Esterase Isozymes Research Articles

Genome-Wide Association Study and Transcriptome Analysis Provide Candidate Genes for Agronomic Traits of Agaricus bisporus

Agaricus bisporus, belonging to the genus Agaricus and the family Agaricaceae, is a popular commercially cultivated mushroom with rich nutritional and medical values. Cultivation of A. bisporus requires superior cultivars. Understanding the differences between wild and cultivated accession at the genetic level is crucial for mining loci and genes associated with cultivation-related traits, informing future breeding directions. Additionally, the identification of loci and genes associated with important agronomic traits (e.g., yield and quality) facilitates mushroom molecular breeding. In this study, we conducted sequencing of 200 strains of A. bisporus and analyzed genomic variations. Population structure and genetic relationships were investigated for 200 strains. Selection signs and genes were also obtained after selection sweep analysis. Thirteen candidate genes in the selective elimination regions had significantly different expression patterns at the fruit body stage. Moreover, six genes were identified for the esterase isozyme type through a combination of GWAS and RNA data. This study provides insight into environmental adaptation at the genetic base, providing valuable genetic resources for button mushroom molecular breeding to improve environmental adaptation, quality, or yield.

First Report of Root-Knot Nematode, Meloidogyne javanica, Infecting Stachys byzantina in São Paulo, Brazil

Stachys byzantina belongs to the Labiatae and is known by the names "peixinho-da-horta" (Brazil) and "lamb's ear" (USA). Its importance is associated with its medicinal properties (Bahadori et al. 2020) and nutritional aspects (Milião et al. 2022). Root-knot nematodes cause severe damage to plants and suppress production. In January 2021, plants of S. byzantina in the municipality of Jaboticabal (21°14'38.7"S, 48°17'10.6"W) showed symptoms of reduced growth, yellowed leaves and the presence of galls in the roots. Initially, samples of roots from a S. byzantina were analyzed at the Nematology Laboratory (LabNema/UNESP), Jaboticabal, Brazil, estimating 20,000 eggs and juveniles of Meloidogyne sp. in 10 g of roots. To confirm the host ability of the species, a pathogenicity test was performed using Koch's postulate. For this purpose, the test was conducted in a greenhouse where 3,000 eggs and second-stage juveniles (J2) were inoculated onto three plants (n=3) of S. byzantina. After 90 days, the inoculated plants showed the same symptoms as those observed in the field. No symptom or nematode was detected in the uninoculated plant (control). Nematodes were extracted from the roots of inoculated plants and quantified. The perineal pattern of females (n=10) (Netscher and Taylor, 1974) and the labial region of males (n=10) (Eisenback and Hirschmann, 1981) were analyzed and compared with the morphological characteristics of the original description of the species (Chitwood, 1949). For analysis based on esterase isozyme phenotype, the α-method of Esbenshade and Triantaphyllou (1990) was used, and females (n=7) were examined. To confirm identification, whole genomic DNA from an adult female (n=1) was extracted using the Qiagen DNeasy® Blood & Tissue Kit and this sample was used for both genetic sequencing and the sequence-characterized amplified region techniques (SCAR). PCR amplifications were performed for the 18s rRNA gene using primers 988F and 1912R from Holterman et al (2006). Our sequence was deposited in GenBank (NCBI) under the identifier OP422209. Finally, species-specific SCAR primers (Fjav/Rjav, Me-F/Me-R, and Finc-F/Finc-R) designed by Zijlstra (2000) were used to identify Meloidogyne spp. Koch's postulate analysis yielded the following results: (n=1) 9,280 eggs and J2 (Reproduction factor, RF = 33.09); (n=2) 111,720 eggs and J2 (RF = 37.24); (n=3) 59,700 eggs and J2 (RF = 19.9) (RF mean = 30.08). The following characteristics were observed in the perineal region of females: Low and rounded trapezoidal dorsal arch with two distinct lateral lines clearly separating the dorsal and ventral arch regions, similar to the morphological features of the species description by Chitwood (1949). Males had a convex labial plate with a non-raised labial disk joining the submedial labia, a non-rugged labial region, the basal tubercles were usually wider than high, and a rounded tail tip (Eisenback and Hirschmann 1981). The α-esterase enzyme profile showed the J3 phenotype typical of M. javanica (Rm [×100] = 46.0, 54.5, and 58.9). The 18s rRNA sequences grouped Meloidogyne sp. with species such as M. enterolobii, M. incognita, and M. javanica. A DNA fragment of about 700 bp was amplified with Mj (Fjav/Rjav) primers, but not with Me (Me-F/Me-R) and Mi (Finc-F/Finc-R) primers, which confirmed the identification of M. javanica. Accurate identification and characterization of the occurrence of new hosts of M. javanica will allow us to determine the range and geographic distribution of the species. This is the first report on the occurrence of M. javanica on S. byzantina in Brazil. This report is important so that management strategies can be applied to prevent the spread of the pest to other areas.

