Background: As phenylalanine dehydrogenase (EC 1.4.1.20, PheDH) activity and stability was still diminished in organic solvent and biocatalytic reaction in non-aqueous media has presented as a great alternative to chemical synthesis and biosynthesis, suggesting that it is crucial to obtain a PheDH with high organic solvent-tolerant.Results: It is the first case when thermo-solvent tolerant PheDH producing bacterium was isolated from a marine sample. The BnPheDH' stability could be significantly improved by 1 M NaCI, which was analyzed by molecular dynamics simulation, and it showed better stability in methanol, ethanol, ethylene glycol, ethylene glycol monoethyl ether and acetone. The k(cat)/Km in 30% (v/v) methanol and ethylene glycol monomethylether was 1.15- and 1.35-fold than that of in the aqueous phase, indicating that the catalytic efficiency was significantly improved by the methanol and ethylene glycol monomethylether.Conclusions: The results indicated that the affinity of enzyme, substrate and co-factor and the catalytic efficiency can be significantly improved by organic solvents, suggesting that the BnPheDH had great application value in organic phase or aqueous-organic phase catalytic reaction, and it would be used as an robust biocatalyst to synthesize synthons in the food and pharmaceutical industries. (C) 2016 Elsevier B.V. All rights reserved.
A combined temperature-pH study of urease kinetics was performed with the primary objective to assign the observed pK(a) values to the ionizable groups of the enzyme active site involved in the reaction. This was done in view of the fact that the identity of the groups detected by pH studies reported in the literature has never been conclusively resolved. Accordingly, herein the urease kinetic parameters K-M and v(max) were measured at eight pHs between 5.0 and 8.3 in noninteracting biological buffers (MES and HEPES), at each pH at five temperatures between 15 and 35 degrees C. The determined values of pK(a)s and of the enthalpies of ionization Delta H-ion(0) allowed the ionizable groups to have the pK(a)s assigned. Unlike previously assumed to have pK(a) approximate to 6.5, the determined acidic pK(2) approximate to 5.0 was assigned to a histidine residue (most likely His320 by Klebsiella aerogenes numbering). By contrast, the basic pK(1) approximate to 8.8 was assigned to the Ni-Ni bridging hydroxyl ion, a feature that supports the mechanism operative in the urease activity in which this hydroxyl is a general acid. Further, the pH-dependent analysis of the obtained K-M and v(max) values revealed that even though both the thermodynamic and activation parameters of the urease reaction were little pH-dependent, their values indicated that the most favorable conditions for the substrate binding are at pH 5.5-6.0, and those for the catalytic step, at pH 6.5-7.0. Taken together, the work brings in new mechanistic information of significance to the understanding of the activity of urease. (C) 2015 Elsevier B.V. All rights reserved.
In this study, the methionine sulfoxide reductase A from a pseudomonas monteilii strain (pmMsrA) was reported to synthesize optically active sulfoxides (R)-1a-4a, through asymmetric biocatalytic reductive resolution. Several biotransformation parameters including the reaction time, cell density, and substrate concentration were optimized. Moreover, Substrate scope of pmMsrA catalyzed asymmetric reductive resolution was investigated, which gave chiral (R)-1a-4a with 61%-97% ee.
In our previous studies, a prolyl endopeptidase (PEP) gene from Aspergillus oryzae (MOH) was cloned and expressed in Pichia pastoris; however, the recombinant protein expression level of MOH was very low. In the present study, the PEP expression level was successfully improved by constructing fusion expression proteins with four fusion partners, namely, Streptomyces violaceoruber Phospholipase A2 (PLA2), cellulose-binding domain (CBD), small ubiquitin-related modifier (SUMO) and maltose binding protein (MBP). The enzyme activities of the recombinant fusion proteins CLMH, SLMH, MLMH and PLMH were increased to 3.8-, 2.7-, 4.9- and 7.4-fold compared with that of the parent MOH. Moreover, the extracellular protein content of CLMH, SLMH, MLMH, PLMH were 1.42-, 1.25-, 1.67- and 1.83-fold higher compared with that of MOH. Both PLMH and MOH showed the highest activity at pH 5.5, the highest stability at pH 6.0 and maximal activity at 40°C.
