棉蚜抗氧化乐果品系的羧酸酯酶基因突变

用氧化乐果对室内敏感品系棉蚜Aphis gossypii (Glover)进行抗性选育,经24代筛选,抗性指数达到124.7倍。以α-乙酸萘酯（α-NA）为底物,比较了氧化乐果敏感和抗性品系棉蚜羧酸酯酶的比活力,发现抗性品系羧酸酯酶比活力明显小于敏感品系。对这两个品系的羧酸酯酶基因进行了克隆,通过对抗性和敏感品系羧酸酯酶基因核苷酸序列及推导的氨基酸序列比较,发现抗性品系有4个氨基酸残基发生了替代 (His¹⁰⁴→Arg, Ala¹²⁸→Val, Thr³³³→Asp, Lys⁴⁸⁴→Arg)。对其蛋白质三维结构分析推测只有His¹⁰⁴→Arg的替代是位于其活性中心。棉蚜氧化乐果敏感和抗性品系羧酸酯酶基因cDNA全长的GenBank登录号分别为AY485216和AY485214。     

云南烟蚜抗药性机制研究

通过比较云南烟蚜敏感品系和抗性品系的解毒酶(α-乙酸萘酯羧酸酯酶、β-乙酸萘酯羧酸酯酶)和靶标酶(乙酰胆碱酯酶)的活力,研究了烟蚜对有机磷、拟除虫菊酯和氨基甲酸酯类杀虫剂抗性的生化机制,并通过酯酶基因扩增检测和钠离子通道突变检测,研究了其抗性的分子机制。结果表明:α-乙酸萘酯羧酸酯酶活力增强是烟蚜对有机磷类、氨基甲酸酯类杀虫剂及拟除虫菊酯类杀虫剂的抗性机制之一;乙酰胆碱酯酶在烟蚜对有机磷杀虫剂抗性中起重要作用;3个抗性品系烟蚜均没有发生酯酶基因扩增,抗拟除虫菊酯品系烟蚜发生了钠离子通道突变。     

麦长管蚜羧酸酯酶基因cDNA片段克隆及吡虫啉胁迫对其表达的影响

羧酸酯酶在昆虫对杀虫剂的解毒代谢过程中发挥重要作用,本研究旨在分析吡虫啉胁迫对麦长管蚜羧酸酯酶基因表达的影响。采用同源克隆方法克隆麦长管蚜羧酸酯酶基因cDNA片段,实时荧光定量PCR技术检测羧酸酯酶基因在不同吡虫啉剂量下的表达量变化。扩增所得麦长管蚜羧酸酯酶基因cDNA片段大小为392 bp,命名为SaEST 3(GenBank登录号KY 441614),该片段编码130个氨基酸残基,分子量14 kD,等电点4.93。序列同源性比对及生物信息学分析表明SaEST3推导的氨基酸序列与豌豆长管蚜、麦双尾蚜、夹竹桃蚜、桃蚜的羧酸酯酶氨基酸序列相似性较高,分别为94%、85%、80%和80%。实时荧光定量PCR结果显示,不同吡虫啉处理剂量下,SaEST3 m RNA的相对表达量均上调。克隆得到的基因片段为麦长管蚜羧酸酯酶基因片段,吡虫啉对麦长管蚜羧酸酯酶基因SaEST3表达有一定的影响。     

棉铃虫羧酸酯酶基因的克隆、序列分析及组织表达

为了从分子水平上研究棉铃虫Helicoverpa armigera (Hübner) 对杀虫剂抗性的产生机理,本文通过RT PCR和RACE方法首次从棉铃虫中肠中克隆了一个羧酸酯酶全长cDNA序列。序列分析表明,该基因包含一个1 794 bp的开放读码框,129 bp的5′UTR和139 bp的3′UTR区域。该基因编码597个氨基酸, 推测编码蛋白质的等电点pI为4.92,分子量为67.1 kD,GenBank登录号为EF547544。通过对氨基酸的同源性分析表明,该羧酸酯酶与斜纹夜蛾Spodoptera litura羧酸酯酶的同源性最高,达60%。半定量RT-PCR分析表明,该基因在中肠组织中表达量最高,在脂肪体和生殖腺中表达量较低,在头部则不表达。推测该羧酸酯酶基因可能主要参与棉铃虫对外源物质的解毒代谢。     

