Biochemical Insights into Siderophore Esterases

Iron is an essential but excessively toxic nutrient. Although iron is rich in nature, the acquisition of iron is a challenge to life. Its solubility is very low because it is mostly in the form of oxidation or hydroxide. In order to overcome this, microorganisms have evolved a variety of iron absorption pathways, the most important of which is the siderophore-dependent iron absorption pathway. Both bacteria and fungi require specific siderophore esterases to encourage the release of iron within the cell. A deeper understanding of siderophore esterases is crucial for the development of new antibacterial and antifungal diagnostic and therapeutic approaches. There have been many recent studies on anti-infectives via siderophore antibiotic couplers in which siderophore esterases have also played an important role, and in this review, we provide an overview of several of the more common iron carriers as well as siderophore esterases in terms of structure as well as function.

Comparative Study of Malathion Toxicity and General Esterases in Larvae and Adults from a Field Population of Oxya chinensis (Thunberg) (Orthoptera: Acridoidea)

The susceptibility of Oxya chinensis to malathion was compared in larvae and adults from a field population, collected from Jinyuan outskirt, Shanxi Province. The results showed that Oxya chinensis was more susceptible to malathion in the adult stage than in the larval stage. The LD50 values for malathion susceptibility of Oxya chinensis were 4.94 and 2.44 mg g-1 body weight in the larvae and adults respectively. The results indicated that the larvae were 2.02-fold less susceptible to malathion than the adults. The general esterases and the kinetics were characterized and compared between the two life stages and between females and males. Larval preparations of Oxya chinensis were more active than adult preparations in females and males. The larvae showed 1.18-, 1.49-, and 1.17- fold higher specific activities than the adults in females with α-NA, α-NB and β-NA respectively. In males, the ratios were 1.34-, 1.70-, and 1.06-fold. Female preparations were more active than those of males in the adults. The reverse results were observed in the larvae where male preparations were more active than female preparations. Kinetic studies showed that Km values of general esterases hydrolyzing α-NA, α-NB, and β-NA in the adult stage were 1.36-, 1.32- and 1.39-fold respectively, higher than those in the larval stage in females. In males, the ratios were 1.24-, 2.14-, and 1.20-fold. The esterase from male insects had a higher affinity (lower Km value) to the substrate than those from females. The results also showed that the Vmax values of general esterase hydrolyzing α-NA, α-NB, and β-NA in the two stages were similar. From the results of bioassays and biochemical analyses, it has been inferred that a higher level of resistance to malathion in larvae than in adults would appear to result from differences in the expression of resistance mechanisms in these two life stages. Enhanced esterase activities appeared to play a major role in resistance to malathion in both larvae and adults. From the analysis of inhibition in vitro, the esterases in the two life stages were B-type, and carboxylesterases were predominant enzymes in the composition of the esterases in the two stages.

Isolation and Characterization of Two Novel Gene Encoded Alkaline Esterases from an Alkaline Soil Metagenomic Library

The development of industrial biotechnology has created an increasing demand for alkaline lipolytic enzymes with functional diversity. In this study, an alkaline soil metagenomic library was constructed to search for new lipolytic enzymes. Two novel gene encoded alkaline esterases（designated as estA and estB） were isolated by functional screening from the library. The estA gene consisted of 834 bp and coded for 277 amino acids with a molecular mass of 29998. Amino acid sequence homology analysis indicates that EstA belongs to α/β hydrolase fold family 4.4（abH4.4）, with EstA being the smallest member of that family yet reported. The estB gene consisted of 1185 bp and encoded 394 amino acids with a theoretical molecular mass of 40090. Its conserved domain analysis shows that EstB belongs to the GDSL hydrolase superfamily. Both EstA and EstB exhibit only moderate identity（〈38%） in amino acid sequence to the known lipolytic enzyme genes in the database. The two genes were respectively expressed in Escherichia coli and the protein products were purified for functional characterization. While the expressed EstA did not exhibit the functional properties that were superior to those of other esterases previously reported, the EstB was stable at temperature up to 45 ℃ and its maximum activity was measured to be 53.6 U/mg at pH=10. Both the en- zymes have further enriched the diversity of the lipolytic enzymes database and also appear to be promising biocatalysts for potential biotechnological application.