Effect of Malathion (An Organophospahte) on Electrophoretic Banding Patterns of Esterase Isozymes in Gill, Liver, Brain Tissue of Fresh Water Fish Channa Punctatus (Bloch)

This investigation was conducted to compare the electrophoretetic banding patterns of esterase isozyme in the gill, liver, and brain tissues of the freshwater fish Channa punctatus (Bloch) at 24 hours, 48 hours, 72 hours, and 96 hours after exposure to Malathion (an Organophosphate) to those at the control group. Quantitative analysis of the esterase isozymes was performed using 7.5 native polyacrylamide gel electrophoresis (PAGE) stained with -naphthyl acetate as substrate. The relative mobilities of three esterase Isozyme bands in gill, liver, and brain tissue were determined to be 0.60.05, 0.40.05, and 0.30.05, respectively; these bands were designated Est-1, Est-2, and Est-3, respectively. Control samples from the gill, liver, and brain all had all three esterase bands. Both Est 2 and Est-3 Esterase Isozyme bands in gill and liver tissue were eliminated when fish were subjected to Malathion (an Organophosphate) for 72 and 96 hours, respectively. Malathion induced greater damage in Est-1 and Est-3 brain tissue than in Est-2.

Genome-wide exploration of the GDSL-type esterase/lipase gene family in rapeseed reveals several BnGELP proteins active during early seedling development.

The Gly-Asp-Ser-Leu (GDSL)-type esterase/lipase proteins (GELP) are one of the most important families of lipolytic enzymes and play prominent roles in seed germination and early seedling establishment through mobilizing the lipids stored in seeds. However, there are no comprehensive studies systematically investigating the GELP gene family in Brassica napus (BnGELP), and their biological significance to these physiological processes are far from understood. In the present study, a total of 240BnGELPgenes were identified in B. napuscultivar "Zhongshuang 11" (ZS11), which is nearly 2.3-fold more GELP genes than in Arabidopsis thaliana. The BnGELPgenes clustered into 5 clades based on phylogenetic analysis. Ten BnGELPs were identified through zymogram analysis of esterase activity followed by mass spectrometry, among which five clustered into the clade 5. Gene and protein architecture, gene expression, and cis-element analyses of BnGELP genes in clade 5 suggested that they may play different roles in different tissues and in response to different abiotic stresses. BnGELP99 and BnGELP159 were slightly induced by cold, which may be attributed to two low-temperature responsive cis-acting regulatory elements present in their promoters. An increased activity of esterase isozymes by cold was also observed, which may reflect other cold inducible esterases/lipases in addition to the ten identified BnGELPs. This study provides a systemic view of the BnGELP gene family and offers a strategy for researchers to identify candidate esterase/lipase genes responsible for lipid mobilization during seed germination and early seedling establishment.

Enzymatic and Molecular Identification of Meloidogyne Species in Tomato Orchards in Paraguay

Tomato is a major crop in Paraguay, where it provides a source of employment and income for households. Tomato production can be affected by root-knot nematodes, especially Meloidogyne spp. The unequivocal identification of Meloidogyne spp. in Paraguay has not been conducted yet. This study aims to identify Meloidogyne species in eight tomato production districts of this country by biochemical and molecular techniques. Females of Meloidogyne spp. were extracted from tomato roots and characterized using esterase isozyme phenotypes. In addition, DNA was extracted from nematode eggs, and species-specific SCARs (sequence-characterized amplified regions) were used to confirm the diagnosis. Nematodes were detected in 100% of studied samples (prevalence), of which M. incognita (Est: I2, Rm: 1.1;1.2) and M. javanica (Est: J3, Rm: 1.0, 1.20, 1.35) were present in 39.13% and 26.08% of samples, respectively. One population (8.69%) of Meloidogyne sp. presenting an atypical esterase profile (Rm: 1.0 and 1.3) was only detected in Julián Augusto Saldívar District. Mixed populations, mostly M. incognita and M. javanica, were observed in 26.08% of samples. The SCAR primers incK14F/incK14R amplified specific fragments for M. incognita (399 bp) and M. javanica (670 bp), confirming the enzymatic results. Here, we present the first study of root-knot nematode identification at the species level in Paraguay.

