multiple rational designs were used to identify the key residues for its thermostability. Based on protein sequence and 3D structure analyses around the catalytic regions. Nine site-mutants were constructed, among which N173Y and S187A were identified as the most thermotolerant and thermolabile ones, with t1/2 values of 61 min and 14.0 min at 90 °C, respectively. Therefore, N173 and S187 were then selected as “hotspots” for site-saturation mutagenesis. Interestingly, most of the N173 and S187 variants exhibited a similar thermostability to that of N173Y and S187A, respectively, confirming their different roles in the thermostability of NFEg16A. In addition, each S187A and its surrounding substitutions (D144 N and T164 N) was independently detrimental to the thermostability of NFEg16A, since the t1/2 (90 °C) of S187A, D144 N and T164 N were 14.0 min, 20.6 min and 27.2 min, respectively. Surprisingly, combinatorial substitution of S187A with D144 N or T164 N showed positive effects on the thermostability, with the increase of t1/2 (90 °C) to 30.9 min and 63.5 min for S187A-D144 N and S187A-T164 N, respectively. More importantly, S187A-T164 N showed higher thermostability than that of wild type. In short, we successfully identified two key sites and their surrounding residues in response to the thermostability of NFEg16A and further improved its thermostability by several rational designs. These findings could be used for the protein engineering of homologous 1,3(4)-β-D-glucanases, as well as other enzyme family members with high similarities.

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

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.

The Generation of Thermostable Fungal Laccase Chimeras by SCHEMA-RASPP Structure-Guided Recombination in Vivo.

Fungal laccases are biotechnologically relevant enzymes that are capable of oxidizing a wide array of compounds, using oxygen from the air and releasing water as the only byproduct. The laccase structure is comprised of three cupredoxin domains sheltering two copper centers-the T1Cu site and the T2/T3 trinuclear Cu cluster-connected to each other through a highly conserved internal electron transfer pathway. As such, the generation of laccase chimeras with high sequence diversity from different orthologs is difficult to achieve without compromising protein functionality. Here, we have obtained a diverse family of functional chimeras showing increased thermostability from three fungal laccase orthologs with ∼70% protein sequence identity. Assisted by the high frequency of homologous DNA recombination in Saccharomyces cerevisiae, computationally selected SCHEMA-RASPP blocks were spliced and cloned in a one-pot transformation. As a result of this in vivo assembly, an enriched library of laccase chimeras was rapidly generated, with multiple recombination events simultaneously occurring between and within the SCHEMA blocks. The resulting library was screened at high temperature, identifying a collection of thermostable chimeras with considerable sequence diversity, which varied from their closest parent homologue by 46 amino acids on average. The most thermostable variant increased its half-life of thermal inactivation at 70 °C 5-fold (up to 108 min), whereas several chimeras also displayed improved stability at acidic pH. The two catalytic copper sites spanned different SCHEMA blocks, shedding light on the recognition of specific residues involved in substrate oxidation. In summary, this case-study, through comparison with previous laccase engineering studies, highlights the benefits of bringing together computationally guided recombination and in vivo shuffling as an invaluable strategy for laccase evolution, which can be translated to other enzyme systems.

Asp305Gly mutation improved the activity and stability of the styrene monooxygenase for efficient epoxide production in Pseudomonas putida KT2440

BackgroundStyrene monooxygenase (SMO) catalyzes the first step of aromatic alkene degradation yielding the corresponding epoxides. Because of its broad spectrum of substrates, the enzyme harbors a great potential for an application in medicine and chemical industries.ResultsIn this study, we achieved higher enzymatic activity and better stability towards styrene by enlarging the ligand entrance tunnel and improving the hydrophobicity through error-prone PCR and site-saturation mutagenesis. It was found that Asp305 (D305) hindered the entrance of the FAD cofactor according to the model analysis. Therefore, substitution with amino acids possessing shorter side chains, like glycine, opened the entrance tunnel and resulted in up to 2.7 times higher activity compared to the wild-type enzyme. The half-lives of thermal inactivation for the variant D305G at 60 °C was 28.9 h compared to only 3.2 h of the wild type SMO. Moreover, overexpression of SMO in Pseudomonas putida KT2440 with NADH regeneration was carried out in order to improve biotransformation efficiency for epoxide production. A hexadecane/buffer (v/v) biphasic system was applied in order to minimize the inactivation effect of high substrate concentrations on the SMO enzyme. Finally, SMO activities of 190 U/g CDW were measured and a total amount of 20.5 mM (S)-styrene oxide were obtained after 8 h.ConclusionsThis study offers an alternative strategy for improved SMO expression and provides an efficient biocatalytic system for epoxide production via engineering the entrance tunnel of the enzyme’s active site.

