Increase In Residual Activity Research Articles

Improving the thermal stability and catalytic activity of ulvan lyase by the combination of FoldX and KnowVolution campaign

Thermal stability is one of the most important properties of ulvan lyases for their application in algae biomass degradation. The Knowledge gaining directed eVolution (KnowVolution) protein engineering strategy could be employed to improve thermostability of ulvan lyase with less screening effort. Herein, the unfolding free energies (ΔΔG) of the loop region were calculated using FoldX and four sites (D103, G104, T113, Q229) were selected for saturation mutagenesis, resulting in the identification of a favorable single-site mutant Q229M. Subsequently, iteration mutation was carried out with the mutant N57P (previously obtained by our group) to further enhance the performance of ulvan lyase. The results showed that the most beneficial variant N57P/Q229M exhibited a 1.67-fold and 2-fold increase in residual activity compared to the wild type after incubation at 40 °C and 50 °C for 1 h, respectively. In addition, the variant produced 1.06 mg/mL of reducing sugar in 2 h, which was almost four times as much as the wild type. Molecular dynamics simulations revealed that N57P/Q229M mutant enhanced the structural rigidity by augmenting intramolecular hydrogen bonds. Meanwhile, the shorter proton transmission distance between the general base of the enzyme and the substrate contributed to the glycosidic bond breakage. Our research showed that in silico saturation mutagenesis using position scan module in FoldX allowed for faster screening of mutants with improved thermal stability, and combining it with KnowVolution enabled a balanced effect of thermal stability and enzyme activity in protein engineering.

Cross-linked phytase aggregates for improved phytate degradation at low pH in animal feed

Phytases are widely used food and feed enzymes to improve phosphate availability and reduce anti-nutritional factors. Despite the benefits, enzyme usage is restricted by the harsh conditions in a gastrointestinal tract (pH 2–6) and feed pelleting conditions at high temperatures (60–90 °C). The commercially available phytase Quantum® Blue has been immobilized as CLEAs using glutardialdehyde and soy protein resulting in a residual activity of 33%. The influence of the precipitating agent, precipitant concentration, cross-linker concentration and cross-linking time, sodium borohydride as well as the proteic feeders gluten, soy protein and bovine serum albumin (BSA) has been optimized. The best conditions were 90% (v/v) ethyl lactate as precipitating reagent, 100 mM glutardialdehyde and a soy protein concentration of 227 mg/L with a cross-linking time of 1 h. The intrinsically stable phytase remained its high thermal stability and temperature optimum. The phytase-CLEA achieved a 425% increase of residual activity under harsh acidic conditions between pH 2.2 and 3.5 compared to the free enzyme. The free and immobilized phytase were deployed in an in vitro assay simulating the acidic conditions in the gizzard of poultry at pH 2. The hydrolysis of phytate was monitored using a novel high-performance thin-layer chromatography (HPTLC) analysis and DAD scanner to study the InsPx fingerprint. All lower inositol phosphate pools (InsP1–InsP6) and free phosphate were separated and analyzed. The phytase-CLEA efficiently released 80% of the total phosphate within 180 min, whereas the free enzyme only released 6% in the same time under the same conditions.

Improving the specific activity and stability of alkaline pectinase PEL3 through SpyTag/SpyCatcher cyclization.

PEL3, an alkaline pectinase, exhibited the highest activity among documented alkaline pectate lyases reported in our early study. Unfortunately, undesired thermal stability hampering its industrial application. The purpose of this study is to enhance the performance of wild-type PEL3 (W-PEL3) based on SpyTag/SpyCatcher-mediated cyclization. The cyclized PEL3 (C-PEL3) was observed to fold correctly and generate a spatial conformation in a head-to-tail manner in E. coli. C-PEL3 exhibited comparable optimum pH and temperature to those of W-PEL3. Moreover, the catalytic activity of C-PEL3 increased by 23% compared to W-PEL3, and the kcat/Km of C-PEL3 was 1.5-fold greater than that of the W-PEL3. Importantly, C-PEL3 showed improved stability compared to W-PEL3. Firstly, C-PEL3 displayed a 65% increase in residual activity after treatment at 55°C for 30min. Secondly, C-PEL3 was prone to resist heat-induced protein aggregation. Thirdly, C-PEL3 exhibited metal ion stability. Circular dichroism analysis revealed that C-PEL3 was more capable of maintaining its secondary structures than W-PEL3 upon heat treatment. C-PEL3, the initial example of a circular pectinase through SpyTag/SpyCatcher cyclization, exhibits superior performance and represents a highly encouraging contender for industrial utilization.