High-throughput Screening of the Enantioselectivity of Hyperthermophilic Mutant Esterases from Archaeon Aeropyrum pernix K1 for Resolution of (R,S)-2-Octanol Acetate

To identify the desired hypertherrnophilic variants within a mutant esterase library for the resolution of （R, S）-2- octanol acetate, a simple, reliable, and versatile method was developed in this study. We built a screening strategy including two steps, first we selected agar plate with substrate to screen the enzymatic activity; secondly we used a pH indicator to screen the enantioselectivity. This method could rapidly detect favorable mutants with high activity and enantioselectivity. A total of 96. 2% of tedious screening work can be precluded using this screening strategy. It is an effective screening for alkyl ester and can be applied to relative screening researches. The four improved mutants were screened from the mutant esterase library. Their enantioselectivities, activities, and structures were investigated at different temperatures.

Characterization of Esterases of Tamarindus indica Seeds

Germinating seeds of Tamarindus indica synthesizes various enzymes which are required for the degradation of seed reserves such as xyloglucans, fatty acid esters and proteins. Among these, esterases, belonging to a group of hydrolytic enzymes catalyze the hydrolysis of various types of esters. They play an important role in cell expansion as well as detoxification of xenobiotics and many agrochemicals and insecticides. The esterases are extracted from the germinating tamarind seeds using 50 mM phosphate buffer, pH 7. The Km with α-naphthyl acetate as the substrate is 19.23 μM and the enzymes are optimally active at pH 7.0 to 7.5 and are stable between pH 5.0 to 9.0. The optimum temperature of esterase activity of tamarind seed is between 37?C - 50?C and is stable up to 40?C. The activity declined by 30% at 60?C and about 90% at 70?C. Highest esterase activity and specific activity are observed on the 21st day of germination. The polyacrylamide gel electrophoresis (PAGE) indicated the presence of nine isozymes of esterases. Band numbers 1, 5 and 6 are the major esterolytic bands present throughout the germination period while band numbers 2 & 3 are minor bands present only during the latter period of the germination. Based on substrate and inhibitor specificity in conjunction with electrophoresis, the esterases 1 to 8 have been classified as carboxylesterases sensitive to organophosphate inhibitor (OP) and PCMB (p-chloromercuribenzoate) while esterase 9 is classified as carboxylesterase sensitive to OP. These esterases are unaffected by carbamate inhibitor, eserine sulphate.

Esterases from the Seeds of an Edible Legume, <i>Phaseolus vulgaris</i>L.;Variability and Stability during Germination

The total protein and esterase were isolated from the seeds of Red Kidney beans (Phaseolus vulgaris). The crude protein content was observed as 15%. The germination of seeds of red kidney bean has been carried out and change in the total protein content and esterase activity was monitored. The protein content was decreased from 15% (24 hours) to 8% (144 hours) during germination. The socked seeds and seedlings of all the days of germination exhibited esterase activity. Maximum ester hydrolyzing activity was observed on 6th day of germination whereas as lowest ester hydrolyzing activity was observed 2nd day germination. Native PAGE was carried out and esterase banding pattern for two different artificial substrates was studied. The esterase banding pattern in presence of 1-Napthyl acetate showed the presence of 4 esterolytic bands while 2 esterolytic bands were observed in presence of 2-Napthyl acetate.

Genome-wide identification, classification, and expression profiling of serine esterases and other esterase-related proteins in the tobacco hornworm, Manduca sexta

Serine esterases(SEs)are hydrolases that catalyze the conversion of carboxylic esters into acids and alcohols.Lipases and carboxylesterases constitute two major groups of SEs.Although over a hundred of insect genomes are known,systematic identification and classification of SEs are rarely performed,likely due to large size and complex composition of the gene family in each species.Considering their key roles in lipid metabolism and other physiological processes,we have categorized 144 M.sexta SEs and SE homologs(SEHs),114 of which contain a motif of GXSXG.Multiple sequence alignment and phylogenetic tree analysis have revealed 39 neutral lipases(NLs),3 neutral lipase homologs(NLHs),11 acidic lipases(ALs),3 acidic lipase homologs(ALHs),a lipase-3,a triglyceride lipase,a monoglyceride lipase,a hormone-sensitive lipase,and a GDSL lipase.Eighty-three carboxylesterase genes encode 29α-esterases(AEs),12 AEHs(e.g.,SEH4-1–3),20 feruloyl esterases(FEs),2 FEHs,2β-esterases(BEs),2 integument esterases(IEs),1 IEH,4 juvenile hormone esterases,2 acetylcholinesterases,gliotactin,6 neuroligins,neurotactin,and an uncharacteristic esterase homolog.In addition to these GXSXG proteins,we have identified 26 phospholipases and 13 thioesterases.Expression profiling of these genes in specific tissues and stages has provided insights into their functions including digestion,detoxification,hormone processing,neurotransmission,reproduction,and developmental regulation.In summary,we have established a framework of information on SEs and related proteins in M.sexta to stimulate their research in the model species and comparative investigations in agricultural pests or disease vectors.