Improvement of phenolic compounds production in callus cultures of Cereus hildmannianus (K.) Schum. by elicitation

Cereus hildmannianus (Cactaceae) is a medicinal plant with various medicinal and industrial applications. Plant biotechnology is an attractive approach for specialized metabolite production under controlled conditions, with the elicitation process being considered a highly effective strategy for increasing the production of bioactive compounds. In this regard, the present study investigated the effects of elicitors, sucrose (S, 1–8 g L−1), UV-C light (UV-C, 10–80 min), salicylic acid (SA, 50–200 µmol), and jasmonic acid (JA, 50–200 µmol), on the profile of esterase isozymes, total phenolic and total flavonoid contents, antioxidant activity (FRAP, DPPH, and ABTS), and the dereplication method by Ultra-High-Performance Liquid Chromatography coupled with Mass Spectrometry (UHPLC-ESI-Q-TOF–MS/MS). The isozyme profile was not significantly changed. The highest accumulations of total phenolics were observed in callus tissues induced with 100 µmol JA (390.8 µg GAE mg−1 DW), 200 µmol SA (355.5 µg GAE mg−1 DW), 20 min UV-C (182.9 µg GAE mg−1 DW), and 6 g L−1 S (122.1 µg GAE mg−1 DW); the highest concentrations of total flavonoids were observed following culture with 200 µmol SA (89.8 µg QE mg−1 DW) and 2 g L−1 S (28.4 µg QE mg−1 DW). Furthermore, the dereplication study allowed putative identification of thirty-six compounds (thirty phenolic acids and organic acids, and six flavonoids). The highest antioxidant activities were obtained with callus tissues induced with 100 µmol JA, 200 µmol SA, 80 min UV-C, and 1 g L−1 S. The elicitors were able to alter the production of phenolic compounds in callus cultures of C. hildmannianus. These results are promising for the clean and sustainable bioproduction of bioactive molecules for pharmaceutical and cosmeceutical purposes, prioritizing the conservation of the species. Elicitation of callus tissues of Cereus hildmannianus with sucrose, UV-C light, salicylic acid, and jasmonic acid enhances the phenolic compound content with antioxidant activity, which is known to be associated with promising antiaging activity.

Electrophoretic banding patterns of esterase isozymes in fresh water fish Channa punctatus

Electrophoretic banding patterns of esterase isozymes in fresh water fish Channa punctatus

Succession of enzymes and microbial biomarkers in the process of vermicomposting: An insight towards valorization of toxic paper mill wastes using Perionyx excavatus (Oligochaeta; Perrier, 1872).

Main insight of the study was to evaluate the potential of Perionyx excavatus, an indigenous earthworm of India towards bioconversion of toxic paper mill wastes. The study had considered a comprehensive study over succession of different enzymes and microbial biomasses alongside removal of heavy metals to reflect the qualitative enrichment of the toxic waste through valorisation procedure. The process of vermicomposting was performed using ratio of paper mill sludge: cow dung: straw as 5:4:1 and Perionyx excavatus for a period of 60days. Qualitative enrichment was considered through detailed evaluation of physicochemical parameters and succession of 19 enzymes using API ZYM technology. Heavy metal loads in terms of Zn, Cr, Cu, Pb along with bioaccumulation factor (BAF) were considered to establish functional role of earthworm in bioremediation. Microbial succession was measured in terms of PLFA biomarkers. Enzymatic enrichment of different isozymes of esterase, aminopeptidase and glycosyl hydrolase were found in the vermicompost. A significant reduction of heavy metals was noticed in vermicomposting of primary sludge as Zn > Pb > Cr > Cu and secondary sludge as Zn > Pb > Cu > Cr. An increase in density of Gram positive bacterial and fungal populations was found at the end phase of vermicomposting process which supports maturity of vermicompost. Presence of different enzyme activities, reduction of heavy metal loads along with rise of eukaryotic PLFA markers, had illustrated the efficiency of vermicomposting as a valorisation procedure in the qualitative enrichment of toxic organic paper mill sludge.