Engineering the Thermostability of the Mono- and Diacylglycerol Lipase SMG1 for the Synthesis of Diacylglycerols.

Diacylglycerols (DAGs) display huge application prospectives in food industries. Therefore, new strategies to produce diacylglycerides are needed. Malassezia globose lipase (SMG1) could be used to synthesize DAGs. However, the poor thermostability of SMG1 seriously hampers its application. Herein, a rational design was used to generate a more thermostable SMG1. Compared with the wild type (WT), the M5D mutant (Q34P/A37P/M176V/G177A/M294R/ G28C-P206C), which contains five single-point mutations and one additional disulfide bond, displayed a 14.0 °C increase in the melting temperature (Tm), 5 °C in the optimal temperature, and 1154.3-fold in the half-life (t1/2) at 55 °C. Meanwhile, the specific activity towards DAGs of the M5D variant was improved by 3.0-fold compared to the WT. Molecular dynamics (MD) simulations revealed that the M5D mutant showed an improved rigid structure. Additionally, the WT and the M5D variants were immobilized and used for the production of DAGs. Compared with the WT, the immobilized M5D-catalyzed esterification showed a 9.1% higher DAG content and a 22.9% increase in residual activity after nine consecutive cycles. This study will pave the way for the industrial application of SMG1.

Enhancement of activity and thermostability of Aspergillus niger ATCC 10864 phytase A through rational design

Aspergillus niger ATCC 10864 phytase A was produced in Penicillium verruculosum. The enzyme was found to have two pH optima of 2.5 and 5.0, as well as a T-optimum of 50–55 °C. Two amino acid substitutions, A76M and S265P, were designed for improvement in thermostability, and two more, N300K and D363N, were designed for improvement in enzyme activity. The most thermostable variant, S265P, was characterized by a 3.8-fold increase in time of half-life at 55 °C and a 1.2-fold increase in residual activity at 90 °C compared to the wild-type. The most active variant, D363N, was 1.7-times more active at 40 °C and retained 1.3-times higher residual activity at 90 °C compared to the wild-type. The obtained results revealed the importance of substitutions with proline in α-helixes for the thermostability improvement of phytases. Also, the importance of sequence motif 361HDN363 was demonstrated with relevance to values of catalytic parameters.

Biodegradation of nitriles derived from glucosinolates in rapeseed meal by BnNIT2: a nitrilase from Brassica napus with wide substrate specificity.

Nitriles derived from glucosinolates (GSLs) in rapeseed meal (RSM) can cause lesions on animal liver and kidneys. Nitrilase converts nitriles to carboxylic acids and NH3, eliminating their toxicity. Here we describe a nitrilase, BnNIT2, from Brassica napus (optimal temperature, 45°C; pH, 7.0) that is stable at 40°C and has a wide substrate specificity. Recombinant BnNIT2 converted the three main nitriles from GSLs (3-hydroxy-4-pentenenitrile, 3-butenenitrile, and 4-pentenenitrile), with the highest specific activity (58.6 U/mg) for 4-pentenenitrile. We used mutagenesis to improve the thermostability of BnNIT2; the resulting mutant BnNIT2-H90M had an ~ 14.5% increase in residual activity at 50°C for 1h. To verify the functionality of BnNIT2, GSLs were extracted from RSM and converted into nitriles at pH 5.0 in the presence of Fe2+. Then, BnNIT2 was used to degrade the nitriles from GSLs; ultimately, ~ 80% of nitriles were removed. Thus BnNIT2 is a potential enzyme for detoxification of RSM. KEY POINTS: • Functional identification of the plant nitrilase BnNIT2. • Identified a mutant, H90M, with improved thermostability. • BnNIT2 was capable of degrading nitriles from transformed GSLs.

