Carboxylic Ester Bonds Research Articles

Assay Analysis of Tannase from Lactobacillus plantarum.

Tannin, which is an astringent taste in the mouth, is a polyphenol compound contained in some plants. Tannin causes denaturation of proteins of the tongue or oral mucosa. Tannase, a hydrolase that cleaves carboxylic ester bonds specifically, is used in many industrial fields. Some tannase (tannin acyl hydrolase, EC3.1.1.20) is used widely to prevent or reduce creaming of some foods and beverages. Because some tannins are formed of insoluble salts combined with protein, they reduce creaming such as the white hazing of iced tea. Moreover, they can clarify beverages such as fruit juices during wine and beer production. Tannase is produced by microorganisms under conditions with tannic acid present, mainly from plants. Tannase characteristics differ according to its microorganism of origin. Therefore, it is important to study the microbes used as lactic acid bacteria (LAB), evaluate new methods of tannase assay, and apply them in food or other industries. In this chapter, assay of tannase in LAB is demonstrated using methyl gallate as substrate, with color development by rhodanine and potassium hydroxide solution, using a spectrophotometer. Actual data of high tannase-producing LAB, Lactobacillus plantarum, and enzyme characteristics in optimum conditions are presented in this chapter.

Microbial detoxification of bifenthrin insecticide by selected fungal strains and optimizing conditions using response surface methodology for agricultural sustainability.

Bifenthrin is a type I broad spectrum pyrethroid insecticide widely employed in urban and agricultural settings with little knowledge about its biodegradation. Bifenthrin was subjected to a 35 days incubation period in which it was degraded by five fungal strains named as Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus, Penicillium chrysogenum, and Lasiodiplodia theobromae. Penicillium chrysogenum was found to be extremely effective in degrading bifenthrin up to 85%. Furthermore, response surface methodology (RSM) with Box-Behnken design is applied to optimize the degradation conditions with varying pH, temperature (°C), and incubation time (days). The p value < 0.05 in the response surface design and analysis of variance showed the significance of the reaction parameters. The ideal conditions for Penicillium chrysogenum to break down bifenthrin (10 mgL-1) were found to be 30 °C, pH 7, and a 24 days incubation period. In eutrophic conditions and a glucose-rich media, this fungus co-metabolized bifenthrin. By hydrolytically cleaving the carboxyl ester bond, the Penicillium chrysogenum breaks down bifenthrin, as shown by the chromatogram of four metabolites from GCMS. The biodegradation of bifenthrin by strain Penicillium chrysogenum and its use in agronomic situations are now well understood as per the findings of this study.

Enzyme-targeted near-infrared fluorescent probe for organophosphorus pesticide residue detection.

Pesticide residues significantly affect food safety and harmhuman health. In this work, a series of near-infrared fluorescent probes were designed and developed by acylating the hydroxyl group of the hemicyanine skeleton with aquenching moiety for monitoring the presence of organophosphorus pesticides in food and live cells. The carboxylic ester bond on the probe was hydrolyzed catalyticallyin the presence of carboxylesterase and thereby the fluorophore was releasedwith near-infrared emission. Notably, the proposed probe 1 exhibited excellent sensitivity against organophosphorus based on the carboxylesterase inhibition mechanism and the detection limit for isocarbophos achieved 0.1734μg/L in the fresh vegetable sample. More importantly, probe 1 allowed for situ visualization of organophosphorus in live cells and bacteria, meaning great potential for tracking the organophosphorus in biological systems. Consequently, this study presents a promising strategy for tracking pesticide residues in food and biological systems.

CES1 Releases Oxylipins from Oxidized Triacylglycerol (oxTAG) and Regulates Macrophage oxTAG/TAG Accumulation and PGE2/IL-1β Production.