Rational Engineering of Bacillus cereus Leucine Dehydrogenase Towards α-keto Acid Reduction for Improving Unnatural Amino Acid Production.

Unnatural amino acids (UAAs) play a key role in modern medicinal chemistry such as small molecules and peptide-based drugs with fast-growing markets. Low efficiency for natural enzymes including leucine dehydrogenase (LeuDH, EC1.4.1.9) are one major challenge for UAA production. Here, rational engineering of LeuDH from Bacillus cereus with a structure-based design approach is studied. The results achieve higher enzymatic activity and stability toward α-keto acid reduction by improving the hydrophobic and rigidity of enzymatic substrate entrance tunnel. High catalytic efficiency for variant E116V is associated with the presence of more hydrophobic tunnels that allows easy substrate diffusion, which is confirmed in absorbance spectroscopy study. For variant T45M/E116V, melting temperature and half-lives of thermal inactivation at 60 °C is 62.8 °C and 29.2 h, respectively, much higher than 48.4 °C and 3.4 h of wild type. Structural analysis indicates that an additional hydrogen bond in β5 fold is formed in variant T45M, which results in a more rigid β5 fold leading to better stability. Furthermore, asymmetric synthesis of α-amino acids with coenzyme regeneration reveals higher productivities for variant T45M/E116V. This study indicates the importance of substrate entrance tunnel for enzymatic activities and stability, the engineered LeuDH would better serve UAA production.

Sandwich fusion of CBM9_2 to enhance xylanase thermostability and activity

Used as model for sandwich fusion, a mesophilic Aspergillus niger GH11 xylanase (Xyn) was fused into C2-Xyn-C2 with a thermophilic Thermotaga maritima GH10 xylanase carbohydrate-binding module CBM9_2 (C2). Linearized plasmids C2-pET20b-C2-Xyn were amplified from template pET20b-Xyn-C2 with a 4.3 kb C2-pET20b megaprimer, ligated into circular plasmids in blunt-end ligation, and transformed into E. coli BL21 (DE3) cells. The C2-Xyn-C2 had optimum activity at 45 °C and pH 4.2, a 2.85 h thermal inactivation half-life at 80 °C and a 8.69 h at 50 °C, with the 8.69 h value 24.8-, 7.5-, and 7.1-fold longer than the Xyn and single terminal fusion enzymes Xyn-C2, and C2-Xyn. Thermodynamics showed that the enzyme had a 1.8 °C higher melting temperature, lower values ΔS, ΔΔG, and a denser structure than the Xyn. Kinetics showed that the C2-Xyn-C2 catalytic efficiency was 1.2–~6-fold and 2.7–~7.9-fold higher on beechwood and oat-spelt xylan than those of the enzymes Xyn, Xyn-C2, and C2-Xyn. The sandwich fusion evolved the xylanase with “armor-hands” to enhance simultaneously thermostability and activity in quality.

Substituting Both the N-Terminal and "Cord" Regions of a Xylanase from Aspergillus oryzae to Improve Its Temperature Characteristics.

To improve the temperature characteristics of AoXyn11A, a mesophilic glycoside hydrolase family (GHF) 11 xylanase from Aspergillus oryzae CICC40186, its N-terminal and "cord" regions were selected to be substituted by means of the computer-aided analysis and calculation. In brief, one mutant, named ATX11A41, possessing the lowest root-mean-square deviation (RMSD) value was designed based on the molecular dynamics (MD) simulation by substituting the N-terminal 41 amino acids of AoXyn11A with the corresponding 42 ones of pXYL11, a thermophilic GHF11 xylanase from Thermobifida fusca. On the basis of the primary structure alignment of pXYL11 with ATX11A41 (or AoXyn11A), another mutant, named ATX11A41/cord, was designed by substituting the cord region (93GTYNPGSGG101) of ATX11A41 with the corresponding one (93GTYRPTG99) of pXYL11. Both mutant-encoding genes, ATx11A41 and ATx11A41/cord, were constructed as designed theoretically by a megaprimer PCR technique and were expressed in Pichia pastoris GS115. The specific activities of recombinant (re) AoXyn11A, ATX11A41, and ATX11A41/cord were 2916.7, 2667.6, and 2457.0U/mg, respectively. The analysis of temperature characteristics displayed that the temperature optimum (Topt) of reATX11A41 or reATX11A41/cord was 65°C, which was 15°C higher than that of reAoXyn11A. The thermal inactivation half-life (t1/2) values of reATX11A41 and reATX11A41/cord at 60°C were 55 and 83min, respectively, whereas that of reAoXyn11A was only 18min at 50°C. The melting temperature (Tm) values of reAoXyn11A, reATX11A41, and reATX11A41/cord were 54.2, 66.7, and 71.9°C, respectively. In conclusion, the above findings indicated that the substitution of both the N-terminal and cord regions of a mesophilic AoXyn11A greatly contributed to its improved temperature characteristics.