•After simulation, a mono sarcosine oxidase (SOX) was expressed in E. coli as inclusion body, and then reconstructed with natural or coenzyme-like ligands.•The ligands containing halogen atoms on the position 7- or -8-site of isoalloxazine ring could lead to new interactions between ligand and enzyme.•The resconstruction could further help to stabilize the enzyme complex and promote the resistance against organic solvents.
To replace fossil resources with biomass, a lot of conversion methods have been studied. Most of each biomass-conversion usually correspond to one specific purpose, such as to produce chemicals, fuels, or energy. However, when a production of chemicals is through one or more oxidation reactions, co-production of electricity is possible through a conversion on an enzymatic bioanode in a biofuel cell. The simultaneous production will reduce the energy required for producing chemicals. According to the coproduction concept, here we show a production of meso-galactaric acid which is considered a platform chemical. meso-Galactaric acid can be obtained from C1 aldehyde oxidation of d-galacturonic acid, which exists in large quantities as pectin in food process residue. d-Galacturonic acid oxidation catalyzed by pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) and subsequent meso-galactaric acid production was confirmed for the first time by NMR measurements. PQQ-GDH is a useful catalyst for in vitro production, especially for electrosynthesis, because it requires neither the expensive cofactor nor O2. Hence, PQQ-GDH was fixed on an electrode to fabricate the PQQ-GDH electrode. The catalytic current from d-galacturonic acid oxidation with the electrode was confirmed in the electrochemical experiments to show the simultaneous production of meso-galactaric acid and the electric current.
Eight new isolated fungi of the genus Penicillium were evaluated for β- fructofuranosidase (FFase) production. From these, Penicillium citreonigrum was selected for FFase and fructooligosaccharides (FOS) production. The influence of temperature, yeast extract concentration, pH and fermentation time on the FFase activity when using the whole microorganism was evaluated by 24 and 23 designs. The pH was set at 6.5 and no yeast extract was used in the optimization experiments since both shown low significant effects on FFase activity. After optimization, temperature and fermentation time, were set to 25.5 °C and 67.8 h. Under these conditions, the model predicted a FFase production of 301.84 U/mL. The scaled-up process in a 2 L bioreactor enhanced the enzyme productivity up to 1.5 times (6.11 U/mL h). A concentration of 58.7 g/L of FOS was obtained, where kestose was the main product. Assays performed for enzyme characterization showed that 50 °C and a pH 5.0 are the optimal conditions for FFase activity. FFase showed to be stable at temperatures between 25 and 30 °C and pH 4.0–10.0 and its activity increased in the presence of ions, especially Cu4+. Results obtained in this primary report are a clear indication on the interest of using P. citreonigrum as a source of FFase for further FOS production.
Laccases create C-C, C-O or C-N bonds and have been investigated intensively as catalysts for green chemistry and white biotechnology. However, little is known about C-S bond formation in laccasecatalyzed reactions. We have used the laccases from Pycnoporus cinnabarinus and Myceliophthora thermophila to create heteromolecular reaction products from p-hydroquinones and aromatic thiols via C-S bond formation. We discovered a broad assortment of different thiolated products, ranging from dimers to tetramers. During laccase-catalysis of the aromatic thiols without p-hydroquinones homomolecular dimers were formed and these were always linked by S-S bonds. The use of laccase is a convenient way to form heteromolecular thiolated products and homomolecular S-n S bonds under eco-friendly environments. (C) 2016 Elsevier B.V. All rights reserved.