Computational Design of Nitrile Hydratase from Pseudonocardia thermophila JCM3095 for Improved Thermostability

High thermostability and catalytic activity are key properties for nitrile hydratase (NHase, EC 4.2.1.84) as a well-industrialized catalyst. In this study, rational design was applied to tailor the thermostability of NHase from Pseudonocardia thermophila JCM3095 (PtNHase) by combining FireProt server prediction and molecular dynamics (MD) simulation. Site-directed mutagenesis of non-catalytic residues provided by the rational design was subsequentially performed. The positive multiple-point mutant, namely, M10 (αI5P/αT18Y/αQ31L/αD92H/βA20P/βP38L/βF118W/βS130Y/βC189N/βC218V), was obtained and further analyzed. The Melting temperature (Tm) of the M10 mutant showed an increase by 3.2 °C and a substantial increase in residual activity of the enzyme at elevated temperatures was also observed. Moreover, the M10 mutant also showed a 2.1-fold increase in catalytic activity compared with the wild-type PtNHase. Molecular docking and MD simulations demonstrated better substrate affinity and improved thermostability for the mutant.

Tyrosinase from Penicillium chrysogenum: Characterization and application in phenol removal from aqueous solution.

The tyrosinase of Penicillium chrysogenum strain AUMC 14100 Accession No. MN219732 was purified to homogeneity and chemically modified by N-ethylmaleimide (NEM) and 5-(dimethylamino)naphthalene-1-sulfonyl chloride (dansyl chloride, DC). The inactivation of the purified enzyme obeyed pseudo-first-order reaction kinetics in the presence of NEM and DC (1-5 mM). The rate constants of the enzyme inactivation by NEM and DC were calculated to be 0.083 mol/min and 0.0013 mol/min, respectively. The recovery of enzyme activity by the protective effect of substrate indicates a non-specific modification of the active center. The order of tyrosinase inactivation kinetics and the substrate protection revealed the essentiality of sulfhydryl and lysyl residues in the enzyme active site and its role in the enzyme catalysis. The immobilized tyrosinase on alginate showed a gradual increase in residual activity over the immobilization time until the fourth hour. The desorptivity of tyrosinase was gradually raised with higher sodium dodecyl sulfate (SDS) concentrations. The immobilized enzyme retained about 70% of its original activity after 8 repeated cycles. Thus, immobilized tyrosinase of Penicillium chrysogenum removed 75% of phenol after 8 cycles and thus seems likely to be a good candidate for phenol removal in aqueous solution.

Engineering of serine protease for improved thermostability and catalytic activity using rational design

The study involves the isolation and characterization of a serine peptidase, named SP, from Pseudomonas aeruginosa. In addition to basic characterization, the protein was engineered, by site-directed mutagenesis of selected non-catalytic residues, to increase its thermal stability and catalytic activity. Among the eight-point mutations, predicted by FireProt, two mutants, A29G and V336I, yielded a positive impact. The Tm of A29G and V336I showed an increase by 5 °C and also a substantial increase in residual activity of the enzyme at elevated temperature. Moreover, the catalytic activity of A29G and V336I also showed an increase of 1.4-fold activity, compared to the wild-type (WT). Moreover, molecular docking simulations also predicted better substrate affinity of the mutants. We have also performed molecular dynamics (MD) simulations at 315 and 345 K, and the MD data at 345 K demonstrates improved thermostability for the mutants, compared to the WT. Our findings not only contribute to a better understanding of the structure-stability-activity relationship of SP but also highlights, that modification of non-catalytic residues could also promote favourable catalytic behaviour.

Engineering thermal stability and solvent tolerance of the soluble quinoprotein PedE from Pseudomonas putida KT2440 with a heterologous whole-cell screening approach.

