Precision Engineering of the Co-immobilization of Enzymes for Cascade Biocatalysis.

The design and orderly layered co-immobilization of multiple enzymes on resin particles remain challenging. In this study, the SpyTag/SpyCatcher binding pair was fused to the N-terminus of an alcohol dehydrogenase (ADH) and an aldo-keto reductase (AKR), respectively. A non-canonical amino acid (ncAA), p-azido-L-phenylalanine (p-AzF), as the anchor for covalent bonding enzymes, was genetically inserted into preselected sites in the AKR and ADH. Employing the two bioorthogonal counterparts of SpyTag/SpyCatcher and azide-alkyne cycloaddition for the immobilization of AKR and ADH enabled sequential dual-enzyme coating on porous microspheres. The ordered dual-enzyme reactor was subsequently used to synthesize (S)-1-(2-chlorophenyl)ethanol asymmetrically from the corresponding prochiral ketone, enabling the in situ regeneration of NADPH. The reactor exhibited a high catalytic conversion of 74 % and good reproducibility, retaining 80 % of its initial activity after six cycles. The product had 99.9 % ee, which that was maintained in each cycle. Additionally, the double-layer immobilization method significantly increased the enzyme loading capacity, which was approximately 1.7 times greater than that of traditional single-layer immobilization. More importantly, it simultaneously enabled both the purification and immobilization of multiple enzymes on carriers, thus providing a convenient approach to facilitate cascade biocatalysis.

Near-infrared light-driven asymmetric photolytic reduction of ketone using inorganic-enzyme hybrid biocatalyst.

Effective photolytic regeneration of the NAD(P)H cofactor in enzymatic reductions is an important and elusive goal in biocatalysis. It can, in principle, be achieved using a near-infrared light (NIR) driven artificial photosynthesis system employing H2O as the sacrificial reductant. To this end we utilized TiO2/reduced graphene quantum dots (r-GQDs), combined with a novel rhodium electron mediator, to continuously supply NADPH in situ for aldo-keto reductase (AKR) mediated asymmetric reductions under NIR irradiation. This upconversion system, in which the Ti-O-C bonds formed between r-GQDs and TiO2 enabled efficient interfacial charge transfer, was able to regenerate NADPH efficiently in 64 % yield in 105 min. Based on this, the pharmaceutical intermediate (R)-1-(3,5-bis(trifluoromethyl)phenyl)ethan-1-ol was obtained, in 84 % yield and 99.98 % ee, by reduction of the corresponding ketone. The photo-enzymatic system is recyclable with a polymeric electron mediator, which maintained 66 % of its original catalytic efficiency and excellent enantioselectivity (99.9 % ee) after 6 cycles.

Enzymatic properties of a non-classical aldoxime dehydratase capable of producing alkyl and arylalkyl nitriles.

Nitriles are of significant interest in the flavor and fragrance industries with potential application in cosmetics due to their higher stability than analogous aldehydes. However, the traditional methods to prepare nitriles need toxic reagents and hash conditions. This work aimed to develop a chemoenzymatic strategy to synthesize nitriles from natural aldehydes with aldoxime as the intermediate. A non-classical aldoxime dehydratase (Oxd) was discovered from the fungus Aspergillus ibericus (OxdAsp) to catalyze the dehydration of aldoximes to corresponding nitriles under mild conditions. The amino acid sequence of OxdAsp exhibits an approximately 20% identity with bacterial Oxds. OxdAsp contains a heme prosthetic group bound with the axial H287 in the catalytic pocket. The structure models of OxdAsp with substrates suggest that its catalytic triad is Y138-R141-E192, which is different from the classically bacterial Oxds of His-Arg-Ser/Thr. The catalytic mechanism of OxdAsp was proposed based on the mutagenesis of key residues. The hydroxyl group of the substrate is fixed by E192 to increase its basicity. Y138 acts as a general acid-based catalyst, and its phenolic proton is polarized by the adjacent R141. The protonated Y138 would donate a proton to the hydroxyl group of the substrate and eliminate a water molecule from aldoxime to produce nitrile. The recombinant OxdAsp can efficiently dehydrate citronellal oxime and cinnamaldoxime to citronellyl nitrile and cinnamonitrile in aqueous media, which are applied as fragrance ingredients in the food and cosmetic fields. KEY POINTS: • A novel aldoxime dehydratase from the Aspergillus genus was first characterized as a heme-binding protein. • The catalytic mechanism was predicted based on the molecular interactions of the catalytic pocket with the substrate. • A chemoenzymatic strategy was developed to synthesize nitriles from natural aldehydes with aldoxime as the intermediate.

