Unique Esterase Research Articles

A Novel Esterase from a Marine Metagenomic Library Exhibiting Salt Tolerance Ability

A putative lipolytic enzyme gene, named as est9x, was obtained from a marine microbial metagenome of the South China Sea. Sequence analysis showed that Est9X shares lower than 27% sequence identities with the characterized lipolytic enzymes, but possesses a catalytic triad highly conserved in lipolytic enzymes of the α/β hydrolase superfamily. By phylogenetic tree construction, Est9X was grouped into a new lipase/esterase family. To understand Est9X protein in depth, it was recombinantly expressed, purified, and biochemically characterized. Within potential hydrolytic activities, only lipase/esterase activity was detected for Est9X, confirming its identity as a lipolytic enzyme. When using p-nitrophenol esters with varying lengths of fatty acid as substrates, Est9X exhibited the highest activity to the C2 substrate, indicating it is an esterase. The optimal activity of Est9X occurred at a temperature of 65°C, and Est9X was pretty stable below the optimum temperature. Distinguished from other salttolerant esterases, Est9X's activity was tolerant to and even promoted by as high as 4 M NaCl. Our results imply that Est9X is a unique esterase and could be a potential candidate for industrial application under extreme conditions.

Identification and isozyme characterisation of Meloidogyne spp. infecting horticultural and agronomic crops, and weed plants in Florida

Abstract A total of 327 root samples collected from horticultural and agronomic crops, and weeds associated with these crops in Florida crop production regions, were examined. Isozyme phenotypes, esterase (EST) and malate dehydrogenase (MDH) phenotypes were used to characterise and identify the species of Meloidogyne present in these samples. At least 26 females from each sample were examined using polyacrylamide gel electrophoresis. Sixteen major bands of EST activity were found, corresponding to 12 phenotypes. A species-specific EST phenotype was consistently associated with each of the Meloidogyne spp. identified: Meloidogyne arenaria, M. floridensis, M. graminicola, M. hapla, M. incognita, M. javanica, M. mayaguensis and M. partityla. Two unique EST phenotypes, which have not been described, were found associated with three unidentified nematode populations. Five bands of MDH activity and four phenotypes were also found among the populations. During this study, new host records were determined for M. floridensis and M. mayaguensis, two recently reported Meloidogyne spp. in Florida. Plant species found as new hosts of M. floridensis were Cucumis sativus, Phaseolus sp., Solanum melongena and one weed species, Emilia sonchifolia, and the M. incognita-resistant peach rootstock ‘Flordaguard’. New hosts for M. mayaguensis were Carica papaya, Capsicum annuum var. Longum, and six weed plants, including Eclipta prostrata, Fatoua villosa, Panicum sp., Poinsettia cyathophora, Solanum americanum and also one unidentified weed species belonging to the family Acanthaceae. To our knowledge, Florida is the only geographic area of North America where M. floridensis and M. mayaguensis have been detected.

Esterase-mediated malathion resistance in the human head louse, Pediculus capitis (Anoplura: Pediculidae)

Resistance in a dual malathion- and permethrin-resistant head louse strain (BR-HL) was studied. BR-HL was 3.6- and 3.7-fold more resistant to malathion and permethrin, respectively, compared to insecticide-susceptible EC-HL. S, S, S-Tributylphosphorotrithioate synergized malathion toxicity by 2.1-fold but not permethrin toxicity in BR-HL. Piperonyl butoxide did not synergize malathion or permethrin toxicity. Malathion carboxylesterase (MCE) activity was 13.3-fold and general esterase activity was 3.9-fold higher in BR-HL versus EC-HL. There were no significant differences in phosphotriesterase, glutathione S-transferase, and acetylcholinesterase activities between strains. There was no differential sensitivity in acetylcholinesterase inhibition by malaoxon. Esterases from BR-HL had higher affinities and hydrolysis efficiencies versus EC-HL using various naphthyl-substituted esters. Protein content of BR-HL females and males was 1.6- and 1.3-fold higher, respectively, versus EC-HL adults. Electrophoresis revealed two esterases with increased intensity and a unique esterase associated with BR-HL. Thus, increased MCE activity and over-expressed esterases appear to be involved in malathion resistance in the head louse.

An acidic esterase as a biochemical marker for somatic embryogenesis in orchardgrass (Dactylis glomerata L.) suspension cultures.

The isoenzyme pattern of esterases (EC 3.1.1.2) secreted into the medium of orchardgrass (Dactylis glomerata L.) embryogenic suspension cultures during defined stages of somatic embryogenesis was compared with that of non-embryogenic suspension cultures during unorganised cell proliferation. Isoelectric focusing revealed the presence of 7-14 predominantly acidic isoforms. Comparison with the corresponding cell-wall isoenzyme pattern showed minor, mainly quantitative differences. The pattern of intracellular soluble esterases did not change markedly during somatic embryo development. A unique esterase whose migration in two-dimensional gel electrophoresis corresponds to an apparent molecular mass of 36 kDa and pI=3.8 was detected only in embryogenic cultures at very early stages of development. Since this isoform appeared long before morphological changes had taken place, it could possibly be used as a biochemical marker for embryogenic potential in D. glomerata L. suspension cultures.