Triacylglycerols (TAGs) are storage forms of fat, primarily found in cytoplasmic lipid droplets in cells. TAGs are broken down to their component free fatty acids by lipolytic enzymes when fuel reserves are required. However, polyunsaturated fatty acid (PUFA)-containing TAGs are susceptible to nonenzymatic oxidation reactions, leading to the formation of oxylipins that are esterified to the glycerol backbone (termed oxTAGs). Human carboxylesterase 1 (CES1) is a member of the serine hydrolase superfamily and defined by its ability to catalyze the hydrolysis of carboxyl ester bonds in both toxicants and lipids. CES1 is a bona fide TAG hydrolase, but it is unclear which specific fatty acids are preferentially released during lipolysis. To better understand the biochemical function of CES1 in immune cells, such as macrophages, its substrate selectivity when it encounters oxidized PUFAs in TAG lipid droplets requires study. We sought to identify those esterified oxidized fatty acids liberated from oxTAGs by CES1 because their release can activate signaling pathways that enforce the development of lipid-driven inflammation. Gaining this knowledge will help fill data gaps that exist between CES1 and the lipid-sensing nuclear receptors, PPARγ and LXRα, which are important drivers of lipid metabolism and inflammation in macrophages. Oxidized forms of triarachidonoylglycerol (oxTAG20:4) or trilinoleoylglycerol (oxTAG18:2), which contain physiologically relevant levels of oxidized PUFAs (<5 mol %), were incubated with recombinant CES1 to release oxylipins and nonoxidized arachidonic acid (AA) or linoleic acid (LA). CES1 hydrolyzed each oxTAG, yielding regioisomers of hydroxyeicosatetraenoic acids (5-, 11-, 12-, and 15-HETE) and hydroxyoctadecadienoic acids (9- and 13-HODE). Furthermore, human THP-1 macrophages with deficient CES1 levels exhibited a differential response to extracellular stimuli (oxTAGs, lipopolysaccharide, and 15-HETE) as compared to those with normal CES1 levels, including enhanced oxTAG/TAG lipid accumulation and altered cytokine and prostaglandin E2 profiles. This study suggests that CES1 can metabolize oxTAG lipids to release oxylipins and PUFAs, and it further specifies the substrate selectivity of CES1 in the metabolism of bioactive lipid mediators. We suggest that the accumulation of oxTAGs/TAGs within lipid droplets that arise due to CES1 deficiency enforces an inflammatory phenotype in macrophages.

Origin, structure and functional transition of sex pheromone components in a false widow spider

Female web-building spiders disseminate pheromone from their webs that attracts mate-seeking males and deposit contact pheromone on their webs that induces courtship by males upon arrival. The source of contact and mate attractant pheromone components, and the potential ability of females to adjust their web’s attractiveness, have remained elusive. Here, we report three new contact pheromone components produced by female false black widow spiders, Steatoda grossa: N-4-methylvaleroyl-O-butyroyl-L-serine, N-4-methylvaleroyl-O-isobutyroyl-L-serine and N-4-methylvaleroyl-O-hexanoyl-L-serine. The compounds originate from the posterior aggregate silk gland, induce courtship by males, and web pH-dependently hydrolyse at the carboxylic-ester bond, giving rise to three corresponding carboxylic acids that attract males. A carboxyl ester hydrolase (CEH) is present on webs and likely mediates the functional transition of contact sex pheromone components to the carboxylic acid mate attractant pheromone components. As CEH activity is pH-dependent, and female spiders can manipulate their silk’s pH, they might also actively adjust their webs’ attractiveness.

Novel mechanism and degradation kinetics of allethrin using Bacillus megaterium strain HLJ7 in contaminated soil/water environments

