Catalytic Efficiency Of Enzyme Research Articles

Layer-by-Layer Engineered All-Liquid Microfluidic Chips for Enzyme Immobilization.

Enzyme immobilization in the confines of microfluidic chips, that promote enzyme activity and stability, has become a powerful strategy to enhance biocatalysis and biomass conversion. Here, based on a newly developed all-liquid microfluidic chip, fabricated by the interfacial assembly of nanoparticle surfactants (NPSs) in a biphasic system, a layer-by-layer assembly strategy to generate polysaccharide multilayers on the surface of a microchannel, greatly enhancing the mechanical properties of the microchannel and offering a biocompatible microenvironment for enzyme immobilization, is presented. Using horseradish peroxidase and glucose oxidase as model enzymes, all-liquid microfluidic enzymatic and cascade reactors have been constructed and the crucial role of polysaccharide multilayers on enhancing the enzyme loading and catalytic efficiency is demonstrated.

Unique Biradical Intermediate in the Mechanism of the Heme Enzyme Chlorite Dismutase.

The heme enzyme chlorite dismutase (Cld) catalyzes O–O bond formation as part of the conversion of the toxic chlorite (ClO2–) to chloride (Cl–) and molecular oxygen (O2). Enzymatic O–O bond formation is rare in nature, and therefore, the reaction mechanism of Cld is of great interest. Microsecond timescale pre-steady-state kinetic experiments employing Cld from Azospira oryzae (AoCld), the natural substrate chlorite, and the model substrate peracetic acid (PAA) reveal the formation of distinct intermediates. AoCld forms a complex with PAA rapidly, which is cleaved heterolytically to yield Compound I, which is sequentially converted to Compound II. In the presence of chlorite, AoCld forms an initial intermediate with spectroscopic characteristics of a 6-coordinate high-spin ferric substrate adduct, which subsequently transforms at kobs = 2–5 × 104 s–1 to an intermediate 5-coordinated high-spin ferric species. Microsecond-timescale freeze-hyperquench experiments uncovered the presence of a transient low-spin ferric species and a triplet species attributed to two weakly coupled amino acid cation radicals. The intermediates of the chlorite reaction were not observed with the model substrate PAA. These findings demonstrate the nature of physiologically relevant catalytic intermediates and show that the commonly used model substrate may not behave as expected, which demands a revision of the currently proposed mechanism of Clds. The transient triplet-state biradical species that we designate as Compound T is, to the best of our knowledge, unique in heme enzymology. The results highlight electron paramagnetic resonance spectroscopic evidence for transient intermediate formation during the reaction of AoCld with its natural substrate chlorite. In the proposed mechanism, the heme iron remains ferric throughout the catalytic cycle, which may minimize the heme moiety’s reorganization and thereby maximize the enzyme’s catalytic efficiency.

Substrate specificity, physicochemical and kinetic properties of a trypsin from the giant Amazonian fish pirarucu (Arapaima gigas)

Specificity studies can contribute to the understanding of how substrate binding and catalysis change among similar enzymes. This study evaluated through the substrate specificity, physicochemical and kinetic properties of a trypsin from the pyloric caeca of pirarucu (Arapaima gigas). For such purposes, two series of FRET-peptides (Abz-RXFK-Eddnp and Abz-XRFK-Eddnp) were used. The mature trypsin molecular mass was 23.5 kDa and N-terminal sequence was IVGGYECPRNSVPYQVSLNVGYH. Moreover, higher trypsin catalytic efficiency was observed for the substrates containing Arg, Tyr and Ser at P1′ and Lys and Val at P2. However, catalytic deficiency was found in the presence of Pro, Trp, Asp, Gly, Glu, Ala at P1’ and Gly, Glu, Trp, Asn, Gln and Tyr at P2. Using z-FR-MCA as a substrate, the optimum pH (9.25) and temperature (45 °C) were determined by pseudo-first-order kinetics. The enzyme retained all of its initial activity after 180 min incubation at temperatures up to 45 °C. Kinetic assays in the presence of Ca2+ showed an increase of approximately 7-fold in enzyme catalytic efficiency (kcat/Km). On the other hand, Na+, K+ and Mg2+ were able to inhibit pirarucu trypsin. Therefore, the knowledge of kinetic and biochemical properties, as well as enzyme specificity, are important for the evaluation of biotechnological use of this enzyme and may contribute to the development of more efficient substrates for fish trypsin.

