Screening ofExiguobacterium acetylicum from soil samples showing enantioselective and alkalotolerant esterase activity

About 3,000 bacterial colonies with esterase activities were isolated from soil samples by enrichment culture and halo-size on Luria broth-tributyrin (LT) plates. The colonies were assayed for esterase activity in microtiter plates using enantiomerically pure (R)- and (S)-2-phenylbutyric acid resorufin ester (2PB-O-res) as substrates. Two enantioselective strains (JH2 and JH13) were selected by the ratio of initial rate of hydrolysis of enantiomerically pure (R)- and (S)-2-PB-O-res. When cell pellets were used, both strains showed hgh apparent enantioselectivity (E _app>100) for (R)-2PB-O-res and were identified asExiguobacterium acetylicum. The JH13 strain showed high esterase activity onp-nitrophenyl acetate (pNPA), but showed low lipase activity onp-nitrophenyl palmitate (pNPP). The esterase was located in the soluble fraction of the cell extract. The crude intracellular enzyme preparation was stable at a pH range from 6.0 to 11.0.     

Balance of Activities of Alcohol Acetyltransferase and Esterase in Saccharomyces cerevisiae Is Important for Production of Isoamyl Acetate

Isoamyl acetate is synthesized from isoamyl alcohol and acetyl coenzyme A by alcohol acetyltransferase (AATFase) in Saccharomyces cerevisiae and is hydrolyzed by esterases at the same time. We hypothesized that the balance of both enzyme activities was important for optimum production of isoamyl acetate in sake brewing. To test this hypothesis, we constructed yeast strains with different numbers of copies of the AATFase gene (ATF1) and the isoamyl acetate-hydrolyzing esterase gene (IAH1) and used these strains in small-scale sake brewing. Fermentation profiles as well as components of the resulting sake were largely alike; however, the amount of isoamyl acetate in the sake increased with an increasing ratio of AATFase/Iah1p esterase activity. Therefore, we conclude that the balance of these two enzyme activities is important for isoamyl acetate accumulation in sake mash.     

Activities of formylmethanofuran dehydrogenase,methylenetetrahydromethanopterin dehydrogenase,methylenetetrahydromethanopterin reductase,and heterodisulfide reductase in methanogenic bacteria

The activities of formylmethanofuran dehydrogenase, methylenetetrahydromethanopterin dehydrogenase, methylenetetrahydromethanopterin reductase, and heterodisulfide reductase were tested in cell extracts of 10 different methanogenic bacteria grown on H₂/CO₂ or on other methanogenic substrates. The four activities were found in all the organisms investigated: Methanobacterium thermoautotrophicum,M. wolfei, Methanobrevibacter arboriphilus, Methanosphaera stadtmanae, Methanosarcina barkeri (strains Fusaro and MS), Methanothrix soehngenii, Methanospirillum hungatei, Methanogenium organophilum, and Methanococcus voltae. Cell extracts of H₂/CO₂ grown M. barkeri and of methanol grown M. barkeri showed the same specific activities suggesting that the four enzymes are of equal importance in CO₂ reduction to methane and in methanol disproportionation to CO₂ and CH₄. In contrast, cell extracts of acetate grown M. barkeri exhibited much lower activities of formylmethanofuran dehydrogenase and methylenetetrahydromethanopterin dehydrogenase suggesting that these two enzymes are not involved in methanogenesis from acetate. In M. stadtmanae, which grows on H₂ and methanol, only heterodisulfide reductase was detected in activities sufficient to account for the in vivo methane formation rate. This finding is consistent with the view that the three other oxidoreductases are not required for methanol reduction to methane with H₂.     