As a common pyrethroid insecticide, allethrin is widely used for various purposes in agriculture and home applications. At present, allethrin residues have been frequently detected worldwide, yet little is known about the kinetics and degradation mechanisms of this insecticide. In this study, a highly efficient allethrin-degrading bacterium, Bacillus megaterium strain HLJ7, was obtained through enrichment culture technology. Strain HLJ7 can remove 96.5% of 50 mg L−1 allethrin in minimal medium within 11 days. The first-order kinetic analysis of degradation demonstrated that the half-life of allethrin degradation by strain HLJ7 was 3.56 days, which was significantly shorter than the 55.89 days of the control. The Box–Behnken design of the response surface method optimized the degradation conditions for strain HLJ7: temperature 32.18 °C, pH value 7.52, and inoculation amount 1.31 × 107 CFU mL−1. Using Andrews equation, the optimal concentration of strain HLJ7 to metabolize allethrin was determined to be 21.15 mg L−1, and the maximum specific degradation rate (qmax), half-rate constant (Ks) and inhibition coefficient (Ki) were calculated to be 1.80 d−1, 1.85 mg L−1 and 68.13 mg L−1, respectively. Gas chromatography-mass spectrometry identified five intermediate metabolites, suggesting that allethrin could be degraded firstly by cleavage of its carboxylester bond, followed by degradation of the five-carbon ring and subsequent metabolism. The results of soil remediation experiments showed that strain HLJ7 has excellent bioremediation potential in the soils. After 15 days of treatment, about 70.8% of the initial allethrin (50 mg kg−1) was removed and converted into nontoxic intermediate metabolites, and its half-life was significantly reduced in the soils. Taken together, these findings shed light on the degradation mechanisms of allethrin and also highlight the promising potentials of B. megaterium HLJ7 in bioremediation of allethrin-comtaminated environment.

Transcriptomic Responses of Fusarium verticillioides to Lactam and Lactone Xenobiotics.

The important cereal crops of maize, rye, and wheat constitutively produce precursors to 2-benzoxazolinone, a phytochemical having antifungal effects towards many Fusarium species. However, Fusarium verticillioides can tolerate 2-benzoxazolinone by converting it into non-toxic metabolites through the synergism of two previously identified gene clusters, FDB1 and FDB2. Inspired by the induction of these two clusters upon exposure to 2-benzoxazolinone, RNA sequencing experiments were carried out by challenging F. verticillioides individually with 2-benzoxazolinone and three related chemical compounds, 2-oxindole, 2-coumaranone, and chlorzoxazone. These compounds all contain lactam and/or lactone moieties, and transcriptional analysis provided inferences regarding the degradation of such lactams and lactones. Besides induction of FDB1 and FDB2 gene clusters, four additional clusters were identified as induced by 2-benzoxazolinone exposure, including a cluster thought to be responsible for biosynthesis of pyridoxine (vitamin B6), a known antioxidant providing tolerance to reactive oxygen species. Three putative gene clusters were identified as induced by challenging F. verticillioides with 2-oxindole, two with 2-coumaranone, and two with chlorzoxazone. Interestingly, 2-benzoxazolinone and 2-oxindole each induced two specific gene clusters with similar composition of enzymatic functions. Exposure to 2-coumranone elicited the expression of the fusaric acid biosynthetic gene cluster. Another gene cluster that may encode enzymes responsible for degrading intermediate catabolic metabolites with carboxylic ester bonds was induced by 2-benzoxazolinone, 2-oxindole, and chlorzoxazone. Also, the induction of a dehalogenase encoding gene during chlorzoxazone exposure suggested its role in the removal of the chlorine atom. Together, this work identifies genes and putative gene clusters responsive to the 2-benzoxazolinone-like compounds with metabolic inferences. Potential targets for future functional analyses are discussed.

Carboxylesterase 1 (Ces1) Releases Oxylipins From Oxidized Triacylglycerols: Examination of Its Substrate Selectivity