Immobilizing Enzymes on Noble Metal Hydrogel Nanozymes with Synergistically Enhanced Peroxidase Activity for Ultrasensitive Immunoassays by Cascade Signal Amplification.

Enzyme immobilization plays an essential role in solving the problems of the inherently fragile nature of enzymes. Although prominent stability and reuse of enzymes can be achieved by enzyme immobilization, their bioactivity and catalytic efficiency will be adversely affected. Herein, PdCu hydrogel nanozymes with a hierarchically porous structure were used to immobilize horseradish peroxidase (HRP) to obtain PdCu@HRP. In addition to the improvement of stability and reusability, PdCu@HRP displayed synergistically enhanced activities than native HRP and PdCu hydrogels. Not only the specific interactions between PdCu hydrogel nanozymes and enzymes but also the enrichment of substrates around enzymes by electrostatic adsorption of hydrogels was proposed to expound the enhanced catalytic activity. Accordingly, by taking advantage of the excellent catalytic performance of the PdCu@HRP and the glucose oxidase encapsulated in zeolitic imidazolate framework-8, colorimetric biosensing of the carcinoembryonic antigen via catalytic cascade reactions for achieving signal amplification was performed. The obtained biosensor enhanced the detection sensitivity by approximately 6.1-fold as compared to the conventional HRP-based enzyme-linked immunosorbent assay, demonstrating the promising potential in clinical diagnosis.

Weaving Enzymes with Polymeric Shells for Biomedical Applications.

Enzyme therapeutics have received increasing attention due to their high biological specificity, outstanding catalytic efficiency, and impressive therapeutic outcomes. Protecting and delivering enzymes into target cells while retaining enzyme catalytic efficiency is a big challenge. Wrapping of enzymes with rational designed polymer shells, rather than trapping them into large nanoparticles such as liposomes, have been widely explored because they can protect the folded state of the enzyme and make post-functionalization easier. In this review, the methods for wrapping up enzymes with protective polymer shells are mainly focused on. It is aimed to provide a toolbox for the rational design of polymeric enzymes by introducing methods for the preparation of polymeric enzymes including physical adsorption and chemical conjugation with specific examples of these conjugates/hybrid applications. Finally, a conclusion is drawn and key points are emphasized.

Improvement Thermal Stability of D-Lactate Dehydrogenase by Hydrophobin-1 and in Silico Prediction of Protein-Protein Interactions.

Hydrophobins are small surface-active proteins. They can connect to hydrophobic or hydrophilic regions and oligomerize in solution to form massive construction. In nature, these proteins are produced by filamentous fungi at different stages of growth. So far, researchers have used them in various fields of biotechnology. In this study, recombinant hydrophobin-1 (rHFB1, 7.5kDa) was used to stabilize recombinant D-lactate dehydrogenase (rD-LDH, 35kDa). rD-LDH is a sensitive enzyme deactivated and oxidized by external agents such as O2 and lights. So, its stabilization with rHFB1 can be the best index to demonstrate the positive effect of rHFB1 on preserving and improving enzyme's activity. The unique ability of rHFB1 for interacting with hydrophobic regions of rD-LDH was predicted by protein-protein docking study with ClusPro and PIC servers and confirmed by fluorescence experiments, and Colorless Native-PAGE. Measurement of thermodynamic parameters allows for authenticating the role of rHFB1 as a thermal stabilizer in the protein-protein complex (rD-LDH@rHFB1). Interaction between rHFB1 and rD-LDH improved half-life of enzyme 2.25-fold at 40°C. Investigation of the kinetic parameters proved that the presence of rHFB1 along with the rD-LDH enhancement strongly the affinity of the enzyme for pyruvate. Furthermore, an increase of Kcat/Km for complex displayed the effect of rHFB1 for improving the enzyme's catalytic efficiency.

