In vitro analysis of the three-component Rieske oxygenase cumene dioxygenase from Pseudomonas fluorescens IP01.

Rieske non-heme iron-dependent oxygenases (ROs) are a versatile group of enzymes traditionally associated with the degradation of aromatic xenobiotics. In addition, ROs have been found to play key roles in natural product biosynthesis, displaying a wide catalytic diversity with typically high regio- and stereo- selectivity. However, the detailed characterization of ROs presents formidable challenges due to their complex structural and functional properties, including their multi-component composition, cofactor dependence, and susceptibility to reactive oxygen species. In addition, the substrate availability of natural product biosynthetic intermediates, the limited solubility of aromatic hydrocarbons, and the radical-mediated reaction mechanism can further complicate functional assays. Despite these challenges, ROs hold immense potential as biocatalysts for pharmaceutical applications and bioremediation. Using cumene dioxygenase (CDO) from Pseudomonas fluorescens IP01 as a model enzyme, this chapter details techniques for characterizing ROs that oxyfunctionalize aromatic hydrocarbons. Moreover, potential pitfalls, anticipated complications, and proposed solutions for the characterization of novel ROs are described, providing a framework for future RO research and strategies for studying this enzyme class. In particular, we describe the methods used to obtain CDO, from construct design to expression conditions, followed by a purification procedure, and ultimately activity determination through various activity assays.

Substrate access tunnel engineering of a Fe-type nitrile hydratase from Pseudomonas fluorescens ZJUT001 for substrate preference adjustment and catalytic performance enhancement.

Substrate access tunnel engineering is a useful strategy for enzyme modification. In this study, we improved the catalytic performance of Fe-type Nitrile hydratase (Fe-type NHase) from Pseudomonas fluorescens ZJUT001 (PfNHase) by mutating residue Q86 at the entrance of the substrate access tunnel. The catalytic activity of the mutant PfNHase-αQ86W towards benzonitrile, 2-cyanopyridine, 3-cyanopyridine, and 4-hydroxybenzonitrile was enhanced by 9.35-, 3.30-, 6.55-, and 2.71-fold, respectively, compared to that of the wild-type PfNHase (PfNHase-WT). In addition, the mutant PfNHase-αQ86W showed a catalytic efficiency (kcat/Km) towards benzonitrile 17.32-fold higher than the PfNHase-WT. Interestingly, the substrate preference of PfNHase-αQ86W shifted from aliphatic nitriles to aromatic nitrile substrates. Our analysis delved into the structural changes that led to this altered substrate preference, highlighting an expanded entrance tunnel region, theenlarged substrate-binding pocket, and the increased hydrophobic interactions between the substrate and enzyme. Molecular dynamic simulations and dynamic cross-correlation Matrix (DCCM) further supported these findings, providing a comprehensive explanation for the enhanced catalytic activity towards aromatic nitrile substrates.

Specific glycine-dependent enzyme motion determines the potency of conformation selective inhibitors of threonyl-tRNA synthetase.

The function of proteins depends on their correct structure and proper dynamics. Understanding the dynamics of target proteins facilitates drug design and development. However, dynamic information is often hidden in the spatial structure of proteins. It is important but difficult to identify the specific residues that play a decisive role in protein dynamics. Here, we report that a critical glycine residue (Gly463) dominates the motion of threonyl-tRNA synthetase (ThrRS) and the sensitivity of the enzyme to antibiotics. Obafluorin (OB), a natural antibiotic, is a novel covalent inhibitor of ThrRS. The binding of OB induces a large conformational change in ThrRS. Through five crystal structures, biochemical and biophysical analyses, and computational simulations, we found that Gly463 plays an important role in the dynamics of ThrRS. Mutating this flexible residue into more rigid residues did not damage the enzyme's three-dimensional structure but significantly improved the thermal stability of the enzyme and suppressed its ability to change conformation. These mutations cause resistance of ThrRS to antibiotics that are conformationally selective, such as OB and borrelidin. This work not only elucidates the molecular mechanism of the self-resistance of OB-producing Pseudomonas fluorescens but also emphasizes the importance of backbone kinetics for aminoacyl-tRNA synthetase-targeting drug development.

