Continuous-flow synthesis of polysubstituted γ-butyrolactones via enzymatic cascade catalysis

Polysubstituted chiral γ-butyrolactones are the core structural units of many natural products and high value-added flavors and fragrances used in the food and cosmetic industry. Current enzymatic cascade synthesis of these molecules faces the problems of low enzyme activity and phase separation in batch reaction, resulting in low productivity. Herein, we report a new continuous-flow process to synthesize the optically pure Nicotiana tabacum lactone(3S,4S)-4a and whisky lactone(3R,4S)-4b from α,β-unsaturatedγ-ketoesters. A new ene reductase(ER) from Swingsia samuiensi(Ss ER) and a carbonyl reductase(Ss CR)were engineered by directed evolution to improve their activity and thermostability. The continuous-flow preparative reactions were performed in two 3D microfluidic reactors, generating(3S,4S)-4a(99% ee and87% de) and(3R,4S)-4b(99% ee and 98% de) with space-time yields 3 and 7.4 times higher than those of the batch reactions. The significant enhancement in the productivity of enzyme cascade catalysis brought by cutting-edge continuous microfluidic technology will benefit the general multi-enzyme catalytic systems in the future.

Understanding the cooperative effects in the catalysis of homodimeric fluoroacetate dehalogenase

Fluoroacetate dehalogenases(FAcD),a homodimeric enzyme,catalyzes the conversion of fluoroacetic acid to glycolic acid(GoA).It has been proved that the enzyme has a half-of-the-site reactivity.Namely,its catalytic(C)subunit converts the first substrate to a covalent intermediate;then,the non-catalytic(NC)subunit binds a second substrate and promotes the conversion of the intermediate in the C subunit into the final product.After the release of the product,the C subunit becomes the NC subunit,and the previous NC subunit becomes the C subunit.To elucidate the detailed mechanism behind this cooperative catalysis,we have conducted microsecond-scale MD simulations along the reaction pathway.The simulations indicate that the substrate in the NC subunit induces W185 and Y141 adopting an open conformation in the C subunit.The opening of W185(C)facilitates the entry of catalytic water,enhancing the catalytic activity for product formation,while the opening of Y141(C)creates an unfavorable environment for product binding,promoting its release.An interaction network analysis reveals that the substrate in the NC subunit can induce conformational changes through a conserved water chain at the interface.

Theoretical Study on GSH Activation Mechanism of a New Type of Glutathione Transferase Gtt2

Glutathione transferases（GSTs） play an important role in the detoxification of xenobiotic/endobiotic toxic compounds. The α-, π-, and/l-classes of cytosolic GSTs have been studied extensively, while Gtt2 from Saccharo- myces cerevisiae, a novel atypical GST, is still poorly understood. In the present study, we investigated the gluta- thione（GSH） activation mechanism of Gtt2 using the density functional theory（DFT） with the hybrid functional B3LYP. The computational results show that a water molecule could assist a proton transfer between the GSH thiol and the N atom of His133. The energy barrier of proton transfer is 46.0 kJ/mol. The GSH activation mechanism and the characteristics of active site are different from those of classic cytosolic GSTs.

Characterization and application of a recombinant dopa decarboxylase from Harmonia axyridis for the efficient biosynthesis of dopamine

Here,a dopa decarboxylase(DDC)from Harmonia axyridis was heterogeneously expressed in Escherichia coli for the efficient biosynthesis of dopamine.For the production of recombinant DDC,the cultivation conditions including IPTG concentration,temperature and induction time were optimized and obtained an optimal specific enzyme activity of 51.72 U·mg^(-1) crude extracts.After the purification of DDC with a recovery yield of 68.79%,its activity was further characterized.The Vmax,Km,Kcat,and Kcat/Km of DDC for d ihyd roxy pheny la la nine(dopa)were 0.02 mmol·ml^(-1)·s^(-1),2.328 mmol·ml^(-1),10435.90 s^(-1) and4482.77 ml,mmol respectively.The highest DDC activity was observed at the condition of pH 7.5 and 45℃.With the purified DDC,the feasibility to produce dopamine from L-dopa was evaluated.The optimal yield was determined at the following bioconversion conditions:pH of 7,0,the reaction temperature of 40℃,0.4 mmol·L^(-1) of PLP and 4 g·L^(-1) of L-dopa,Subsequently,a fed-batch process for the production of dopamine was developed and the effect of oxygen was evaluated.The titer,yield and productivity of dopamine reached up to 21.99 g·L^(-1)80.88%and 14.66 g·L^(-1)·h^(-1) at 90 min under anaerobic condition.