羧酸酯酶A2在大肠杆菌中的表达及特征分析

本研究采用RT-PCR法从抗性蚊虫(Culex quinquefasciatus)中克隆了羧酸酯酶A2的全长cDNA,对其进行了序列测定,并构建了融合表达质粒pET-ESTA2。转化大肠杆菌BL21后,在异丙基硫代牛乳糖苷(IPTG)的诱导下,使羧酸酯酶A2在大肠杆菌内得到表达。表达产物经亲和层析纯化获得了1条带的重组蛋白。与报道的从蚊虫体内纯化的羧酸酯酶相比,从表达产物中纯化的羧酸酯酶Km与其一致,但从表达产物纯化的羧酸酯酶的Vm比蚊虫中纯化的羧酸酯酶的Vm高。表明用亲和层析纯化的羧酸酯酶A2比从蚊虫中提取的纯度高。羧酸酯酶A2表达纯化及特征分析为其应用奠定了基础。     

白纹伊蚊溴氰菊酯抗性和敏感品系羧酸酯酶性质比较

本文对白纹伊蚊Aedes albopictus溴氰菊酯抗性品系和敏感品系羧酸酯酶的生物化学性质进行了比较。白纹伊蚊抗性品系和敏感品系羧酸酯酶随底物浓度(α-乙酸萘酯或β-乙酸萘酯)的变化比活力变化趋势一致,但抗性品系对这2种底物的比活力均高于敏感品系,抗性品系羧酸酯酶的米氏常数和最大反应速度与敏感品系有显著差异。胆碱酯酶抑制剂测定结果表明,抗性品系羧酸酯酶对敌敌畏和磷酸三苯酯的敏感性高于敏感品系,对残杀威的敏感性低于敏感品系。2个品系羧酸酯酶对脱叶磷的敏感性差异不大。说明羧酸酯酶可能与白纹伊蚊对溴氰菊酯抗性有关。     

羧酸酯酶A2在大肠杆菌中的表达及特征分析

本研究采用RT-PCR法从抗性蚊虫（Culex quinquefasciatus)中克隆了羧酸酯酶A2的全长cDNA,对其进行了序列测定。并构建了融合表达质粒pET-ESTA2,转化大肠杆菌BL21后,在异丙基硫代半乳糖苷（IPTG）的诱导下,使羧酸酯酶A2在大肠杆菌内得到表达,表达产物经亲和层析纯化获得了1条带的重组蛋白,与报道的从蚊虫体内纯化的羧酸酯酶相比,从表达产物中纯化的羧酸酯酶Km与其一致,但从表达产物纯化的羧酸酯酶的Vm比蚊虫中纯化的羧酸酯酶的Vm高,表明用亲和层析纯化的羧酸酯酶A2比从蚊虫中提取的纯度高,羧酸酯酶A2表达纯化及特征分析为其应用奠定了基础。     

溴氰菊酯对飞蝗羧酸酯酶基因表达的影响

【目的】研究溴氰菊酯作用下飞蝗羧酸酯酶基因的mRNA表达特性,为溴氰菊酯的代谢解毒及飞蝗Locusta migratoria防治中抗性风险的评估提供基础资料。【方法】本文采用不同剂量溴氰菊酯处理3龄飞蝗,提取总RNA,体外反转录合成cDNA模板,采用Real-time PCR技术分析飞蝗羧酸酯酶基因在溴氰菊酯不同浓度和不同时间处理后的表达模式。【结果】飞蝗经不同浓度溴氰菊酯处理12 h后,LmCesA3和LmCesE1表现为诱导效应;除LmCesA2外,其余羧酸酯酶基因经溴氰菊酯LD30剂量处理后分别在不同的时间点表现为诱导效应。【结论】5个羧酸酯酶基因LmCesA1、LmCesA3、LmCesD1、LmCesE1和LmCesI1可以被溴氰菊酯诱导,表明其可能参与飞蝗对溴氰菊酯的代谢解毒及抗性产生。     