Insight into Substrate Preference of Two Chimeric Esterases by Combining Experiment and Molecular Simulation

Better understanding of the relationship between the substrate preference and structural module of esterases is helpful to novel enzyme development. For this purpose, two chimeric esterases AAM7 and PAR, constructed via domain swapping between two ancient thermophilic esterases, were investigated on their molecular simulation（including homology modeling, substrates docking and substrate binding affinity validation） and enzymatic assay（specific activities and activation energies calculating）. Our results indicate that the factors contributing to the substrate preference of many enzymes especially the broad-specificity enzymes like esterases are multiple and complicated, the substrate binding domains or binding pockets are important but not the only factor for substrate preference.

Inhibitory effect of procyanidin B2 and tannin acid on cholesterol esterase and their synergistic effect with orlistat

This study was aimed to analyze the effect of procyanidin B2(PC)and tannin acid(TA)on the activities of cholesterol esterase(CEase)and the inhibitory mechanisms of enzymatic activity.The interaction mechanisms were investigated by enzymatic kinetics,multi-spectroscopy methods,thermodynamics analysis,molecular docking,and dynamic simulations.PC and TA could bind with CEase and inhibit the activity of enzyme in a mixed-competitive manner and non-competitive manner,which was verified by molecular docking simulations and dynamics simulations.Also,PC and TA showed the synergistic inhibition with orlistat.Fluorescence,UVvis and the thermodynamic analysis revealed that the complexes were formed from CEase and inhibitors by noncovalent interaction.As revealed by the circular dichroism results,both PC and TA decreased enzymatic activities by altering the conformations of CEase.The inhibition of PC and TA on CEase might be one mechanism for its cholesterol-lowering effect.

Emerging significance of butyrylcholinesterase

Butyrylcholinesterase(BChE;EC 3.1.1.8),an enzyme structurally related to acetylcholinesterase,is widely distributed in the human body.It plays a role in the detoxification of chemicals such as succinylcholine,a muscle relaxant used in anesthetic practice.BChE is well-known due to variant forms of the enzyme with little or no hydrolytic activity which exist in some endogamous communities and result in prolonged apnea following the administration of succinylcholine.Its other functions include the ability to hydrolyze acetylcholine,the cholinergic neurotransmitter in the brain,when its primary hydrolytic enzyme,acetylcholinesterase,is absent.To assess its potential roles,BChE was studied in relation to insulin resistance,type 2 diabetes mellitus,cognition,hepatic disorders,cardiovascular and cerebrovascular diseases,and inflammatory conditions.Individuals who lack the enzyme activity of BChE are otherwise healthy,until they are given drugs hydrolyzed by this enzyme.Therefore,BChE is a candidate for the study of loss-of-function mutations in humans.Studying individuals with variant forms of BChE can provide insights into whether they are protected against metabolic diseases.The potential utility of the enzyme as a biomarker for Alzheimer’s disease and the response to its drug treatment can also be assessed.