Microbial Community Profiles Related to Volatile Fatty Acids Production in Mesophilic and Thermophilic Fermentation of Waste Activated Sludge Pretreated by Enzymolysis

Mesophilic and thermophilic anaerobic fermentation performance of waste activated sludge(WAS)pretreated by enzymes catalysis associated with microbial community shifts were investigated.WAS disintegration was boosted considerably by enzymolysis with 8750 mg/L of soluble COD release within 180 min.Mesophilic anaerobic fermentation(MAF)produced nearly equal VFA accumulation with over 3200 mg COD/L compared with that of thermophilic fermentation(TAF).Bacterial community consortia showed great shifting differences in dynamics of main T⁃RFs between MAF and TAF.Moreover,MAF was conducive to form intermediate bacterial community evenness compared to TAF,which preserved a robust function of VFA production.The enzymes catalysis prompted bio⁃energy(electricity)recovery potential of WAS organics via anaerobic fermentation(MAF/TAF)with evaluating electricity conversion efficiency of 0.75-0.82 kW·h/kg VSS(3.9 times higher than control test).Finally,this study proposed some novel thinking on future WAS treatment/management towards energy recovery coupled with energy⁃sufficient wastewater treatment by co⁃locating WAS anaerobic fermentation,MFC plant with wastewater treatment plant(s).

Application of Enzyme Engineering in Pharmacy

Enzyme engineering is an important part of modern biotechnology.Due to its high reaction specificity,high efficiency,mild reaction conditions,and low pollution,it is also an important method widely used in the pharmaceutical field.The application of enzymes in medicine is diverse,such as:diagnosis,prevention and treatment of diseases with enzymes,manufacture of various drugs with enzymes,etc.,mainly through manual operations,to obtain enzymes required by the pharmaceutical industry,and through various means Enzymes perform their catalytic functions.This article mainly introduces the application of enzyme engineering in the pharmaceutical field,and also prospects the development trend of enzyme engineering in the pharmaceutical field.

Characterization of HpnG as a Purine Nucleoside Phosphorylase in Bacteriohopanepolyol Biosynthesis

Comprehensive Summary HpnG plays a crucial role in the production of ribosylhopane,a key intermediate in the biosynthesis of bacteriohopanepolyol.Despite early extensive studies,the precise function of HpnG has remained elusive.Here,we report functional characterization of HpnG as a purine nucleoside phosphorylase,which converts adenosylhopane to phosphoribosylhopane in the presence of phosphate.HpnG demonstrates broad substrate specificity and impressive stability,making it a valuable enzymatic tool for applications in nucleoside processing and related biotechnology.

Bioactive Hydrogels with Pro-coagulation Effect for Hemostasis

Hemostatic hydrogels are widely applied for wound management of damaged tissues,traumatic wounds,and surgical incisions.Some hydrogels composed of bioactive components,including fibrin and thrombin,showed great promise in the clinic due to their good pro-coagulation effect.With the expanding knowledge of cascade reaction of blood coagulation and emerging bioactive substances,massive bioactive hydrogels consisting of peptides,hemocoagulase,polyphosphate(polyP),etc.,have been developed as effective hemostatic materials.Based on the coagulation process and mechanism,we summarize the role of reported bioactive hydrogels in hemostasis in this review.We conclude the key points in the coagulation process,including activation of coagulation factors,fibrinogen polymerization,etc.,then discuss how to design bioactive hydrogels to accelerate coagulation targeted to these points.Finally,we conclude the progress and propose a perspective of bioactive hydrogels with a pro-coagulation effect for hemostasis.