Engineering ketoreductases for the enantioselective synthesis of chiral alcohols.

The use of engineered ketoreductases (KREDS), both as whole microbial cells and isolated enzymes, in the highly enantiospecific reduction of prochiral ketones is reviewed. The homochiral alcohol products are key intermediates in, for example, pharmaceuticals synthesis. The application of sophisticated protein engineering and enzyme immobilisation techniques to increase industrial viability are discussed.

Engineered aldoxime dehydratase to enable the chemoenzymatic conversion of benzyl amines to aromatic nitriles.

A chemoenzymatic strategy has been implemented to synthesize nitriles from benzyl amines under mild conditions. Aldoxime dehydratase (Oxd) plays a decisive role to convert aldoximes into corresponding nitriles. However, natural Oxds commonly exhibit extremely low catalytic capacity toward benzaldehyde oximes. Here, we engineered the OxdF1 from Pseudomonas putida F1 to enhance its catalytic efficiency toward benzaldehyde oximes by a semi-rational design strategy. The protein structure-based CAVER analysis indicates that M29, A147, F306, and L318 are located adjacent to the substrate tunnel entrance of OxdF1, which were responsible for the transportation of substrate into the active site. After two rounds of mutagenesis, the maximum activities of the mutants L318F and L318F/F306Y were 2.6 and 2.8 U/mg respectively, which were significantly higher than the wild OxdF1 of 0.7 U/mg. Meanwhile, the lipase type B from Candida antarctica was functionally expressed in Escherichia coli cells to selectively oxidize benzyl amines to aldoximes using urea-hydrogen peroxide adduct (UHP) as an oxidant in ethyl acetate. To merge the oxidation and dehydration reactions, a reductive extraction solution was added to remove the residue UHP, which is critical to eliminate its inhibition on the Oxd activity. Consequently, nine benzyl amines were efficiently converted into corresponding nitriles by the chemoenzymatic sequence.

Putting precision and elegance in enzyme immobilisation with bio-orthogonal chemistry.

The covalent immobilisation of enzymes generally involves the use of highly reactive crosslinkers, such as glutaraldehyde, to couple enzyme molecules to each other or to carriers through, for example, the free amino groups of lysine residues, on the enzyme surface. Unfortunately, such methods suffer from a lack of precision. Random formation of covalent linkages with reactive functional groups in the enzyme leads to disruption of the three dimensional structure and accompanying activity losses. This review focuses on recent advances in the use of bio-orthogonal chemistry in conjunction with rec-DNA to affect highly precise immobilisation of enzymes. In this way, cost-effective combination of production, purification and immobilisation of an enzyme is achieved, in a single unit operation with a high degree of precision. Various bio-orthogonal techniques for putting this precision and elegance into enzyme immobilisation are elaborated. These include, for example, fusing (grafting) peptide or protein tags to the target enzyme that enable its immobilisation in cell lysate or incorporating non-standard amino acids that enable the application of bio-orthogonal chemistry.

Programmable Polyproteams of Tyrosine Ammonia Lyases as Cross-Linked Enzymes for Synthesizing p-Coumaric Acid.