Effect of soil drenching with humic acid, L-methionine and phosphoric acid on Fusarium wilt and induction of enzymes related to oxidative stress and defense in tomato plants

The main objective of the present study was to evaluate the efficacy of humic acid, L-methionine and phosphoric acid as a soil application for controlling Fusarium wilt disease of tomato and their effect on gene expression alterations using protein and isozyme profiling. Two isolates from each of Fusarium oxysporum f. sp. lycopersici (FOL) and F. solani (FS) were obtained from diseased tomato plants. According to pathogenicity tests, all tested isolates were able to infect tomato cultivars; cv. Super Jackal and cv. 745 under greenhouse conditions. In in vitro studies, the addition of humic acid, L-methionine and phosphoric acid to the medium at different concentrations reduced the growth of the tested isolates of the pathogens. Under greenhouse conditions, the addition of humic acid at 2%, 2 g/L of L-methionine and 0.6% of phosphoric acid to infested soil after sowing significantly decreased percentage disease severity of Fusarium wilt at both seasons (2016/17 and 2017/18). Protein bands at molecular weight 47.2 KD, 38.2 KD, 16.3 KD and 15.6 KD appeared only in treated plants. New esterase, peroxidase, chitinase and superoxide dismutase isozyme bands were detected in all treatments while not appear in the control. This results revealed that these treatments were able to induced new protein which increases resistance in tomato plants to Fusarium wilt disease.

Do Metabolic Enzymes Explain Osmoregulation in Juvenile American Eels

During the spring, juvenile eels (Anguilla rostrata) (5-6 cm; 50-200 mg) gather in creeks along the Hudson River Tributary Estuary after migration from the Atlantic Ocean. This requires them to quickly adapt to environments with lower salt concentrations. We wondered how this is achieved. To study eels in a lab setting, we transferred them into Instant Ocean® sea salt (100% artificial sea water [ASW]) or dilutions thereof for 2 weeks; mortality was only observed in 100% ASW (37% expired in 3 hours). Interestingly, after 4 hours, 10 or 50% ASW eels showed little change in body mass, suggesting osmoregulation. While much progress has been made in understanding the mature eel response, few studies have reported enzyme activities in young eels. We hypothesized that dehydrogenases and acetylcholine esterase activities might provide a clue to explain osmoregulation and survival. Supernatants of whole juvenile eel extracts obtained from these experiments (IACUC-approved protocol) were run on 7.5% Tris-glycine PAGE gel (150V, 50 min) and the activities of non–denatured isozyme bands were analyzed with Bio-Rad ChemiDoc™ XRS+ Imager. Denatured supernatant extracts were also run on TGX 4-15% Tris-glycine PAGE gel to assess total protein banding. There was little difference in number of protein bands between eels exposed to 10% vs 50% ASW(2 and 3 weeks); however, band intensities were significantly higher for 50% ASW. Further studies revealed that two MDH (malate dehydrogenase) isozyme bands were present in extracts of eels exposed to a broad range of salinities: a relatively heavier band as mitochondrial (m) MDH and a lighter cytoplasmic (c) MDH. Activities of the mMDH bands were more robust than the cMDH bands at 50% and 10% ASW. Although the mMDH activities in eels exposed to either salinity were similar, the cMDH activities in eels exposed to 50% ASW were greater than in eels exposed to 10%. Additional experiments identified four acetylcholine esterase (AChE) isozyme bands in eel extracts; the activity of only one was significantly prominent in all exposures. Transferring the eels from 50% to 10% ASW reduced activity of the relatively heaviest isozyme band. Notably, we could not discern any fluctuations in lactate dehydrogenase (LDH) levels. In conclusion, while these preliminary findings do not reveal any concrete trends in metabolic regulation tied to survival, it does appear several key enzymes have a translational correlation tied to saline homeostasis. We acknowledge that the interpretation of these experimental results is complicated by a lack of genetic validation (i.e., sequences information and parental lines) - “nature” . In addition, the investigators were unable to track or define the environmental stresses faced during migration by individual juvenile eels before capture, which may manifest itself in unique stress responses - “nurture.”