Timing of appearance of versiconal hemiacetal acetate esterase and versiconal cyclase activity in cultures ofAspergillus parasiticus

The sequence of steps versiconal hemiacetal acetate (VHA) to versiconal (VL) catalyzed by an esterase and VL to versicolorin B (VB) catalyzed by VL cyclase has been previously demonstrated in cell-free systems fromAspergillus parasiticus. VHA esterase and VL cyclase activities were estimated by determining the amounts of VL and VB after incubation of VHA in cell-free extracts from mycelia that were either synthesizing or not synthesizing aflatoxins. VHA esterase activity but not VL cyclase activity was present in extracts from cells grown in a nonaflatoxin-producing medium. VHA esterase activity was present in extracts from mycelia grown in aflatoxin-producing medium harvested after one to six days of incubation. VL cyclase activity was absent at one day, low at two days, maximal at three to five days, and lower at six days. VL cyclase activity appears in the later part of the growth period which is also the period of aflatoxin biosynthesis. This supports a role for VL cyclase in the aflatoxin biosynthetic pathway.     

The Aspergillus parasiticus estA-Encoded Esterase Converts Versiconal Hemiacetal Acetate to Versiconal and Versiconol Acetate to Versiconol in Aflatoxin Biosynthesis

In aflatoxin biosynthesis, the pathway for the conversion of 1-hydroxyversicolorone to versiconal hemiacetal acetate (VHA) to versiconal (VHOH) is part of a metabolic grid. In the grid, the steps from VHA to VHOH and from versiconol acetate (VOAc) to versiconol (VOH) may be catalyzed by the same esterase. Several esterase activities are associated with the conversion of VHA to VHOH, but only one esterase gene (estA) is present in the complete aflatoxin gene cluster of Aspergillus parasiticus. We deleted the estA gene from A. parasiticus SRRC 2043, an O-methylsterigmatocystin (OMST)-accumulating strain. The estA-deleted mutants were pigmented and accumulated mainly VHA and versicolorin A (VA). A small amount of VOAc and other downstream aflatoxin intermediates, including VHOH, versicolorin B, and OMST, also were accumulated. In contrast, a VA-accumulating mutant, NIAH-9, accumulated VA exclusively and neither VHA nor VOAc were produced. Addition of the esterase inhibitor dichlorvos (dimethyl 2,2-dichlorovinylphosphate) to the transformation recipient strain RHN1, an estA-deleted mutant, or NIAH-9 resulted in the accumulation of only VHA and VOAc. In in vitro enzyme assays, the levels of the esterase activities catalyzing the conversion of VHA to VHOH in the cell extracts of two estA-deleted mutants were decreased to approximately 10% of that seen with RHN1. Similar decreases in the esterase activities catalyzing the conversion of VOAc to VOH were also obtained. Thus, the estA-encoded esterase catalyzes the conversion of both VHA to VHOH and VOAc to VOH during aflatoxin biosynthesis.     

Increase of acetate ester-hydrolysing esterase activity in mixed cultures of Saccharomyces cerevisiae and Pichia anomala

Aim: To examine the efficacy of mixed cultures with Saccharomyces cerevisiae and Pichia anomala on flavour profiles of alcoholic beverages, a Pichia mutant with low levels of ethyl acetate that negatively impact on the sensory quality was isolated. Methods and Results: A petite mutant isolated from P. anomala NBRC 10213 treated with ethidium bromide had the lower activity of ethyl acetate‐hydrolysing esterase (EAHase) than the wild‐type in crude extracts. In the fermentation tests of pure cultures, the P. anomala mutant produced less ethanol, acetate and ethyl acetate than the wild‐type. In mixed cultures with S. cerevisiae, the P. anomala mutant died quicker and produced lower amounts of ethyl acetate than the wild‐type. Mixed cultures of S. cerevisiae and P. anomala showed higher activities of EAHase than pure culture of S. cerevisiae throughout the fermentation periods. The transition to the formation of acetate esters was considerably analogous to the transition to the activity of acetate ester‐hydrolysing esterase with little time lag. Conclusions: The P. anomala mutant was superior to the wild‐type in flavour profiles. The higher ethyl acetate concentrations formed mainly by P. anomala in mixed cultures are the primary stimulus for the EAHase in S. cerevisiae and the activity of acetate ester‐hydrolysing esterase is crucial to the formation of acetate esters in mixed cultures of S. cerevisiae and P. anomala. Significance and Impact of the Study: An application of non‐Saccharomyces yeast, P. anomala to enhance the sensory quality in alcoholic beverage and a mechanism of the formation of acetate esters in mixed cultures with S. cerevisiae and P. anomala were offered.     