Ideal immobilization with enhanced biocatalyst activity and thermostability enables natural enzymes to serve as a powerful tool to yield synthetically useful chemicals in industry. Such an enzymatic method strategy becomes easier and more convenient with the use of genetic and protein engineering. Here, we developed a covalent programmable polyproteam of tyrosine ammonia lyases (TAL-CLEs) by fusing SpyTag and SpyCatcher peptides into the N-terminal and C-terminal of the TAL, respectively. The resulting circular enzymes were clear after the spontaneous isopeptide bonds formed between the SpyTag and SpyCatcher. Furthermore, the catalytic performance of the TAL-CLEs was measured via a synthesis sample of p-Coumaric acid. Our TAL-CLEs showed excellent catalytic efficiency, with 98.31 ± 1.14% yield of the target product-which is 4.15 ± 0.08 times higher than that of traditional glutaraldehyde-mediated enzyme aggregates. They also showed over four times as much enzyme-activity as wild-type TAL does and demonstrated good reusability, and so may become a good candidate for industrial enzymes.

Programing a cyanide-free transformation of aldehydes to nitriles and one-pot synthesis of amides through tandem chemo-enzymatic cascades.

Nitriles are broadly applied to synthesize pharmaceuticals, agrochemicals, and materials because of their versatile transformation. Although various methods have been developed for introducing a nitrile group into organic molecules, most of them entail the use of highly toxic chemicals, transition metals, or harsh conditions. In this work, we reported a greener chemo-enzymatic cascade to synthesize alky and aryl nitriles from readily accessible aldehydes, that were further transformed into corresponding amides via an artificial enzyme cascade. A biphasic reaction system was designed to bridge chemical synthesis and enzymatic catalysis through simple phase separation. The biphasic system mainly perfectly avoided the inactivation of hydroxylamine on aldoxime dehydratase from Pseudomonas putida (OxdF1) and nitrile hydratase from Aurantimonas manganoxydans ATCC BAA-1229 (NHase1229). For the synthesis of various nitriles, moderate isolation yields of approximately 60% were obtained by the chemo-enzymatic cascade. Interestingly, two seemingly conflicting reactions of dehydration and hydration were sequentially proceeded to synthesize amides by the synergistic catalysis of OxdF1 and NHase1229 in E. coli cells. An isolation yield of approximately 62% was achieved for benzamide at the one-liter scale. In addition, the shuttle transport of substrates and products between two phases is convenient for the product separation and n-hexane recycling. Thus, the chemo-enzymatic cascade shows a potential application in the cyanide-free and large-scale synthesis of nitriles and amides.

Controllably crosslinked dual enzymes enabled by genetic-encoded non-standard amino acid for efficiently enantioselective hydrogenation.

In traditional method for preparing crosslinked enzymes aggregates using glutaraldehyde, random linkage is inevitable, which often destroys the enzyme active sites and severely decreases the activity. To address this issue, using genetic encode expanding, nonstandard amino acids (NSAAs) were inserted into enzyme proteins at the preselected sites for crosslinking. When aldehyde ketone reductase (AKR), alcohol dehydrogenase (ADH) and glucose dehydrogenase (GDH) were utilized as model enzymes, their mutants containing p-azido-L-phenylalanine were bio-orthogonally crosslinked with diyne to form crosslinked dual enzymes (CLDEs) acting as a cascade biological oxidation and reduction system. Then, the resultant self-purified CLDEs were characterized using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM), etc. In the asymmetric synthesis of (S)-1-(2,6-dichloro-3-fluorophenyl) ethanol using CLDEs, high product yield (76.08%), ee value (99.99%) and reuse stability were achieved. The yield and ee value were 12.05 times and 1.39 times higher than those using traditional crosslinked enzyme aggregates, respectively. Thus, controllable insertion NSAAs in number and location can engender reasonable linkage and metal-free self-purification for target enzyme proteins. This facile and sustainable method could be further expanded to other dual and multienzyme systems for cascade biocatalysis.

Catalytically active inclusion bodies (CatIBs) induced by terminally attached self-assembling coiled-coil domains: To enhance the stability of (R)-hydroxynitrile lyase.