Triacylglycerols (TAGs) are the most quantitatively important storage form of fat and are found primarily in cytoplasmic lipid droplets within cells. TAGs are broken down to its component free fatty acids by lipolytic enzymes when fuel reserves are required. However, when TAGs possess large quantities of polyunsaturated fatty acids (PUFAs) they are prone to nonenzymatic oxidation reactions, leading to formation of oxylipins (i.e., oxidized forms of fatty acids) that are esterified to the glycerol backbone (termed oxTAGs). Human carboxylesterase (CES1) is a member of the serine hydrolase superfamily and defined by its ability to catalyze the hydrolysis of carboxyl ester bonds in both toxicants and lipids. Although it is known that CES1 is a bona fideTAG hydrolase, it is unclear which specific fatty acids are preferentially released during lipolysis. Moreover, to better understand the biochemical function of CES1 in macrophages, we need to determine its substrate selectivity when it encounters oxidized PUFAs in TAG lipid droplets. Thus, the goal of this study is to systematically identify those oxidized fatty acids that are liberated from oxTAGs by CES1, because their release activates signaling pathways important for the development of lipid‐driven inflammation. Gaining this knowledge will fill data gaps that exist between CES1 and the lipid‐sensing nuclear receptors, PPARγ and LXRα, that are abundantly expressed in macrophages and are important drivers of lipid metabolism and inflammation. Oxidized forms of triarachidonoylglycerol (oxTAG C20:4) and trilinoleoylglycerol (oxTAG C18:2) – containing physiologically relevant levels of oxidized PUFAs (&lt;5 mol%) – were incubated with recombinant CES1, or Pseudomonaslipase (a positive control), to assess the release of oxylipins and non‐oxidized arachidonic acid (AA) and linoleic acid (LA), which were quantified by LC‐MS. CES1 was shown to efficiently metabolize oxTAG C20:4 and oxTAG C18:2, yielding several regioisomers of hydroxyeicosatetraenoic acid (5‐, 12‐, and 15‐HETE) and hydroxyoctadecadienoic acid (9‐ and 13‐HODE), respectively. The CES1‐catalyzed cumulative release of HODEs was faster than that of HETEs from the respective oxTAGs (7 pmol HODEs/min vs. 1 pmol HETEs/min). Thus, for oxTAGs, hydroxy fatty acids derived from LA are preferred by CES1 to those derived from AA. A similar trend was noted for the release of non‐oxidized PUFAs from the respective oxTAGs (470 pmol LA/min vs. 83 pmol AA/min). CES1 also preferentially liberated 5‐HETE over 12‐HETE and 15‐HETE from oxTAG C20:4, whereas no differences were noted between 9‐HODE and 13‐HODE. This study indicates that CES1 can metabolize oxTAG lipids to release oxylipins and PUFAs. It further specifies the substrate selectivity of CES1 in the metabolism of bioactive lipid mediators that can regulate inflammatory activities of immune cells.

Indigenous bacterial consortium-mediated cypermethrin degradation in the presence of organic amendments and Zea mays plants

Cypermethrin is a toxic pyrethroid insecticide that is widely used in agricultural and household activities. One of the most serious issues is its persistence in the environment, because it is easily transported to the soil and aquatic ecosystem. The biodegradation of cypermethrin is emerging as an environmentally friendly method for large-scale treatment. This study examined the application of a novel binary bacterial combination-based (Bacillus thuringiensis strain SG4 and Bacillus sp. strain SG2) approach used for the enhanced degradation of cypermethrin from the environment. The bacterial strains degraded cypermethrin (80% and 85%) in the presence of external nitrogen sources (KNO3 and NaNO3). Furthermore, when immobilized in agar disc beads, the co-culture degraded cypermethrin (91.3%) with a half-life (t1/2) of 4.3 days compared to 4.9 days using sodium alginate beads. Cereal straw, farmyard manure, press mud compost, fresh cow dung, and gypsum were used as organic amendments in the soil to stimulate cypermethrin degradation. Cereal straw promoted the fastest cypermethrin degradation among the different organic amendments tested, with a t1/2 of 4.4 days. The impact of cypermethrin-degrading bacterial consortium on cypermethrin rhizoremediation was also investigated. Bacterial inoculums exhibited beneficial effects on plant biomass. Moreover, Zea mays and the bacterial partnership substantially enhanced cypermethrin degradation in soil. Six intermediate metabolites were detected during the degradation of cypermethrin, indicating that cypermethrin could be degraded first by the hydrolysis of its carboxyl ester bond, followed by the cleavage of the diaryl linkage and subsequent metabolism. Our findings highlight the promising potential and advantages of the bacterial consortium for the bioremediation of a cypermethrin-contaminated environment.