SummaryDue to their ability for direct electron transfer to electrodes, the utilization of rare earth metals as cofactor, and their periplasmic localization, pyrroloquinoline quinone‐dependent alcohol dehydrogenases (PQQ‐ADHs) represent an interesting class of biocatalysts for various biotechnological applications. For most biocatalysts protein stability is crucial, either to increase the performance of the protein under a given process condition or to maximize robustness of the protein towards mutational manipulations, which are often needed to enhance or introduce a functionality of interest. In this study, we describe a whole‐cell screening assay, suitable for probing PQQ‐ADH activities in Escherichia coli BL21(DE3) cells, and use this assay to screen smart mutant libraries for increased thermal stability of the PQQ‐ADH PedE (PP_2674) from Pseudomonas putida KT2440. Upon three consecutive rounds of screening, we identified three different amino acid positions, which significantly improve enzyme stability. The subsequent combination of the beneficial mutations finally results in the triple mutant R91D/E408P/N410K, which not only exhibits a 7°C increase in thermal stability but also a twofold increase in residual activity upon incubation with up to 50% dimethyl sulfoxide (DMSO), while showing no significant difference in enzymatic efficiency (k cat/KM).

Liquefied petroleum gas as solvent medium for the treatment of immobilized pectinases

Abstract This work is aimed at assessing the influence of pressurized fluids treatment on the enzymatic activity of pectin lyase (Pectinex® Ultra SP-L and Pectinex® Mash) immobilized in hybrid polymer-inorganic of alginate/gelatin/calcium oxalate (AGOCa). The effects of system pressure (30–190 bar), exposure time (1–6 h) and depressurization rate (20–100 bar/min), using liquefied petroleum gas (LPG) on the enzymatic activity of pectin lyase and pectin methyl esterase have been evaluated through Central Composite Rotational Design (CCRD) 2 3 . The pectin lyase Pectinex® Ultra SP-L presented a 14% increase in residual activity using LPG at 30 bar during 1 h exposure with a 100 bar/min depressurization rate and the commercial Pectinex ® Mash had a 38% increment observed at 190 bar for 1 h treatment at 20 bar/min depressurization rate. Enzyme extracts immobilized on alginate-gelatin remained at approximately 60% of pectin lyase (Pectinex® Mash) activity on the sixth recycle after treatment LPG. However, approximately 53% of the pectin lyase (Pectinex® Ultra SP-L) remained until the third recycle. The treatment with pressurized LPG increases the catalytic power of pectinases, hence constituting an interesting solvent to improve the enzyme activity before its use in the process or to carry out reactions using LPG as reaction medium in a enzymatic gas–solid system.

Enhanced thermostability of a fungal alkaline protease by different additives.

A fungal strain (Conidiobolus brefeldianus MTCC 5184) isolated from plant detritus secreted a high activity alkaline protease. Thermostability studies of the fungal alkaline protease (FAP) revealed that the protease is stable up to 50°C with 40% residual activity after one hour. Effect of various additives such as sugars, sugar alcohols, polyols, and salts, on the thermostability of FAP was evaluated. Among the additives tested, glycerol, mannitol, xylitol, sorbitol, and trehalose were found to be very effective in increasing the stability of FAP, which was found to be concentration dependent. Fivefold increase in residual activity of FAP was observed in the presence of trehalose (50%) and sorbitol (50%) at 50°C for 4 h, compared to FAP without additive. Other additives like calcium at 20 mM and 10–15% ammonium sulphate showed lower stability improvement than trehalose and sorbitol. NaCl, MgCl2, K2HPO4, and glycine were found to be poor stabilizers and showed only a marginal improvement. PEG 6000 did not show any increase in stability but was found to be slightly inhibitory.