The low solubility of glycosylated flavonoids represents a hurdle to conduct efficient enzymatic deglycosylations in aqueous media. To overcome this drawback, environmentally-unfriendly dimethylsulfoxide (DMSO) is typically used as co-solvent. Using a specific diglycosidase from Acremonium sp. DSM24697 for the deglycosylation of the rutinosylated flavonoid (hesperidin) as model reaction, this communication explores the use of (non-hazardous and biodegradable) deep eutectic solvents (DESs) as co-solvents in flavonoid biocatalysis. The enzymatic deglycosylation was observed when DES composed of choline chloride and glycerol or ethylene-glycol was used at proportions of up to 40% (DES-Buffer, v/v), displaying a promising framework to combine enhanced flavonoid solubilities and high enzymatic activities. The deglycosylation activity significantly increased when the single DES components – glycerol and ethylene-glycol – were added (e.g. 140% of enzyme activity at glycerol at 40% v/v), whereas deleterious effects were observed when choline chloride was solely added, presumably due to its chaotropic effect. Future research opportunities may be envisaged in the genetic design to evolve more robust biocatalysts, and in tailoring DES to deliver more enzyme-compatible solvents.
Monolignol oxidoreductases from the berberine bridge enzyme-like (BBE-like) protein family (pfam 08031) catalyze the oxidation of monolignols to the corresponding aldehydes. In this report, we explore the potential of a monolignol oxidoreductase from Arabidopsis thaliana (AtBBE-like protein 15) as biocatalyst for oxidative reactions. For this study we employed a variant with enhanced reactivity towards oxygen, which was obtained by a single amino acid exchange (L182V). The pH and temperature optima of the purified AtBBE-like protein 15 L182V were determined as well as the tolerance toward organic co-solvents; furthermore the substrate scope was characterized. The enzyme has a temperature optimum of 50 °C and retains more than 50% activity between pH 5 and pH 10 within 5 min. The enzyme shows increased activity in the presence of various co-solvents (10–50% v/v), including acetonitrile, 2-propanol, 1,4-dioxane, and dimethyl sulfoxide. Primary benzylic and primary or secondary allylic alcohols were accepted as substrates. The enantioselectivity E in the oxidation of secondary alcohols was good to excellent (E>34 to >200).
During enzymatic hydrolysis of proteins, some cleavage sites will be accessible in the intact protein. Others may only become accessible after demasking of the cleavage site, i.e. after cleavage of other bonds in the protein. Studies on demasking so far have used data of the release of few peptides in a hydrolysate. To obtain a more complete understanding of the hydrolysis kinetics and to validate the demasking model, this study used the absolute quantification of all peptides formed during hydrolysis of whey protein isolate by Bacillus licheniformis protease. This allowed the determination of the rate constants of hydrolysis of each individual cleavage site in the substrate protein. For five cleavage sites, no cleavages were found. For nine cleavage sites, the hydrolysis was best described with first order kinetics addition. The other 13 cleavage sites were better described with the two-step demasking model, which consists of two consecutive first order reaction equations. In this way, the demasking concept was validated, showing that some peptide bonds are only cleaved when present in intermediate peptides.
Recently, tyrosol has gained attention as a result of its many pharmacological properties and due to the fact that it can be isolated from cheap and abundant resources. Lipophilic tyrosyl esters, which are scarce in nature, have proven in certain cases to acquire improved biological activity compared to tyrosol itself, increasing their potential use in the food and cosmeceutical industries. The enzymatic approach for the synthesis of such esters has prevailed, as it is “green”, compared to chemical practices. We hereby report the enzymatic synthesis of tyrosyl esters of various aliphatic fatty acids performed by a recombinant cutinase from Fusarium oxysporum (FoCut5a). The reaction system used consists of an aqueous phase saturated with the corresponding fatty-acid vinyl ester, which played the role of the acyl donor. We also proceeded to the study of several parameters on the yield of the tyrosyl butyrate ester synthesis. The maximum yield achieved was 60.7% after 4h at 20°C, in pH 7.0, with initial tyrosol concentration of 12.5mM and using 5μg FoCut5a mL−1 reaction as catalyst. The optimum reaction conditions can be considered mild, highlighting the environmentally friendly nature of this reaction, along with the fact that there are not any harmful reagents involved. Additionally, the use of two thermodynamic models, Conductor-like Screening Model for Real Solvents (COSMO-RS) and UNIquac Functional-group Activity Coefficients (UNIFAC), were employed for the prediction of reactants’ and products’ solubilities and their distribution in the reaction biphasic system, aiming to correlate the reaction yields with these important thermodynamic quantities and understand the ability of this enzymatic reaction in synthesizing tyrosyl esters.