Recent Trends in Omics-Based Enzyme Discovery to Combat Global Plastic Waste Crisis

The worldwide rapidly increasing amount of plastic waste is considered a major concern in the environmental crisis today. Recycling post-consumer plastics is believed to be a viable approach to mitigate the global plastic waste challenge. Plastic recycling is considered attractive due to the reason that it could help achieve waste valorization while meeting the goals of environmental quality standards. To this end, the current research efforts are heavily focused towards developing innovative technologies for new enzyme discoveries for effective plastic waste management. Recent research has shown biocatalytic depolymerization mediated by enzymes as an efficient and sustainable alternative for plastic treatment and recycling. Using this approach, researchers have discovered different plastic-degrading enzymes that have been derived from microbial sources. Further, the concept of protein engineering has been implemented to modify and optimize plastic-degrading enzymes. In this brief opinion, we describe some of the recent trends and notable advances in mining novel plastic-degrading enzymes through cutting-edge omics-based techniques in order to improve the enzyme catalytic efficiency.

Structural and functional study on cysteine 495, coordinating ligand to T1Cu site in multicopper oxidase CopA

Excessive intake of manganese seriously affects human health. Manganese oxidizing bacteria can efficiently remove manganese, among which manganese oxidase plays a decisive role. Multicopper oxidase, one of the manganese oxidases, has 4 copper binding sites, among them, T1Cu coordinates with two histidine, one cysteine and one axial residue, mainly transferring electrons from the substrate to T2Cu and T3Cu. Here, we conducted site-directed mutagenesis on T1Cu coordinating 495 amino acid site from cysteine to aspartic acid, histidine and methionine in multicopper oxidase CopA from Brevibacillus panacihumi MK-8, through the enzyme kinetics and structure models, finding that the enzyme catalytic efficiency (kcat/Km) of the mutated C495H with Mn2+ and ABTS reached 9.03 min−1 mM−1 and 8863 s−1 mM−1, 1.47 times and 1.67 times that of CopA. And it was found strain Rosetta-pET-copAC495H could remove 91.67% manganese after 7-day culture, which was 11.65% higher than the original strain. To sum up, these results provide a vision for the future application of protein engineering in biological manganese removal.

A general Ca-MOM platform with enhanced acid-base stability for enzyme biocatalysis

Co-precipitation of enzymes in metal-organic frameworks is a unique enzyme-immobilization strategy but is challenged by weak acid-base stability. To overcome this drawback, we discovered that Ca2+ can co-precipitate with carboxylate ligands and enzymes under ambient aqueous conditions and form [email protected] material composites stable under a wide range of pHs (3.7–9.5). We proved this strategy on four enzymes with varied isoelectric points, molecular weights, and substrate sizes—lysozyme, lipase, glucose oxidase (GOx), and horseradish peroxidase (HRP)—as well as the cluster of HRP and GOx. Interestingly, the catalytic efficiency of the studied enzymes was found to depend on the ligand, probing the origins of which resulted in a correlation among enzyme backbone dynamics, ligand selection, and catalytic efficiency. Our approach resolved the long-lasting stability issue of aqueous-phase co-precipitation and can be generalized to biocatalysis with other enzymes to benefit both research and industry.

Biocatalytic Yarn for Peroxide Decomposition with Controlled Liquid Transport

AbstractA robust biocatalytic yarn with controllable liquid transport properties is created by coating thin layers of chitosan containing catalase onto a cellulosic yarn. The resulting material integrates enzyme catalytic functionality with protective coating properties of chitosan and structural functionality of the textile. Mild immobilization conditions and good affinity between the two polysaccharides minimize enzyme inactivation during the preparation steps and prevent enzyme from leaching during peroxide decomposition testing and washing, providing a novel and versatile enzyme immobilization strategy. The catalytic efficiency of enzymes in a reaction containing solid, liquid, and gas phases is facilitated when dissolved enzyme substrate is transported by liquid flowing through the coated textile structure. The flow‐through configuration decomposes at least two times more peroxide in a twenty‐times smaller reaction zone volume compared to a stirred tank configuration. Liquid transport through the yarn and liquid spatial distribution within the yarn are investigated by in situ neutron radiography and neutron computed tomography, revealing a constrained wicking mechanism that benefits biocatalytic yarn performance. This new class of sustainable and flexible biocatalytic textile matrices has beneficial multifunctional properties, not previously described, that are applicable for numerous small‐ and large‐scale applications including controlled flow reactors and reactive filtration.