Characterization of a Novel Esterase Belonging to Family V from Marinobacter flavimaris

Lipolytic enzymes have attracted enormous attentions because of their ability in ester hydrolysis,ester synthesis,transesterification and other biochemical reactions.Bacteria are important sources of lipolytic enzymes applied in industry.Here,a novel lipolytic enzyme encoded by esterase gene est1347 was identified in Marinobacter flavimaris WLL162,and was purified and characterized.The lipolytic enzyme Est1347 consisted of 312 amino acid residues and a 21-amino-acids N-terminal signal peptide with a predicted molecular weight of 34.2 kDa.It belongs to family V of bacterial lipolytic enzymes based on the amino acid sequence homology analysis.Est1347 is a mesophilic and alkali-resistant enzyme with the highest activity at 45℃and pH 8.5;it is stable at temperatures below 50℃and pH 7.5–11.0.Est1347 showed a preference for middle-length chain substrate p-NPC10 and a wide range of other substrates.The Km,Vmax,Kcat and Kcat/Km values of Est1347 for p-NPC10 in pH 8.5 at 45℃were 0.9411 mmol L^(−1),1285μmol min^(−1)mg^(−1),698.91 s^(−1)and 743.65 s^(−1)(mmol L^(−1))^(−1),respectively.It is also tolerant to the metal ions,organic solvents and detergents.In conclusion,the esterase Est1347 laid a foundation for further study of bacterial lipolytic enzyme family V.

Isoenzymes for Four Coreid Species Identification (Hemiptera: Coreidae)

The vertical slab polyacrylamide gel electrophoretic technique is used to examine the esterase isozyme of four species (Hemiptera: Coreidae), the analysis has revealed that esterase zymogram can be divided into four zones. Esterase zymogram of intergeneric species show distinct difference, while some intrageneric common features are discovered. The clustering analysis shows that the taxonomic status of 4 species, and sex variance is bigger than individuals and is smaller than species.

Diagnosis of spontaneous bacterial peritonitis: An update on leucocyte esterase reagent strips

Ascites remain the commonest complication of decompensated cirrhosis. Spontaneous bacterial peritonitis (SBP) is defined as the infection of ascitic fluid (AF) in the absence of a contiguous source of infection and/or an intraabdominal inflammatory focus. An AF polymorphonuclear (PMN) leucocyte count ≥ 250/mm 3 -irrespective of the AF culture resultis universally accepted nowadays as the best surrogate marker for diagnosing SBP. Frequently the results of the manual or automated PMN count do not reach the hands of the responsible medical personnel in a timely manner. However, this is a crucial step in SBP management. Since 2000, 26 studies (most of them published as full papers) have checked the validity of using leukocyte esterase reagent strips (LERS) in SBP diagnosis. LERS appear to have low sensitivity for SBP, some LERS types more than others. On the other hand, though, LERS have consistently given a high negative predictive value (> 95% in the majority of the studies) and this supports the use of LERS as a preliminary screening tool for SBP diagnosis. Finally, an AF-tailored dipstick has been developed. Within the proper setting, it is set to become the mainstream process for handling AF samples.

Mating Genotype Analysis of Ganoderma lucidum Populations

[Objective] The mating genotype was studied and compared with esterase isozyme of Ganoderma lucidum populations between groups in order to clarify their differences in genetic relationship analysis. [Method] OWE-SOJ technique was applied to identify standard mating types and determinate mating genotype between groups of monokaryons isolates from 24 G. lucidum stains. Genetic relationships were analyzed by combined group mating genotype determination with esterase isozyme assay. [Result] All strains of G. lucidum could be divided into 7 large groups of the mating genotype. Four alleles of A factor, four alleles of B factor and one mixed alleles of A factor were found in this study. Distorted segregation ratio among monokaryon mycelia of G. lucidum had been observed in four kinds of mat- ing types to some extent. Twenty-eight different types of enzyme bands were determined in esterase isozyme test, Twenty-four strains of G. lucidum could be divided into 9 large groups through the cluster analysis when the genetic similarity coefficient was 0.73214. Comparing the results of mating genotype analysis and esterase isozyme analysis, it showed great similarity. [Conclusion] Mating genotype analysis could be used as an important supplementary method for strain identification and genetic diversity research.