Enhanced Enzymatic Performance of Immobilized Pseudomonas fluorescens Lipase on ZIF-8@ZIF-67 and Its Application to the Synthesis of Neryl Acetate with Transesterification Reaction.

In this study, hybrid skeleton material ZIF-8@ZIF-67 was synthesized by the epitaxial growth method and then was utilized as a carrier for encapsulating Pseudomonas fluorescens lipase (PFL) through the co-precipitation method, resulting in the preparation of immobilized lipase (PFL@ZIF-8@ZIF-67). Subsequently, it was further treated with glutaraldehyde to improve protein immobilization yield. Under optimal immobilization conditions, the specific hydrolytic activity of PFL@ZIF-8@ZIF-67 was 20.4 times higher than that of the free PFL. The prepared biocatalyst was characterized and analyzed by scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared (FT-IR). Additionally, the thermal stability of PFL@ZIF-8@ZIF-67 at 50 °C was significantly improved compared to the free PFL. After 7 weeks at room temperature, PFL@ZIF-8@ZIF-67 retained 78% of the transesterification activity, while the free enzyme was only 29%. Finally, PFL@ZIF-8@ZIF-67 was applied to the neryl acetate preparation in a solvent-free system, and the yield of neryl acetate reached 99% after 3 h of reaction. After 10 repetitions, the yields of neryl acetate catalyzed by PFL@ZIF-8@ZIF-67 and the free PFL were 80% and 43%, respectively.

Engineering cascade biocatalysis in whole cells for syringic acid bioproduction.

BACKGROUND: Syringic acid (SA) is a high-value natural compound with diverse biological activities and wide applications, commonly found in fruits, vegetables, and herbs. SA is primarily produced through chemical synthesis, nonetheless, these chemical methods have many drawbacks, such as considerable equipment requirements, harsh reaction conditions, expensive catalysts, and numerous by-products. Therefore, in this study, a novel biotransformation route for SA production was designed and developed by using engineered whole cells. RESULTS: An O-methyltransferase from Desulfuromonas acetoxidans (DesAOMT), which preferentially catalyzes a methyl transfer reaction on the meta-hydroxyl group of catechol analogues, was identified. The whole cells expressing DesAOMT can transform gallic acid (GA) into SA when S-adenosyl methionine (SAM) is used as a methyl donor. We constructed a multi-enzyme cascade reaction in Escherichia coli, containing an endogenous shikimate kinase (AroL) and a chorismate lyase (UbiC), along with a p-hydroxybenzoate hydroxylase mutant (PobA**) from Pseudomonas fluorescens, and DesAOMT; SA was biosynthesized from shikimic acid (SHA) by using whole cells catalysis. The metabolic system of chassis cells also affected the efficiency of SA biosynthesis, blocking the chorismate metabolism pathway improved SA production. When the supply of the cofactor NADPH was optimized, the titer of SA reached 133 µM (26.2 mg/L). CONCLUSION: Overall, we designed a multi-enzyme cascade in E. coli for SA biosynthesis by using resting or growing whole cells. This work identified an O-methyltransferase (DesAOMT), which can catalyze the methylation of GA to produce SA. The multi-enzyme cascade containing four enzymes expressed in an engineered E. coli for synthesizing of SA from SHA. The metabolic system of the strain and biotransformation conditions influenced catalytic efficiency. This study provides a new green route for SA biosynthesis.

Structures of the alkanesulfonate monooxygenase MsuD provide insight into C-S bond cleavage, substrate scope, and an unexpected role for the tetramer.