Simultaneous directed evolution of coupled enzymes for efficient asymmetric synthesis of l-phosphinothricin.

The traditional strategy to improve the efficiency of an entire coupled enzyme system relies on separate direction of the evolution of enzymes involved in their respective enzymatic reactions. This strategy can lead to enhanced single-enzyme catalytic efficiency but may also lead to loss of coordination among enzymes. This study aimed to overcome such shortcomings by executing a directed evolution strategy on multiple enzymes in one combined group that catalyzes the asymmetric biosynthesis of l-phosphinothricin. The genes of a glutamate dehydrogenase from Pseudomonas moorei (PmGluDH) and a glucose dehydrogenase from Exiguobacterium sibiricum (EsGDH), along with other gene parts (promoters, ribosomal binding sites (RBSs), and terminators) were simultaneously evolved. The catalytic efficiency of PmGluDH was boosted by introducing the beneficial mutation A164G (from 1.29 s-1mM-1 to 183.52 s-1mM-1), and the EsGDH expression level was improved by optimizing the linker length between the RBS and the start codon of gdh. The total turnover numbers of the bioreaction increased from 115 (GluDH WTNADPH) to 5846 (A164GNADPH coupled with low expression of EsGDH), and to 33950 (A164GNADPH coupled with high expression of EsGDH). The coupling efficiency was increased from ∼30% (GluDH_WT with low expression of GDH) to 83.3% (GluDH_A164G with high expression of GDH). In the batch production of l-phosphinothricin utilizing whole-cell catalysis, the strongest biocatalytic reaction exhibited a high space-time yield (6410 g·L-1·d-1) with strict stereoselectivity (>99% enantiomeric excess).Importance: The traditional strategy to improve multienzyme-catalyzed reaction efficiency may lead to enhanced single-enzyme catalytic efficiency but may also result in loss of coordination among enzymes. We describe a directed evolution strategy of an entire coupled enzyme system to simultaneously enhance enzyme coordination and catalytic efficiency. The simultaneous evolution strategy was applied to a multienzyme-catalyzed reaction for the asymmetric synthesis of l-phosphinothricin, which not only enhanced the catalytic efficiency of GluDH but also improved the coordination between GluDH and GDH. Since this strategy is enzyme-independent, it may be applicable to other coupled enzyme systems for chiral chemical synthesis.

High‐Level Production of Phenylacetaldehyde using Fusion‐Tagged Styrene Oxide Isomerase

AbstractAn order‐of‐magnitude improvement in the production of phenylacetaldehyde to 3.37 M (405 g L−1) from the enzymatic isomerisation of styrene oxide was achieved. A small ubiquitin‐related modifier (SUMO) tag increases the productivity of the whole‐cell biocatalytic system by enhancing the expression of active membrane‐bound styrene oxide isomerase (SOI) while retaining enzyme catalytic efficiency and broad natural substrate scope. The isomerisation was performed by using Escherichia coli expressing SUMO‐tagged SOI in an organic‐aqueous biphasic system to yield 96% of phenylacetaldehyde.magnified image

Improving Enzyme Catalytic Efficiency by Co-operative Vibrational Strong Coupling of Water.

Here, we report enhancement of catalytic efficiency of an enzymatic reaction by co-operative vibrational strong coupling (VSC) of water and the enzyme α-chymotrypsin. Selective strong coupling of the O-H stretching mode of water along with O-H and N-H stretching modes of the enzyme modify the rate of the enzymatic ester hydrolysis, increasing the catalytic efficiency by more than 7 times. This is specifically achieved by controlling the rate-determining proton-transfer process through a co-operative mechanism. Here, VSC is also used as a spectroscopic tool to understand the mechanism of the enzymatic reaction, suggesting its potential applications in chemistry.