Carboxylic Esterase and Its Associations With Long-term Effects of Organophosphorus Pesticides

To examine a） the effect of organophosphorus pesticide exposure on activity of carboxylic esterases, namely butyrylcholinesterase （BChE）, carboxylesterase （CarbE） and paraoxonase （PonE）; and b） the association of polymorphisms of BChE and PonE with individual genetic susceptibility to organophosphorus pesticide exposure. Methods A cross-sectional study was conducted in 75 workers exposed to organophosphorus pesticides and 100 non-exposed controls. The serum activity of these enzymes was measured. Variant forms of BCHE-K, PON-192, and PON-55 were detected. A symptom score was developed as a proxy measure of clinical outcomes. Results Activities of both BChE and CarbE were lower in exposed workers （27.3±21.65 runol.hl.mL^-l and 235.6±104.03 nmol-min^-l.mL^-l） than in non-exposed workers （78.313±30.354 nmol.h^-l.mL^-1 and 362.681_＋194.997 nmol.min^-1.mL^-1）. The activity of PonE was not associated with exposure status. The AChE activity in the exposed workers with BCHE-K genotype UU （61 cases）, genotype UK （12 cases） and genotype KK （2 cases） was 105.05, 84.42 and 79.00 mmol-h^-1.mL^-1, respectively and the accumulative symptom scores were 3.74, 9.17, and 12.50 accordingly. The AChE activity in the exposed workers with PON-192 genotype BB （37）, genotype AB （27） and genotype AA （11） was 116.8, 91.2, and 72,3 mmol-h^-1.mL^-1, respectively and the symptom scores were 2.00, 6.74, and 9.73 accordingly. The AChE activity in those with PON-55 genotype LL （70） and genotype LM （5） was 102.4 and 82.8 mmol-h^-1.mL^-1 and the symptom scores were 4.53 and 9.20. The symptom score was the highest in individuals with abnormal homozygote for each of the three gene loci. Condusions Long-term exposure to organophosphorus pesticides can inhibit BChE and CarbE activity, but exerts no inhibitory effect on PonE activity. Different genotypes of BCHE-K, PON-192, and PON-55 may be related to the severity of adverse health effects of organophosphorus pesticide exposure. Implications of potentially higher susceptibility of workers with mutant homozygotes should be evaluated to reduce health risks.

Genome-wide identification and co-expression analysis of GDSL genes related to suberin formation during fruit russeting in pear

Glycine-aspartic acid–serine-leucine(GDSL)type lipases/esterases genes play critical roles in plant development and are related to the responses to abiotic and biotic stress.However,little is known about the GDSL family in pear(Pyrus spp.).Studies have shown GDSL-domain proteins play key roles in suberin deposition.Suberin deposition in the fruit epidermis,also called russeting,is an important defect that negatively affects consumer's appeal in some fruit species,such as pear,apple and grapevine.Fruit russeting is mainly associated with cuticle microcracking and suberin accumulation in the inner part of the epidermal cell walls.To gain insight into the role of the GDSL gene family in suberin deposition and russet development in pear,we performed a genome-wide characterization of the GDSL family,including their identification,chromosomal localization,phylogenetic relationships,and expression patterns,in different tissues/organs in pear.One hundred and thirteen GDSL-type lipases/esterases genes were identified in the pear genome,and a phylogenetic analysis revealed that GDSL family can be classified into four distinct groups.Thirty GDSL genes were co-expressed with five homolog pear genes of three well-known suberin biosynthesis Arabidopsis genes(AtGPAT5,AtASFT,and AtCYP86B1)in the transcriptional co-expression network during pear fruit development.Among the 30 co-expressed GDSL genes,twelve genes were further analyzed by quantitative Real-time PCR,and the results showed the expression levels of the 12 genes were different between the russet exocarp and green exocarp of sand pear at different fruit development stages.Our study provides a detailed overview of the GDSL gene family and lays the foundation for future functional characterization of GDSL genes in P.bretschneideri.

A Photosensitivity Insecticide, 5-Aminolevulinic Acid, Exerts EffectiveToxicity to Oxya chinensis (Orthoptera: Acridoidea)

