Improving the enzymatic activity of l-amino acid α-ligase for imidazole dipeptide production by site-directed mutagenesis.

Imidazole dipeptides, histidine-containing dipeptides, including carnosine (ß-alanyl-l-histidine), anserine (ß-alanyl-3-methyl-l-histidine), and balenine (ß-alanyl-1-methyl-l-histidine) in animal muscles have physiological functions, such as significant antioxidant and antifatigue effects. They are obtained by extraction from natural raw materials, including chicken and fish meat. However, using natural raw materials entails stable supply and mass production limitations. l-amino acid α-ligase (Lal) catalyzes the formation of various dipeptides from unprotected l-amino acids by conjugating with adenosine 5'-triphosphate (ATP) hydrolysis reaction. In this study, site-directed mutagenesis of Lal was applied to establish an efficient method for producing imidazole dipeptides by the enzymatic process. We significantly improved the conversion rate from substrate amino acids compared with wild-type Lal.

Tuning Immobilized Enzyme Features by Combining Solid-Phase Physicochemical Modification and Mineralization.

Lipase B from Candida antarctica (CALB) and lipase from Thermomyces lanuginosus (TLL) were immobilized on octyl agarose. Then, the biocatalysts were chemically modified using glutaraldehyde, trinitrobenzenesulfonic acid or ethylenediamine and carbodiimide, or physically coated with ionic polymers, such as polyethylenimine (PEI) and dextran sulfate. These produced alterations of the enzyme activities have, in most cases, negative effects with some substrates and positive with other ones (e.g., amination of immobilized TLL increases the activity versus p-nitro phenyl butyrate (p-NPB), reduces the activity with R-methyl mandate by half and maintains the activity with S-isomer). The modification with PEI increased the biocatalyst activity 8-fold versus R-methyl mandelate. Enzyme stability was also modified, usually showing an improvement (e.g., the modification of immobilized TLL with PEI or glutaraldehyde enabled to maintain more than 70% of the initial activity, while the unmodified enzyme maintained less than 50%). The immobilized enzymes were also mineralized by using phosphate metals (Zn2+, Co2+, Cu2+, Ni2+ or Mg2+), and this affected also the enzyme activity, specificity (e.g., immobilized TLL increased its activity after zinc mineralization versus triacetin, while decreased its activity versus all the other assayed substrates) and stability (e.g., the same modification increase the residual stability from almost 0 to more than 60%). Depending on the enzyme, a metal could be positively, neutrally or negatively affected for a specific feature. Finally, we analyzed if the chemical modification could, somehow, tune the effects of the mineralization. Effectively, the same mineralization could have very different effects on the same immobilized enzyme if it was previously submitted to different physicochemical modifications. The same mineralization could present different effects on the enzyme activity, specificity or stability, depending on the previous modification performed on the enzyme, showing that these previous enzyme modifications alter the effects of the mineralization on enzyme features. For example, TLL modified with glutaraldehyde and treated with zinc salts increased its activity using R-methyl mandelate, while almost maintaining its activity versus the other unaltered substrates, whereas the aminated TLL maintained its activity with both methyl mandelate isomers, while it decreased with p-NPB and triacetin. TLL was found to be easier to tune than CALB by the strategies used in this paper. In this way, the combination of chemical or physical modifications of enzymes before their mineralization increases the range of modification of features that the immobilized enzyme can experienced, enabling to enlarge the biocatalyst library.

ScCobB2-mediated Lysine Desuccinylation Regulates Protein Biosynthesis and Carbon Metabolism in Streptomyces coelicolor.

As a recently discovered protein posttranslational modification in eukaryotes, lysine succinylation has attracted increasing interest due to its ability to regulate several critical cellular processes, including catabolism, ß-oxidation, and ketogenesis. Nevertheless, understanding of the regulatory mechanisms is still at an early stage due to the lack of identified specific desuccinylases in microorganisms. Here, in the model soil bacterium Streptomyces coelicolor, we biochemically characterized a sirtuin-like protein ScCobB2 as a divergent desuccinylase. Based on it, we were able to identify a total of 673 unique succinylated sites, of which 470 sites in 317 proteins were quantified by comparing the ΔScCobB2 to the wild-type succinylome via LC-MS/MS analysis. Further analyses of the quantitative succinylome revealed that at least 114 proteins representing two major pathways, protein biosynthesis and carbon metabolism, are obviously hypersuccinylated in ΔScCobB2 cells. We experimentally examined the regulatory roles of ScCobB2 on 13 hypersuccinylated proteins, including glyceraldehyde-3-phosphate dehydrogenase, aconitate hydratase, and several ribosomal proteins, the results of which suggested a high confidence in our quantitative data. This work provided the first discovery of a specific desuccinylase in bacteria and demonstrated it has pivotal regulatory roles in multiple biological processes of S. coelicolor, laying the foundation for future research of succinylation regulation in other microorganisms.