Establishing Modular Cell-Free Expression System for the Biosynthesis of Bicyclomycin from a Chemically Synthesized Cyclodipeptide

Cell-free expression systems have emerged as a versatile and powerful platform for metabolic engineering,biosynthesis and synthetic biology studies.Nevertheless,successful examples of the synthesis of complex natural products using this system are still limited.Bicyclomycin,a structurally unique and complex diketopiperazine alkaloid,is a clinically promising antibiotic that selectively inhibits the transcription termination factor Rho.Here,we established a modular cell-free expression system with cascade catalysis for the biosynthesis of bicyclomycin from a chemically synthesized cyclodipeptide.The six cell-free expressed biosynthetic enzymes,including five iron-andα-ketoglutarate-dependent dioxygenases and one cytochrome P450 monooxygenase,were active in converting their substrates to the corresponding products.The co-expressed enzymes in the cell-free module were able to complete the related partial pathway.In vitro biosynthesis of bicyclomycin was also achieved by reconstituting the entire biosynthetic pathways(i.e.,six enzymes)using the modular cell-free expression system.This study demonstrates that the modular cell-free expression system can be used as a robust and promising platformforthe biosynthesis of complex antibiotics.

Dendrimer-induced synthesis of porous organosilica capsules for enzyme encapsulation

Organic matter-induced mineralization is a green and versatile method for synthesizing hybrid nanostructured materials,where the material properties are mainly influenced by the species of natural biomolecules,linear synthetic polymer,or small molecules,limiting their diversity.Herein,we adopted dendrimer poly(amidoamine)(PAMAM)as the inducer to synthesize organosilica-PAMAM network(OSPN)capsules for mannose isomerase(MIase)encapsulation based on a hard-templating method.The structure of OSPN capsules can be precisely regulated by adjusting the molecular weight and concentration of PAMAM,thereby demonstrating a substantial impact on the kinetic behavior of the MIase@OSPN system.The MIase@OSPN system was used for catalytic production of mannose from Dfructose.A mannose yield of 22.24% was obtained,which is higher than that of MIase in organosilica network capsules and similar to that of the free enzyme.The overall catalytic efficiency(kcat/Km)of the MIase@OSPN system for the substrate D-fructose was up to 0.556 s^(-1)·mmol^(-1)·L.Meanwhile,the MIase@OSPN system showed excellent stability and recyclability,maintaining more than 50% of the yield even after 12 cycles.

A H_(2)O_(2)generation-detection-regulation integrated platform for boosting the efficiency of peroxygenase-catalysed C-H oxidative hydroxylation

The peroxygenases are ideal biocatalysts for the selective oxyfunctionalisation of stable C-H bonds.However,the catalytic efficiency of this approach is limited due to enzyme lability toward oxidant H_(2)O_(2).Although the reported in-situ H_(2)O_(2) generation system enables the stable biocatalytic process without deactivating the enzyme,the greatest catalytic potential of peroxygenases still cannot be fulfilled effectively.To address the above issue,a H_(2)O_(2) generation-detection-regulation platform that integrated an effective organocatalyst-driven H_(2)O_(2) generation system,a precise electrochemical H_(2)O_(2) real-time detection device,and a convenient H_(2)O_(2) regulation strategy was first developed.The suitable range of H_(2)O_(2) generation rate for maximizing the catalytic efficiency of peroxygenases while minimizing inactivation of the enzyme was firstly obtained by simply adjusting the amount of organocatalyst.According to the determined suitable range,the C-H oxyfunctionalisation efficiency of peroxygenases for each substrate was significantly boosted,achieving~3-fold of the reported highest turnover frequency.

Integration of chemoselective ligation with enzymespecific catalysis： Saccharic colorimetric analysis using aminooxy/hydrazine-functionalized gold nanoparticles

Here we developed a saccharic colorimetric method based on the combination of chemoselective ligation and enzyme-specific catalysis using aminooxy/ hydrazine-functionalized gold nanoparticles （AO/AuNPs or H/AuNPs）. In the detection of galactose （Gal）, galactohexodialdose （GHDA）, the galactose oxidase （GalOx）-catalyzed product, has an aldehyde group, which allows it to chemoselectively react with an aminooxy or hydrazine group at the outer layer of AO/AuNPs or H/AuNPs by oxime/hydrazone click chemistry to form oxime or hydrozone. Consequent134 through the specific recognition of 1,4-phenylenediboronic acid （PDBA） on cis-diols, GHDA, which contains two pairs of hydroxyls in the cis form, can bind not only with AO/AuNPs or H/AuNPs, but also with PDBA to form boronate diester, thereby triggering the aggregation of AuNPs and causing the corresponding color change. As GalOx catalyzed specific substrates, the amount of Gal correlated with the production of GHDA and the extent of AuNPs aggregation, thus allowing a simple and easily operatable colorimetric method for Gal detection to be developed. Under the optimized experimental conditions, the ratios of absorbance at a wavelength of 617 nm to that at 536 nm vary linearly with the logarithmic values of Gal concentrations within a wide range of 500 nM to 5 mM. Moreover, this colorimetric method shows anti-interference capability and high sensitivity with a detection limit of 21 nM. Thus, a universal platform for accurate and specific colorimetric analysis can be established through the integration of chemoselective ligation with enzyme specific catalysis.