5-Aminolevulinic acid （ALA）, a major photosensitivity insecticide, has attracted increasing attention as a new type of highly efficient, environmental friendly pesticide to be used to control the pest. To examine whether or not ALA acts effectively to grasshopper, Oxya chinensis and elucidate the detoxification mechanism of ALA, the susceptibility to ALA was assessed in O. chinensis and two major metabolic detoxification enzymes including glutathione S-transferases （GSTs） and general esterases （ESTs）-specific activities were compared in different development stages and different body sections of O. chinensis treated by ALA and the control. The results showed that the ALA exhibited obvious toxicity to the grasshopper in different development stages. In the low-dose treatment （0.0597 mmol L-1）, the mortalities of O. chinensis reached a significant level （55.5% in the 1st instar nymphs, 61.4% in the 2nd instar nymphs, 71.4% in the 3rd instar nymphs, and 64.4% in the 4th instar nymphs. But, there was no dose-dependent toxic effect. Thereby, we proposed that ALA has the potential for acting as photosensitivity insecticide for controlling O. chinensis. GSTs activity assays using CDNB and DCNB as substrates indicated that the thorax and abdomen of the different instar nymphs treated by ALA showed 1.52-5.56 fold significantly increased GSTs activities compared with the control. However, for the ESTs-specific activity assay, there was no significant difference between O. chinensis treated by ALA and the control within different instar nymphs, when a-NA, a-NB and b-NA were used as substrates. Therefore, GSTs-mediated metabolic detoxification as evidenced by significantly increased GSTs activities might contribute to protect against oxidative damage and oxidative stress by ALA in O. chinensis.

Morphological changes of cholinergic nerve fibers in the urinary bladder after establishment of artificial somatic-autonomic reflex arc in rats

Objective To establish an artificial somatic-autonomic reflex arc in rats and observe the following distributive changes of neural fibers in the bladder. Methods Adult Sprague-Dawley rats were randomly divided into three groups： control group, spinal cord injury （SCI） group, and reinnervation group. DiI retrograde tracing was used to verify establishment of the model and to investigate the transport function of the regenerated efferent axons in the new reflex arc. Choline acetyltransferase （CHAT） in the DiI-labeled neurons was detected by immunohistochemistry. Distribution of neural fibers in the bladder was observed by acetylcholine esterase staining. Results DR-labeled neurons distributed mainly in the left ventral horn from L3 to L5, and some of them were also CHAT-positive. The neural fibers in the bladder detrusor reduced remarkably in the SCI group compared with the control （P 〈 0.05）. After establishment of the somatic-autonomic reflex arc in the reinnervation group, the number of ipsilateral fibers in the bladder increased markedly compared with the SCI group （P 〈 0.05）, though still much less than that in the control （P 〈 0.05）. Conclusion The efferent branches of the somatic nerves may grow and replace the parasympathetic preganglionic axons through axonal regeneration. Acetylcholine is still the major neurotransmitter of the new reflex arc. The controllability of detrusor may be promoted when it is reinnervated by the pelvic ganglia efferent somatic motor fibers from the postganglionic axons.

Study on Characters of Major Hydrolase Isozymes in Wheat Seeds

ObjectiveThis study aimed to investigate the characters of major hydrolase isozymes in wheat seeds. MethodThe spectra of amylase, esterase and proteolytic enzyme isozymes in the germ, endosperm and radicle were studied in detail during the germination process using the polyacrylamide gel electrophoresis（PAGE）. ResultThe results indicated that isozymes of amylase and esterase changed obviously in the endosperm during the germination, while isozymes of proteolytic enzyme exhibited distinct variation in the germ and radicle. ConclusionThis study provides basic data for effeciently improving the germination rate of crop seeds in the future.

Serum Esterase Polymorphisms and Their Associations with Prolificacy in Small Tail Han Sheep

[ Objective] The aim of this research was to provide a theoretical basis for the.breeding and identification of multiparous Small Tail Han sheep. [ Method] The polymorphisms of serum esterase （Es） locus in Small Tail Han sheep were detected by vertical discontinuous polyacrylamide gel electrophoresis （PAGE）. According to the statistical analysis of different genotypes and their litter size, the correlation between Es polymor- phisms and litter size was analyzed. [ Result] There were three genotypes in Es locus, and Es^＋- was the dominate genotype. The differences of ewe litter size in the 1 ,t parity among these three genotypes were nonsignificant （ P 〉 0.05）, As far as the 2^nd and 3rd parity was considered, the litter size of genotype Es＋＋ was significantly （P〈0.05） higher than that of Es-- and Es^＋- ; the average litter size of Es＋＋ was extremely significant （ P 〈 0.01 ） higher than that of Es - - and Es ＋ - while there was no significant difference （ P 〉 0.05） between the latter two. [ Conclusion] The Es lo- cus could be regarded as a genetic marker locus for early selection of high-yielding ewes and Es ＋＋ is the high-yielding genotype of Small Tail Han sheep.