Genetic variation as a tool for histochemical localization of a nonspecific esterase

A prominent esterase activity was demonstrated histochemically in the straight portion of the proximal tubules in kidney of the mouse strain DBA/2J after inhibition with bis-p-nitrophenyl phosphate and subsequent staining, using 5-bromoindoxyl acetate as substrate. In the strain PUC/1Fre, the corresponding esterase was only weakly expressed. By comparing data from the literature (von Deimling et al. 1981) with the characteristic features of this kidney esterase including substrate preference, sensitivity to inhibitors, solubility, histochemical location, and strain differences, it was concluded that it was identical with the previously electrophoretically defined esterase-16.     

Novel ferulic acid esterases are induced by growth of Aspergillus niger on sugar-beet pulp

 A ferulic acid esterase (FAE-III), which was induced by growth of Aspergillus niger CBS 120.49 on oat-spelts xylan, was capable of releasing ferulic acid from wheat bran but not from sugar-beet pulp (SBP) [Faulds CB, Williamson G (1994) Microbiology 140:779–787]. Growth of this strain on SBP gave low levels of ferulic acid esterase activity (using methyl ferulate as substrate). A similar growth with a different A. niger strain (CS 180) gave tenfold higher levels of esterase activity. Assaying culture filtrates obtained from A. niger CS 180 grown on SBP over a 3 to 10-day period against four simple phenolic methyl esters demonstrated that at least two esterases were produced, and, by comparison of substrate specificity, FAE-III was either absent or present only at low levels. Furthermore, immunodetection of proteins did not detect the presence of FAE-III in culture supernatants of SBP-grown cultures, whereas it did in cultures grown on oat-spelts xylan. These results show that SBP does not contain the inducer for FAE-III, but does induce novel esterases. When A. niger CS 180 cultures were grown on different carbon sources, esterase activity was induced on SBP, sugar-beet arabinan and oat-spelts xylan, but not on simple sugars or de-esterified sugar-beet pectin. Further, SBP-grown cultures co-inoculated with arabinanase, galactanase or xylanase did not exhibit increased levels of extracellular FAE activity or an earlier appearance of esterase activity, although there was an increase in esterase activity with added polygalacturonase. These results show that novel esterases are induced by growth of A. niger on SBP. Received: 11 September 1995/Received revision: 5 December 1995/Accepted: 11 December 1995     

新型甘露聚糖酶-乙酰酯酶双功能酶的功能和结构域协同研究

[目的] 分析鉴定高效木质纤维素降解菌群EMSD5来源的新型甘露聚糖酶-乙酰酯酶双功能酶44884,解析催化域间的协同关系,以及碳水化合物结合模块（CBM）对催化域特性的影响,拓展对该类双功能酶的认识,为甘露聚糖酶的升级改造和应用提供依据。[方法] 通过大肠杆菌异源表达甘露聚糖酶-乙酰酯酶双功能酶44884,并构建截短和定点突变的突变体,利用TLC和DNS法比较野生型和突变体的酶学性质。[结果] 成功对44884全长和突变蛋白进行克隆表达,并发现44884中2个催化域能够彼此促进各自产物的释放,而且以双功能酶形式存在时,这种促进效果更为明显。44884中的2个CBM65均有甘露聚糖和结晶纤维素结合活性,且CBM65的存在并不改变甘露聚糖酶和乙酰酯酶的最适反应条件和水解模式。虽然CBM65显著降低了2个催化域的热稳定性,但水解天然底物时,2个CBM65对各自临近催化域的水解具有明显的促进效果。[结论] 本研究首次发现并探究了新型甘露聚糖酶和乙酰酯酶形成的双功能酶44884的功能,解析了催化域之间高效的协同效应,以及新型甘露聚糖结合模块CBM65对双功能酶水解的促进作用。     

3-Acetyl deoxynivalenol and esterase production in a fungicideinsensitive strain ofFusarium culmorum

