Characterization of four esterase genes and esterase activity from the gut of the termite Reticulitermes flavipes

Four esterase genes and general esterase activity were investigated in the gut of the termite Reticulitermes flavipes. Two genes (RfEst1 and RfEst2) share significant translated identity with a number of insect JH esterases. The two remaining genes (RfEst3 and RfEst4) apparently code for much shorter proteins with similarity to fungal phenolic acid esterases involved in hemicellulose solubilization. All four genes showed consistently high midgut expression. This result was further supported by colorimetric activity assays and Native polyacrylamide gel electrophoresis, which showed significant esterase activity and a number of isoforms in the midgut. The greatest esterase activity and isoform composition were detected when α‐naphthyl propionate was used as a substrate. Moreover, esterase activity and diverse isoforms were present in gut mitochondrial, microsomal, and cytosolic sub‐cellular protein fractions, as well as in the hindgut lumen. These findings reveal an agreement between gut esterase gene expression and activity distributions, and support the idea that R. flavipes gut esterase activity is host (not symbiont)‐derived. In addition, these findings support the hypotheses that termite gut esterases may play important roles in lignocellulose digestion and caste differentiation. This study provides important baseline data that will assist ongoing functional‐genomic efforts to identify novel genes with roles in semiochemical, hormone, and lignocellulose processing in the termite gut. © 2009 Wiley Periodicals, Inc.     

鸭血清胆碱酯酶的纯化及性质研究

首次采用新技术双水相萃取方法作为鸭血清胆碱酯酶(EC.3.1.1.8 CHE) 纯化的第一步,后经 DEAE-Sephadex A50,sephadex G200 柱层析,获得电泳纯鸭血清胆碱酯酶,提纯倍数1018倍,酶活力回收43.4%,比活274.9U/mg。鸭血清胆碱酯酶性质研究表明:此酶是糖蛋白和酸性蛋白水解酶,等电点 4.2 左右,最适 pH7.5 左右;对底物碘化硫代丁酰胆碱的 K_m=9.8×10^-5mol/L;SDS-PAGE 电泳和聚丙烯酰胺梯度电泳表明,鸭血清胆碱酯酶以相同亚基组成的不同聚合体形式存在,亚基分子量 78000,具有完整的酶活性.不同聚合体带电状态相同.     

Changes in the Activity and Pattern of Hemolymph Esterases in the Larvae of Greater Wax Moth Galleria mellonellaL. (Lepidoptera, Pyralidae) during Mycosis

The extended pattern of multiple esterase forms has been revealed in the hemolymph of wax moth Galleria mellonellalarvae infected by the fungi Metarhizium anisopliae, Beauveria bassianaor Paecilomyces fumoso-roseus. The total esterase activity of the hemolymph also increases during mycosis. Mechanical damage of the cuticle, treatment with deltamethrin, and chilling of the caterpillars induced similar changes in the hemolymph pattern of esterase activity. Presumably, the changed spectrum and activity of the hemolymph esterases during mycosis is due to the damaged cuticle and epidermis cells.     

A novel geminal diol as a highly specific and stable in vivo inhibitor of insect juvenile hormone esterase

Thio-containing and acetylenic trifluoromethyl ketones were potent inhibitors of insect juvenile hormone (JH) esterase with greater inhibitory activity than aliphatic and α,β-unsaturated homologs. Octylthio-1,1,1-trifluoropropan-2-one was the most potent inhibitor with the greatest equilibrium hydration constant in pure water. However, a keto/hydrate equilibrium was not necessary for JH esterase inhibition. The carbonyl tautomer of 1-octyl [1-(3,3,3-trifluoropropan-2,2- dihydroxy)] sulfone (OTPdOH-sulfone) was not detectable, and yet OTPdOH-sulfone was a potent in vitro inhibitor of JH esterase with an I₅₀ of 1.2 nM. The mechanism of JH esterase inhibition by these compounds is discussed. OTPdOH-sulfone inhibited JH esterase with minimal activity toward insect 1-naphthyl acetate esterase and electric eel acetylcholinesterase. The inhibitor was also active in vivo, selective for JH esterase, and persistent for over 32 h. OTPdOH-sulfone when topically applied to larval and adult cabbage loopers, Trichoplusia ni, elicited juvenoid activity apparently because of the specific in vivo inhibition of JH metabolism. Arch. Insect Biochem. Physiol. 36:165–179, 1997. © 1997 Wiley-Liss, Inc.     