Electrochemical characteristics of a gold nanoparticle-modified controlled enzyme-electrode contact junction electrode.

Biodevices in which biomolecules such as enzymes and antibodies are immobilized on the surface of electrode materials are capable of converting chemical energy into electrical energy, and are expected to contribute to solving energy problems and developing medical measurements especially as biobatteries and biosensors. Device performance depends on the interface formed between the biomolecule layer and electrode material, and the interface is required to simultaneously achieve a highly efficient enzymatic reaction and electron transfer. However, when enzymes were immobilized on a material surface, the enzymes undergoes a structural change due to the interaction between the enzyme and the electrode surface, making it difficult to maximize the function of the enzyme molecule on the material surface. In this study, we postulate that the structural change of the enzyme would be reduced and the electrochemical performance improved by making the contact area between the enzyme and the electrode extremely small and adsorbing it as a point. Therefore, we aimed to develop a high-power biodevice that retains enzyme structure and activity by interposing gold nanoparticles (AuNPs) between the enzyme and the electrode. The enzymatic and electrochemical properties of pyrroloquinoline quinone-dependent glucose dehydrogenase adsorbed on AuNPs of 5-40 nm diameter were investigated. We found that the characteristics differed among the particles, and the enzyme adsorbed on 20 nm AuNPs showed the best electrochemical characteristics.

An assay for DNA polymerase ß lyase inhibitors that engage the catalytic nucleophile for binding.

DNA polymerase ß (Pol ß) repairs cellular DNA damage. When such damage is inflicted upon the DNA in tumor cells treated with DNA targeted antitumor agents, Pol ß thus diminishes their efficacy. Accordingly, this enzyme has long been a target for antitumor therapy. Although numerous inhibitors of the lyase activity of the enzyme have been reported, none has yet proven adequate for development as a therapeutic agent. In the present study, we developed a new strategy to identify lyase inhibitors that critically engage the lyase active site primary nucleophile Lys72 as part of the binding interface. This involves a parallel evaluation of the effect of the inhibitors on the wild-type DNA polymerase ß (Pol ß) and Pol ß modified with a lysine analogue at position 72. A model panel of five structurally diverse lyase inhibitors identified in our previous studies (only one of which has been published) with unknown modes of binding were used for testing, and one compound, cis-9,10-epoxyoctadecanoic acid, was found to have the desired characteristics. This finding was further corroborated by in silico docking, demonstrating that the predominant mode of binding of the inhibitor involves an important electrostatic interaction between the oxygen atom of the epoxy group and Nε of the main catalytic nucleophile, Lys72. The strategy, which is designed to identify compounds that engage certain structural elements of the target enzyme, could find broader application for identification of ligands with predetermined sites of binding.

Lysosomal Proteome and Secretome Analysis Identifies Missorted Enzymes and Their Nondegraded Substrates in Mucolipidosis III Mouse Cells.