Genetic Diversity of Egyptian Cowpea (Vigna unguiculata (L.) Walp.) Landraces and their Genetic Relationships Based on Seed Storage Proteins and Isozymes

THE CURRENT research was performed to study the genetic diversity of 19 cowpea landraces (Vigna unguiculata (L.) Walp.) from 7 localities in Egypt using seed storage protein and three isozymes (α-esterase, β-esterase, and peroxidase) gel electrophoresis. The results of the seed protein pattern of the studied landraces revealed a total number of 29 bands with molecular weights ranged from 7 to 265kD (one unique, 26 polymorphic, and 2 monomorphic bands) were detected. The unique band with a molecular weight of 205kD was detected in only one landrace. The number of bands in different 19 landraces ranged from 9-14 bands per landrace. The highest number of bands (14 bands) was detected in one landrace only (Sohag number 2) and the lowest number of bands (9 bands) was detected in 3 landraces (numbers 4,7 and 8 from Sohag, Luxor, and Dakahlia, respectively).Banding patterns of three isozymes (α- esterase, β-esterase and peroxidase) in Egyptian cowpea landraces showed that seven loci were present in all studied landraces of cowpea, one locus of α-esterase, five loci of β-esterase and one locus peroxidase. The number of loci varied in different landraces and ranged from 2-6 loci for α-esterase isozyme, 5-7 loci of β -esterase isozyme and 1-5 loci for peroxidase. The α-esterase and peroxidase banding patterns showed higher polymorphism in cowpea landraces compared with β-esterase.By using the combined protein and isozyme data, high variability among landraces was obtained and analyzed by the NTSYS-pc software program.

Leaves peroxidase and esterase isozymes in sunflower crops exposed to saline environment

Water deficit stress is one of the main problems determining the yield of many crop plants. The study of the effects of salinity on plant growth and development and the search for biochemical stress markers are of great importance in the selection of resistant species. Accumulation and antioxidant activities in response to salinity at different sunflower varieties constitute markers for genetic selection and improvement of plants in the face of tolerance to salinity. Electrophoretic analysis of zymograms of different sunflower varieties grown in a saline environment for 40 days revealed zymograms of peroxidases and esterases specific to varieties tolerant to the saline environment. Peroxidases activity extracted from leaves is augmented in relation to their involvement in the physiological process related to salinity tolerance. In susceptible varieties, peroxidases activity is decreased compared to normal conditions. Variability of activity and polymorphism of isozyme peroxidases and esterases in response to salinity at different sunflower varieties constitute markers for genetic selection and plant improvement in the face of salinity tolerance.

POLYMORPHISM OF ESTERASE ISOENZYMES OF RIPE SEEDS OF SAMPLES OF RADISH (Raphanus sativus L.)