3-Acetyl deoxynivalenol (3-ADON) and esterase production were determined in a strain ofFusarium culmorum insensitive to the fungicide, difenoconazole. Following further exposure to this fungicide for different periods of time, the initiation of 3-ADON production was observed to be accelerated in the insensitive strain compared to a control (sensitive) strain of the phytopathogen. In particular, 3-ADON appeared in insensitive cultures within 21 days of incubation with fungicide levels of 1 to 4 μg/ml media and at 43 days with difenoconazole levels of 6 and 10 μg/ml. However, in the control strain, 3-ADON production was delayed until 28 and 57 days for the respective doses of fungicide. For these times and fungicide levels, the overall production of 3-ADON by the insensitive strain was significantly different from the zero values recorded with the control strain (P<0.05). Moreover, although difenoconazole was generally effective in depressing 3-ADON synthesis in both strains, the suppression was not significant (P>0.05) for the insensitive strain at 43 and 57 days of incubation with fungicide added at 1 μg/ml. In a parallel experiment, total esterase production was observed to increase progressively over time and at consistently higher levels for the insensitive strain from day 14 onwards so that by day 35 the strain difference was significant (P<0.05). Although the increase with time occurred in both strains, this enhancement appeared at 14 days in the insensitive strain (P<0.05) but delayed until 21 days in the control strain (P<0.05). In conclusion, these investigations have demonstrated the persistence of 3-ADON production and enhanced levels of total esterases in a strain ofF. culmorum insensitive to difenoconazole. Furthermore, it is proposed that changes in esterase profiles might be of diagnostic value in identifying toxigenic strains of differentFusarium species.     

一株高产酯酶中度嗜盐菌的分离、鉴定及酯酶部分酶学性质的研究

利用平板筛选法从盐场卤水池中筛选到一株高效降解Tween 20的中度嗜盐菌DF-B6菌株。通过形态学、生理生化及16S rDNA序列分析确定该菌株为Idiomarina属的成员。底物特异性实验确定DF-B6菌株分泌的脂裂解酶为酯酶, 而非脂肪酶。在含8% NaCl的0.05 mol/L Tris-HCl (pH 8.0)缓冲液中, 50°C条件下作用, 酶活性最高。当反应液中金属离子浓度为10 mmol/L时, Mg2+、Ca2+和Mn2+对酶活有激活作用, 而Zn2+、Fe3+和Cu2+则对酶反应有抑制作用。     

Isolation and Characterization of a GDSL Esterase from the Metagenome of a Marine Sponge-associated Bacteria

Using a metagenome library constructed from a bacterial associated with a marine sponge Hyrtios erecta, we identified a novel esterase that belongs to the SGNH hydrolase superfamily of esterases. The substrate specificity of EstHE1 was determined using p-nitrophenyl (pNP) ester (C2: acetate, C4: butylate, C6: caproate, C12: laurate, C16: palmitate). EstHE1 exhibited activity against C2 (5.6 U/mg), C4 (5.1 U/mg), and C6 (2.8 U/mg) substrates. The optimal temperature for EstHE1 esterase activity of the pNP acetate substrate was 40°C, and EstHE1 retained 60% of its enzymatic activity in the 30–50°C range. This esterase showed moderate thermostability, retaining 58% of its activity even after preincubation for 12 h at 40°C. EstHE1 also maintained activity in high concentrations of NaCl, indicating that this esterase is salt-tolerant. Thus, EstHE1 has the thermal stability and salt tolerance necessary for use as an industrial enzyme.     

Screening and identification of a novel esterase EstPE from a metagenomic DNA library

Esterases represent a large family of hydrolases with broad substrate specificity and functional sequence space. Although many attempts to screen new esterases have been conducted, there have been few reports conducted to discriminate unique enzymes from typical ones based on novel structure and function. In this study, we discovered an esterase and a novel family through a successive assay of whole cells and crude lysates (oxidative open condition). The screened putative esterases from the metagenomic DNA of salted shrimp consisted of 753 bp encoding 27 kDa of polypeptide, namely PE esterase. Sequence analyses revealed that an identical gene was reported from whole genome sequencing of Stenotrophomonas maltophilia K279a. However, its biochemical and phylogenetic characteristics have not yet been evaluated. PE esterase was overexpressed only by the MBP fusion state in E. coli and was easily purified using an affinity column. This enzyme showed a typical spectrum of substrate specificity and possessed the consensus motifs, Ser-Asp-His and GXSXG, which are essential for most esterase/lipase superfamilies. Interestingly, the entire organization of the ORF and consensus sequence around the active site were distinct from the related enzymes, and its structure could be affected by a reducing agent, DTT.     

Esterase Activities of Brevihacterium sp. R312 and Brevihacterium linens 62

Esterase activity of Brevihacterium linens 62 and Brevibacterium sp. R312 was detected. Each strain had esterase activities that hydrolyzed p-nitrophenyl acetate and α-naphthyl acetate. Biosynthesis and optimum pH and temperature of the two esterase activities showed that the latter were caused by different esterases. The influence of the culture medium and the growth substrate on biosynthesis of the esterase systems were studied. Hydrolysis of methylthioacetate and phenethyl acetate by cell extracts of the two strains was done. No enzymatic ester synthesis reaction was observed. However, transfer reactions by cell extracts of the two strains were done.     