Targeting of soluble lysosomal enzymes requires mannose 6-phosphate (M6P) signals whose formation is initiated by the hexameric N-acetylglucosamine (GlcNAc)-1-phosphotransferase complex (α2ß2γ2). Upon proteolytic cleavage by site-1 protease, the α/ß-subunit precursor is catalytically activated but the functions of γ-subunits (Gnptg) in M6P modification of lysosomal enzymes are unknown. To investigate this, we analyzed the Gnptg expression in mouse tissues, primary cultured cells, and in Gnptg reporter mice in vivo, and found high amounts in the brain, eye, kidney, femur, vertebra and fibroblasts. Consecutively we performed comprehensive quantitative lysosomal proteome and M6P secretome analysis in fibroblasts of wild-type and Gnptgko mice mimicking the lysosomal storage disorder mucolipidosis III. Although the cleavage of the α/ß-precursor was not affected by Gnptg deficiency, the GlcNAc-1-phosphotransferase activity was significantly reduced. We purified lysosomes and identified 29 soluble lysosomal proteins by SILAC-based mass spectrometry exhibiting differential abundance in Gnptgko fibroblasts which was confirmed by Western blotting and enzymatic activity analysis for selected proteins. A subset of these lysosomal enzymes show also reduced M6P modifications, fail to reach lysosomes and are secreted, among them α-l-fucosidase and arylsulfatase B. Low levels of these enzymes correlate with the accumulation of non-degraded fucose-containing glycostructures and sulfated glycosaminoglycans in Gnptgko lysosomes. Incubation of Gnptgko fibroblasts with arylsulfatase B partially rescued glycosaminoglycan storage. Combinatorial treatments with other here identified missorted enzymes of this degradation pathway might further correct glycosaminoglycan accumulation and will provide a useful basis to reveal mechanisms of selective, Gnptg-dependent formation of M6P residues on lysosomal proteins.

Complexes of glucose oxidase with chitosan and dextran possessing enhanced stability.

In this study, the different mole ratios of glucose oxidase/chitosan/dextran-aldehyde and glucose oxidase/chitosan/dextran-sulfate complexes were synthesized. The modification of glucose oxidase by non-covalent complexation with dextran and chitosan in different molar ratios was studied in order to increase the enzyme activity. The enzyme/polymer complexes obtained were investigated by UV spectrophotometer and dynamic light scattering. Activity determination of synthesized complexes and free enzyme were performed at a temperature range. The best results were obtained by Cchitosan/Cdextran-aldehyde = 10/1 ratio and Cchitosan/Cdextran-sulfate = 1/5 ratio that were used in thermal stability, shelf life, salt stress, and ethanol effect experiments. The results demonstrated that both complexes were thermally stable at 60 °C and had superior storage stability compared to the free glucose oxidase. Complexes showed higher enzymatic activity than free enzyme in the organic solvent environment using 10% ethanol. The complexes were resistant to salt stress containing 0.1 M NaCl or CaCl2. The particle size distribution results of the triple complex evaluated the complexation of the chitosan, dextran derivative, and glucose oxidase. The average size of the triple complex in diameter was found to be 325.8 ± 9.3 nm. Overall findings suggest that the complexes of glucose oxidase, chitosan, and dextran showed significant enhancement in the enzyme activity.

Synthetic Biology Perspectives of Microbial Enzymes and Their Innovative Applications.

Microbial enzymes are high in demand and there is focus on their efficient, cost effective and eco-friendly production. The relevant microbial enzymes for respective industries needs to be identified but the conventional technologies don't have much edge over it. So, there is more attention towards high throughput methods for production of efficient enzymes. The enzymes produced by microbes need to be modified to bear the extreme conditions of the industries in order to get prolific outcomes and here the synthetic biology tools may be augmented to modify such microbes and enzymes. These tools are applied to synthesize novel and efficient enzymes. Use of computational tools for enzyme modification has provided new avenues for faster and specific modification of enzymes in a shorter time period. This review focuses on few important enzymes and their modification through synthetic biology tools including genetic modification, nanotechnology, post translational modification.

[Enzyme engineering in microbial biosynthesis of terpenoids: progress and perspectives].

Terpenoids, known for their structural and functional diversity, are highly valued, especially in food, cosmetics, and cleaning products. Microbial biosynthesis has emerged as a sustainable and environmentally friendly approach for the production of terpenoids. However, the natural enzymes involved in the synthesis of terpenoids have problems such as low activity, poor specificity, and insufficient stability, which limit the biosynthesis efficiency. Enzyme engineering plays a pivotal role in the microbial synthesis of terpenoids. By modifying the structures and functions of key enzymes, researchers have significantly improved the catalytic activity, specificity, and stability of enzymes related to terpenoid synthesis, providing strong support for the sustainable production of terpenoids. This article reviews the strategies for the modification of key enzymes in microbial synthesis of terpenoids, including improving enzyme activity and stability, changing specificity, and promoting mass transfer through multi-enzyme collaboration. Additionally, this article looks forward to the challenges and development directions of enzyme engineering in the microbial synthesis of terpenoids.

