Heterologous production of the Piromyces equi cinnamoyl esterase in Trichoderma reesei for biotechnological applications

Aims:  The objective of the study was to produce and characterize the cinnamoyl esterase EstA from the anaerobic fungus Piromyces equi for potential industrial applications.
Methods and Results:  The catalytic domain EstA was produced in Trichoderma reesei. Because the two fungi displayed different genome features, including different codon usage and GC content, a synthetic gene was designed and expressed, leading to the production of the corresponding protein at around 33 mg per litre in the T. reesei culture medium. After the recombinant protein was purified, biochemical characterization showed that EstA presents peak activity at pH 6·5 and at 50–60°C. Furthermore, EstA remained stable at pH 6–8 and below 50°C. EstA was compared to cinnamoyl esterases FaeA and FaeB from Aspergillus niger in terms of ferulic acid (FA) release from wheat bran (WB), maize bran (MB) and sugar beet pulp (SBP).
Conclusion:  The synthetic gene was successfully cloned and overexpressed in T. reesei. EstA from P.   equi was demonstrated to efficiently release FA from various natural substrates.
Significance and Impact of the Study:  Recombinant EstA produced in an industrial enzyme producer, T. reesei , was biochemically characterized, and its capacity to release an aromatic compound (FA) for biotechnological applications was demonstrated.     

An excess of homozygotes at a serum esterase locus in the Atlantic mackerel Scomber scombrus

A serum esterase polymorphism in the Atlantic mackerel Scomber scombrus revealed a significant excess of homozygotes in seven of eleven area samples collected in the N.E. Atlantic. Breaking down the data by sex, year class, size within year class and haul did not remove the homozygous excess. The presence of a null allele is unlikely due to the high frequency required and the lack of null allele homozygotes. It is suggested that the homozygous excess might arise from a shifting selection on different batches of larvae produced over the spawning area and season.     

Insecticide resistance and detoxifying enzyme activity in the principal bancroftian filariasis vector, Culex quinquefasciatus, in northeastern India

The insecticide resistance status of Culex quinquefasciatus Say (Diptera: Culicidae) to DDT and deltamethrin across army cantonments and neighbouring villages in northeastern India was investigated. In India, DDT is still the insecticide of choice for public health programmes. In military stations, pyrethroids, especially deltamethrins, are used for insecticide‐treated nets (ITNs). Recent information on the levels of resistance to DDT and deltamethrin in mosquito populations of northeastern India is scare. Continued monitoring of insecticide resistance status, identification of the underlying mechanisms of resistance in local mosquito populations and the establishment of a baseline data bank of this information are of prime importance. Insecticide susceptibility assays were performed on wild‐caught adult female Cx. quinquefasciatus mosquitoes to the discriminating doses recommended by the World Health Organisation (WHO) to DDT (4%) and deltamethrin (0.05%). Across all study sites, mortality as a result of DDT varied from 11.9 to 50.0%, as compared with 91.2% in the susceptible laboratory strain (S‐Lab), indicating that Cx. quinquefasciatus is resistant to DDT. The species was found to be 100% susceptible to deltamethrin in all study sites except Benganajuli and Rikamari. Knock‐down times (KDT) in response to deltamethrin varied significantly between study sites (P < 0.01) from 8.3 to 17.8 min for KDT₅₀ and 37.4 to 69.5 min for KDT₉₀. All populations exceeded the threshold level of alpha‐esterase, beta‐esterase and glutathion S‐transferase (GST) established for the S‐Lab susceptible strain, and all populations had 100% elevated esterase and GST activity, except Missamari and Solmara. Beta‐esterase activity in Field Unit II (96.9%) was less than in any of the other populations. Benganajuli had the highest activity level for all the enzymes tested. There was a significant correlation between all enzyme activity levels and insecticide resistance phenotype by populations (P < 0.05). The results presented here provide the first report and baseline information of the insecticide resistance status of Cx. quinquefasciatus in northeastern India, and associated information about biochemical mechanisms that are essential for monitoring the development of insecticide resistance in the area.     

Relationship between esterase gene frequencies and length in juvenile snapper Chrysophrys auratus Forster

Samples of 0-group snapper Chrysophrys auratus were collected in the Hauraki Gulf of New Zealand in 1979,1980 and 1981. Each fish was measured and the liver examined for an esterase polymorphism. A similar change in allele frequency with length was observed in each year class. In the total data set there is a significant relationship between allele frequency and length. The relationships between allele frequencies and temperature and growth, along with implications for genetic-stock separation studies are discussed.     

Isoelectric focusing of horse serum esterase isozymes and detection of new phenotypes

A new method for separating the isozymes of horse serum esterase is described. The improved resolution has enabled us to detect several previously undescribed phenotypes. This method has also been used to detect two different apparently 'silent' alleles.     