Enhancing the activity and thermal stability of a phthalate-degrading hydrolase by random mutagenesis

Our previous work has reported that EstJ6 was a phthalate-degrading hydrolase. In the study, a random mutant library was constructed by two rounds of error-prone PCR, three mutants (ET1.1, ET2.1, and ET2.2) with enhanced hydrolytic activity against dibutyl phthalate (DBP) were obtained. The best mutant ET2.2, accumulated three amino acid substitutions (Thr91Met, Ala67Val, and Val249Ile) and exhibited 2.8-fold increase enzyme activity and 2.3-fold higher expression level. Meanwhile, compared with EstJ6, ET2.2 showed over 50% improvement in thermostability (at 50 °C for 1 h) and 1.2-fold increase in 50% methanol tolerance. Kinetic parameters analysis revealed that the Km value for ET2.2 decreased by 60% and the kcat/Km value increased by 166%. The molecular docking indicated that the shortening of hydrogen bond between Ser146-OH and DBP-CO, which may led to an increase in enzyme activity and catalytic efficiency, the enhancement of hydrophobicity of hydrophobic pocket was related to the improvement of organic solvents tolerance, and three hydrophobic amino acid substitutions Thr91Met, Ala67Val, and Val249Ile facilitated to improve the thermal stability and organic solvents tolerance. These results confirmed that random mutagenesis was an effective tool for improving enzyme properties and lay a foundation for practical applications of phthalate-degrading hydrolase in biotechnology and industrial fields.

Engineering of a highly thermostable endoglucanase from the GH7 family of Bipolaris sorokiniana for higher catalytic efficiency.

In a previous study, we reported an alkaliphilic and thermostable endoglucanase (BsGH7-3) of glycoside hydrolase family 7 (GH7) from the hemibiotrophic plant pathogen Bipolaris sorokiniana. However, the catalytic efficiency of the enzyme was lower than for some other endoglucanases of the GH7 family reported in the literature. To engineer a more active enzyme, we identified conserved residues in the substrate-binding tunnel and on the surface of the protein that could play a role in charge-charge interaction and stabilize the structure. The mutants D257W and Q225H in the substrate-binding tunnel and Y222R and Q401N on the protein surface showed a 2-fold increase in specific activity and a 1.5-fold increase in turnover number and were active over a broader range of pH. The mutants also showed a higher tolerance to NaCl. The rational design of the BsGH7-3 mutants helped in increasing the catalytic efficiency of the thermostable enzyme and may be useful in combination with other cellulases like cellobiohydrolase and β-glucosidase towards complete saccharification of cellulose into glucose.

Soil enzyme kinetics indicate ecotoxicity of long-term arsenic pollution in the soil at field scale.

Information on the kinetic characteristics of soil enzymes under long-term arsenic (As) pollution in field soils is scarce. We investigated Michaelis-Menten kinetic properties of four soil enzymes including β-glucosidase (BG), acid phosphatase (ACP), alkaline phosphatase (ALP), and dehydrogenase (DHA) in field soils contaminated by As resulting from long-term realgar mining activity. The kinetic parameters, namely the maximum reaction velocity (Vmax), enzyme-substrate affinity (Km) and catalytic efficiency (Vmax/Km) were calculated. Results revealed that the enzyme kinetic characteristics varied in soils and were significantly influenced by total nitrogen (N) and total As, which explained 31.8% and 30.7% of the variance in enzyme kinetics respectively. Enzyme pools (Vmax) and catalytic efficiency (Vmax/Km) of BG, ACP and DHA decreased with elevated As pollution, while the enzyme affinity for substrate (Km) was less affected. Redundancy analysis and stepwise regression suggested that the adverse influence of As on enzyme kinetics may offset or weakened by soil total N and soil organic matter (SOM). Concentration-response fitting revealed that the specific kinetic parameters expressed as the absolute enzyme kinetic parameters multiplied by normalized soil total N and SOM were more relevant than the absolute ones to soil total As. The arsenic ecological dose values that cause 10% decrease (ED10) in the specific enzyme kinetics were 20-49mgkg-1, with a mean value of 35mgkg-1, indicating a practical range of threshold for As contamination at field level. This study concluded that soil enzymes exhibited functional adaptation to long-term As stress mainly through the reduction of enzyme pools (Vmax) or maintenance of enzyme-substrate affinity (Km). Further, this study demonstrates that the specific enzyme kinetics are the better indicators of As ecotoxicity at field-scale compared with the absolute enzyme parameters.