Efficient thermophilic polyethylene terephthalate hydrolase enhanced by cross correlation-based accumulated mutagenesis strategy.

Polyethylene terephthalate (PET) has caused significant pollution issues. Compared to chemical degradation with high energy consumption and cost, enzymatic degradation offers a sustainable solution for PET waste recycling. However, the hydrolytic activity of current PET hydrolases still requires improvement. In this study, a cross-correlation-based accumulated mutagenesis (CAM) strategy was developed to enhance the hydrolysis activity. By mitigating epistatic effect and combinational mutations, we achieved a highly active variant LCC-YGA (H183Y/L124G/S29A) with 2.1-fold hydrolytic activity on amorphous PET films of LCC-ICCG. Conformational analysis elucidated how the introduction of distal mutations enhanced activity. The dynamic correlation among different regions facilitated a synergistic effect, enhancing binding pocket flexibility through remote interactions. Totally, this work offers novel insights and methods for PET hydrolases engineering and provides an efficient enzyme for PET degradation and recycling.

Improving paste stabilities of cassava starch through molecular density after maltogenic amylase and transglucosidase.

To improve paste stability of cassava starch, including acid resistance, high-temperature shear resistance and freeze-thaw stability, cassava starch was modified by sequential maltogenic amylase and transglucosidase to form an optimally denser structure, or branched density (12.76 %), molecular density (15.17 g/mol/nm3), and the proportions of short-branched chains (41.41 % of A chains and 44.01 % of B1 chains). Viscosity stability (88.52 %) of modified starch was higher than that (64.92 %) of native starch. After acidic treatment for 1 h, the viscosity of modified starch and native starch decreased by 56.53 % and 65.70 %, respectively. Compared to native starch, modified starch had lower water loss in freeze-thaw cycles and less viscosity reduction during high-temperature and high-shear processing. So, the appropriate molecular density and denser molecule structure enhanced paste stabilities of modified starch. The outcome expands the food and non-food applications of cassava starch.

Diverse models of cavity engineering in enzyme modification: Creation, filling, and reshaping.

Most enzyme modification strategies focus on designing the active sites or their surrounding structures. Interestingly, a large portion of the enzymes (60%) feature active sites located within spacious cavities. Despite recent discoveries, cavity-mediated enzyme engineering remains crucial for enhancing enzyme properties and unraveling folding-unfolding mechanisms. Cavity engineering influences enzyme stability, catalytic activity, specificity, substrate recognition, and docking. This article provides a comprehensive review of various cavity engineering models for enzyme modification, including cavity creation, filling, and reshaping. Additionally, it also discusses feasible tools for geometric analysis, functional assessment, and modification of cavities, and explores potential future research directions in this field. Furthermore, a promising universal modification strategy for cavity engineering that leverages state-of-the-art technologies and methodologies to tailor cavities according to the specific requirements of industrial production conditions is proposed.

Stabilization of enzymes in ionic liquids via modification of enzyme charge.

Due to the propensity of ionic liquids (ILs) to inactivate enzymes, the development of strategies to improve enzyme utility in these solvents is critical to fully exploit ILs for biocatalysis. We have developed a strategy to broadly improve enzyme utility in ILs based on elucidating the effect of charge modifications on the function of enzymes in IL environments. Results of stability studies in aqueous-IL mixtures indicated a clear connection between the ratio of enzyme-containing positive-to-negative sites and enzyme stability in ILs. Stability studies of the effect of [BMIM][Cl] and [EMIM][EtSO4 ] on chymotrypsin specifically found an optimum ratio of positively-charged amine-to-negatively-charged acid groups (0.39). At this ratio, the half-life of chymotrypsin was increased 1.6- and 4.3-fold relative to wild-type chymotrypsin in [BMIM][Cl] and [EMIM][EtSO4 ], respectively. The half-lives of lipase and papain were similarly increased as much as 4.0 and 2.4-fold, respectively, in [BMIM][Cl] by modifying the ratio of positive-to-negative sites of each enzyme. More generally, the results of stability studies found that modifications that reduce the ratio of enzyme-containing positive-to-negative sites improve enzyme stability in ILs. Understanding the impact of charge modification on enzyme stability in ILs may ultimately be exploited to rationally engineer enzymes for improved function in IL environments.