A Temporal Evolution Perspective of Lipase Production by Yarrowia lipolytica in Solid-State Fermentation

Lipases are enzymes that, in aqueous or non-aqueous media, act on water-insoluble substrates, mainly catalyzing reactions on carboxyl ester bonds, such as hydrolysis, aminolysis, and (trans)esterification. Yarrowia lipolytica is a non-conventional yeast known for secreting lipases and other bioproducts; therefore, it is of great interest in various industrial fields. The production of lipases can be carried on solid-state fermentation (SSF) that utilizes solid substrates in the absence, or near absence, of free water and presents minimal problems with microbial contamination due to the low water contents in the medium. Moreover, SSF offers high volumetric productivity, targets concentrated compounds, high substrate concentration tolerance, and has less wastewater generation. In this sense, the present work provides a temporal evolution perspective regarding the main aspects of lipase production in SSF by Y. lipolytica, focusing on the most relevant aspects and presenting the potential of such an approach.

Self‐Polymerization of Monomer and Induced Interactions with Perovskite for Highly Performed and Stable Perovskite Solar Cells

AbstractWhile there is promising achievement in terms of the power conversion efficiency (PCE) of perovskite solar cells (PSCs), long‐term stability has been considered the main obstacle for their practical application. In this work, the authors demonstrate the small monomer 2‐(dimethylamino) ethyl methacrylate (DMAEMA) with unsaturated carboxylic acid ester bond in the antisolvent for perovskite formation to improve the PCE and stability. The results show that DMAEMA is self‐polymerized and uniformly distributed in the film, contributing to the improved crystallinity of the perovskites. Equally important, it is found that there are newly established interactions of Pb2+ and DMAEMA, and iodine and ternary amine between DMAEMA and perovskites, which improves the uniformity of the lead (II) iodide vertical distribution along with the films and thus phase stability, as well as largely decreases defects density in the film. Overall, the inverted PSCs with DMAEMA exhibit a open‐circuit voltage of 1.10 V, short‐circuit current of 23.86 mA cm−2, fill factor of 0.82, and finally PCE reaches 21.52%. Meanwhile, the PSC stability is significantly improved due to the inhibition of the formation of iodine, reduction of the uncoordinated Pb2+, and suppression of phase segregation.

Novel Mechanism and Kinetics of Tetramethrin Degradation Using an Indigenous Gordonia cholesterolivorans A16.

Tetramethrin is a pyrethroid insecticide that is commonly used worldwide. The toxicity of this insecticide into the living system is an important concern. In this study, a novel tetramethrin-degrading bacterial strain named A16 was isolated from the activated sludge and identified as Gordonia cholesterolivorans. Strain A16 exhibited superior tetramethrin degradation activity, and utilized tetramethrin as the sole carbon source for growth in a mineral salt medium (MSM). High-performance liquid chromatography (HPLC) analysis revealed that the A16 strain was able to completely degrade 25 mg·L−1 of tetramethrin after 9 days of incubation. Strain A16 effectively degraded tetramethrin at temperature 20–40 °C, pH 5–9, and initial tetramethrin 25–800 mg·L−1. The maximum specific degradation rate (qmax), half-saturation constant (Ks), and inhibition constant (Ki) were determined to be 0.4561 day−1, 7.3 mg·L−1, and 75.2 mg·L−1, respectively. The Box–Behnken design was used to optimize degradation conditions, and maximum degradation was observed at pH 8.5 and a temperature of 38 °C. Five intermediate metabolites were identified after analyzing the degradation products through gas chromatography–mass spectrometry (GC-MS), which suggested that tetramethrin could be degraded first by cleavage of its carboxylester bond, followed by degradation of the five-carbon ring and its subsequent metabolism. This is the first report of a metabolic pathway of tetramethrin in a microorganism. Furthermore, bioaugmentation of tetramethrin-contaminated soils (50 mg·kg−1) with strain A16 (1.0 × 107 cells g−1 of soil) significantly accelerated the degradation rate of tetramethrin, and 74.1% and 82.9% of tetramethrin was removed from sterile and non-sterile soils within 11 days, respectively. The strain A16 was also capable of efficiently degrading a broad spectrum of synthetic pyrethroids including D-cyphenothrin, chlorempenthrin, prallethrin, and allethrin, with a degradation efficiency of 68.3%, 60.7%, 91.6%, and 94.7%, respectively, after being cultured under the same conditions for 11 days. The results of the present study confirmed the bioremediation potential of strain A16 from a contaminated environment.

