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.

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.

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.

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.

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.

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).

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.

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.

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.

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%.

Theoretical Study of Iron Porphyrin Imine Specie of P411 Enzyme:Electronic Structure and Regioselectivity of C(sp^(3))-H Primary Amination

The cytochrome P411 enzyme is a variant of cytochrome P450_(BM3) from Bacillus megaterium whose active site is an iron porphyrin imine([Fe(Por)(NH)]^(-))specie.This specie has been reported to successfully promote the primary amination of benzylic and allylic C(sp^(3))-H bonds.We employed density functional theory to study the electronic structure of the active site of P411 enzyme and the primary amination of C-H bond reaction that it catalyzes.The calculated spin densities and orbital values indicate the existence of resonance in this specie;namely,[(por)(–OH)Fe^(Ⅳ)–N^(2-)–H]^(-)↔[(por)(–OH)Fe^(Ⅲ)–N^(·-)–H]^(-).The amination of C(sp^(3))-H bonds consists of two main reaction steps:hydrogen-atom abstraction and radical recombination,and the former is demonstrated to be the rate-determining step.Furthermore,we studied the regioselectivity of the amination of primary and secondary C(sp^(3))-H bonds.Our calculations indicated that the secondary C(sp^(3))-H bonds of the substrate would be more favored for the activation by P411 enzyme.These results provide valuable information for understanding the properties and selectivity of C-H/C-N bond-activation reactions catalyzed by the P411 enzyme or other similar enzymes.

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.

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.

Enzymatic Synthesis of a Diastereomer of Neoabyssomicin Derivative Using the Diels-Alderase AbyU

Main observation and conclusion The enzyme AbyU catalyses a Diels-Alder(DA)reaction during abyssomicin C biosynthesis.In this study,AbyU is shown to convert the native substrate of another Diels-Alderase(DAase),AbmU,to a new abyssomicin derivative,abyssomicin 7.

DNA-Templated Silver Nanoclusters Formation at Gold Electrode Surface and Its Application to Hydrogen Peroxide Detection

In this work, we have prepared Ag nanoclusters （Ag NCs） at gold electrode surface by using thiol-modified oligodeoxynucleotide consisting of eighteen cytosine deoxyribonucleotides （polyC18） as template and NaBH4 as reducing agent. Experimental results show that Ag nanoclusters （Ag NCs） can be formed around the template polyC18, while the formation can be characterized with electrochemical method. Further studies reveal that the fab- ricated Ag NCs may display high catalytic activity for the reduction of hydrogen peroxide （H2O2）, which can be further used for the detection of H20〉

Melatonin biosynthesis pathways in nature and its production in engineered microorganisms

Melatonin is a biogenic amine that can be found in plants,animals and microorganism.The metabolic pathway of melatonin is different in various organisms,and biosynthetic endogenous melatonin acts as a molecular signal and antioxidant protection against external stress.Microbial synthesis pathways of melatonin are similar to those of animals but different from those of plants.At present,the method of using microorganism fermentation to produce melatonin is gradually prevailing,and exploring the biosynthetic pathway of melatonin to modify microorganism is becoming the mainstream,which has more advantages than traditional chemical synthesis.Here,we review recent advances in the synthesis,optimization of melatonin pathway.L-tryptophan is one of the two crucial precursors for the synthesis of melatonin,which can be produced through a four-step reaction.Enzymes involved in melatonin synthesis have low specificity and catalytic efficiency.Site-directed mutation,directed evolution or promotion of cofactor synthesis can enhance enzyme activity and increase the metabolic flow to promote microbial melatonin production.On the whole,the status and bottleneck of melatonin biosynthesis can be improved to a higher level,providing an effective reference for future microbial modification.