Enhancement of Escherichia coli Ribonuclease R Cytosine-Sensitive Activity by Single Amino Acid Substitution.

Exoribonucleases are frequently used as nuclei acids detection tools for their sequences, modifications, and structures. Escherichia coli ribonuclease R (EcR) is the prototypical exoribonuclease of the RNase II/RNB family degrading RNA in the 3'-5' direction. Different from RNase II, EcR is capable of degrading structured RNA efficiently, which makes it a potential analysis tool for various RNA species. In this work, we examined the nuclease activity of EcR degrading a series of RNA substrates with various sequences. Our biochemical work reveals that EcR is significantly sensitive to cytosine compared with other bases when catalyzing RNA degradation. EcR shows higher cytosine sensitivity compared to its homolog RNase II when degrading RNAs, and the hydrolysis process of EcR is transiently halted and produces apparent intermediate product when the 1-nt upstream of C is A or U, or G. Furthermore, the substitution of glycine with proline (G273P) in EcR enhances its cytosine sensitivity. These findings expand our understanding of EcR enzymatic activities. The EcR G273P mutant bearing higher cytosine sensitivity could help enrich cytosine trails in RNAs and will have potential implications in the detection and analysis of various RNA species especially small RNAs in biological and clinical samples.

Effects of Enzymatic Modification and Cross-Linking with Sodium Phytate on the Structure and Physicochemical Properties of Cyperus esculentus Starch.

In this study, C. esculentus porous starch (PS) and C. esculentus cross-linked porous starch (CPS) were prepared by enzymatic modification and sodium phytate cross-linking, and their physicochemical and structural properties were determined. The results showed that the adsorption and emulsification capacities of PS were 1.3606 g/g and 22.6 mL/g, respectively, which were significantly higher than 0.5419 g/g and 4.2 mL/g of C. esculentus starch (NS). The retrogradation curves of starch paste showed that the stability of PS was inferior to that of NS. In addition, the results of texture analysis showed that the gel strength of PS was also significantly reduced relative to NS. The PS exhibited a rough surface with pores and low molecular order and crystallinity according to scanning electron microscope (SEM), fourier infrared spectroscopy (FTIR), and X ray diffractometer (XRD) analyses. As compared to PS, CPS still presented a high adsorption capacity of 1.2744 g/g and the steadiness of starch paste was significantly better. XPS demonstrated the occurrence of the cross-linking reaction. Our results show that enzyme modification and dual modification by combining enzymatic treatment with sodium phytate cross-linking can impart different structures and functions to starch, creating reference material for the application of modified starch from C. esculentus.

Enzymatic Hydrolysis Modifies Emulsifying Properties of Okra Pectin.

Okra pectins (OKPs) with diverse structures obtained by different extraction protocols have been used to study the relationship between their molecular structure and emulsifying properties. A targeted modification of molecular structure offers a more rigorous method for investigating the emulsifying properties of pectins. In this study, three glycoside hydrolases, polygalacturonase (PG), galactanase (GL), and arabinanase (AR), and their combinations, were used to modify the backbone and side-chains of OKP, and the relationships between the pectin structure and emulsion characteristics were examined by multivariate analysis. Enzymatic treatment significantly changed the molecular structure of OKP, as indicated by monosaccharide composition, molecular weight, and structure analysis. GL- and AR- treatments reduced side-chains, while PG-treatment increased side-chain compositions in pectin structure. We compared the performance of hydrolyzed pectins in stabilizing emulsions containing 50% v/v oil-phase and 0.25% w/v pectin. While the emulsions were stabilized by PG (93.3% stability), the emulsion stability was reduced in GL (62.5%), PG+GL+AR (37.0%), and GL+AR (34.0%) after 15-day storage. Furthermore, microscopic observation of the droplets revealed that emulsion destabilization was caused by flocculation and coalescence. Principal component analysis confirmed that neutral sugar side-chains are key for long-term emulsion stabilization and that their structure explains the emulsifying properties of OKP. Our data provide structure-function information applicable to the tailored extraction of OKP with good emulsification performance, which can be used as a natural emulsifier.

Structural and functional modification of kudzu starch using α-amylase and transglucosidase.