Bacterial two-component flavin-dependent monooxygenases cleave the stable C-S bond of environmental and anthropogenic organosulfur compounds. The monooxygenase MsuD converts methanesulfonate (MS-) to sulfite, completing the sulfur assimilation process during sulfate starvation, but the mechanism of this conversion remains unclear. To explore the mechanism of C-S bond cleavage, we report a series of crystal structures of MsuD from Pseudomonas fluorescens in different liganded states. This report provides the first crystal structures of an alkanesulfonate monooxygenase with a bound flavin and alkanesulfonate, elucidating the roles of the active site lid, the protein C terminus, and an active site loop in flavin and/or alkanesulfonate binding. These structures position MS- closest to the flavin N5 position, consistent with an N5-(hydro)peroxyflavin mechanism rather than a classical C4a-(hydro)peroxyflavin mechanism. A fully enclosed active site is observed in the ternary complex, mediated by interchain interaction of the C terminus at the tetramer interface. These structures identify an unexpected function of the protein C terminus in this protein family in stabilizing tetramer formation and the alkanesulfonate-binding site. Spurred by interest from the crystal structures, we conducted biochemical assays and molecular docking that redefine MsuD as a small- to medium-chain alkanesulfonate monooxygenase. Functional mutations verify the sulfonate-binding site and reveal the critical importance of the protein C terminus for monooxygenase function. These findings reveal a deeper understanding of MsuD's functionality at the molecular level and consequently how it operates within its role as part of the sulfur assimilation pathway.

Intramolecular Stereoselective Stetter Reaction Catalyzed by Benzaldehyde Lyase.

The reliable design and prediction of enzyme promiscuity to access transformations not observed in nature remains a long-standing challenge. Herein, we present the first example of an intramolecular stereoselective Stetter reaction catalyzed by benzaldehyde lyase, guided by the rational structure screening of various ThDP-dependent enzymes using molecular dynamics (MD) simulations. After optimization, high productivity (up to 99 %) and stereoselectivity (up to 99:1 e.r.) for this novel enzyme function was achieved.

Enzymatically Triggered Jack-in-the-Box-like Hydrogels.

Enzymes can support the synthesis or degradation of biomacromolecules in natural processes. Here, we demonstrate that enzymes can induce a macroscopic-directed movement of microstructured hydrogels following a mechanism that we call a "Jack-in-the-box" effect. The material's design is based on the formation of internal stresses induced by a deformation load on an architectured microscale, which are kinetically frozen by the generation of polyester locking domains, similar to a Jack-in-the-box toy (i.e., a compressed spring stabilized by a closed box lid). To induce the controlled macroscopic movement, the locking domains are equipped with enzyme-specific cleavable bonds (i.e., a box with a lock and key system). As a result of enzymatic reaction, a transformed shape is achieved by the release of internal stresses. There is an increase in entropy in combination with a swelling-supported stretching of polymer chains within the microarchitectured hydrogel (i.e., the encased clown pops-up with a pre-stressed movement when the box is unlocked). This utilization of an enzyme as a physiological stimulus may offer new approaches to create interactive and enzyme-specific materials for different applications such as an optical indicator of the enzyme's presence or actuators and sensors in biotechnology and in fermentation processes.

Active-site loop variations adjust activity and selectivity of the cumene dioxygenase.

Active-site loops play essential roles in various catalytically important enzyme properties like activity, selectivity, and substrate scope. However, their high flexibility and diversity makes them challenging to incorporate into rational enzyme engineering strategies. Here, we report the engineering of hot-spots in loops of the cumene dioxygenase from Pseudomonas fluorescens IP01 with high impact on activity, regio- and enantioselectivity. Libraries based on alanine scan, sequence alignments, and deletions along with a novel insertion approach result in up to 16-fold increases in activity and the formation of novel products and enantiomers. CAVER analysis suggests possible increases in the active pocket volume and formation of new active-site tunnels, suggesting additional degrees of freedom of the substrate in the pocket. The combination of identified hot-spots with the Linker In Loop Insertion approach proves to be a valuable addition to future loop engineering approaches for enhanced biocatalysts.