Chaperone‐like activity of β‐casein and its effect on residual in vitro activity of horseradish peroxidase

SummaryIn this study, the residual activity horseradish peroxidase was used as a novel marker of chaperone‐like activity of β‐casein under elevated temperature. It was shown that β‐casein does affect residual activity of horseradish peroxidase (HRP) depending on the concentration and molar ratio between proteins. Incubating HRP (0.1 mg mL−1) for 10 min at 72 °C resulted in residual activity of 59 ± 5%, while addition of 1 mg mL−1 β‐casein resulted in increase in residual activity up to 85 ± 1%. Increased residual activity is not merely attributed to an effect of higher total protein concentration, as similar experiment with bovine serum albumin resulted in residual activity of horseradish peroxidase that was significantly lower than without any addition. The effect of β‐casein on HRP disappears when pH is below the isoelectric point of β‐casein. It was also proven by light scattering studies that β‐casein interacts with horseradish peroxidase when the temperature was increased from 25 to 70 °C whereas interactions seem to cease when temperature was lowered back to 25 °C. This study highlights how specific proteins can influence enzyme activity, which is of potential importance for various industries such as enzyme manufacturers and food industry.

Atividade residual de herbicidas pré-emergentes aplicados em solos contrastantes

This study investigated the residual activity of different rates of the herbicides alachlor, oxyfluorfen, prometryne and S-metolachlor applied in pre-emergence in soils of contrasting textures. For that, eight simultaneous experiments were conducted with samples of PSAMENT (sandy texture) and RED LATOSOL (clay texture), which received alachlor (0; 2.40 and 3.36 kg ha -1 ), oxyfluorfen (0; 0.48 and 0.72 kg ha -1 ), prometryne (0; 0.75 and 1.50 kg ha -1 ) and S-metolachlor (0; 1.20 and 1.44 kg ha -1 ) applications. The treatments monitoring was made through sowing of bio-indicator ( Brachiaria decumbens or Cucumis sativus ). Control was assessed by scores from 0 to 100% at 0, 25, 50, 75, 100 days after application (DAA). Regardeless of rate, alachlor provided control around 80% to 66 DAA, in clay soil and up to 50 DAA in sandy. Oxyfluorfen showed control around 80% to 56 DAA to clay soil and up to 36 DAA to sandy, in both rates. Both rates of prometryne in clay soil provided control over 80% to 28 DAA, with residual activity higher in recommended rate (1.50 kg ha -1 ). In samples sandy, S-metolachlor provided the same control for both rates, controlling the bio-indicator at 80% up to 52 DAA, and for clay soil in rate at 1.44 kg ha -1 , presented 96% control the 80 DAA. Thereby, there was no increase in residual activity in sandy soil, even with the application of the recommended rate for clay soil.

Moisture-induced solid state instabilities in α-chymotrypsin and their reduction through chemical glycosylation

BackgroundProtein instability remains the main factor limiting the development of protein therapeutics. The fragile nature (structurally and chemically) of proteins makes them susceptible to detrimental events during processing, storage, and delivery. To overcome this, proteins are often formulated in the solid-state which combines superior stability properties with reduced operational costs. Nevertheless, solid protein pharmaceuticals can also suffer from instability problems due to moisture sorption. Chemical protein glycosylation has evolved into an important tool to overcome several instability issues associated with proteins. Herein, we employed chemical glycosylation to stabilize a solid-state protein formulation against moisture-induced deterioration in the lyophilized state.ResultsFirst, we investigated the consequences of moisture sorption on the stability and structural conformation of the model enzyme α-chymotrypsin (α-CT) under controlled humidity conditions. Results showed that α-CT aggregates and inactivates as a function of increased relative humidity (RH). Furthermore, α-CT loses its native secondary and tertiary structure rapidly at increasing RH. In addition, H/D exchange studies revealed that α-CT structural dynamics increased at increasing RH. The magnitude of the structural changes in tendency parallels the solid-state instability data (i.e., formation of buffer-insoluble aggregates, inactivation, and loss of native conformation upon reconstitution). To determine if these moisture-induced instability issues could be ameliorated by chemical glycosylation we proceeded to modify our model protein with chemically activated glycans of differing lengths (lactose and dextran (10 kDa)). The various glycoconjugates showed a marked decrease in aggregation and an increase in residual activity after incubation. These stabilization effects were found to be independent of the glycan size.ConclusionWater sorption leads to aggregation, inactivation, and structural changes of α-CT as has been similarly shown to occur for many other proteins. These instabilities correlate with an increase in protein structural dynamics as a result of moisture exposure. In this work, we present a novel methodology to stabilize proteins against structural perturbations in the solid-state since chemical glycosylation was effective in decreasing and/or preventing the traditionally observed moisture-induced aggregation and inactivation. It is suggested that the stabilization provided by these chemically attached glycans comes from the steric hindrance that the sugars conveys on the protein surface therefore preventing the interaction of the protein internal electrostatics with that of the water molecules and thus reducing the protein structural dynamics upon moisture exposure.