STABILITY OF ORGANOPHOSPHATE RESISTANCE IN CULEX PIPIENS PALLENS COQUILLETT UNDER RELAXATION OF INSECITICDE SELECTION*

Abstract The stability of resistance in the organophosphate resistant strain of Culex pipiens pallens with amplification of single gene coding for esterase B₂ was determined under relaxation of insecticide selection. Insecticide resistance and amplification of esterase were both lost when strains were not homozygous for the presence of amplified gene, probably due to biological disadvantage of the esterase gene. LC₅₀ and LC₉₅ of chlorpyrifos for this strain were 0. 2099 mg/L and 0.9036 mg/L in the F₀ generation respectively, but 0. 0207 mg/L and 2. 8027 mg/L respectively in the F₁₆ generation. In the homozygous strain, insecticide resistance was still stable and amplification of esterase remained after 16 generations under relaxation of insecticide selection, which indicated that copy of esterase gene was not lost in the offspring of this strain. The evolution of insecticide resistance in mosquitoes is discussed.     

Identification of a type-D feruloyl esterase from<Emphasis Type="Italic"> Neurospora crassa</Emphasis>

Feruloyl esterases constitute an interesting group of enzymes that have the potential for use over a broad range of applications in the agri–food industries. In order to expand the range of available enzymes, we have examined the presence of feruoyl esterase genes present in the genome sequence of the filamentous fungus Neurospora crassa. We have identified an orphan gene (contig 3.544), the translation of which shows sequence identity with known feruloyl esterases. This gene was cloned and the corresponding recombinant protein expressed in Pichia pastoris to confirm that the enzyme (NcFaeD-3.544) exhibits feruloyl esterase activity. Unusually the enzyme was capable of p-coumaric acid release from untreated crude plant cell wall materials. The substrate utilisation preferences of the recombinant enzyme place it in the recently recognised type-D sub-class of feruloyl esterase.     

A novel esterase from a psychrotrophic bacterium, Acinetobacter sp. strain no. 6, that belongs to the amidase signature family

A novel esterase that belongs to the amidase signature family was found in a psychrotrophic bacterium, Acinetobacter sp. strain no. 6, isolated from Siberian soil. The gene coding for the esterase, named EstA8, was cloned, and an open reading frame of 1488 bp corresponding to 496 amino acid residues was identified. EstA8 showed 30% sequence identity with 6-aminohexanoate-cyclic-dimer hydrolases from Pseudomonas sp. strain NK87 and Flavobacterium sp. strain K172, which degrade a by-product of the nylon-6 industry. EstA8 was overproduced in Escherichia coli JM109 under the control of the lac promoter of pUC118 and purified. Consistent with the fact that the source microorganism is cold-adapted, the enzyme was unstable at moderate temperatures. It lost 75% of its original activity by incubation at 40 °C for 30 min. Despite its structural similarity to 6-aminohexanoate-cyclic-dimer hydrolase, 6-aminohexanoate cyclic dimer did not serve as the substrate. EstA8 is a member of the amidase signature family, but its esterase activity toward p-nitrophenyl esters, such as p-nitrophenyl acetate, was much higher than its amidase activity toward p-nitroanilides, such as p-nitroacetanilide.     

Characterization of a novel esterase conferring insecticide resistance in the mosquito Culex tarsalis

Resistance to the organophosphate insecticide, malathion, in a strain of Culex tarsalis mosquitoes is due to increased activity of a malathion carboxylesterase (MCE). To determine whether resistance was due to a qualitative or quantitative change in the MCE, the enzyme was purified from both malathion-resistant and -susceptible mosquitoes. Enzyme kinetic measurements revealed that the two strains have one MCE in common, but resistant mosquitoes also have a unique MCE which hydrolyses malathion 18 times faster. Interestingly, this MCE does not hydrolyse α-naphthyl acetate, a substrate commonly used to detect increased levels of esterases in other organophosphate-resistant insects. Unlike the over-produced esterase of some related mosquito species, each MCE in C. tarsalis accounts for only a small fraction (0.015%) of the total extractable protein in either strain. Therefore, resistance in these insects is due to the presence of a qualitatively different enzyme, and not to a quantitative increase of a non-specific esterase. This study therefore demonstrates that the underlying biochemical mechanisms of insecticide resistance in one insect cannot necessarily be predicted from those of another, even closely related species. © 1995 Wiley-Liss, Inc.     