Integral Stereoselectivity of Lipase Based on the Chromatographic Resolution of Enantiomeric/Regioisomeric Diacylglycerols.

Stereoselectivity, a distinctive characteristic of lipase (EC 3.1.1.3), refers to the ability to differentiate between enantiomeric positions (sn-1 and sn-3) in triacylglycerol (TAG). This property has been determined based on the time course of enantiomeric excess of diacylglycerol (DAG) considering several consecutive steps of lipase-catalyzed hydrolysis of TAG; however, this concept is insufficient to represent the true nature of lipases which are capable of hydrolyzing the sn-2 position of TAG under the condition acyl migration occurs. Here, we suggest "integral stereoselectivity" to capture the preference of lipases for all ester groups of both TAG and DAG, as a novel index of the stereochemistry of lipase. To determine integral stereoselectivity, we established an analytical system based on the chromatographic resolution of dioleoylglycerol (DO) enantiomers and regioisomers. DO enantiomers were derivatized with 4-nitrophenyl isocyanate, and subsequently, resolved by chiral-phase high-performance liquid chromatography-ultraviolet. Regioisomers of monooleoylglycerol and DO were analyzed by HPLC with an evaporative light-scattering detector. Time-course analysis of three model lipases involved in the hydrolysis of trioleoylglycerol validated the analytical system designed to determine the integral stereoselectivity. As an accurate indicator of lipase stereochemistry reflecting all hydrolysis steps, integral stereoselectivity can expedite the development of lipases with unique stereochemistry from agricultural sources and their application to the food industry.

Diflunisal Derivatives as Modulators of ACMS Decarboxylase Targeting the Tryptophan-Kynurenine Pathway.

In the kynurenine pathway for tryptophan degradation, an unstable metabolic intermediate, α-amino-ß-carboxymuconate-ε-semialdehyde (ACMS), can nonenzymatically cyclize to form quinolinic acid, the precursor for de novo biosynthesis of nicotinamide adenine dinucleotide (NAD+). In a competing reaction, ACMS is decarboxylated by ACMS decarboxylase (ACMSD) for further metabolism and energy production. Therefore, the inhibition of ACMSD increases NAD+ levels. In this study, an Food and Drug Administration (FDA)-approved drug, diflunisal, was found to competitively inhibit ACMSD. The complex structure of ACMSD with diflunisal revealed a previously unknown ligand-binding mode and was consistent with the results of inhibition assays, as well as a structure-activity relationship (SAR) study. Moreover, two synthesized diflunisal derivatives showed half-maximal inhibitory concentration (IC50) values 1 order of magnitude better than diflunisal at 1.32 ± 0.07 µM (22) and 3.10 ± 0.11 µM (20), respectively. The results suggest that diflunisal derivatives have the potential to modulate NAD+ levels. The ligand-binding mode revealed here provides a new direction for developing inhibitors of ACMSD.

Gene cloning, heterologous expression, and partial characterization of a novel cold-adapted subfamily I.3 lipase from Pseudomonas fluorescence KE38.

A novel cold-active true lipase from Pseudomonas sp. KE38 was cloned, sequencing and expressed in E. coli by degenerate PCR and genome walking technique. The open reading frame of the cloned gene encoded a polypeptide chain of 617 amino acids with a confirmed molecular weight of 64 kD. Phylogenetic analysis of the deduced amino acid sequence of the lipase indicated that it had high similarity with lipases of subfamily Ι.3 of bacterial lipases. Recombinant lipase was purified in denatured form as inclusion bodies, which were then renatured by urea followed by dialysis. Lipase activity was determined titrimetrically using olive oil as substrate. The enzyme showed optimal activity at 25 °C, pH 8.5 and was highly stable in the presence of various metal ions and organic solvents. Low optimal temperature and high activity in the presence of methanol and ethanol make this lipase a potential candidate for transesterification reactions and biodiesel production.