Differences in ecological and genetic traits between Panonychus citri (Acari: Tetranychidae) populations infesting citrus and Osmant hus

Comparative studies of the ecological and genetic traits of Panonychus citri (McGregor) populations on citrus and other host plants were conducted. Panonychus citri on Osmanthus trees has a unique esterase allele and host range in comparison with populations on other host plants, for example Citrus unshiu and Ilex crenata. Reproductive incompatibility between P. citri populations on C. unshiu and Osmanthus fragrans was found. Therefore, in Japan, P. citri differentiates into two groups one of which associates with Osmanthus, and the other with other host plants.

Isozyme Profile During Somatic Embryogenesis and In Vitro Flowering of Bambusa vulgaris

Peroxidase and esterase isozymes were investigated during plant regeneration via somatic embryogenesis in Bambusa vulgaris, The transition of non-embryogenic calli to embryogenic calli, somatic embryo development, germination and subsequent flowering of somatic embryo derived shoots were associated with selective expression or repression of isoforms of peroxidase and esterase. Non-embryogenic callus showed six peroxidase and four esterase bands. During somatic embryogenesis and germination of somatic embryos, some bands were suppressed and new isoforms of peroxidase and esterase appeared. During flowering, in addition to four peroxidase bands, a new unique esterase band ‘a’ appeared. Each developmental stage was thus associated with a definite isozyme profile.

Identification and isolation of a unique esterase from the medium of non-embryogenic cell line of cultured carrot cells

A unique esterase isozyme ‘z’ with very low electrophoretic mobility on the anionic polyacrylamide gel (PAGE) was found in the medium of a non-embryogenic (Ca-4) line of cultured carrot (Daucus carota L.) cells. The protein corresponding to this esterase isozyme ‘z’ was purified by electroelution from preparative PAGE and the esterase migrated as a single band with an apparent Mr of 35 000 on SDS-PAGE. The purified esterase isozyme ‘z’ exhibited at least 350-fold higher specific activity than that in the total medium proteins.

Species differences in the hydrolysis of meperidine and its inhibition by organophosphate compounds

The hydrolysis of meperidine was assayed in washed, unfortified liver microsomal fractions of guinea pig, rat, mouse, dog, and human, by following substrate disappearance as quantitated by high-performance liquid chromatography. Using the method of Lineweaver-Burk plots, the velocity of the meperidine hydrolysis reaction was not detectable in guinea pig, very low in human, and extremely high in dog. Hydrolysis of p-nitrophenyl acetate was also monitored in liver microsomal preparations from the same animal species, with guinea pig showing greatest hydrolytic activity and rat showing least hydrolytic activity for this substrate. The data in the above two assays suggested that meperidine hydrolysis is mediated by a unique esterase not present in guinea pig and very low in human, but present with high activity in dog liver microsomes. From these comparative studies we concluded that liver microsomes from different species may contain different carboxylesterases having different affinities for meperidine. To further characterize meperidine carboxylesterase of dog and rat liver microsomes, inhibitory studies in vitro with two organophosphate compounds—paraoxon (diethyl- p-nitrophenyl phosphate) and soman (pinacolyl methylphosphonofluoridate)—indicated a varied pattern of enzyme inhibition. These results suggested that liver microsomal carboxylesterases are involved in the metabolism of meperidine and that interference with these enzymes by organophosphate compounds may alter pharmacologic and toxicologic effects of meperidine.

A comparison of muscle esterases in the fish genus trachurus by vertical gel electrophoresis

1. 1. The multiple forms of soluble muscle esterases of Trachurus trachurus, T. picturatus and T. mediterraneus have been analysed by slab polyacrylamide electrophoresis. 2. 2. Seven bands of esterase activity were observed for each species and were classified according to their substrate and inhibitor specificities. 3. 3. Four of the bands were shared by the three species, but each one had its own unique esterase profile. 4. 4. Sialic acid was found attached to two carboxylesterases.

Comparative analysis of tissue esterases of the zebra danio ( Brachydanio rerio) and the pearl danio ( B. albolineatus) by disc gel electrophoresis

1. 1. The multiple forms of soluble esterases from liver, ovary, skeletal muscle and intestine of Brachydanio rerio and Brachydanio albolineatus have been analyzed by polyacrylamide (disc) gel electrophoresis. 2. 2. Nineteen separable zones of soluble esterase activity were observed in B. rerio and 10 separable zones in B. albolineatus. 3. 3. Although each tissue had its own unique esterase profile, different tissues within a species shared several esterase isozymes in common. The same tissues of the two species had fewer esterases in common. 4. 4. Esterase isozymes were identified in liver and intestinal tissues that showed unusual patterns of inhibition with DFP, eserine sulfate and sulfhdryl-group inhibitors.