The catalytically-active inclusion bodies (CatIBs) represent a promising strategy for immobilizing enzyme without additional carriers and chemicals, which has aroused great attention in academic and industrial communities. In this work, we discovered two natural parallel right-handed coiled-coil tetramer peptides from PDB database by a structural mining strategy. The two self-assembling peptides, NSPdoT from rotavirus and HVdoT from human Vasodilator-stimulated phosphoprotein, efficiently induced the CatIBs formation of a (R)-Hydroxynitrile lyase from Arabidopsis thaliana (AtHNL) in Escherichia coli cells. This is convenient to simultaneously purify and immobilize the target proteins as biocatalysts. As expected, HVdoT-AtHNL and NSPdoT-AtHNL possessed drastically increased tolerance toward lower pH values, which will be very critical to synthesize cyanohydrins under acidic condition for suppressing the non-enzymatic side reaction. In addition. AtHNL-CatIBs are produced at high yield in host cells as bioactive microparticles, which exhibited high thermal and pH stabilities. Therefore, the CatIBs method represent a promising application for the immobilization of enzymes in the biocatalysis field.

Cyanide-free synthesis of aromatic nitriles from aldoximes: Discovery and application of a novel heme-containing aldoxime dehydratase.

Aromatic nitriles are important structural motifs that frequently existed in pharmaceutical drugs. Due to the convenient synthesis of aldoximes from aldehydes, the dehydration of aldoximes to corresponding nitriles by aldoxime dehydratases (Oxds) is considered as a safe and robust enzymatic production route. Although the Oxd genes are widely distributed in microbial kingdom, so far less than ten Oxds were expressed and further characterized. In this study, we found 26 predicted putative Oxd genes from the GenBank database using a genome mining strategy. The Oxd gene from Pseudomonas putida F1 was cloned and functionally expressed in Escherichia coli BL21 (DE3). The amino acid sequence of OxdF1 shows high identities of 33∼85 % to other characterized Oxds, and contained a ferrous heme as the catalytic site. The optimum reaction pH and temperature of recombinant OxdF1 were 7.0 and 35 °C, respectively. OxdF1 was stable in pH 7.0 potassium phosphate buffer at 30 °C, and its half-life was approximately 3.8 h. OxdF1 can efficiently dehydrate aromatic and heterocyclic aldoximes to nitriles, such as 2-bromobenzaldoxime, 2-chloro-6-fluorobenzaldoxime, thiophene-2-carboxaldoxime, and pyridine-3-aldoxime. Therefore, the recombinant OxdF1 shows a potential application in the cyanide-free synthesis of aromatic nitriles.

Toward Environmental Sustainability, Health, and Equity: How the Psychological Characteristics of College Students Are Reflected in Understanding Sustainable Development Goals.

This study aimed to identify how the psychological characteristics of college students are reflected in understanding sustainable development goals (SDGs) by examining college students' psychological characteristics, including attitude, interests, motivations, and self-efficacy, through the Sustainable Development Goals Psychological Inventory (SDGPI). The relationships among SDGs attitude, SDGs interest, SDGs motivation, and SDGs self-efficacy were analyzed by Pearson product-moment correlation coefficients. In addition, the Mann-Whitney U test and Kruskal-Wallis one-way analysis of variance were used to explore the differences among the college student groups in terms of gender, grade, and major in relation to attitude and personal characteristics. Attitude scores based on regression analysis were used to predict college students' interest, motivation, and self-efficacy in relation to the SDGs. According to the results, (a) the college students considered the three most important SDGs to be good health and well-being (SDG 3) (49.72%), quality education (SDG 4) (41.39%), and no poverty (SDG 1) (32.22%), while the three least important SDGs were decent work and economic growth (SDG 8) (41.11%), partnerships for the goals (SDG 17) (38.06%), and response consumption and institutions (SDG 12) (30.83%); (b) the SDGPI had a high reliability, with a Cronbach's alpha of 0.942; (c) there was a significant positive correlation between attitude and the variables of interest, motivation, and self-efficacy; (d) differences in attitudes, interest, and motivation between men and women were distinct and women scored much higher in these three subscales than men; (e) attitude could explain significant variance in interest, motivation and self-efficacy. In addition, attitude, interest, and motivation could account for self-efficacy. This study supports the development of sustainability education (SE) at the college level by providing new insights into college students' psychological characteristics in relation to the SDGs.

Efficiently Enantioselective Hydrogenation Photosynthesis of (R)-1-[3,5-Bis(trifluoromethyl)phenyl] ethanol over a CLEs-TiO2 Bioinorganic Hybrid Materials.