Cloning and biochemical characterization of a novel lipolytic gene from activated sludge metagenome,and its gene product

In this study, a putative esterase, designated EstMY, was isolated from an activated sludge metagenomic library. The lipolytic gene was subcloned and expressed in Escherichia coli BL21 using the pET expression system. The gene estMY contained a 1,083 bp open reading frame (ORF) encoding a polypeptide of 360 amino acids with a molecular mass of 38 kDa. Sequence analysis indicated that it showed 71% and 52% amino acid identity to esterase/lipase from marine metagenome (ACL67845) and Burkholderia ubonensis Bu (ZP_02382719), respectively; and several conserved regions were identified, including the putative active site, GDSAG, a catalytic triad (Ser203, Asp301, and His327) and a HGGG conserved motif (starting from His133). The EstMY was determined to hydrolyse p-nitrophenyl (NP) esters of fatty acids with short chain lengths (≤C8). This EstMY exhibited the highest activity at 35°C and pH 8.5 respectively, by hydrolysis of p-NP caprylate. It also exhibited the same level of activity over wide temperature and pH spectra and in the presence of metal ions or detergents. The high level of stability of esterase EstMY with unique substrate specificities makes it highly valuable for downstream biotechnological applications.     

Pirimiphos-methyl resistance in two stored product mites, Acarus siro and Acarus farris, as detected by impregnated paper bioassay and esterase activity assays

The response to pirimiphos-methyl, in one strain of Acarus farris and two strains of Acarus siro, was assessed using an impregnated filter paper bioassay and by the selection of adults following exposure to pirimiphos-methyl. It was concluded that one of the strains of A. siro was resistant to pirimiphos-methyl and that a major resistance mechanism was involved. The second strain of A. siro gave a response similar to that of a laboratory strain unexposed to organophosphates and was considered to be susceptible. The A. farris strain responded to selection at the ED50 but not at the ED99, and it was concluded that a minor resistance mechanism is present in this strain. Assays of esterase activity were used to attempt to identify the biochemical mechanisms involved in the resistance detected by the bioassays. The A. farris and susceptible A. siro strains showed similar levels of esterase activity but the esterase activity of the resistant A. siro strain was significantly greater. An increase in esterase activity followed selection of both the A. farris strain and the resistant A. siro strain. An acetylcholinesterase assay showed no significant difference between the susceptible and pirimiphos-methyl selected strains of A. siro. The results suggest that esterases are involved in the resistance to pirimiphos-methyl found in A. siro and A. farris but that in A. siro, at least, other mechanisms may also be present.     

Studies on esterase histochemistry of amphibian kidney

Summary In the hope that the histochemical picture of the kidney may help to understand its role in excretion and osmoregulation, an effort is here made to study the distribution of esterases in amphibian kidney.The kidneys of adults and tadpoles of the frog, Rana tigrina and the toad, Bufo melanostictus were used for this study. Some of these animals were subjected to dehydration for 3–4 days and to the effect of 150 mM NaCl for 8–12 days before their kidneys were used. The esterases were visualised using tweens, naphthol esters and 5-bromoindoxyl acetate as substrates. These were accompanied by activator/inhibitor studies.Very interesting results were obtained in the distribution of the esterases. Tween esterase and -naphthyl acetate esterase were found in the proximal tubules of the adult frog kidney only while 5-bromoindoxyl acetate esterase was found to be present in all the animals tested. On the other hand, naphthol AS acetate esterase was absent in the tadpole stages of the frog and toad. Further 5-bromoindoxyl acetate esterase and naphthol AS acetate esterase were demonstrated in the glomeruli of frogs and toads subjected to NaCl solution. Activator/ inhibitor studies helped in characteristically differentiating these different esterases.There seems to be a relationship between the distribution of the different esterases and the excretory and osmoregulatory adaptations of these animals which differ in the adult and tadpole stages and in the experimental conditions mentioned. The possible implications of the esterase distribution is discussed in considerable details.U.G.C. Research Scholar.     

