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 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.
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.
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.
Palm kernel cake (PKC), mainly composed of mannan, lignin and protein, is abundant renewable resource with commercial value. To develop clean and efficient way for PKC refinery, the method based on the synergism of hot water pretreatment (HWP), steam pretreatment (SP) and enzymatic hydrolysis were developed. HWP of 180 °C, 20 min and SP of 121 °C, 20 min showed similar performance for sugar release from PKC. The main saccharides produced from PKC by HWP and SP were mannose and manno-oligosaccharides, while no furfural formed. The surface structure analyzed by SEM showed that HWP enhanced the microporosity of PKC, and the accessibility of which was increased thereafter. When HWP pretreated PKC was further hydrolyzed with enzyme cocktail (cellulase, xylanase, endo-mannanase), 45% of PKC was solubilized compared with the control. The manno-oligosaccharides produced by HWP and SP were converted to mannose and mannobiose by endo-mannanase. The results suggested that both HWP and SP promote enzymatic hydrolysis of PKC by releasing oligosaccharides and enhancing microporosity, and the synergism of which was effective for PKC decomposition.
This study focused on the production and immobilisation of the crude enzyme extract of recombinant monoamine oxidase (EC 1.4.3.4), originating from Aspergillus niger (MAO-N-D5) and expressed in Escherichia coli, in PVA gel using the LentiKats® technique. MAO-Ns are important enzymes in the chemical industry due to their stereoselectivity and they are often used for the deracemisation of non-optically pure mixtures of amines. Biomass production, enzyme preparation, enzyme immobilisation, process parameters for the immobilised enzyme and characterisation of the enzyme are described in detail here. The biomass was prepared in laboratory bioreactors, and two different disruption techniques were compared. The activity of the enzyme was determined by biotransformation with secondary amine 3-azabicyclo [3,3,0] octane as a substrate. The crude enzyme extract showed 61.5% of the whole cell activity and the immobilised enzyme showed a wider optimum pH and temperature ranges than the free enzyme. The initial specific activity of the immobilised monoamine oxidase crude enzyme extract remained at 80% after 12 repeated biotransformations. For the first time, the full kinetic parameters of an immobilised MAO-N-D5 were obtained based on a ping-pong bi–bi reaction mechanism. The specific activity was 0.29Ug−1(Lentikats) and the Km was 7.31mM, which were similar in comparison to whole cell MAO-N-D5. Characterisation of immobilised MAO-N-D5 showed particular benefits in terms of activity and stability in comparison with free and whole cell MAO-N-D5; therefore, the immobilisation of this enzyme is very suitable for industrial applications.
A one-pot enzymatic reaction sequence for the synthesis of optically pure d-glyceraldehyde 3-phosphate (d-GAP) and l-glycerol 3-phosphate (sn-G3P) was designed using fructose-bisphosphate aldolase from rabbit muscle (RAMA), sn-glycerol 3-phosphate dehydrogenase (sn-G3PDH) and formate dehydrogenase from Candida boidinii (FDH). The reaction sequence significantly improves the aldol cleavage of d-fructose 1,6-bisphosphate (d-F16BP) catalyzed by RAMA and yields 100% conversion of d-F16BP by overcoming thermodynamic limitation. The degradation kinetics of d-GAP under reaction conditions was investigated and a reaction kinetics model defining the entire cascade was developed. Validation of the model shows 98.5% correlation between experimental data and numerically simulated data matrices. The evaluation of different types of reactor was performed by combining the reaction kinetics model, mass balances and kinetics of the non-enzymatic degradation of d-GAP. Batch-wise operation in a stirred tank reactor (STR) is the most convenient procedure for the one-pot enzymatic syntheses of d-GAP and sn-G3P. The separation of the two products d-GAP and sn-G3P has been achieved using polyethylenimine (PEI)-cellulose TLC.