The lipase catalysed synthesis of xylose caproate ester was performed by condensation of xylose, an aldopentose and caproic acid in organic solvents. A dual-solvent system containing DMSO and acetone (1:10 v/v) was used to determine the optimal conditions for the reaction. Different reaction parameters (solvent system, reaction time, substrate molar ratio and the amount of enzyme loaded) were studied. The highest conversion rate (64%) was obtained within 24h with the optimal conditions of 16% (w/v) Novozym 435 and a molar ratio of xylose to caproic acid of 1:4. (C) 2016 Elsevier B.V. All rights reserved.
A chitosanolytic activity found in a commercial α-amylase from Bacillus amylolyquefaciens (BAN) was covalently immobilized onto glyoxal agarose beads (25% recovery of activity) and assessed for the continuous production of chitooligosaccharides (COS). The immobilization did not change the reaction profile (with chitotriose and chitobiose as major products, using chitosans of different polymerization and deacetylation degrees), but significantly increased the enzyme thermostability. A two-step process was proposed, in which chitosan was first hydrolyzed in a batch reactor to a viscosity that could flow through a packed-bead reactor (PBR), thus avoiding clogging of the column. The relationship between hydrolysis degree of chitosan (1% w/v) and viscosity of the solution was assessed in a batch reactor. A 50% hydrolyzed chitosan did not cause any clogging of the PBR. Under these conditions, the productivity of the PBR at the lowest dilution rate was 37gCOSL−1h−1, with a conversion yield of 73%. In contrast, at the highest dilution rate, the productivity was nearly 200gCOSL−1h−1, but the conversion yield dropped to around 40%.
The thermal stabilization of enzymes is a critical factor in the development and reliability of enzyme-based processes and functional materials. Using a simple amine coupling approach for enzyme immobilization onto magnetic microbeads, followed by encasement of the beads in a hydrogel, we demonstrate that the thermal stability of the enzyme acetylcholinesterase can be increased dramatically. For example, when free and microbead-immobilized enzyme ("EM Conjugate") are incubated overnight in a dry state at 63 °C (140 °F), the catalytic efficiency (kcat/Km) of the latter is higher than the former by six orders of magnitude (a factor of 2.16 × 106). This effect arises mostly through a ∼29,700-fold decrease in Km experienced by the EM Conjugate, relative to that of the free enzyme. Encapsulation of the EM Conjugate in a hydrogel based on poly(N-(3-aminopropyl methacrylamide)), which contains a primary amine, affords the enzyme additional stability when incubated overnight at 63 °C in an aqueous state. For example, its catalytic efficiency is four orders of magnitude higher than that of both the free enzyme (a factor of 4.34 × 104) and that of the EM Conjugate alone (a factor of 1.78 × 104) after all are incubated overnight at 63 °C. The presence of the hydrogel also caused the Michaelis constant to decrease by 1.38 × 104 relative to that of the EM Conjugate, reaching a value of 2.18 × 10−3 M. Thus the hydrogel enables the AChE substrate binding site to retain a significant amount of its natural affinity for the substrate, after heating. This effect may occur via ion-pairing by the primary amines in the hydrogel polymer repeat unit, which are protonated and positively-charged at the assay pH. To the best of our knowledge, this simple method for enzyme thermal stabilization is novel and has not yet been investigated.