Engineering of biological pathways with man-made materials provides inspiring blueprints for sustainable drug production. (R)-1-[3,5-Bis(trifluoromethyl)phenyl]ethanol [(R)-3,5-BTPE], as an important artificial chiral intermediate for complicated pharmaceutical drugs and biologically active molecules, is often synthesized through a hydrogenation reaction of 3,5-bis(trifluoromethyl)acetophenone (3,5-BTAP), in which enantioselectivity and sufficient active hydrogen are the key to restricting the reaction. In this work, a biohybrid photocatalytic hydrogenation system based on an artificial cross-linked enzymes (CLEs)-TiO2-Cp*Rh(bpy) photoenzyme is developed through a bottom-up engineering strategy. Here, TiO2 nanotubes in the presence of Cp*Rh(bpy) are used to transform NADP+ to NADPH during the formation of chiral alcohol intermediates from the catalytic reduction of a ketone substrate by alcohol dehydrogenase CLEs. Hydrogen and electrons, provided by water and photocatalytic systems, respectively, are transferred to reduce NADP+ to NADPH via [Cp*Rh(bpy)(H2O)]2+. With the resulting NADPH, [(R)-3,5-BTPE] is synthesized using our efficient CLEs obtained from the cell lysate by nonstandard amino acid modification. Through this biohybrid photocatalytic system, the photoenzyme-catalyzed combined reductive synthesis of [(R)-3,5-BTPE] has a yield of 41.2% after reaction for 24 h and a very high enantiomeric excess value (>99.99%). In the case of reuse, this biohybrid system retained nearly 95% of its initial catalytic activity for synthesizing the above chiral alcohol. The excellent reusability of the CLEs and TiO2 nanotubes hybrid catalytic materials highlights the environmental friendliness of (R)-3,5-BTPE production.

Succinic anhydride-based chemical modification making laccase@Cu3(PO4)2 hybrid nanoflowers robust in removing bisphenol A in wastewater.

To prepare a robust biocatalyst and enhance the removal of bisphenol A in wastewater, succinic anhydride was reacted with laccase to obtain succinic anhydride-modified laccase (SA-laccase) and then co-crystallized with Cu3(PO4)2 to form SA-laccase@Cu3(PO4)2 hybrid nanoflowers (hNFs). The activity of SA-laccase@Cu3(PO4)2 reached 5.27 U/mg, 1.86-, 2.88- and 2.15-fold those of bare laccase@Cu3(PO4)2, laccase@Ca3(PO4)2 and laccase@epoxy resin, respectively. Compared with free laccase, the obtained hNFs present enhanced activity and tolerance to pH and high temperature in the removal of BPA. Under the optimum conditions of pH 6.0 and 35 °C, BPA removal reached 93.2% using SA-laccase@Cu3(PO4)2 hNFs, which was 1.21-fold of that using free laccase. In addition, the obtained SA-laccase@Cu3(PO4)2 hNFs retained nearly 90% of their initial catalytic activity for BPA removal after 8 consecutive batch cycles. This efficient method for preparing immobilized laccase can also be further developed and improved to acquire green biocatalysts for removing persistent organic pollutants in wastewater.

Predictive value of baseline 18F-FDG PET/CT and interim treatment response for the prognosis of patients with diffuse large B-cell lymphoma receiving R-CHOP chemotherapy.