Isolation of the Volatile Components of Fresh Onion by Thermal Desorption Cold Trap Capillary Gas Chromatography

A new esterase activity from Bacillus licheniformis was characterized from an Escherichia coli recombinant strain. The protein was a single polypeptide chain with a molecular mass of 81 kDa. The optimum pH for esterase activity was 8-8.5 and it was stable in the range 7-8.5. The optimum temperature for activity was 45°C and the half-life was 1 h at 64°C. Maximum activity was observed on p-nitrophenyl caproate with little activity toward long-chainfatty acid esters. The enzyme had a K_M of 0.52 mM for p-nitrophenyl caproate hydrolysis at pH 8 and 37°C. The enzyme activity was not affected by either metal ions or sulfydryl reagents. Surprisingly, the enzyme was only slightly inhibited by PMSF. These characteristics classified the new enzyme as a thermostable esterase that shared similarities with lipases. The esterase might be useful for biotechnological applications such as ester synthesis.     

General esterases of Bemisia tabaci (Hemiptera: Aleyrodidae) change in response to feed on cotton varieties

The esterase enzymes are a major component of insect detoxification systems and play a crucial role in hydrolyzing lots of xenobiotic compounds. Among insect, generalist herbivores can exhibit developed biochemical defences as a result of exposing to a wide range of plant chemical compounds. To overcome this ability, host plants may affect the level of hydrolases in herbivore insects feeding on. To examine this hypothesis, in the present study total esterase activity was investigated in a highly polyphagous whitefly, Bemisia tabaci, reared on six different varieties of cotton, Gossypium hirsutum. Results showed significant differences in esterase activity of B. tabaci feeding on the host plant varieties. The highest esterase activities were detected in whiteflies feeding on Sk-Tb and Siokra varieties, whereas those whiteflies that feed on Hopicala variety exhibited the least esterase activities. Our findings highlight the important role of host plants in detoxification ability of herbivore insects. The importance of these findings in biology of insect pests and their applications in integrated pest management programmes of B. tabaci have been discussed in detail.     

Effect of culture conditions on growth and esterase production by the moderate thermophile Bacillus circulans MAS2

Growth and esterase production (activity on p-nitrophenyl caprylate) by the newly isolated Bacillus circulans MAS2 bacterial strain were studied. The growth rate at 50°C was high (0.9 h^-1) on LB medium with glucose added. Esterase production followed growth with the majority of activity being intracellular during exponential growth phase. During stationary phase, the esterase activity was released in the culture medium. The strain was able to grow at 35– 55°C with maximum growth rate at 50°C, showing a pattern typical of a moderate thermophile. Growth occurred at pH 6–9 with a maximum at 8, with a similar pattern for the esterase production. Addition of glucose, fructose, sucrose or sodium acetate greatly promoted both growth and esterase production while starch, inulin, tributyrin or glycerol showed no effect. Complex nitrogen sources such as tryptone or yeast extract increased growth and esterase production while mineral sources (ammonium chloride or sulfate), glycine or glutamate showed no effect. An increase of tryptone plus yeast extract and glucose concentrations stimulated growth and esterase production which reached 160 U L⁻¹. Received 17 March 1999/ Accepted in revised form 25 June 1999     

Kinetic characterization and fed-batch fermentation for maximal simultaneous production of esterase and protease from Lysinibacillus fusiformis AU01

The simultaneous production of intracellular esterase and extracellular protease from the strain Lysinibacillus fusiformis AU01 was studied in detail. The production was performed both under batch and fed-batch modes. The maximum yield of intracellular esterase and protease was obtained under full oxygen saturation at the beginning of the fermentation. The data were fitted to the Luedeking–Piret model and it was shown that the enzyme (both esterase and protease) production was growth associated. A decrease in intracellular esterase and increase in the extracellular esterase were observed during late stationary phase. The appearance of intracellular proteins in extracellular media and decrease in viable cell count and biomass during late stationary phase confirmed that the presence of extracellular esterase is due to cell lysis. Even though the fed-batch fermentation with different feeding strategies showed improved productivity, feeding yeast extract under DO-stat fermentation conditions showed highest intracellular esterase and protease production. Under DO-stat fed-batch cultivation, maximum intracellular esterase activity of 820?×?10³ U/L and extracellular protease activity of 172?×?10³ U/L were obtained at the 16th?hr. Intracellular esterase and extracellular protease production were increased fivefold and fourfold, respectively, when compared to batch fermentation performed under shake flask conditions.     