A biochemical approach was used to assess the genetic variability of the seed radish (Raphanus sativus L.) accessions which are distinguished by a wide variety of morphological characters. It is known that the esterase complex in plants has intraspecific specificity; in addition, these enzymes are characterized by tissue specificity. Earlier, the samples of the collections of the genetic resources of the radish were never evaluated for the presence of isozyme forms of esterases in mature seeds of this culture. The establishment of the general variability of isoenzyme systems and the identification of their genetic control make it possible to reveal the subtle mechanisms of the organism's relationship with the environment and homeostasis, which is especially important for long-term storage of samples in genetic seed collections. The development of effective biochemical markers for the rapid assessment of collection, as well as genetically and breeding significant material is also essential. This work allows us to fill the gap that exists in relation to the samples of genetic resources of the radish. From the collection of the Federal Research Center Vavilov All-Russian Institute of Plant Genetic Resources (VIR), 49 radish accessions were selected, belonging to three subspecies, divided according to geographic principle as Chinese, Japanese and European radish. All esterase isozymes of seeds were separated using native vertical electrophoresis in polyacrylamide gel followed by processing for nonspecific esterase. According to their esterase composition, all accessions were subdivided into 7 zymotypes, differing from each other by the presence or absence of certain zones. In total, in the esterase complex of radish seeds, 5 main isozymes with different molecular weights varying from 45.3 kDa to 35.0 kDa were found. All five zones were characterized by a high level of polymorphism among the samples. Based on the composition of isozymes, all genotypes formed 7 zymotypes. Zymotype No. 1, represented by the maximum number of esterases (5 zones), comprised of 43 % of the total number of genotypes. Zymotype No. 2 constituted 33 % of all samples. The rarest zymotypes No. 5 and No. 7 (4 %) differed in the minimum amount of esterase enzymes (2 zones each). Zymotypes No. 2 and No. 4 were characterized by 4 zones. Representatives of two groups, No. 3 and No. 6 had 3 zones in their esterase complex. The quantitative ratio of all esterase zones varied greatly in the studied samples. The minimum content (4.78 %) was found for the B5 zone, the maximum amount (67.44 %) was found for the B1 zone. The prevalence of each zone among all studied samples ranged from 13 to 23 %. Zones B3 (Mr = 39.7 kDa) and B4 (Mr = 37.1 kDa) were the most common among all esterase isozymes; these zones were observed in 23 % of genotypes. For 22 % of representatives, the B2 zone was characteristic (Mr = 42.9 kD). Zones B1 (Mr = 45.3 kD) and B5 (Mr = 35 kD) were less common, 19 % and 13 %, respectively. The average heterozygosity of isozygous forms of esterases of the studied radish samples was Htotal = 0.212, with variance for the same samples Var(Htotal) = 0.0007. Cluster analysis of esterase enzymes divided the studied set of radish samples into European and Asian subspecies and varieties, and together with phenotypic traits, it allowed constructing a dendrogram corresponding to the botanical, agrobiological and geographical location of the samples. It should be noted that the accessions of the European subspecies radish are located in two clusters, and the accessions of Russian origin form a separate group in the first cluster, and the samples of European origin are grouped in the third cluster which also includes Japanese radishes of European origin. Perhaps this division is associated with the peculiarities of the selection process in creating these samples. Based on the data obtained, esterase enzymes are recommended as biochemical markers in genetic selection experiments.

Biochemical studies on electrophoretic banding pattern among the different isolates of Colletotrichum capsici causing fruit rot in chilli

Ten isolates of Colletotrichum capsici collected from different regions of Vidarbha and Marathwada (Maharashtra), Bidar district (Karnataka) and Guntur district (Andra Pradesh) showing varied type of pathogenic ability against chilli variety Jayanti and designated as strongly, moderately and weakly pathogenic on the basis of per cent leaf area covered by the disease. Electrophoresis pattern of polyphenol oxidase, esterase and protein have been used to estimate the polymorphism in different fungal populations or species. Therefore, studies were undertaken to know the biochemical variation among the isolates of C. capsici in the form of appearance of isozyme and protein banding pattern on polyacrylamide gel. Electrophoretic protein banding pattern revealed that, Rm values ranged between 0.208 to 0.725 possessing 1 to 4 bands. Isolates among weakly to highly pathogenic, 5 isolates exhibited one band each, while two bands each were existed in 3 isolates. Three bands of Cc6 (Rm value 0.208, 0.350 and 0.725) relates to strongly pathogenic. Polyphenol oxidase isozyme was estimated and Rm values were observed from 0.057 to 0.192, possessing 2 to 3 bands. Eight isolates showed presence of 3 bands each whereas, 2 isolates exhibited 2 bands with Rm values 0.142 and 0.192. Esterase isozyme pattern was observed in all the test isolates of C. capsici and Rm values ranged from 0.048 to 0.544 possessing 3 to 4 bands.

A Moraxella Virulence Factor Catalyzes an Essential Esterase Reaction of Biotin Biosynthesis.