The present study aimed to investigate the prognostic value of baseline 18F-FDG PET/CT quantitative parameters and interim treatment response, and to assess whether the combination of these could improve the predictive efficacy in patients with diffuse large B-cell lymphoma (DLBCL) receiving R-CHOP chemotherapy. PET/CT images and clinical data of 64 patients with DLBCL who had undergone 18F-FDG PET/CT scan before and after 3 or 4 cycles of R-CHOP chemotherapy were retrospectively reviewed. The quantitative parameters including standardized uptake value (SUV), metabolic tumor volume (MTV), total lesion glycolysis (TLG), and maximum diameter of the maximum lesion (Dmax) were measured on baseline PET/CT images. Cox proportional hazards model was used to evaluate the influence of baseline PET/CT parameters, clinical indicators and interim treatment response on prognosis. Survival analysis was performed using Kaplan-Meier method. Receiver operating characteristic (ROC) curve analysis was performed to estimate the predictive efficacy of the combination of baseline PET/CT parameters and interim treatment response. Ann Arbor stage, International Prognostic Index (IPI), lactate dehydrogenase (LDH), necrosis, MTVmax, TLGmax, Dmax and interim treatment response showed association with 2-year progression-free survival (PFS, P<0.05). LDH, necrosis, MTVmax, MTVsum, TLGmax, TLGsum, Dmax and interim treatment response showed association with 2-year overall survival (OS, P<0.05). Ann Arbor stage, Dmax and interim treatment response were found to be independent predictors of 2-year PFS (P<0.05), while Dmax and interim treatment response were found to be independent predictors of 2-year OS (P<0.05). The PFS and OS curves of Dmax <5.7 cm group and Dmax ≥5.7 cm group, complete response (CR) group and non-CR group were significantly different, respectively (P<0.05). The baseline 18F-FDG PET/CT parameters and interim treatment response have important prognostic values in DLBCL patients who received R-CHOP chemotherapy. Combined application of Dmax and interim treatment response improved the predictive efficacy of 2-year PFS. It may be helpful to identify patients who are at high-risk of relapse and to guide early clinical intervention of these patients.

What Lies Behind Teaching and Learning Green Chemistry to Promote Sustainability Education? A Literature Review.

In this qualitative study, we aim to identify suitable pedagogical approaches to teaching and learning green chemistry among college students and preservice teachers by examining the teaching methods that have been used to promote green chemistry education (GCE) and how these methods have supported green chemistry learning (GCL). We found 45 articles published in peer-reviewed scientific journals since 2000 that specifically described teaching methods for GCE. The content of the articles was analyzed based on the categories of the teaching methods used and the revised version of Bloom's taxonomy. Among the selected articles, collaborative and interdisciplinary learning, and problem-based learning were utilized in 38 and 35 articles, respectively. These were the most frequently used teaching methods, alongside a general combination of multiple teaching methods and teacher presentations. Developing collaborative and interdisciplinary learning skills, techniques for increasing environmental awareness, problem-centered learning skills, and systems thinking skills featuring the teaching methods were seen to promote GCL in 44, 40, 34, and 29 articles, respectively. The results showed that the integration of green chemistry teaching (GCT), e.g., with sustainable education, promoted GCL by fostering environmental consciousness and behavioral change and cognitive processes in a sustainable direction.

Stable immobilization of aldehyde ketone reductase mutants containing nonstandard amino acids on an epoxy resin via strain-promoted alkyne-azide cycloaddition.

To avoid random chemical linkage and achieve precisely directed immobilization, mutant enzymes were obtained and immobilized using an incorporated reactive nonstandard amino acid (NSAA). For this purpose, aldehyde ketone reductase (AKR) was used as a model enzyme, and 110Y, 114Y, 143Y, 162Q and 189Q were each replaced with p-azido-l-phenylalanine (pAzF). Then, the mutant AKR was coupled to the functionalized support by strain-promoted alkyne-azide cycloaddition (SPAAC). The effects of the incorporation number and site of NSAAs on the loading and thermal stability of the immobilized AKR were examined. The results show that the mutant enzymes presented better specific activity than the wild type, except for AKR-110Y, and AKR-114Y showed 1.16-fold higher activity than the wild type. Moreover, the half-life (t 1/2) of the five-point immobilized AKR reached 106 h and 45 h, 13 and 7 times higher than that of the free enzyme at 30 °C and 60 °C, respectively. Comparison of these three types of enzymes shows that multi-point immobilization provides improved loading and thermal stability and facilitates one-step purification. We expect this platform to facilitate a fundamental understanding of precisely oriented and controllable covalent immobilization and enable bio-manufacturing paradigms for fine chemicals and pharmaceuticals.

Enhanced reductive removal of ciprofloxacin in pharmaceutical wastewater using biogenic palladium nanoparticles by bubbling H2.