Site-directed mutagenesis of myofibril-bound serine proteinase from Crucian carp: possible role of Pro95, A127 and I130 on thermal stability

The myofibril-bound serine proteinase (MBSP) from the skeletal muscle of fish belongs to a class of trypsin-like serine proteinases with higher thermal stability than trypsin. To investigate the mechanism responsible for the thermal stability of MBSP from Crucian carp, mutagenesis of key residues were identified by amino acid sequence alignments and Protein Mutant Stability Prediction online prediction software. Meanwhile, five mutants (MutP95T, MutR125N, MutA127S, MutI130D, and MutA127S/I130D) were obtained using site-directed mutagenesis. The mutants and wild-type enzymes were expressed in Pichia pastoris (SMD1168), the secondary structure of the mutants of MBSP was approximately similar to that of wild-type MBSP by circular dichroism spectroscopy analysis. The thermal Inactivation parameters showed that the half-life for thermal inactivation (t1/2) of MutP95T was reduced by approximately 10min, whereas MutA127S, MutI130D and MutA127S/I130D were more stable than the wild-type and increased by approximately 18min, 15min and 24min, respectively. The unfolding free energy difference (ΔΔG) between the variants and wild-type of MutA127S, MutI130D and MutA127S/I130D was enhanced when compared with the wild-type. As a result, the mutants were efficiently produced with altered thermal stability, and MBSP could potentially be used as novel tool enzyme for mass spectrometric identification.

Improving the temperature characteristics and catalytic efficiency of a mesophilic xylanase from Aspergillus oryzae, AoXyn11A, by iterative mutagenesis based on in silico design

To improve the temperature characteristics and catalytic efficiency of a glycoside hydrolase family (GHF) 11 xylanase from Aspergillus oryzae (AoXyn11A), its variants were predicted based on in silico design. Firstly, Gly21 with the maximum B-factor value, which was confirmed by molecular dynamics (MD) simulation on the three-dimensional structure of AoXyn11A, was subjected to site-saturation mutagenesis. Thus, one variant with the highest thermostability, AoXyn11AG21I, was selected from the mutagenesis library, E. coli/Aoxyn11AG21X (X: any one of 20 amino acids). Secondly, based on the primary structure multiple alignment of AoXyn11A with seven thermophilic GHF11 xylanases, AoXyn11AY13F or AoXyn11AG21I–Y13F, was designed by replacing Tyr13 in AoXyn11A or AoXyn11AG21I with Phe. Finally, three variant-encoding genes, Aoxyn11AG21I, Aoxyn11AY13F and Aoxyn11AG21I–Y13F, were constructed by two-stage whole-plasmid PCR method, and expressed in Pichia pastoris GS115, respectively. The temperature optimum (Topt) of recombinant (re) AoXyn11AG21I–Y13F was 60 °C, being 5 °C higher than that of reAoXyn11AG21I or reAoXyn11AY13F, and 10 °C higher than that of reAoXyn11A. The thermal inactivation half-life (t1/2) of reAoXyn11AG21I–Y13F at 50 °C was 240 min, being 40-, 3.4- and 2.5-fold longer than those of reAoXyn11A, reAoXyn11AG21I and reAoXyn11AY13F. The melting temperature (Tm) values of reAoXyn11A, reAoXyn11AG21I, reAoXyn11AY13F and reAoXyn11AG21I–Y13F were 52.3, 56.5, 58.6 and 61.3 °C, respectively. These findings indicated that the iterative mutagenesis of both Gly21Ile and Tyr13Phe improved the temperature characteristics of AoXyn11A in a synergistic mode. Besides those, the catalytic efficiency (kcat/Km) of reAoXyn11AG21I–Y13F was 473.1 mL mg−1 s−1, which was 1.65-fold higher than that of reAoXyn11A.