Atypical micromegakaryocytes,promegakaryoblasts and megakaryoblasts: a critical evaluation by immunohistochemistry,cytochemistry and morphometry of bone marrow trephines in chronic myeloid leukemia and myelodysplastic syndromes

A morphometric analysis of bone marrow trephine biopsies has been performed to study the frequency and planimetric characteristics of so-called atypical micromegakaryocytes in chronic myeloid leukemia (CML) and myelodysplastic syndromes (MDS). In addition, an attempt was made to discriminate this particular cell population from small immature elements of megakaryocytopoiesis, such as promegakaryoblasts and megakaryoblasts. The staining reactions employed included periodic acid-Schiff (PAS), alpha-naphthyl acetate esterase (ANAE) and immunohistochemistry with a monoclonal antibody against platelet glycoprotein IIIa (Y₂/ 51-CD61). Comparison of the various staining reactions applied to the different megakaryocytic elements together with morphometric measurements resulted in a clearcut identification of promegakaryoblasts. These were defined as the earliest immature and exclusively CD61-positive precursors. Atypical micromegakaryocytes were characterized by their dysplastic features and strong ANAE reactivity in addition to their positive CD61 staining. When stringent diagnostic criteria (diameter ranging between 10 to 15 μm, mean size about 12 urn) were applied, this abnormal cell population comprised less than 10% of total megakaryocytopoiesis in CML and MDS. It may be assumed that dysmegakaryocytic features in the latter disorders are partially generated by small to medium-sized megakaryocytes (diameter less than 30 μm). In conclusion, the relative frequency of promegakaryoblasts in the normal bone marrow (range 6–8%) is confirmed by evaluation of the immunohistochemical and cytochemical staining methods (CD61 and ANAE). Furthermore, the ANAE reaction facilitates the recognition of atypical micromegakaryocytes as well as small megakaryocytes. Thus cytochemistry provides a better insight into alterations of these cell lineages in various pathological conditions. This work was supported by the Deutsche Forschungsgemeinschaft (DFG-Th 390/1–2)     

Enantioselective production of levofloxacin by immobilized porcine liver esterase

Porcine liver esterase, which cleaves ofloxacin butyl ester enantioselectively to levofloxacin, was successfully immobilized in calcium alginate and polyacrylamide gel. Immobilized esterase in 5% (w/v) calcium alginate exhibited 58% immobilization efficiency and could be reused five times without severe loss of enzyme activity. On the other hand, entrapped esterase in polyacrylamide gel, composed of 20% of total monomer and 8.3% of cross-linking agent, could be reused 10 times, and 51% of enzyme activity remained after the 10th batch without decrease of enantioselectivity. Compared with entrapped methods, significant reduction of enzyme activity was found in the case of physical adsorption on to QAE-Sephadex.     

Esterase activity assay by flow injection analysis (FIA)

An automatic flow injection analysis (FIA) system for on-line determination of esterase activity has been developed. It is based on a colorimetric method using p-nitrophenyl propionate as substrate. The system permits a linear range analysis up to 0.18 U ml^–1, although the range can be extended up to 1 U ml^–1 without external dilution of the sample. The sampling frequency is of 4 samples per h with a relative standard deviation of 0.9%.     

Simultaneous secretion of seven lignocellulolytic enzymes by an industrial second-generation yeast strain enables efficient ethanol production from multiple polymeric substrates

A major hurdle in the production of bioethanol with second-generation feedstocks is the high cost of the enzymes for saccharification of the lignocellulosic biomass into fermentable sugars. Simultaneous saccharification and fermentation with Saccharomyces cerevisiae yeast that secretes a range of lignocellulolytic enzymes might address this problem, ideally leading to consolidated bioprocessing. However, it has been unclear how many enzymes can be secreted simultaneously and what the consequences would be on the C6 and C5 sugar fermentation performance and robustness of the second-generation yeast strain. We have successfully expressed seven secreted lignocellulolytic enzymes, namely endoglucanase, β-glucosidase, cellobiohydrolase I and II, xylanase, β-xylosidase and acetylxylan esterase, in a single second-generation industrial S. cerevisiae strain, reaching 94.5 FPU/g CDW and enabling direct conversion of lignocellulosic substrates into ethanol without preceding enzyme treatment. Neither glucose nor the engineered xylose fermentation were significantly affected by the heterologous enzyme secretion. This strain can therefore serve as a promising industrial platform strain for development of yeast cell factories that can significantly reduce the enzyme cost for saccharification of lignocellulosic feedstocks.     

Mutations in the equine plasma transferrin and esterase systems

Eleven apparent mutations of the equine plasma transferrin and esterase gene (10 in TF and one in ES ) were found in an analysis of approximately 240000 thoroughbred horses. Eight of the transferrin mutations produced variants not previously recognized in horses. In the two remaining transferrin mutations and the esterase mutation, reduced plasma concentrations of the proteins were demonstrated by immunological techniques and together with the family data indicated the existence of 'null' alleles.