The oligosaccharide-producing multifunctional amylase (OPMA-N) has both hydrolytic and transglycosyl activities. Our previous reports demonstrated that the function and catalytic versatility of OPMA-N is closely related to its oligomerization, and its oligomeric state is affected by several conserved residues, such as Trp358, and by the cooperation of its small, noncatalytic N-terminal module and the catalytic module. We have demonstrated that the residue Trp358 exposed on the surface of OPMA-N molecule and has an obvious impact on OPMA-N oligomerization mainly by the charge effects. In this study, we investigated the effects of module recombination on the functional integration of OPMA-N. A series of module recombinants of the N-terminal and catalytic modules revealed that the intramolecular, semi-intramolecular and semi-intermolecular interactions of the N-terminal module with two or more catalytic modules enhanced the substrate affinity of the catalytic modules and facilitated the transglycosyl activity and functional integration of the enzyme by mediating positive cooperativity between the catalytic modules. Free N-terminal module alone did not contribute to these effects. Based on the results of this study, we speculated that the substrate affinity, but not the maximal catalytic activity, was the primary driving force in the natural evolution of enzymes, and that molecules of natural or modular enzymes may have occupied some evolutionary spaces that could be expanded or exhumed through module recombination to increase their overall catalytic efficacies or eventual catalytic efficiencies. All of the results in this study could be applied to integration of enzyme function and for creation of novel enzymes. Additionally, our results may provide important insights into the evolution of enzymes or organisms.
Cytochromes P450 (P450s) are valuable enzymes that can generate a range of useful compounds via biocatalytic oxidations that complement traditional synthetic chemistry. In this study three bacterial P450s, P450cam (CYP101A1), CYP101B1 and the mutant N242A-P450cin (N242A-CYP176A1), were used to produce a range of products from the oxidation of the monoterpenes (1R)- and (1S)-camphor and 1,8-cineole. We demonstrate that both in vitro and in vivo catalytic turnover with these P450s can produce a complement of up to seven hydroxycamphors and seven hydroxycineoles, in addition to compounds produced from further oxidation. The CYP101B1 whole cell catalytic system was found to produce 300–600mg/L of culture of oxidation products that could be easily separated chromatographically. The CYP101B1 in vitro oxidation of 1,8-cineole primarily produced (1S)-5α-hydroxycineole, which was 78% of the total product formed. However, the amount of (1S)-5α-hydroxycineole was reduced to 42% of the total products when isolated from the CYP101B1 whole cell system. (1S)-6α-Hydroxycineole (96% ee) could be isolated from a whole cell catalytic turnover of 1,8-cineole by N242A-P450cin in a yield of 46mg/L (98% of the total product). However, the amount of product isolated ((1R)-5-endo-hydroxycamphor, 75% of the total products) from the whole cell catalytic oxidation of (1R)-camphor with N242A-P450cin was much lower (6mg/L) due to the inefficient use of reducing equivalents (3.5±0.5%) for substrate oxidation. These compounds will assist in the identification of specific structures in mechanistic investigations and structure elucidation, but further optimisation is required to generate larger quantities for synthetic applications.
In order to obtain a deep insight into the effects of spacer arms on performing CB modification and dye-affinity adsorption, poly(methyl methacrylate) (PMMA) magnetic microspheres were prepared and modified respectively with poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA) and ethylenediamine (EDA) as spacer arms. Then, an affinity dye-ligand, Cibacron Blue F3GA (CB), was coupled with these spacer arms to synthesize three kinds of CB-attached magnetic microsphere including CB-PEG-PMMA, CB-PVA-PMMA and CB-EDA-PMMA. CB density of the resulting microspheres was determined to be 138.1μmol/g for CB-PEG-PMMA, 209.7μmol/g for CB-PVA-PMMA, and 266.0μmol/g for CB-EDA-PMMA, respectively. The affinity adsorption performances of the resulting microspheres were evaluated using bovine serum albumin (BSA). The results showed that the adsorption capacity of BSA increased with the increase in CB density, and CB-EDA-PMMA microspheres exhibited the highest adsorption capacity. Furthermore, the effects of pH, BSA concentration and ionic strength on BSA adsorption were investigated and the maximum adsorption capacity was found to be 114.0mg/g CB-EDA-PMMA microspheres. Moreover, the reusability of the microspheres was also studied.