花生羧酸酯酶AhCXE的克隆与原核表达

前期功能研究发现当倒置离体培养的果针发生向地性弯曲时,花生羧酸酯酶(AhCXE)基因表达量显著增加。本研究以花生果针为材料,利用RT-PCR与RACE克隆技术,克隆了AhCXE基因并通过生物信息学技术分析了AhCXE基因的基本特征,利用原核诱导技术分析目标蛋白的结构与蛋白表达特征。测序结果表明AhCXE基因ORF全长为1 068 bp,编码355个氨基酸;生物信息学分析显示AhCXE蛋白属于α/β蛋白水解酶家族,具有酯酶活性,参与体内脂类代谢,与大豆羧酸酯酶具有较高的相似度,最后将该蛋白编码的基因命名为AhCXE。     

棉蚜抗吡虫啉品系和敏感品系主要解毒酶活性比较

通过生物测定和生物化学方法比较了棉蚜 Aphis gossypii 对吡虫啉抗性（约为7倍）和敏感品系几种主要解毒酶的活性。结果表明：氧化胡椒基丁醚对两个品系均无明显增效作用。抗性品系中羧酸酯酶和谷胱甘肽S-转移酶的比活力均明显高于敏感品系,抗性品系中羧酸酯酶的K_m值也显著高于敏感品系,说明抗性品系羧酸酯酶与底物的亲和力明显高于敏感品系。上述结果证明羧酸酯酶和谷胱甘肽S-转移酶活力增强在棉蚜对吡虫啉的抗性中起重要作用。     

Molecular comparisons of the Culex pipiens (L.) complex esterase gene amplicons

The amplification of carboxylesterase genes is a mechanism of organophosphate resistance in Culex mosquitoes. Amplified carboxylesterase genes from an insecticide resistant Culex pipiens strain collected in Cyprus were analysed and compared to other Culex amplified carboxylesterase alleles. A 12 kb section of genomic DNA containing two gene loci coding for carboxylesterase alleles A5 and B5 was cloned and sequenced. A comparison between this amplicon and one from a strain with co-amplified carboxylesterase alleles A2 and B2 revealed a number of differences. The intergenic spacer was 3.7 kb in length in the A5-B5 amplicon (2.7 kb in A2-B2) and contained putative Juan and transposable elements upstream of B5. A fragment of a gene with high homology to aldehyde oxidase was also present immediately downstream of A5. The comparison revealed no differences that would explain the successful spread of the A2-B2 amplicon worldwide whilst the A5-B5 amplicon is restricted to the Mediterranean.     

Paralogous gene analysis reveals a highly enantioselective 1,2-O-isopropylideneglycerol caprylate esterase of Bacillus subtilis.

Carboxylesterase NP of Bacillus subtilis Thai I-8, characterized in 1992 as a very enantioselective (S)-naproxen esterase, was found to show no enantiopreference towards (S)-1,2-O-isopropylideneglycerol (IPG) esters. The ybfK gene was identified by the B. subtilis genome project as an unknown gene with homology to carboxylesterase NP. The purpose of the present study was to characterize the ybfK gene product in order to determine whether this paralogue of carboxylesterase NP had an altered or enhanced stereospecificity. The ybfK gene was cloned and expressed in B. subtilis using a combination of two strong promoters in a multicopy vector. The enzyme was purified from the cytoplasm of B. subtilis by means of anion exchange and hydrophobic interaction chromatography. The purified YbfK is an enzyme of 296 amino acids and shows an apparent molecular mass of 32 kDa (SDS/PAGE). Comparison of the activities of YbfK and carboxylesterase NP towards caprylate esters of IPG revealed that YbfK produces (S)-IPG with 99.9% enantioselectivity. Therefore, we conclude that we have isolated a paralogue of carboxylesterase NP that can be used for the enantioselective production of (S)-IPG.     