The bacterial 4S pathway – an economical alternative for crude oil desulphurization that reduces CO2 emissions

We discuss structural and mechanistic aspects of the Dsz enzymes in the 4S pathway, with a focus on rational molecular strategies for enzyme engineering, aiming at enzyme catalytic rate and efficiency improvement to meet industrial demands.

Tailoring Digestibility of Starches by Chain Elongation Using Amylosucrase from Neisseria polysaccharea via a Zipper Reaction Mode.

Amylosucrase from Neisseria polysaccharea (NpAS) was applied to modify waxy corn starch (WCS) and irradiated WCS, whose attenuated digestibility was studied. Herein, the mobility of the reaction mixture did not affect the enzyme catalytic efficiency, and the reaction kinetics suggest that the enzyme elongated the starch chain via a zipper reaction mode. The A-type crystalline structure of native and irradiated WCS was changed to B type after NpAS treatment, and a longer chain length led to a further increase in the gelatinization temperature. Chain elongation increased the content of resistant starch (RS) from 22.3% (native WCS) to be as much as 64.4% for NpAS-modified WCS, accordingly decreasing the contents of both rapidly and slowly digestible starches. Pearson correlation analysis implies that the RS content of NpAS-modified starches was positively and negatively correlated to the proportion of intermediate chains [13 ≤ degree of polymerization (DP) ≤ 24] and short chains (DP ≤ 12), respectively.

Alleviation of chlorimuron-ethyl toxicity to soybean by branched-chain amino acids or naphthalic anhydride

Soybean seeds, soaked in branched-chain amino acids [Valine, Isoleucine and Leucine (VIL)] mixture or dressed in naphthalic anhydride (NA), were germinated for 10 days and treated with chlorimuron-ethyl for 120 h. Chlorimuron-ethyl significantly inhibited acetolactate synthase (ALS) activity in shoots of all samples during the first 72 h. Moreover, it increased Km but decreased Vmax, Kcat, Kcat/Km and Vmax/Km as well as the endogenous levels of valine, isoleucine and leucine particularly during the first 72 h. In concomitance, there were significant decreases in protein content and activities of nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), glutamate synthase (GOGAT) as well as growth parameters during the first 72 h. However, VIL or NA mitigated the herbicide effect. So, the drop in Vmax, Kcat and Kcat/Km by chlorimuron-ethyl declares decreases in enzyme concentration, catalytic rate and catalytic efficiency, respectively. Whereas the increase in Km could indicate that chlorimuron-ethyl induced an inhibition for ALS of the mixed-type which would affect synthesis and structural integrity of the enzyme. This inhibition would diminish the formation of branched-chain amino acids and protein, particularly with the inhibition of N incorporation enzyme (NR, NiR, GS and GOGAT). Consequently, retardation and disturbances in formation of structural and functional protein would arise. Nonetheless, VIL mixture overcame chlorimuron-ethyl toxicity via compensating the drop in the endogenous branched-chain amino acids while NA might cause mitigation by detoxifying the herbicide.

Activation of Prodrugs by NIR‐Triggered Release of Exogenous Enzymes for Locoregional Chemo‐photothermal Therapy

AbstractEnzymes have been used to direct the conversion of prodrugs in cancer therapy. However, non‐specific distribution of endogenous enzymes seriously hinders their bioapplications. Herein, we developed a near‐infrared‐triggered locoregional chemo‐photothermal therapy based on the exogenous enzyme delivery and remolded tumor mivroenvironment. The catalytic efficiency of enzymes was enhanced by the hyperthermia, and the therapeutic efficacy of photothermal therapy (PTT) was improved owing to the inhibition of heat shock protein 90 by chemotherapeutics. The locoregional chemo‐phototherapy achieved a one‐time successful cure in 4T1 tumor‐bearing mice model. Thus, a mutually reinforcing feedback loop between PTT and chemotherapy can be initiated by the irradiation, which holds a promising future in cancer therapy.