The large agglomeration of starch paste in hot water, and fast retrogradation tendency and low transparency of starch gel restrict widespread application of kudzu starch. To improve the above defects, kudzu starch was modified with sequentially α-amylase (AA) and transglucosidase (TG), the latter for varying times. The results indicated that, compared to kudzu starch, amylose content and molecular weight of AA/TG-treated starches reduced by 20.07% and 69.50%, respectively. The proportion of A chain increased by 68.68%, whereas B1, B2 and B3 chains decreased by 14.28%, 48.29% and 23.44%, respectively. The degree of branching dramatically increased by 128.3%. After AA→TG treatment, the changes of starch structure enhanced the functional properties of kudzu starch. The solubility, paste clarity and gelatinization temperature increased, whereas the relative crystallinity, viscosity, storage and loss moduli decreased. Overall, the AA→TG modification would be desirable to improve the functional properties of kudzu starch to expand more large-scale application.

Post-Synthetic Enzymatic and Chemical Modifications for Novel Sustainable Polyesters.

Because of their biodegradability, compostability, compatibility and flexible structures, biodegradable polymers such as polyhydroxyalkanoates (PHA) are an important class of biopolymers with various industrial and biological uses. PHAs are thermoplastic polyesters with a limited processability due to their low heat resistance. Furthermore, due to their high crystallinity, some PHAs are stiff and brittle. These features result sometimes in very poor mechanical characteristics with low extension at break values which limit the application range of some natural PHAs. Several in vivo approaches for PHA copolymer modifications range from polymer production to enhance PHA-based material performance after synthesis. The methods for enzymatic and chemical polymer modifications are aiming at modifying the structures of the polyesters and thereby their characteristics while retaining the biodegradability. This survey illustrates the efficient use of enzymes and chemicals in post-synthetic PHA modifications, offering insights on these green techniques for modifying and improving polymer performance. Important studies in this sector will be reviewed, as well as chances and obstacles for their stability and hyper-production.

Rational design of electroactive redox enzyme nanocapsules for high-performance biosensors and enzymatic biofuel cell.

The potential application of biodevices based on enzymatic bioelectrocatalysis are limited by poor stability and electrochemical performance. To solve the limitation, modifying enzyme with functional polymer to tailor enzyme function is highly desirable. Herein, glucose oxidase (GOx) was chosen as a model enzyme, and according to the chemical structure of GOx cofactor (flavin adenine dinucleotide, FAD), we customize a biomimetic cofactor containing vinyl group (SFAD) for GOx, and prepared an GOx nanocapsule via in-situ polymerization. The characterization of particle size distribution, TEM, fluorescence and electrochemical performance indicated the successful formation of electroactive GOx nanocapsule with SFAD-containing polymeric network (n (GOx-SFAD-PAM)). The network can act as an electronic "highway" to link the active site with electrode, with capability to accelerate electron transfer as well as enhanced GOx stability. Further investigation of bioelectrocatalysis shows that n (GOx-SFAD-PAM)-based biosensor has low detection potential (-0.4 vs. Ag/AgCl), high sensitivity (64.97 µAmM-1cm-2), good anti-interference performance, quick response (3⁓5s) and excellent stability, and that n (GOx-SFAD-PAM)-based enzymatic biofuel cell (EBFC) has the high maximum power density (1011.21 µWcm-2), which is a 385-fold increase over that of native GOx-based EBFC (2.62 µWcm-2). This study suggests that novel enzyme nanocapsule with electroactive polymeric shell might provide a prospective solution for the performance improvement of enzymatic bioelectrocatalysis-based biodevices.

Dual modification of various starches: Synthesis, properties and applications.

The problems associated with native starches (NSs) and single modified starches were stated in order to justify dual modification of various starches. Broadly, there are two types of dual modification, i.e., homogeneous dual modification and heterogeneous dual modification. The combination of two physical modifications, e.g., (extrusion/annealing); two chemical modifications, e.g., (succinylation/cross-linking) and two enzymes modification (α-amylase/pullulanase) falls under the former classification and the latter classification is the combination of two of each of the differently stated modifications, e.g., acetylation/annealing, extrusion/succinylation, and microwave-assisted phosphorylation, etc. The classification, synthesis, properties and applications of dually modified starches were discussed. There is an attempt to elucidate the problems of each of the single modification in order to justify dual modifications. In dual modifications, the order of reactions, the reaction conditions, the medium of reaction, and the botanical sources of the various starches are very important parameters.