Substrate specificity and reaction directionality of a three-residue cyclophane forming enzyme Pau B

Three-residue cyclophane-forming enzymes(3-Cy FEs) are a group of radical S-adenosylmethionine(SAM)enzymes involved in the biosynthesis of ribosomally synthesized and posttranslationally modified peptides(Ri PPs). 3-Cy FE catalyzes the crosslinking between an aromatic residue(Ω1) and a non-aromatic residue(X3) in a Ω1-X2-X3 motif to produce a cyclophane ring, a key step in the biosynthesis of the Ri PP natural product triceptide. In this study, we perform a genome-wide search for the Xye-type triceptides, showing these Ri PPs are likely class-specific and only present in gamma-proteobacteria. The 3-Cy FE Pau B from Photorhabdus australis exhibits a relaxed substrate specificity on the X3 position, but glycine in this position is not suitable for cyclophane formation. We also reconstituted the activity of Pau B in vitro,showing it produces the N-terminal cyclophane firstly, and then the C-terminal ring, whereas the middle cyclophane is produced in the last step.

A Bacterial Cytochrome P450 Enzyme Catalyzes Multistep Oxidation Reactions in Pyrroindomycin Biosynthesis

Cytochrome P450 enzymes (P450s) belong to a large family of oxidative hemeproteins and catalyze highly diverse oxygenation reactions that are involved in the biosynthesis of various natural products. Here, we report a multifunctional cytochrome P450 enzyme, PyrE2, which catalyzes the regioselective, successive 6-electron oxidation of an inert methyl group to produce a carboxyl product through formation of the hydroxyl and aldehyde intermediates in pyrroindomycin biosynthesis. The time-course biotransformation was characterized by the presence of the hydroxyl and aldehyde intermediates, the lag of the formation of the carboxyl product, and the subsequent loss of both intermediates, indicating that each 2-electron oxidation exhibits the distributive mechanism that requires substrate binding and product releasing. Bioinformatics analysis shows that the homologs of pyrE2 are common in the gene clusters of the spirotetronates varying in the oxidative state of the corresponding exocyclic carbon, indicating the generality and diversity of P450-catalyzed oxygenation in related biosynthetic pathways.

Mechanism of allosteric activation of SIRT6 revealed by the action of rationally designed activators

The recent discovery of activator compounds binding to an allosteric site on the NAD+-dependent protein lysine deacetylase,sirtuin 6(SIRT6)has attracted interest and presents a pharmaceutical target for aging-related and cancer diseases.However,the mechanism underlying allosteric activation of SIRT6 by the activator MDL-801 remains largely elusive because no major conformational changes are observed upon activator binding.By combining molecular dynamics simulations with biochemical and kinetic analyses of wild-type SIRT6 and its variant M136 A,we show that conformational rotation of 2-methyl-4-fluoro-5-bromo substituent on the right phenyl ring(R-ring)of MDL-801,which uncovers previously unseen hydrophobic interactions,contributes to increased activating deacetylation activity of SIRT6.This hypothesis is further supported by the two newly synthesized MDL-801 derivatives through the removal of the 5-Br atom on the R-ring(MDL-801-D1)or the restraint of the rotation of the R-ring(MDL-801-D2).We further propose that the 5-Br atom serves as an allosteric driver that controls the ligand allosteric efficacy.Our study highlights the effect of allosteric enzyme catalytic activity by activator binding and provides a rational approach for enhancing deacetylation activity.