Enhancement of lipase stability and productivity through chemical modification and its application to latex-based polymer emulsions

Abstract The primary focus of this research was to employ amino-group specific chemical modifications for improving the productivity and stability of two commercially produced lipases, Lipase-A from Candida antarctica (CAL UM ) and Greasex from Humicola lanuginosa (HLL UM ), for application in a latex-based paint formulation. The modified lipases showed higher percentage increase (benzoic anhydride-modified, HLL BA , 150%; PEG-modified, HLL PEG ,162% at 75 °C) as well as higher absolute productivities 41, 50, 52 and 53 μmole substrate mg −1 lipase for unmodified, CAL PEG , HLL PEG and HLL BA , respectively at 37 °C. The half-lives of thermal inactivation for all modified variants were improved from 40 to 166% at 50, 60 and 70 °C relative to unmodified lipases. The higher thermal stability and catalytic efficiency (k cat /K m ) with concomitant lower activity (k cat ) indicates that enhanced productivity is likely to be due to the modified enzymes being better able to resist thermal denaturation over the time course of the productivity experiments. Importantly, both lipases, CAL BA (60%) and HLL BA (55%) retained the highest activity in paint compared with CAL UM (36%) and HLL UM (39%) after 20 weeks incubation at 25 °C. The long term stability of the modified lipases illustrates their potential value for commercial paint and other industrial applications.

Characterization of a Novel α-l-Arabinofuranosidase from Ruminococcus albus 7 and Rational Design for Its Thermostability.

An α-l-arabinofuranosidase (Abf) encoding gene was obtained via genomic mining from a Ruminococcus albus strain. The specific activity of this GH 51 Abf was 73.3 U/mg at pH 6.0 and 50 °C. The modification of Abf, aimed at improving thermostability, was performed through different strategies. Structure-based rational design using the PoPMuSiC and the Enzyme Thermal Stability System (ETSS) predicted thermal stability of Abf and enhanced the half-life of thermal inactivation (t1/2) at 50 °C for K208W more than 11.1 times versus the wild-type (WT). Sequence-based rational design was also conducted by substituting histidine with lysine at various sites. Among eight mutants, the t1/2 at 50 °C of H337K was prolonged by 5.0-fold, and the specific activity of this mutant was increased to 121.8 U/mg. In addition, the mutant H337K was utilized with some enzymes to extract pectin from apple pomace. The enzymatically produced pectin got less moisture and ash, milder pH, and higher viscosity than its acid-extracted counterpart, indicating that Abf has an application prospect in pectin production.

Enhancement of the thermostability of Streptomyces kathirae SC-1 tyrosinase by rational design and empirical mutation

This study aimed to improve the thermostability of a newly cloned tyrosinase from Streptomyces kathirae SC-1. The POPMuSiC algorithm was applied to predict the folding free energy change (ΔDG) of amino acid substitution. Site-directed mutagenesis was used to construct mutants (Q7K, G234P, and Q7K/G234P), and the mutant, and wild-type enzymes were expressed in Escherichia coli (DE3). Compared to the wild-type tyrosinase, all three mutant enzymes showed improved thermal properties. The mutant with combined substitution (Q7K/G234P) showed the most pronounced shifts in temperature optima, about 10°C upward, and the half-life for thermal inactivation at 60°C, and melting temperatures were increased by 3 times and approximately 10°C, respectively. Finally, the mechanisms responsible for the increased thermostability were analyzed through comparative analysis of structure models. The structure-based rational design strategies in this study may also provide further insight into the thermostability of other industrial enzymes and suggest further potential industrial applications.

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.

Stabilization of fungi-derived recombinant FAD-dependent glucose dehydrogenase by introducing a disulfide bond.

To improve the stability of E. coli-produced non-glycosylated fungal FAD-glucose dehydrogenase induced a disulfide bond by site-directed mutagenesis based on structural comparisons with glucose oxidases. The FAD-glucose dehydrogenase (GDH) mutant Val149Cys/Gly190Cys, which was constructed based on a comparison with the three dimensional structure of glucose oxidase, showed a 110 min half-life of thermal inactivation at 45 °C, which is 13-fold greater than that of the wild-type enzyme. The considerable increase in thermal stability was further supported by Eyring plot analysis. The kinetic parameters of Val149Cys/Gly190Cys (k cat = 760 s(-1), Km = 35 mM, and catalytic efficiency (k cat/Km) = 22 s(-1 )mM(-1)) were almost identical to those of the wild-type enzyme (k cat = 780 s(-1), Km = 35 mM, k cat/Km = 22 s(-1 )mM(-1)). The substrate specificity of Val149Cys/Gly190Cys is indistinguishable from that of the wild type. The constructed mutant, Val149Cys/Gly190Cys, had significantly increased structural stability without changing the catalytic activity and kinetic parameters of FAD-GDH, including its characteristic substrate specificity.