Distribution of acetyl esterase in wood-rotting fungi

The distribution of acetyl esterase was studied in 30 strains of wood-rotting fungi. A screening test on agar plates using glucose β-d-pentaacetate as a substrate indicated that all tested fungi produced acetyl esterase to form a clear zone on the culture. All fungi also showed positive responses in an agar test using carboxymethyl cellulose acetate. Enzyme assay showed that extracellular acetylxylan esterase activity was present in the filtrates of wood-meal culture of all these fungi. The ratio of fungal acetylxylan esterase activity to 4-nitrophenyl acetyl esterase activity were higher than that of porcine liver esterase, indicating that fungal esterases have high affinity for acetylated carbohydrates. Acetyl esterase is suggested to be distributed widely in wood-rotting fungi for degradation of native acetylated hemicelluloses.     

Partial purification and characterization of thermostable esterase from the hyperthermophilic archaeonSulfolobus solfataricus

A thermostable esterase from the hyperthemophilic archaeonSulfolobus solfataricus was partially purified 590-fold with 16.2% recovery. The partially purified esterase had a specific activity of 29.5μmol min⁻¹ mg⁻¹ when the enzyme activity was determined usingp-nitrophenyl butyrate as a substrate. The apparent molecular weight was about 100 kDa, while the optimum temperature and pH for esterase were 75°C and 8.0, respectively. The enzyme showed high thermal stability and solvent tolerance in comparison to its mesophilic counterpart. The enzyme also showed chiral resolution activity for (S)-ibuprofen, indicating thatS. solfataricus esterase can be used for the production of commercially important chiral drugs.     

Role of water activity on the synthesis of geranyl butyrate by a Mucor miehei esterase in a solvent-free system

The initial activity rate for the synthesis of geranyl butyrate by an esterase from Mucor miehei increased with increasing water activity and decreased above 0.5 water activity. The conversion yield after 75 h was about 75% for different, but low, initial water activities and decreased above 0.5 water activity. This behaviour is related to water-enzyme interactions.     

Characterization of the esterase isozymes of Ips typographus (coleoptera,scolytidae)

Four esterase isozymes hydrolyzing α-naphthyl acetate (α-NA) were detected screening whole body homogenates of larvae and adults of Ips typographus by electrophoresis. Two of the four isozymes (isozymes 3 and 4) were not detected by α-NA staining in the pupal stage, but topical application of juvenile hormone III (JH III) on the pupa induced these isozymes. The JH esterase (JHE) activity on the gel was associated with the proteins of isozyme 2. The compounds OTFP, PTFP, and DFP inhibited this catalytic activity of isozyme 2 on the gel at low concentrations, whereas the proteins of isozyme 3 and 4 were affected only at higher concentrations. A quantitative developmental study was performed to characterize which of the esterases hydrolyzed JH III, using a putative surrogate substrate for JH (HEXTAT) and α-NA. The I₅₀ of several esterase inhibitors and the JH metabolites were also defined. All findings supported the results that a protein associated with isozyme 2 is catabolizing JH and that isozymes 3 and 4 are the main contributors to the general esterase activity on α-NA. The JHE from Tenebrio molitor was purified by affinity chromatography. Although the recovery was low, an analytical isoelectric focusing gel showed that the JHE activity of the purified enzyme. T. molitor cochromatographed at the same pl as the JHE activity of I. typographus. Arch. Insect Biochem. Physiol. 34:203–221, 1997. © 1997 Wiley-Liss, Inc.     

Production of recombinant extracellular cholesterol esterase using consistently active promoters in Burkholderia stabilis

ABSTRACT

Burkholderia stabilis FERMP-21014 produces highly active cholesterol esterase in the presence of fatty acids. To develop an overexpression system for cholesterol esterase production, we carried out RNA sequencing analyses to screen strongly active promoters in FERMP-21014. Based on gene expression consistency analysis, we selected nine genes that were consistently expressed at high levels, following which we constructed expression vectors using their promoter sequences and achieved overproduction of extracellular cholesterol esterase under fatty acid-free conditions. Of the tested promoters, the promoter of BSFP_0720, which encodes the alkyl hydroperoxide reductase subunit AhpC, resulted in the highest cholesterol esterase activity (24.3 U mL^?1). This activity level was 243-fold higher than that of the wild-type strain under fatty acid-free conditions. We confirmed that cholesterol esterase was secreted without excessive accumulation within the cells. The gene expression consistency analysis will be useful to screen promoters applicable to the overexpression of other industrially important enzymes.     