The Promiscuous Activity of the Radical SAM Enzyme NosL toward Two Unnatural Substrates

The radical S-adenosyImethionine(SAM)enzyme NosL catalyzes the conversion of L-tryptophan((-Trp,1)to 3-methyl-2-indolic acid(MIA,2),a key in termediate in the biosynthesis of the peptide an tibiotic no siheptide.Previous study showed that this remarkable recombination reaction starts from the cleavage of the Cα-COO^(-) bond to result in a·CO_(2)^(-) radical migration.In contrast to the radical SAM tyrosine lyases,NosL appears unable to cleave the Cα-Cβ bond,which is intrinsically more favorable to be cleaved than the Cα-COO^(-) bond.In this study,we investigate the NosL activity with tryptamine(11)and tryptophol(12),two L-Trp analogues lacking a carboxylate moiety.We showed that NosL cleaves the C1-C2 bond of these two substrates to produce 3-methylindole(7),suggesting that the enzyme can still catalyze a β-scission when the carboxyl group of Trp is absent.We also showed the enzyme exhibits a promiscuous activity,initiating the reaction by abstracting hydrogen atoms from two different sites to produce two sets of products.

Biocatalytic direct asymmetric aldol reaction using proteinase from Aspergillus melleus

The direct asymmetric aldol reaction of aromatic aldehydes with cyclic or acyclic ketones was catalyzed by proteinase from Aspergillus melleus (AMP) in acetonitrile in the presence of water. A wide range of substrates could be transformed into the corresponding aldol products in yields up to 89%, enantioselectivities up to 91% ee and diastereoselectivities up to >99:1 (anti/syn). This work provided an example of enzyme catalytic promiscuity that widens the applicability of this biocatalyst in organic synthesis without the need for additional cofactors or special equipment.

Peroxidase-like properties of Ruthenium nanoframes

This work reports the inherent peroxidase-like properties of Ruthenium （Ru） nanoframes. After templating with Palladium （Pd） seeds, Ru nanoframes with an octa- hedral shape, average edge length of 6.2 nm, and thickness of 1.8 nm were synthesized in high purity （〉95 %） and good uniformity. Using the oxidation of 3,3t,5,5＇-tetram- ethylbenzidine （TMB） by H202 as a model catalytic reac- tion, the Ru frames were demonstrated to be approximately three times more active than natural peroxidases in cat- alyzing the formation of colored products. As compared to their natural counterparts, Ru frames have a stronger binding affinity to TMB as well as a weaker binding affinity to hydrogen peroxide during the catalysis. The Ru frames as peroxidase mimics proved to be chemically and thermally stable. This work represents the first demonstration of Ru nanostructure-based peroxidase mimics and is therefore expected to inspire future research on bio-applications of Ru nanomaterials.

Divergent Synthesis of Core m1,Core m2 and Core m3 O-Mannosyi Glycopeptides via a Chemoenzymatic Approach

O-Mannosylation plays a vital role in the regulation of a variety range of biological processes,for instance,brain and muscle development.However,the precise function remains largely unknown due to its innate heterogeneity.In this regard,it is still welcome to develop efficient methods to access diverse structurally-defined glycopeptides.In this study,a diversity-oriented assembly of O-mannosylα-dystroglycan(α-DG)glycopeptides has been achieved via a chemoenzymatic strategy.This strategy features(i)gram scale divergent synthesis of core m1,core m2 and core m3 mannosylated amino acids from judiciously designed protecting group strategies and chemical glycosidation;(i)efficient glycopeptide assembly via the optimized microwave-assisted solid phase peptide synthesis(SPpS);and(ii)enzymatic elaboration of the core glycan structures to install galactosyl and sialyl-galactosyl moieties.The efficiency and flexibility of this chemoenzymatic approach was demonstrated with the construction of 12 glycopeptides with different core m1,core m2 and core m3 mannosyl glycans,including a core m2 glycopeptide bearing a heptasaccharide for the first time.

Sequential One-Pot Three-Enzyme Synthesis of the Tetrasaccharide Repeating Unit of Group B Streptococcus Serotype Ⅷ Capsular Polysaccharide

The biochemical property and functional identification of three recombinant glycosyltransferases,includingβ-1,4-rhamnosyltransferase(Cps8R),β-1,4-galactosyltransferase(Cps8) and α-2,3-sialyltransferase(Cps8K)involved in the biosynthesis of the tetrasaccharide repeating unit of serotypeⅧcapsular polysaccharide(CPS)of Group B Streptococci(GBS),were systematically investigated.Subsequently,these recombinant enzymes were employed for one-pot three-enzyme efficient synthesis of the tetrasaccharide repeating unit of GBS serotypeⅧCPS using the chemically synthesized Glca-PP-(CH2)ir-OPh as the starting substrate in a satisfying yieldof 87%.