Thermoalkalophilic recombinant esterase entrapment in chitosan/calcium/alginate‐blended beads and its characterization

AbstractBACKGROUNDEsterases (EC 3.1.1.1), a class of hydrolases, degrade the ester bonds of lipids into alcohol and carboxylic acids and synthesize carboxylic ester bonds. They are used in a variety of biotechnological, industrial, environmental, and pharmaceutical applications due to their many valuable properties. Particularly, extremophilic esterases with many superior properties are of great interest for various reactions. Immobilization of enzymes may provide some advantages over free enzymes not only to improve the properties of enzymes but also to increase the reusability of biocatalyst in industrial applications. Therefore, many different immobilization applications for enzymes have been reported in various studies. To our knowledge, a thermophilic esterase has not so far been immobilized by entrapment using chitosan/calcium/alginate‐blended beads. Here, we reported the immobilization of thermoalkalophilic recombinant esterase by entrapment using chitosan/calcium/alginate‐blended beads, and then the entrapped esterase was characterized biochemically in details.RESULTSIn the present study, a thermophilic recombinant esterase was immobilized by entrapment in chitosan/calcium/alginate‐blended beads for the first time. The 0.5 mg mL−1 purified recombinant esterase was entrapped in 1% chitosan, 2% alginate, and 0.7 M CaCl2 blended beads. The results showed that immobilization yield and entrapment efficiency of the entrapped esterase were 69.5% and 80.4%, respectively. SEM micrograph showed that the surface of the beads resembled a mesh and very compact structures. Chitosan/calcium/alginate‐blended beads exhibited an 18.8% swelling ratio and had a moderate porous structure. The entrapment technique highly enhanced the thermostability of the esterase and shifted its optimum temperature from 65 to 80 °C. The immobilized esterase was very stable in a wide range of pH (8.5–11) displaying maximum activity at pH 9. ZnCl2 slightly increased the activity of immobilized esterase whereas several metal ions reduced the enzyme activity. When the enzyme was immobilized in chitosan/calcium/alginate‐blended beads, its Km increased about 2 times and Vmax value decreased almost 1.5 times. Immobilization allowed repeated uses of the esterase having good operational stability in a continuous process.CONCLUSIONThe results revealed that the immobilization of a thermophilic recombinant esterase by entrapment in chitosan/calcium/alginate‐blended beads exhibited considerably better compared to other immobilization processes with various entrapment strategies. © 2021 Society of Chemical Industry (SCI).

L-tryptophan-histidine synthesis by Pseudomonas serine peptidase, an amino acid ester hydrolase of the peptidase family S9

Pseudomonas sp. KM1 produces an amino acid ester hydrolase (KM1AEH) that catalyzes peptide bond formation by acting on carboxylic ester bonds. The KM1AEH gene was cloned from genomic DNA and expressed in Escherichia coli. The recombinant enzyme (rKM1AEH) was purified, and gel filtration showed that it is a 68 kDa monomeric protein. rKM1AEH can synthesize the vasoactive dipeptide tryptophan-histidine from tryptophan methyl ester and histidine as acyl donor and acceptor, respectively. The enzyme showed maximum activity at pH 9.5 and 45 °C and was specifically inhibited by silver (Ag+). Mutation of the catalytic Ser459 residue in the active site of rKM1AEH with Ala, Cys, or Thr eliminated all catalytic activity. The enzyme is a novel ester hydrolase that belongs to the peptidase family S9 based on the phylogenetic analysis.