Determination of metabolic resistance mechanisms in pyrethroid‐resistant and fipronil‐tolerant brown dog ticks

Rhipicephalus sanguineus (Latreille) (Ixodida: Ixodidae) is a three‐host dog tick found worldwide that is able to complete its' entire lifecycle indoors. Options for the management of R. sanguineus are limited and its' control relies largely on only a few acaricidal active ingredients. Previous studies have confirmed permethrin resistance and fipronil tolerance in R. sanguineus populations, commonly conferred by metabolic detoxification or target site mutations. Herein, five strains of permethrin‐resistant and three strains of fipronil‐tolerant ticks were evaluated for metabolic resistance using synergists to block metabolic enzymes. Synergist studies were completed with triphenyl phosphate (TPP) for esterase inhibition, piperonyl butoxide (PBO) for cytochrome P450 inhibition, and diethyl maleate (DEM) for glutathione‐S‐transferase inhibition. Additionally, increased esterase activity was confirmed using gel electrophoresis. The most important metabolic detoxification mechanism in permethrin‐resistant ticks was increased esterase activity, followed by increased cytochrome P450 activity. The inhibition of metabolic enzymes did not have a marked impact on fipronil‐tolerant tick strains.     

Activity of glutathione S‐transferase and esterase enzymes in Helicoverpa armigera (Hübner) after exposure to entomopathogenic fungi

Helicoverpa armigera, a polyphagous insect of crops and vegetables, is acquiring resistance against many commercial insecticides. The present study shows variations in the activity of two detoxification enzymes, namely esterase and glutathione S‐transferase (GST), in H. armigera after exposure to different isolates of entomopathogenic fungi. After treatment of larvae with the different isolates (Day 0), samples were collected on three days (Days 3, 5 and 7) for enzyme analysis. High GST activity was found in samples of hemolymph, intestine and fat bodies of H. armigera following treatment with Beauveria bassiana (isolate Bb‐08), Metarhizium anisopliae (isolates Ma‐11.1 and Ma‐4.1), and Isaria fumosorosea (isolates If‐02 and If‐2.3). High esterase activity was recorded in samples of the intestine and fat bodies on various days after treatment, whereas increased esterase activity in hemolymph was noted only in samples from Day 5 after treatment with M. anisopliae (Ma‐4.1). The detection of high GST and esterase activity demonstrates the possibility of the development of resistance against these microbial control agents in H. armigera.     

Seasonal differences in methyl farnesoate esterase activity in tissues of the spider crab Libinia emarginata

Summary

Methyl farnesoate (MF), an unepoxidated form of insect JH III, is present in Crustacea. MF is synthesized by the mandibular organs and is degraded to fomesoic acid (FA) by peripheral tissues. In this study we investigated MF degradation by esterases in hepatopancreas, ovary, testes and hemolymph of the spider crab Libinia emarginata collected at different times of the year to determine seasonal differences. The conversion of MF to FA varied among the tissues. In the summer, the hepatopancreas showed the greatest esterase activity (52.8% conversion in females and 59.16% in males), and it was twice as high (28.86%) in ovaries than in the testes (12.16%), but was low in the hemolymph of both sexes (10.84% in males, and 6.97% in females). In the fall, the conversion of MF to FA was significantly reduced in all tissues (ovary 8.55%, testes 6.21%, hepatopancreas 10.22%, hemolymph 3.96%). Eyestalk ablation of animals in the fall restored MF esterase activity to summer levels. When tissues from these animals were incubated with OTFP, a specific inhibitor of JH esterase, MF metabolism was significantly reduced. These results suggest that MF esterase activity depends on direct induction by MF, and its degradation is by a specific esterase(s).     

Enantioselectivity suggests a cytosolic origin for a commercial pig liver esterase preparation

A widely utilized pig liver esterase preparation has been found to be derived essentially exclusively from the cytosolic fraction of pig livers. Esterases in cytosol and microsomes prepared from a fresh pig liver hydrolyzed the S- and R-enantiomers of racemic oxazepam 3-acetate (rac-OXA) with specific activity ratios of approximately 2.3:1 and 1:62, respectively. Product formations were analyzed by chiral stationary phase high-performance liquid chromatography. The commercial pig liver esterase preparation showed greater activity toward S-OXA than did the esterases in the cytosolic fraction prepared from fresh pig liver. The results established that (i) esterases contained in microsomes and cytosol of pig liver have opposite enantioselectivity in the hydrolysis of rac-OXA and (ii) the commercial pig liver esterase preparation has a cytosolic origin. © 1995 Wiley-Liss, Inc.     