To treat waste with waste and efficiently remove the organic pollutant, waste palladiums(ii) were adsorbed and reduced on microorganism surface to catalyze the reductive removal of ciprofloxacin in pharmaceutical wastewater. By optimizing conditions such as pH and temperature, the amount of biogenic palladium adsorbed and reduced on E. coli reached 139.48 mg g-1 (Pd/microorganisms). Moreover, most of the Pd(ii) was reduced to nanometer-sized Pd(0) as characterized by TEM and SEM with EDXA. Using the obtained biogenic palladium, the reductive removal of ciprofloxacin is up to 87.70% at 25 °C, 3.03 folds of that achieved in the absence of H2. The results show that waste E. coli microorganisms can efficiently adsorb and remove waste Pd(ii) and produce Bio-Pd nanoparticle catalysts in the presence of H2. This biogenic palladium presents high catalytic activity and great advantages in the reductive degradation of ciprofloxacin. Our method can also be applied to other waste metal ions to prepare the biogenic metals, facilitate their recovery and reuse in degrading organic pollutants in wastewater to achieve "treating waste using waste".

Peptidomics analysis of myometrium tissues in term labor compared with term nonlabor.

Preterm birth (PTB) is a major cause of neonatal mortality, with a poorly understood etiology. The regular contraction of the myometrium was considered as contributing to the etiology of the onset of labor, especially PTB. Thus, studying the mechanism of myometrium contraction is very important for understanding the initiation of labor and also for preventing PTB. Using liquid chromatography-mass spectrometry, we found 322 significantly differential peptides in myometrium tissues between term nonlabor and term labor groups (absolute fold change ≥ 2 and P < .05). We next analyzed length, molecular weights, isoelectric point, and cleavage site of all the different peptides. We, next, analyzed the functions of different peptides through their precursor proteins by Gene Ontology, enrichment and canonical pathway analysis. The results indicated that the extracellular matrix (ECM) played a major role in biological process, the cellular component, and molecular function categories, and revealed that ECM remodeling played a vital role in myometrial contraction. In addition, some known signaling, such as corticotropin-releasing hormone signaling and calcium signaling were proven to be involved in this process. Ingenuity Pathways Analysis upstream regulator analysis suggested that some of the known molecules, which reportedly were very important in labor onset, were included, for example, nuclear factor κB, tubulin, and phosphoinositide 3-kinase. We also identified 23 peptides derived from the precursor protein TITIN, of which 21 peptides sequences from TITIN were located in functional domains. These results suggested that peptides play an important role in labor onset and provide further insight into PTB therapy.

Discovery of a new NADPH-dependent aldo-keto reductase from Candida orthopsilosis catalyzing the stereospecific synthesis of (R)-pantolactone by genome mining.

(R)-Pantolactone (PL) is a key chiral intermediate for the synthesis of calcium (R)-pantothenate and (R)-panthenol used as food additives. The commercial production of (R)-pantothenate is performed by the resolution of racemic pantothenate, which is synthesized through an aldol condensation and a cyanation reaction. In this study, we investigated another synthetic method of (R)-pantothenate through the stereoselective reduction of ketopantoyl lactone (KPL) by aldo-keto reductase (AKR). A series of conjugated polyketone reductases (CPRs) were discovered from GenBank database by genome mining approach. The putative CPR gene from Candida orthopsilosis Co 90-125 (CorCPR) was cloned and functionally expressed in Escherichia coli BL21 (DE3). The optimum pH and temperature of recombinant CorCPR were 6.0-7.0 and 40 ℃, respectively. The Km and vmax toward KPL were1.3 mM and 227.3 µmol/min/mg protein, respectively. The conserved sequences suggest that CorCPR belongs to AKR3C family of AKR superfamily. Furthermore, a catalytic tetrad was proposed, and the detailed mechanism was clarified by molecular docking. In a batch reaction, 50 mM KPL was reduced to (R)-PL with 99% conversion and > 99% enantiomeric excess within 5 h. The recombinant CorCPR from C. orthopsilosis shows potential application in the asymmetric synthesis of (R)-pantothenate preparation.