Pimeloyl-acyl carrier protein (ACP) methyl ester esterase catalyzes the last biosynthetic step of the pimelate moiety of biotin, a key intermediate in biotin biosynthesis. The paradigm pimeloyl-ACP methyl ester esterase is the BioH protein of Escherichia coli that hydrolyses the ester bond of pimeloyl-ACP methyl ester. Biotin synthesis in E. coli also requires the function of the malonyl-ACP methyltransferase gene (bioC) to employ a methylation strategy to allow elongation of a temporarily disguised malonate moiety to a pimelate moiety by the fatty acid synthetic enzymes. However, bioinformatics analyses of the extant bacterial genomes showed that bioH is absent in many bioC-containing bacteria. The genome of the Gram-negative bacterium, Moraxella catarrhalis lacks a gene encoding a homolog of any of the six known pimeloyl-ACP methyl ester esterase isozymes suggesting that this organism encodes a novel pimeloyl-ACP methyl ester esterase isoform. We report that this is the case. The gene encoding the new isoform, called btsA, was isolated by complementation of an E. coli bioH deletion strain. The requirement of BtsA for the biotin biosynthesis in M. catarrhalis was confirmed by a biotin auxotrophic phenotype caused by deletion of btsA in vivo and a reconstituted in vitro desthiobiotin synthesis system. Purified BtsA was shown to cleave the physiological substrate pimeloyl-ACP methyl ester to pimeloyl-ACP by use of a Ser117-His254-Asp287 catalytic triad. The lack of sequence alignment with other isozymes together with phylogenetic analyses revealed BtsA as a new class of pimeloyl-ACP methyl ester esterase. The involvement of BtsA in M. catarrhalis virulence was confirmed by the defect of bacterial invasion to lung epithelial cells and survival within macrophages in the ΔbtsA strains. Identification of the new esterase gene btsA exclusive in Moraxella species that links biotin biosynthesis to bacterial virulence, can reveal a new valuable target for development of drugs against M. catarrhalis.

Androgynomonoecious Jatropha curcas: Chromosomes, Isozymes, and Flowers Gender

Jatropha curcas (J. curcas) is usually monoecious plants, which have male and female flowers on the same inflorescence. However, J. curcas can be found as an androgynomonoecious plant (have male, female, and hermaphrodite flowers), even though very rare. Androgynomonoecious J. curcas can be identified after six months of planting when it had started flowering. Therefore, it is important to identify the characteristics of androgynomonoecious J. curcas that can differentiate between androgynomonoecious and monoecious plants in earlier stages of growth. The objectives of the research were to observe isozymes, chromosome and flowers gender of androgynomonoecious and monoecious J. curcas Banten and Lampung accessions. Seeds from five genotypes of J. curcas were used in the research. The observation was carried out on the chromosome and isozymes (Peroxidase and Esterase isozymes) could be used as markers to differentiate androgynomonoecious and monoecious plants. Observations about the flower gender from offsprings derived from different seeds were important to know the inheritance of flower gender. The androgynomonoecious and monoecious J. curcas were diploid with number of chromosomes 2n=2x=22. The chromosomes of androgynomonoecious have longer than that of monoecious J. curcas. The isozymes of androgynomonoecious J. curcas had four alleles and monoecious J. curcas (Banten female monoecious) had three alleles. The flower inflorescence and gender derived from androgynomonoecious plants were unstable, due to androgynomonoecious is intermediate state.

Overexpression of an alternative allele of carboxyl/choline esterase 4 (CCE04) of Tetranychus urticae is associated with high levels of resistance to the keto-enol acaricide spirodiclofen.

Spirodiclofen is an acaricide that targets lipid biosynthesis by inhibiting acetyl-coenzyme A carboxylase. Spirodiclofen resistance in spider mites has been previously documented and was associated with overexpression of CYP392E10, a cytochrome P450 mono-oxygenase that metabolizes spirodiclofen. However, additional mechanisms have been suggested in several studies and a carboxyl/choline esterase gene, CCE04, was shown to be overexpressed in two genetically different strains, SR-VP and SR-TK, both exhibiting high spirodiclofen resistance levels. We identified two different CCE04 alleles in both resistant strains, CCE04SR-VP and CCE04London , with CCE04SR-VP being highly overexpressed. Isoelectric focusing analysis confirmed the overexpression of a single esterase isozyme, while copy number and random fragment length polymorphism analysis revealed that CCE04SR-VP overexpression was more likely due to selection for the CCE04SR-VP allele rather than gene amplification. Both CCE04 alleles were functionally expressed using the Pichia expression system. Functional enzyme assays revealed only limited kinetic differences between CCE04 isoforms for model substrates. In addition, inhibition/competition experiments with spirodiclofen suggested a similar interaction with both enzymes, whereas its active metabolite, spirodiclofen enol, did not inhibit enzyme activity. Our study suggests that selection with spirodiclofen results in enrichment of a specific allele of CCE04 (CCE04SR-VP ) in two genetically independent strains, which is highly overexpressed. Based on kinetic enzyme data, however, quantitative rather than qualitative differences between CCE04SR-VP and CCE04London seem more likely to be involved in resistance. Our findings are discussed in the light of a possible spirodiclofen resistance mechanism, with sequestration of spirodiclofen by CCE04SR-VP being a likely hypothesis. © 2019 Society of Chemical Industry.