Exploiting Mannuronan C-5 Epimerases in Commercial Alginate Production.

Alginates are one of the major polysaccharide constituents of marine brown algae in commercial manufacturing. However, the content and composition of alginates differ according to the distinct parts of these macroalgae and have a direct impact on the concentration of guluronate and subsequent commercial value of the final product. The Azotobacter vinelandii mannuronan C-5 epimerases AlgE1 and AlgE4 were used to determine their potential value in tailoring the production of high guluronate low-molecular-weight alginates from two sources of high mannuronic acid alginates, the naturally occurring harvested brown algae (Ascophyllum nodosum, Durvillea potatorum, Laminaria hyperborea and Lessonia nigrescens) and a pure mannuronic acid alginate derived from fermented production of the mutant strain of Pseudomonas fluorescens NCIMB 10,525. The mannuronan C-5 epimerases used in this study increased the content of guluronate from 32% up to 81% in both the harvested seaweed and bacterial fermented alginate sources. The guluronate-rich alginate oligomers subsequently derived from these two different sources showed structural identity as determined by proton nuclear magnetic resonance (1H NMR), high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and size-exclusion chromatography with online multi-angle static laser light scattering (SEC-MALS). Functional identity was determined by minimum inhibitory concentration (MIC) assays with selected bacteria and antibiotics using the previously documented low-molecular-weight guluronate enriched alginate OligoG CF-5/20 as a comparator. The alginates produced using either source showed similar antibiotic potentiation effects to the drug candidate OligoG CF-5/20 currently in development as a mucolytic and anti-biofilm agent. These findings clearly illustrate the value of using epimerases to provide an alternative production route for novel low-molecular-weight alginates.

The role of ACC deaminase producing bacteria in improving sweet corn (Zea mays L. var saccharata) productivity under limited availability of irrigation water.

Accumulation of stress ethylene in plants due to osmotic stress is a major challenge for the achievement of optimum sweet corn crop yield with limited availability of irrigation water. A significant increase in earth's temperature is also making the conditions more crucial regarding the availability of ample quantity of irrigation water for crops production. Plant growth promoting rhizobacteria (PGPR) can play an imperative role in this regard. Inoculation of rhizobacteria can provide resistance and adaptability to crops against osmotic stress. In addition, these rhizobacteria also have potential to solve future food security issues. That's why the current study was planned to examine the efficacious functioning of Pseudomonas fluorescens strains on yields and physiological characteristics of sweet corn (Zea mays L. var saccharata) under different levels of irrigation. Three irrigation levels i.e., 100% (I100 no stress), 80% (I80), and 60% (I60) were used during sweet corn cultivation. However, there were four rhizobacteria strains i.e., P. fluorescens P1, P. fluorescens P3, P. fluorescens P8, P. fluorescens P14 which were used in the experiment. The results showed that severe water stress (60% of plant water requirement) decreased chlorophyll a, chlorophyll b, and total chlorophyll contents, Fv/Fm ratio and nutrients uptake. A significant increase in F0, Fm, proline, total soluble sugars, catalase (CAT) and peroxidase (POX) activity led to less ear yield and canned seed yield. Combination of four strains significantly increased the yield traits of sweet corn i.e., ear and (44%) and canned seed yield (27%) over control. The highest promoting effect was observed in the combination of four strains treatment and followed by P1 strain in reducing the harmful effects of drought stress and improving sweet corn productivity. However, P14 gave minimum improvement in growth and yield indices under limited availability of water. In conclusion, combination of four strains inoculation is an efficacious approach for the achievement of better yield of sweet corn under osmotic stress.

PEG-modified lipase immobilized onto NH2-MIL-53 MOF for efficient resolution of 4-fluoromandelic acid enantiomers.