Molecular cloning and analysis of a novel teratocyte-specific carboxylesterase from the parasitic wasp, Dinocampus coccinellae

Teratocytes derived from the embryonic membrane (serosa) of parasitoids are released into the host hemocoel when the parasitoid eggs hatch, where they perform several functions during the post-embryonic stage. A full-length cDNA encoding a putative carboxylesterase was isolated from the teratocytes of Dinocampus coccinellae and was designated as teratocyte-specific carboxylesterase (TSC). It contained an open reading frame of 2571 bp coding for a protein of 857 amino acids with a calculated molecular mass of 89 kDa. The deduced amino acid sequence had many structural features that are highly conserved among serine hydrolases including Ser, Glu and His as a catalytic triad, carboxylesterase type-B (FGGNPNSVTLLGYSAG)/ lipase-serine (VTLLGYSAGA) active sites, and six N-glycosylation sites. Interestingly, the mRNA encoding the TSC gene was expressed exclusively in teratocytes but not in the parasitoid larva or in the non-parasitized host. Most notably, the TSC protein was distinguished by an insertion of 294 amino acids towards the N-terminal region and was flanked by carboxylesterase domains. Furthermore, sequence alignment and homology search revealed these additional amino acids to be unique to TSC and the insertion contributed significantly to its molecular mass resulting in a larger protein than other esterases. In addition to sequence analysis, the possible role of TSC in relation to the host (Coccinella septempunctata) and parasitoid (D. coccinellae) system is discussed.     

Identification and development of a genetically closely-linked carboxylesterase family of the mouse liver

Six carboxylesterase isozymes (viz. ES-1, ES-6, ES-9, ES-20, ES-22 and ES-24), governed by esterase gene cluster 1 on chromosome 8 of the house mouse, were identified electrophoretically in liver supernatants using their biochemical, genetic and developmental characteristics. ES-1 and ES-20 were expressed as liver-specific forms. The peri- and postnatal development of the six isozymes indicated that they were individually regulated at the genetic level, although the isozymes were regulated as a group when compared to genetically unrelated esterases. The concept of evolutionary divergence following repeated gene duplication of an ancestral esterase structural gene was extended to cover divergence of the temporal (regulatory) genes associated with the multigene family. Allelic variation of the temporal genes was more limited than that of the corresponding structural genes.     

Phage display of an intracellular carboxylesterase of Bacillus subtilis: comparison of Sec and Tat pathway export capabilities

Using the phage display technology, a protein can be displayed at the surface of bacteriophages as a fusion to one of the phage coat proteins. Here we describe development of this method for fusion of an intracellular carboxylesterase of Bacillus subtilis to the phage minor coat protein g3p. The carboxylesterase gene was cloned in the g3p-based phagemid pCANTAB 5E upstream of the sequence encoding phage g3p and downstream of a signal peptide-encoding sequence. The phage-bound carboxylesterase was correctly folded and fully enzymatically active, as determined from hydrolysis of the naproxen methyl ester with Km values of 0.15 mM and 0.22 mM for the soluble and phage-displayed carboxylesterases, respectively. The signal peptide directs the encoded fusion protein to the cell membrane of Escherichia coli, where phage particles are assembled. In this study, we assessed the effects of several signal peptides, both Sec dependent and Tat dependent, on the translocation of the carboxylesterase in order to optimize the phage display of this enzyme normally restricted to the cytoplasm. Functional display of Bacillus carboxylesterase NA could be achieved when Sec-dependent signal peptides were used. Although a Tat-dependent signal peptide could direct carboxylesterase translocation across the inner membrane of E. coli, proper assembly into phage particles did not seem to occur.     