Effect of mutations on the thermostability of Aspergillus aculeatus β-1,4-galactanase

New variants of β-1,4-galactanase from the mesophilic organism Aspergillus aculeatus were designed using the structure of β-1,4-galactanase from the thermophile organism Myceliophthora thermophila as a template. Some of the variants were generated using PROPKA 3.0, a validated pKa prediction tool, to test its usefulness as an enzyme design tool. The PROPKA designed variants were D182N and S185D/Q188T, G104D/A156R. Variants Y295F and G306A were designed by a consensus approach, as a complementary and validated design method. D58N was a stabilizing mutation predicted by both methods. The predictions were experimentally validated by measurements of the melting temperature (Tm) by differential scanning calorimetry. We found that the Tm is elevated by 1.1°C for G306A, slightly increased (in the range of 0.34 to 0.65°C) for D182N, D58N, Y295F and unchanged or decreased for S185D/Q188T and G104D/A156R. The Tm changes were in the range predicted by PROPKA. Given the experimental errors, only the D58N and G306A show significant increase in thermodynamic stability.Given the practical importance of kinetic stability, the kinetics of the irreversible enzyme inactivation process were also investigated for the wild-type and three variants and found to be biphasic. The half-lives of thermal inactivation were approximately doubled in G306A, unchanged for D182N and, disappointingly, a lot lower for D58N. In conclusion, this study tests a new method for estimating Tm changes for mutants, adds to the available data on the effect of substitutions on protein thermostability and identifies an interesting thermostabilizing mutation, which may be beneficial also in other galactanases.

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.

Identification and characterization of a novel “thermophilic-like” Old Yellow Enzyme from the genome of Chryseobacterium sp. CA49

Abstract A putative Old Yellow Enzyme (OYE) homologue, Chr-OYE3, was identified from the genome of Chryseobacterium sp. CA49 as a new member of the “thermophilic-like” OYE subfamily. Chr-OYE3 is most closely related to YqjM from Bacillus subtilis with 51% identity. The heterologously expressed enzyme adopted two oligomeric states in solutions, dimers and tetramers. It can reduce a spectrum of activated alkenes, and displayed increased thermal resistance with half-lives of thermal inactivation (t1/2) of 233.5 and 21.8 h at 35 °C and 55 °C, respectively. The enzyme also exhibited high resistance toward various organic solvents, especially water-miscible solvents with higher polarity, such as ethylene glycol and DMSO, which would facilitate its application in biocatalysis where such reaction conditions are often encountered.

Characterization of a robust anti-Prelog short-chain dehydrogenase/reductase ChKRED20 from Chryseobacterium sp. CA49

ChKRED20 is a short-chain dehydrogenase/reductase (SDR) cloned from Chryseobacterium sp. CA49 for the anti-Prelog bioreduction of 3,5-bis(trifluoromethyl)acetophenone to produce the chiral alcohol intermediate for aprepitant. Purified ChKRED20 showed broad pH adaptability and stability with 91% of the maximal activity retained at pH 10.0. The temperature dependence of activity reached the maxima at 50 degrees C. Its half-lives of thermal inactivation were 163 and 9.8 h at 40 degrees C and 50 degrees C, respectively. The enzyme was resistant to a variety of metal ions, additives, and organic solvents. The enzymatic activity could be enhanced by the addition of particular metal ions or detergents to up to 168%. ChKRED20 also displayed good activity and excellent an ti-Prelog stereoselectivity toward a spectrum of acetophenone derivatives, providing chiral alcohols with >99% ee for the majority of the substrates. (C) 2014 Elsevier B.V. All rights reserved.

Directed evolution of phloroglucinol synthase PhlD with increased stability for phloroglucinol production

Phloroglucinol synthase PhlD is a type III polyketide synthase capable of directly converting three molecules of malonyl-CoA to an industrially important chemical--phloroglucinol (1, 3, 5-trihydroxylbenzene). Although this enzymatic process provides an attractive biosynthetic route to phloroglucinol, the low productivity of PhlD limits its further practical application. Here we used protein engineering coupled with in situ product removal to improve the productivity of phoroglucinol biosynthesis in recombinant Escherichia coli. Specifically, directed evolution was used to obtain a series of thermostable PhlD mutants with the best one showing over 24-fold longer half-life of thermal inactivation than the wild-type enzyme at 37 °C. When introduced into a malonyl-CoA overproducing E. coli strain, one of the mutants showed 30% improvement in phloroglucinol productivity compared to the wild-type enzyme in a shake-flask study and the final phloroglucinol concentration reached 2.35 g/L with 25% of theoretical yield. A continuous product extraction strategy was designed to remove the toxic phloroglucinol product from the cell media, which further increased the titer of phloroglucinol to 3.65 g/L, which is the highest phloroglucinol titer ever reported to date.