A new heterogeneous bio-catalyst was prepared by the immobilization of lipase from Pseudomonas fluorescents (PFL) onto metal-organic frameworks (MOF), NH2-MIL-53(Fe), using covalent cross-linking. The immobilized lipase [PEG-PFL@NH2-MIL-53(Fe)] was firstly applied in enantioselective resolution of 4-fluoromandelic acid (4-FMA) enantiomers. After optimization of the immobilization PFL onto NH2-MIL-53, its loading capacity is 224.5 mg PFL/g MOF. The optimal enzymatic conditions are temperature of 50 °C, VA/4-FMA substrate ratio of 6:1, immobilized lipase loading of 60 mg and reaction time of 12 h. Experimental results show that the catalytic activity and thermal stability of PFL are significantly improved by polyethylene glycol (PEG) modification and immobilization. At 65 °C, the catalytic activity of immobilized lipase retains 86.0% of initial activity. Under the optimal conditions, the excellent results were obtained with conversion of 49.6% and enantiomer excess of 98.0% for the immobilized PFL catalyzed transesterification reaction. Furthermore, the immobilized lipase exhibits excellent cycle stability with 83% of its initial activity after four cycle.

Stabilization of ω-transaminase from Pseudomonas fluorescens by immobilization techniques.

Transaminases are a class of enzymes with promising applications for the preparation and resolution of a vast diversity of valued amines. Their poor operational stability has fueled many investigations on its stabilization due to their biotechnological relevance. In this work, we screened the stabilization of the tetrameric ω-transaminase from Pseudomonas fluorescens (PfωTA) through both carrier-bound and carrier-free immobilization techniques. The best heterogeneous biocatalyst was the PfωTA immobilized as cross-linked enzyme aggregates (PfωTA-CLEA) which resulted after studying different parameters as the precipitant, additives and glutaraldehyde concentrations. The best conditions for maximum recovered activity (29 %) and maximum thermostability at 60 ºC and 70 ºC (100 % and 71 % residual activity after 1 h, respectively) were achieved by enzyme precipitation with 90% acetone or ethanol, in presence of BSA (100 mg/mL) and employing glutaraldehyde (100 mM) as cross-linker. Studies on different conditions for PfωTA-CLEA preparation yielded a biocatalyst that exhibited 31 and 4.6 times enhanced thermal stability at 60 °C and 70 °C, respectively, compared to its soluble counterpart. The PfωTA-CLEA was successfully used in the bioamination of 4-hydroxybenzaldehyde to 4-hydroxybenzylamine. To the best of our knowledge, this is the first report describing a transaminase cross-linked enzyme aggregates as immobilization strategy to generate a biocatalyst with outstanding thermostability.

The Influence of Calcium toward Order/Disorder Conformation of Repeat-in-Toxin (RTX) Structure of Family I.3 Lipase from Pseudomonas fluorescens AMS8.

Calcium-binding plays a decisive role in the folding and stabilization of many RTX proteins, especially for the RTX domain. Although many studies have been conducted to prove the contribution of Ca2+ ion toward the folding and stabilization of RTX proteins, its functional dynamics and conformational structural changes remain elusive. Here, molecular docking and molecular dynamics (MD) simulations were performed to analyze the contribution of Ca2+ ion toward the folding and stabilization of the RTX lipase (AMS8 lipase) structure. AMS8 lipase contains six Ca2+ ions (Ca1-Ca6). Three Ca2+ ions (Ca3, Ca4, and Ca5) were bound to the RTX parallel ß-roll motif repeat structure (RTX domain). The metal ion (Ca2+) docking analysis gives a high binding energy, especially for Ca4 and Ca5 which are tightly bound to the RTX domain. The function of each Ca2+ ion is further analyzed using the MD simulation. The removal of Ca3, Ca4, and Ca5 caused the AMS8 lipase structure to become unstable and unfolded. The results suggested that Ca3, Ca4, and Ca5 stabilized the RTX domain. In conclusion, Ca3, Ca4, and Ca5 play a crucial role in the folding and stabilization of the RTX domain, which sustain the integrity of the overall AMS8 lipase structure.