Thermostable esterase from Thermoanaerobacter tengcongensis: high-level expression, purification and characterization

The lipA gene encoding a thermostable esterase was cloned from Thermoanaerobacter tengcongensis and overexpressed in Escherichia coli. The recombinant esterase, with a molecular mass of approx. 43 kDa determined by SDS-PAGE, was purified to homogeneity through Sephadex G-100 gel filtration. The purified enzyme actively hydrolyzed tributyrin but not olive oil. Maximum activity was observed on p-nitrophenyl (NP)-propionate (C3) and p-NP-butyrate (C4), with little activity towards p-NP-palmitate (C16). The esterase was optimally active at 70 °C (over 15 min) and at pH 9. It is highly thermostable, with a residual activity greater than 80% after incubation at 50 °C for more than 10 h. The activity was not inhibited by 5 mM EDTA and PMSF, indicating the esterase is not a metalloenzyme and may contain a specific structure around the catalytic serine residue. In addition, it was stable for 1 h at 37 °C in 1% CHAPS and Triton X-100 but not stable in 1% Tween 20 or SDS.     

Screening ofExiguobacterium acetylicum from soil samples showing enantioselective and alkalotolerant esterase activity

About 3,000 bacterial colonies with esterase activities were isolated from soil samples by enrichment culture and halo-size on Luria broth-tributyrin (LT) plates. The colonies were assayed for esterase activity in microtiter plates using enantiomerically pure (R)- and (S)-2-phenylbutyric acid resorufin ester (2PB-O-res) as substrates. Two enantioselective strains (JH2 and JH13) were selected by the ratio of initial rate of hydrolysis of enantiomerically pure (R)- and (S)-2-PB-O-res. When cell pellets were used, both strains showed hgh apparent enantioselectivity (E _app>100) for (R)-2PB-O-res and were identified asExiguobacterium acetylicum. The JH13 strain showed high esterase activity onp-nitrophenyl acetate (pNPA), but showed low lipase activity onp-nitrophenyl palmitate (pNPP). The esterase was located in the soluble fraction of the cell extract. The crude intracellular enzyme preparation was stable at a pH range from 6.0 to 11.0.     

不同种群棉铃虫三龄幼虫酯酶活性频率分布与抗药性的关系

选用室内饲养的棉铃虫Helicoverpa armigera偃师和湖北2个敏感品系、对辛硫磷高抗的PCP20品系、对氰戊菊酯高抗的YG45品系及1999或2000年采自山东阳谷、河北邯郸和河南安阳的田间高抗种群,江苏徐州、湖北武汉的田间中等抗性水平种群和新疆沙湾的田间敏感种群,采用酶标板酶动力学法测定了各品系（种群）的3龄幼虫个体酯酶活性频率分布和平均酯酶活性。结果表明,偃师敏感品系、湖北敏感品系和新疆沙湾田间敏感种群的酯酶活性个体频率分布相似,三个品系（种群）的平均酯酶活性相近,分别为991、1 138、1 055 mOD·min^-1·larva^-1。室内选育的PCP20抗性品系、YG45抗性品系及山东阳谷、河北邯郸 、河南安阳田间高抗种群的高酯酶活性（活性在1 800 mOD·min^-1·larva^-1以上）个体频率明显高于三个敏感品系（种群）,平均酯酶活性在1 510～2 482 mOD·min^-1·larva^-1之间。江苏徐州、湖北武汉的田间中抗水平种群高酯酶活性个体频率及平均酯酶活性都介于敏感和高抗品系（种群）之间,平均酯酶活性为1 258～1.404 mOD·min^-1·larva^-1。棉铃虫各品系（种群）平均酯酶活性与对拟除虫菊酯类杀虫剂抗性个体频率的相关性要比对有机磷类的高,相关系数分别为0.82和0.42。分析各品系（种群）高酯酶活性个体频率与棉铃虫对拟除虫菊酯类、有机磷类杀虫剂抗性个体频率的相关性,得到相似的结果。考虑到酯酶并不是棉铃虫对拟除虫菊酯抗性的主要机理,建议酯酶活性可作为棉铃虫抗药性生化检测的一个参考指标。本文还讨论了酯酶与棉铃虫对拟除虫菊酯类杀虫剂及有机磷类杀虫剂抗性的关系。