Overexpressed esterases in a fenvalerate resistant strain of the cotton bollworm, Helicoverpa armigera

Enhanced detoxification is the major mechanism responsible for pyrethroid resistance in Chinese populations of Helicoverpa armigera. Previous work has shown that enhanced oxidation contributes to resistance in the fenvalerate-selected Chinese strain, YGF. The current study provides evidence that enhanced hydrolysis by esterase isozymes also contributes to the resistance in this strain. The average esterase activity of third instar YGF larvae was 1.9-fold compared with that of a susceptible SCD strain. Much of this difference was attributed to isozymes at two zones which hydrolysed the model carboxylester substrate 1-naphthyl acetate and also a 1-naphthyl analogue of fenvalerate. A preparation enriched for enzymes migrating to one of these zones from YGF was shown to hydrolyse fenvalerate with a specific activity of about 2.9 nmol/min/mg. This material was also matched by mass spectrometry with four putative carboxylesterase genes, all of which clustered within a phylogenetic clade of secreted midgut esterases. Quantitative PCR on these four genes showed several-fold greater expression in tissues of YGF compared to SCD but no differences was found in the number of copies of the genes between the strains.     

A new variant of the Es-4 locus in the rat

A new variant of kidney esterase in the DK/Nac rat strain is reported. The new esterase was tentatively named ES-4C determined by a third allele of the Es-4 locus of Linkage Group V (LGV). Strain distribution was surveyed using 17 inbred strains, but no strain except for the DK/Nac strain possessed the ES-4C type. Although we surveyed outbred stocks (Jcl: Wistar and Jcl: SD) we could not find rats carrying the ES-4C type. Genetic analysis of the ES-4C type was carried out using mating experiments between DK/Nac and BUF/Nac (ES-4B). The results indicated that the new variant was controlled by the Es-4 locus and it was named the Es-4c allele.     

Promiscuous esterase activities of the C–C hydrolases from <Emphasis Type="Italic">Dyella ginsengisoli</Emphasis>

A C–C hydrolase gene (bphD _LA-4 ) from strain Dyella ginsengisoli LA-4 was cloned and expressed in Escherichia coli BL21 (DE3). BphD_LA-4 together with another hydrolase MfphA_LA-4, which derived from the same strain, possessed esterase activities. p-Nitrophenyl butyrate was the best substrate for both enzymes. BphD_LA-4 had high catalytic efficiency to p-nitrophenyl benzoate, whereas MfphA_LA-4 had no activity. Homology modeling and docking studies demonstrated that the proper hydrogen bond interaction was important for the reactivity of specific substrate.     

Purification and properties of a highly enantioselective l-menthyl acetate hydrolase from Burkholderia cepacia

A highly enantioselective l-menthyl acetate esterase was purified to homogeneity from Burkholderia cepacia ATCC 25416, with a recovery of 4.8% and a fold purification of 22.7. The molecular weight of the esterase was found to be 37 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The N-terminal amino acid sequence was “MGARTDA”, and there was no homology in contrast to other Burkholderia sp. esterases. This enzyme preferentially hydrolyzed short-chain fatty acid esters of menthol with high stereospecificity and high hydrolytic activity, while long-chain l-menthyl esters were poor substrates. Considered its substrate specificity and N-terminal sequence, this esterase was concluded as a new enzyme belonging to the carboxylesterase group (EC 3.1.1.1) of esterase family. The optimum temperature and pH for enzyme activity using racemic menthyl acetate as substrate were 30 °C and 7.0, respectively. The esterase was more stable in the pH range of 7.0–9.0 and temperature range of 30–40 °C. Hydrolytic activity was enhanced by Ca²⁺, K⁺ and Mg²⁺, but completely inhibited by Hg²⁺, Cu²⁺, ionic detergents and phenylmethylsulfonyl fluoride (PMSF) at 0.01 M concentration.     

Specificity of the sex-influenced esterase (ES-SI) isozyme in rat liver

A carboxylesterase which shares common antigenicity with sex-influenced esterase (ES-SI) was found in both the male and the female liver of the rat but not in the following tissues: erythrocyte, heart, kidney, lung, spleen, small intestine, testis, thymus, and lymph node. Subcellular fractionation showed the esterase localizes in the microsome-rich fraction. The strain distribution of the presence or absence of the esterase in inbred rats was identical to that of ES-SI, although in adult males a considerable amount of the esterase exists, unlike ES-SI. The esterase had a higher isoelectric point than ES-SI but after neuraminidase treatment the difference disappeared, suggesting that the esterase has a sialic acid moiety. Because this esterase has different properties from those previously reported, it is proposed that it is designated liver-ES-SI. The common antigenicity and similar strain distribution between ES-SI and liver-ES-SI suggest that liver-ES-SI is a precursor molecule of ES-SI and therefore the two esterases are products of a single gene.This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, and Culture of Japan.