Effect of Pseudomonas fluorescens proteases on the quality of Cheddar cheese.

The objective of this study was to investigate the effect of adding different levels of a thermoresistant protease produced by a Pseudomonas fluorescens strain to milk on the manufacture and quality of Cheddar cheese. Fresh raw milk was collected, standardized, and pasteurized at 72°C for 15 s, and the enzyme was added to give a protease activity of 0.15 or 0.60 U/L (treatments P1 and P4, respectively), while one sample had no enzyme added (control). Milk was stored at 4°C for 48 h and Cheddar cheese was manufactured after 0 and 48 h of storage. Results indicated that the protease was active in milk during 48 h of storage; however, its effect on milk composition was minimal. The protein that was preferentially hydrolyzed by the protease over storage was ß-casein, followed by κ-casein. The mean cheese yield and recovery of fat and protein obtained for all cheeses were not affected by protease activity. The protease showed low activity during cheese manufacture, possibly because of unfavorable conditions, including low pH. One of the factors that might have influenced protease activity was the pH of the curd (approximately 6.55 after acidification and 5.35 at milling), which was lower than that at which the enzyme would have optimum activity (pH 7 to 9). Consequently, the composition, pH, patterns of proteolysis, and hardness of all cheeses produced were similar and in accordance with values expected for that type of cheese, independently of the protease activity level. However, slight increases in proteolysis were observed in P4 cheeses and produced using milk stored for 48 h. Both the P1 and P4 cheeses had higher concentrations of free amino acids (FAA) compared with the control, whereas urea-PAGE electrophoretograms indicated a greater breakdown of caseins in the P4 cheese samples, which may be related to possible increases in numbers of proteolytic bacteria in milk during storage. Therefore, the thermoresistant psychrotrophic bacterial protease(s) tested in this study may affect the manufacture or quality of Cheddar cheese during ripening to a relatively limited extent. However, controlling initial levels of proteolytic bacteria in raw milk remains essential, because proteolysis affects the development of flavor and texture in cheese.

Coupling Two Sequential Biocatalysts with Close Proximity into Metal-Organic Frameworks for Enhanced Cascade Catalysis.

The encapsulation of multiple enzyme/nanoenzyme systems within mental-organic frameworks (MOFs) shows great promise for a myriad of practical applications. Herein, two sequential biocatalysts, oxidase and hemin, were coupled together with close proximity using a bifunctional polymer, poly(1-vinylimidazole) (PVI), and encapsulated into MOFs. As a demonstration of the power of such a protocol, glucose oxidase&PVI-hemin encapsulated in ZIF-8 showed significant enhancement of bioactivity for a cascade reaction compared to its counterpart without PVI. For the colorimetric assay of glucose, it showed a low limit of detection of 0.4 µM (S/N = 3), high selectivity, and excellent stability. Because there are numerous biocatalysts that can readily be coupled and encapsulated into MOFs, a myriad of interesting properties can be simply realized by encapsulating different sequential biocatalysts.

A new strategy for the chemoenzymatic synthesis of glycopeptides by De-O-acetylation with an esterase and glycosylations with glycosyltransferases.

Glycopeptides are fragments of glycoproteins and are important in evaluating the biological roles of carbohydrates in glycoproteins. Fmoc solid-phase peptide synthesis using acetyl-protected glycosylated amino acids is a common strategy for the preparation of glycopeptides, but this approach normally requires chemical de-O-acetylation with a base that ß-eliminates sugar residues and epimerizes the peptide backbone. Here we demonstrate a facile new chemoenzymatic synthetic strategy for glycopeptides, using an esterase for the de-O-acetylation of sugar residues and glycosyltransferases for successive sugar elongations at neutral pH.