The aim of this study was to characterize the stability and activity of laccase from Trametes versicolor in the presence of three different surfactants, namely sodium di-2-ethylhexylsulfosuccinate (AOT), Triton X-100 (TX-100), and cetyltrimethylammonium bromide (CTAB). The kinetic parameters (such as Km, kcat, kcat/Km ratio), optimal pH and temperature and the thermostability of the enzyme at different temperatures were determined and compared in the absence and presence of the three surfactants. Results revealed that the catalytic activity of the enzyme was greatly improved in the presence of low concentrations of AOT, whereas the activity declined in the presence of TX-100 and CTAB inactivated it almost completely. Results also depicted that, in general, the presence of the surfactants affected the enzyme optimum pH and temperature. In terms of stability, TX-100-induced stabilization and AOT and CTAB-mediated destabilization of the enzyme were observed. Laccase-mediated bioconversion of indole to 2,2-bis(3′-indolyl)-indoxyl in the presence of TX-100 as the effective stabilizing surfactant and TEMPO as the enzyme mediator was also investigated.
It is of great significance to investigate the amino acids in different solvent accessibility parts of β-agarase responsible for its thermal stability, which may help for understanding the structural basis of thermophilic β-agarase and developing practical strategies for reengineering new one. We systematically analyzed the amino acids distributed in the internal, intermediate and external states of β-agarases and found 21 significant differences in the three states by t-test. Among them, Glu, His and charged residues in the internal part and Tyr in the external part are critical for the stability at high temperature. Based on it, we developed a support vector regression model to predict the optimal temperature of β-agarases with the 21 significant factors as input vector and the results are encouraging. The mean-absolute error (MAE) and root-mean-square error (RMSE) in the 10-fold cross-validation are 3.80°C and 5.46°C, respectively. In addition, we experimentally verified three β-agarases with the optimal temperature as 45°C, 50°C and 50°C, and the predicted temperatures were 47.2°C, 47.4°C, and 49.9°C, respectively.
Phenylpropanoid glucosides (PPGs) are naturally occurring and bioactive phenolic derivatives, largely distributed in plants. In this work different PPGs have been chemically or enzymatically synthesized from the lignols coniferyl and p-coumaryl alcohols as substrates for a laccase-catalyzed oxidative coupling. The biooxidation of these PPGs has been investigated here and novel dihydrobenzofuran-based structurally modified analogues have been isolated and characterized. Specifically, the presence of a carbohydrate moiety increased the water solubility of these compounds and reduced the number of dimeric products, as pinoresinol-like structures could not be formed. Looking for a possible sugar-promoted stereochemical enrichment of the obtained diastereomeric mixtures of dimers, different carbohydrate moieties (d-glucose, l-glucose and the disaccharide rutinose) were considered and the respective d.e. values of the dimeric products were measured by 1H NMR and HPLC. However, it was found that the sugar substituent had a minor effect on the stereochemical outcome of the radical coupling reactions, the best measured result being a d.e. value of 21%.
Pepsin from porcine gastric mucous shown catalytic promiscuity was first discovered to catalyze the Morita–Baylis–Hillman (MBH) reaction between aromatic aldehydes with 2-cyclohexen-1-one or 2-cyclopenten-1-one in a two-phase medium of phosphate buffer/cyclohexane in the presence of 1,4-diazabicyclo[2.2.2]octane (DABCO). The best results of the corresponding MBH products up to 77% yield and 38% ee were achieved.
The unspecific peroxygenase from the fungus Agrocybe aegerita (AaeUPO) is an up-and-coming biocatalyst that is able to perform specific oxyfunctionalizations of various substrates. Due to inactivation at excess concentrations of its co-substrate H2O2, AaeUPO’s technical application is still limited. This study aims to promote catalyst efficiency via electrochemical in situ supply of H2O2, using an evolved variant of AaeUPO on the example of ethylbenzene hydroxylation. Total turnover numbers of up to 400,000molproductmolAaeUPO−1 and space-time-yields of up to 25gL−1d−1 were achieved in the electro-enzymatic system. These numbers are in the upper range of published data. The presented system stands out by its very high atom economy. Thus, combining electrochemistry and biocatalysis is one step closer towards the first application of peroxygenases in an industrial process.