Principles of lipid–enzyme interactions in the limbus region of the catalytic site of Candida antarctica Lipase B

Lipases (E.C. 3.1.1.3) are ubiquitous hydrolases for the carboxyl ester bond of water-insoluble substrates such as triacylglycerols and phospholipids. Candida antarctica Lipase B (CALB) acts in aqueous as well as in low-water media, thus being of considerable biochemical significance with high interest also for its industrial applications.The hydrolysis reaction follows a two-step mechanism, or ‘interfacial activation’, with adsorption of the enzyme to a heterogeneous interface and subsequent enhancement of the lipolytic activity. Once positioned within the catalytic triad, substrates are then hydrolysed, and products released. However, the intermediate steps of substrate transfer from the lipidic-aqueous phase to the enzyme surface and then down to the catalytic site are still unclear.By inhibiting CALB with ethyl phosphonate and incubating with glyceryl tributyrate (2,3-di(butanoyloxy)propyl butanoate), the crystal structure of the lipid-enzyme complex, at 1.55 Å resolution, shows the tributyrin in the limbus region of active site. The substrate is found 10 Å above the catalytic Ser, with the glycerol backbone pre-aligned for further processing by key interactions via an extended water network with α-helix10 and α-helix5.The findings offer new elements to elucidate the mechanism of substrate recognition, transfer and catalysis of Candida antarctica Lipase B (CALB) and lipases in general.

Crystal Structure of Poly(Aspartic Acid) Hydrolase‐1

Poly(aspartic acid) (PAA) is a biodegradable synthetic polymer that is easily produced through heating aspartic acid followed by the subsequent addition of sodium hydroxide. Polymer applications range from drug delivery and biomimetics to hygiene products. Partial biodegradation of PAA is accomplished using poly(aspartic acid) hydrolase‐1 (PAAH‐1) which selectively cleaves a specific cross‐link. E. coli expressions of recombinant PAAH‐1 showed the production of significant amounts of protein which could be purified using a single Ni‐NTA column. Analysis of the PAAH‐1 sequence using the Phyre2 Protein Fold Recognition Server resulted in a 33% similarity to a “putative” poly(3‐hydroxybutyrate) depolymerase belonging to a family of enzymes that hydrolyze carboxylic ester bonds; however, no known structure of PAAH‐1 has been reported. Protein crystallization screening at the Hauptman‐Woodward Research Institute resulted in numerous protein crystallization hits being identified. Crystallization experiments were optimized in‐house and the structure was determined at 2.45Å using molecular replacement. The progress of PAAH‐1 characterization and its structure will be discussed.Support or Funding InformationNSF‐IUSE Grant: DUE 1611988This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Microbial lipases and their applications – a review

This review focuses on the key aspects of lipases. Lipases (EC 3.1.1.3) are triacylglycerol acylhydrolases that act on carboxylic ester bonds. They breakdown triacylglycerides into glycerides (diglycerides or monoglycerides), fatty acids and glycerol. Their mass ranges from 19 kDa for B. stratosphericus to 92 kDa for P. gessardii. Their optimum temperature and pH ranges from 15 °C to 80 °C for Acinetobacter sp. and Janibacter sp. and 5 to 11 for P. gessardii and E. faecium respectively. Lipases chemo-, regio-, and enantio- specific features make them first choice of enzymes in research. Their kinetics for substrate hydrolysis depends on different esters. Mostly lipases are extracellular. Type 1 secretory system (T1SS) and Type 2 secretory system (T2SS) are involved in secreting lipases to external medium. They are found in eukaryotes and prokaryotes including animals, plants and microorganisms. Moreover, bacterial and fungal enzymes have diverse industrial applications in food, health, pharmaceutical, medical, textile, detergent, cosmetic and paper industries. Genetic engineering is employed to improve the properties of lipases. Their increasing demand in market has made them a hot topic in scientific research. Scientists are trying to discover novel lipase producing microorganisms due to their expanding commercial value. Keywords: Lipases, esterification, transesterification, biochemical and physicochemical properties, recombinant DNA technology