Atividade residual de herbicidas aplicados ao solo em relação ao controle de quatro espécies de Amaranthus

Herbicidas aplicados em pré-emergência normalmente apresentam atividade residual no solo, controlando os primeiros fluxos germinativos das plantas daninhas e prevenindo a matocompetição inicial. O objetivo deste trabalho foi verificar o período de atividade residual proporcionado por doses de herbicidas suficientes para o controle pontual de 95% (C95) das espécies Amaranthus hybridus, A. lividus, A. spinosus e A. viridis, além de avaliar doses recomendadas desses herbicidas. O trabalho foi realizado em casa de vegetação, em solo de textura franco-argiloarenosa (20% de argila e 1,9 de matéria orgânica), e as doses dos herbicidas alachlor, diuron, oxyfluorfen, pendimethalin, prometryne, oxyfluorfen, S-metolachlor, trifluralin 450 e trifluralin 600 foram aplicadas aos 30, 20, 10 e 0 dias antes da semeadura das plantas daninhas. Avaliou-se o controle das plantas daninhas após a permanência dos herbicidas no solo por períodos de 0, 10, 20 e 30 dias depois da aplicação dos tratamentos (DAA). A atividade residual de alachlor e prometryne, na dose C95, não foi suficiente para o controle eficiente (>80%) das espécies por períodos de até 30 DAA. Quanto ao alachlor, o emprego da dose recomendada não se refletiu em aumento considerável da atividade residual, exceto em relação a A. viridis. A dose recomendada de prometryne proporcionou controle eficiente das espécies até 30 DAA, exceto de A. hybridus. A dose recomendada de oxyfluorfen controlou eficientemente A. hybridus e A. spinosus até 30 DAA, espécies estas que não haviam sido eficientemente controladas pela dose C95. Trifluralin 450 promoveu controle residual eficiente de 30 DAA somente em relação a A. hybridus. Trifluralin 600 foi eficiente no controle de A. hybridus e A. viridis até os 30 DAA e até 29 e 28 DAA para A. lividus e A. spinosus, respectivamente. Clomazone não promoveu controle eficiente das espécies até 30 DAA, exceto de A. viridis. Diuron, pendimethalin e S-metolachlor foram eficientes para todas as espécies até 30 DAA, em ambas as doses, demonstrando atividade residual consistente para o solo estudado.

Calcined layered double hydroxides as a “biomolecular vessel” for bromelain: Immobilization, storage and release

Calcined layered double hydroxide (LDO) has been used as the novel biomolecular vessel for the immobilization, storage as well as release of bromelain. The immobilization is attributed to physical adsorption without affecting the structure of LDO, and the optimum initial amount of bromelain was 20 ml of 4 mg/ml for 1 g LDO and the immobilized enzyme activity express was 33.4% at pH 6.5. The adsorption isotherm of bromelain/LDO can be well described with the Langmuir model with a R 2 of 0.9946, a maximum adsorption amount of 157 mg/g and the Langmuir adsorption equilibrium constant of 0.07 ml/mg. Significant improvements have been found in the stability of immobilized bromelain upon heat treatment (increase in residual activity and denaturation temperature) as well as storage time compared with those of free bromelain. In addition, the release maximum of bromelain reached to 70% and the residual activity maintained 81% in water solution at pH 7.