Variasi Biokimia Genetik Populasi Ikan Betutu (Oxyeleotris marmorata, BLKr.) di Waduk Penjalin Brebes

Penjalin water reservoir in Brebes Regency, Central Java, is one of the habitats of the sand goby. A study on its genetic diversity using approaches of isozyme analysis was needed to support domestication of the fish in this area. This study was aimed at the biochemical-genetic variation of sand goby population in Penjalin water reservoir based on esterase (EST), peroxidase (PER), malate dehydrogenase (MDH), aspartate amino-transferase (AAT), and acid phosphatase (ACP) polymorphisms. Visualization of the isozymes was carried out employing horizontal electrophoretic technique with potato starch gel and buffer system of L-histidin monohydrate. Of the five isozymes, ACP was not well-visualized in all samples tested while the remaining four showed no polymorphisms. It could be concluded that there is no biochemical-genetic variation of sand goby population in Penjalin water reservoir based on isozymes of EST, PER, MDH, and AAT.

Properties and Extraction of Crude Pectin Esterase from Potato (Solanum Tuberosum)

In order to study the enzymatic properties of potato esterase, the crude enzyme of potato esterase was extracted with phosphate buffer solution, and the extraction conditions were optimized. The optimal condition is: the optimal condition for phosphate buffer solution pH 7.0, water bath time for 30 min, extraction temperature 35°C, and solid-liquid ratio 1:4 (g/mL). The property analysis showed that the potato esterase extracted from phosphate buffer solution was not a single esterase, but a kind of esterase isozyme. At 273nm and 325nm, the potato esterase crude enzyme had two characteristics uv absorption peak, its isoelectric point was near pI 4.4, and its molecular weight was roughly distributed at 16.4, 20.866, 22.73 and 40.286 KDa. In 40 °C ∼ 45 °C, under the condition of pH 7.0, better able to maintain its activity, and seven kinds of metal ions are inhibitory effect to it. This study provides a theoretical basis for further understanding the enzymatic characteristics of potato esterase and its application in pesticide residue detection.

Morphology and isozyme variation among madeira vine (Anredera cordifolia) accessions from southeastern part of Central Java, Indonesia

Abstract. Restykania, Suratman, Pitoyo A, Suranto. 2019. Morphology and isozyme variation among madeira vine (Anredera cordifolia) accessions from southeastern part of Central Java, Indonesia. Biodiversitas 20: 3024-3032. The genetic diversity among madeira vine accessions from southeastern part of Central Java has not been investigated in detail. Information on genetic diversity and relationship among accessions of this plant will provide important input into determining resourceful management strategies and guiding the improvement of plants through plant breeding programs. The objective of this study was to evaluate variation of morphology and isozyme among madeira vine (Anredera cordifolia (Ten.) Steenis) accessions from southeastern part of Central Java. A total of 13 madeira vine accessions were used for morphological observations and isozyme analysis. Morphological diversity was characterized based on measurements and observations of vegetative structures such as stems, leaves, and aerial tubers. Identification of biochemical markers was done using peroxidase and esterase isozyme systems. A relationship dendrogram among accessions was constructed based on the genetic similarity matrix by applying a cluster analysis. The analysis of quantitative and qualitative morphological characters revealed that the majority of the tested traits showed variation, indicating that there was variability among the tested madeira vine accessions. Polymorphism was observed using isozyme of peroxidase (i.e. two banding patterns) and esterase (i.e. five banding patterns). Based on the dendrogram at a level of 63 % similarity, it showed distinct separation of 13 madeira vine accessions into two major clusters. Cluster I consisted of nine accessions and the closest relationship was shown between KLT 3 and KLT 1 accessions that had 94 % of similarity coefficient. The remaining four accessions were clustered separately as Cluster II with similarity coefficient of 73 %. Therefore, genetic characterization based on morphological and isozyme markers obtained in this study is valuable to understand genetic variability and relationship among madeira vine accessions in southeastern part of Central Java.