Highly pathogenic strains of Escherichia coli revealed by the distinct electrophoretic patterns of carboxylesterase B

One hundred and ninety one strains of Escherichia coli isolated from extra-intestinal infections and 85 strains isolated from the stools of healthy human beings were compared for electrophoretic mobility and isoelectric point of carboxylesterase B, and for production of alpha-haemolysin and the presence of mannose resistant haemagglutinin. Fast and slow electrophoretic mobilities were distinguished among the strains. The frequency of strains showing slow mobilities was considerably higher when they originated from extra-intestinal infections (40%) than when they were obtained from the stools of healthy individuals (7%). In a two-dimensional electrophoretic profile, the fast and slow mobility variants of carboxylesterase B were resolved into two patterns, B1 and B2, respectively. The frequency of pathogenic strains that concomitantly produced alpha-haemolysin and mannose resistant haemagglutinin was 48.7% for strains of pattern B2 but only 2.8% for strains of pattern B1. Thus, the electrophoretic pattern B2 of carboxylesterase B appears to be a molecular marker for a group of highly pathogenic E. coli strains which are frequently implicated in extra-intestinal infections.     

Comparison and functional characterisation of three homologous intracellular carboxylesterases of Bacillus subtilis

Enzymatic hydrolysis of racemic mixtures may provide an attractive method for the enantiopure production of chiral pharmaceuticals. For example, the carboxylesterase NP of Bacillus subtilis Thai I-8 is an excellent biocatalyst in the kinetic resolution of NSAID esters, such as naproxen and ibuprofen methyl esters. Two homologues of this enzyme were identified when the genome sequence of B. subtilis 168 was revealed in 1997. We characterised one of the homologous, YbfK, as a very enantioselective 1,2-O-isopropylidene-sn-glycerol caprylate esterase, while only modest enantioselectivity towards the naproxen ester was observed. The other homologue, the carboxylesterase NA has not been characterised yet. The purpose of the present study was to fully characterise these three highly homologous esterases with respect to their applicability towards the enantiospecific hydrolysis of a wide range of compounds. The esterase genes were cloned and expressed in B. subtilis using a combination of two strong promotors in a multi-copy vector. After purification of the enzymes from the cytoplasm of B. subtilis, the biochemical and enantioselective properties of the enzymes were determined. Although all carboxylesterases have similar physico-chemical properties, comparison of their specific activities and enantioselectivities towards several compounds revealed rather different substrate specificities. We conclude that carboxylesterase NP and carboxylesterase NA are particularly suited for the enzymatic conversion of naproxen esters, while YbfK offers enantiopure (+)-IPG from its caprylate ester. Given the carboxylesterase activities of the esterases it has been proposed to rename the nap gene of B. subtilis 168 into cesA and the ybfK gene into cesB.     

Genetic control of two esterases of rat plasma (Rattus norvegicus)

Three electrophoretic variants of plasma esterase in the albumin zone, presumably carboxylesterase, have been demonstrated in 250 rats representing a laboratory population of Wistar rats. Electrophoretic variants of the enzyme are believed to be controlled by two codominant alleles at the autosomal locus referred to as Es-2. The variant of carboxylesterase represented by a fast-migrating single band on starch gel electrophoresis is determined by the gene named Es-2 ^a, whereas the slow-migrating variant, represented by two bands, is under control of the allelic gene Es-2 ^b. Animals with Es-2 ^a/Es-2 ^b genotype have three bands of carboxylesterase in the albumin zone. Genetically determined polymorphism of plasma esterase, presumably carboxylesterase, in the prealbumin zone was shown in both laboratory and wild populations of rats. Breeding tests suggest that the gene referred to as Es-1 ^a, responsible for the presence of carboxylesterase in the prealbumin zone, is inherited dominantly, whereas animals homozygous for the allele Es-1 ^b locked this esterase fraction.