Compatibilization of highly sustainable polylactide/almond shell flour composites by reactive extrusion with maleinized linseed oil

Highly sustainable composites were produced by melt compounding polylactide (PLA) with almond shell flour (ASF), a processed by-product of the food industry, at a constant weight content of 30wt.-%. However, due to the lack of miscibility between PLA and ASF, both being raw materials obtained from crops, resultant green composite presented poor ductility and low thermal stability. To overcome this limitation, maleinized linseed oil (MLO), a multi-functionalized plant-derived additive, was originally incorporated as a reactive compatibilizer during the extrusion process. Both chemical and physical characterizations showed that 1–5 parts per hundred resin (phr) of MLO successfully serve to obtain PLA/ASF composites with improved mechanical, thermal, and thermomechanical properties. The enhancement achieved was particularly related to a dual compatibilizing effect of plasticization in combination with melt grafting. The latter process was specifically ascribed to the formation of new carboxylic ester bonds through the reaction of the multiple maleic anhydride functionalities present in MLO with the hydroxyl groups of both the PLA terminal chains and cellulose on the ASF surface. The fully bio-based and biodegradable composites described herein give an efficient sustainable solution to upgrade agro-food wastes as well as contributing to reducing the cost of PLA-based materials.

Isolation of a fluoroglycofen-degrading KS-1 strain and cloning of a novel esterase gene fluE.

The bacterium KS-1, capable of degrading fluoroglycofen, was isolated from sludge collected at a herbicide factory. The isolate was identified as Lysinibacillus sp. according to its phenotypic features and 16S rDNA phylogeny. KS-1 degraded 85.25% of the fluoroglycofen (50 mg L-1) within 3 days of incubation. The optimum temperature and pH for fluoroglycofen degradation were 30°C and 7.0, respectively. Furthermore, Zn2+ and Cu2+ could significantly decrease the degradation rate. Three degradation products, which appeared during KS-1-mediated fluoroglycofen metabolism, were identified as deethyl-fluoroglycofen, acifluorfen and decarboxylate-acifluorfen. The fluE gene, which encodes a novel esterase that catalyzes the cleavage of carboxyl ester bonds of fluoroglycofen, was cloned from the KS-1 strain. Sequence alignment reveals that FluE shares 30%-40% amino acid sequence identity with members of the hormone sensitive lipase family. FluE was expressed in Escherichia coli BL21 and purified by Ni-NTA affinity chromatography. Purified FluE could efficiently hydrolyze fluoroglycofen and short-chain p-nitrophenol esters. However, no lipolytic activity was observed with esters containing acyl chains longer than 10 carbon atoms, thereby indicating that this enzyme is an esterase.

Imaging and Detection of Carboxylesterase in Living Cells and Zebrafish Pretreated with Pesticides by a New Near-Infrared Fluorescence Off–On Probe

A new near-infrared fluorescence off-on probe was developed and applied to fluorescence imaging of carboxylesterase in living HepG-2 cells and zebrafish pretreated with pesticides (carbamate, organophosphorus, and pyrethroid). The probe was readily prepared by connecting (4-acetoxybenzyl)oxy as a quenching and recognizing moiety to a stable hemicyanine skeleton that can be formed via the decomposition of IR-780. The fluorescence off-on response of the probe to carboxylesterase is based on the enzyme-catalyzed spontaneous hydrolysis of the carboxylic ester bond, followed by a further fragmentation of the phenylmethyl unit and thereby the fluorophore release. Compared with the only existing near-infrared carboxylesterase probe, the proposed probe exhibits superior analytical performance, such as near-infrared fluorescence emission over 700 nm as well as high selectivity and sensitivity, with a detection limit of 4.5 × 10-3 U/mL. More importantly, the probe is cell membrane permeable, and its applicability has been successfully demonstrated for monitoring carboxylesterase activity in living HepG-2 cells and zebrafish pretreated with pesticides, revealing that pesticides can effectively inhibit the activity of carboxylesterase. The superior properties of the probe make it of great potential use in indicating pesticide exposure.