Phytochemical, antimicrobial, antioxidant and antigenotoxic potentials of Cyperus rotundus extracts

The aqueous, ethyl acetate, methanolic and Total Oligomer Flavonoids (TOF) enriched extracts, obtained from the aerial parts of Cyperus rotundus, were investigated for their contents in phenolic compounds. Antioxidative activity using the NBT/riboflavin assay system, antimicrobial activity against Gram positive and Gram negative bacterial reference strains as well as antigenotoxic activity tested with the SOS chromotest assay were also studied. Significant antibacterial activity against reference strains; Staphylococcus aureus, Enterococcus faecalis, Salmonella enteritidis and Salmonella typhimurium, was detected in the presence of ethyl acetate and TOF enriched extracts. In addition to their antimicrobial activity, the same extracts showed a significant ability to inhibit nitroblue tetrazolium reduction by the superoxide radical in a non enzymatic O₂^.− generating system, and were also able to reduce significantly the genotoxicity induced by nifuroxazide and Aflatoxin B1. The antioxidant, antimicrobial and antigenotoxic activities exhibited by C. rotundus depend on the chemical composition of the tested extracts.     

Acetate formation from CO and CO2 by cell extracts of Peptostreptococcus productus (strain Marburg)

Cell extracts of Peptostreptococcus productus (strain Marburg) obtained from CO grown cells mediated the synthesis of acetate from CO plus CO₂ at rates of 50 nmol/min × mg of cell protein. ¹⁴CO was specifically incorporated into C₁ of acetate. No label exchange occurred between ¹⁴C₁ of acetyl-CoA and CO, indicating that ¹⁴CO incorporation into acetate was by net synthesis rather than by an exchange reaction. In acetate synthesis from CO plus CO₂ the latter substrate could be replaced to some extent by formate or methyl tetrahydrofolate as the methyl donor. The methyl group of methyl cobalamin was incorporated into acetate ony at very low activities. The cell extracts contained high levels of enzyme activities involved in acetate or cell carbon synthesis from CO₂. The following enzymic activities were detected: CO: methyl viologen oxidoreductase, formate dehydrogenase, formyl tetrahydrofolate synthetase, methenyl tetrahydrofolate cyclohydrolase, methylene tetrahydrofolate dehydrogenase, methylene tetrahydrofolate reductase, phosphate acetyltransferase, acetate kinase, hydrogenase, NADPH: benzyl viologen oxidoreductase, and pyruvate synthase. Some kinetic and other properties were studied.     

The effect of growth temperature on esterase patterns in psychrotrophic Bacillus species and other Gram-positive genera

The growth temperature of Bacillus stearothermophilus was previously reported to affect the esterase band pattern obtained after gel electrophoresis. In this study gel electrophoresis of esterases was done on a group of Antarctic bacterial strains cultivated at different temperatures to investigate whether band shift due to a change in growth temperature was a general phenomenon or limited only to select groups. Most strains studied were in the Bacillus genus. Standard strains of known Bacillus species and other Gram-positive genera were examined for comparative purposes. A change in the esterase band pattern was observed as a result of variation in growth temperature. Two major esterase band were dominant in this diverse group of species.     

不同种群棉铃虫三龄幼虫酯酶活性频率分布与抗药性的关系

选用室内饲养的棉铃虫Helicoverpa armigera偃师和湖北2个敏感品系、对辛硫磷高抗的PCP20品系、对氰戊菊酯高抗的YG45品系及1999或2000年采自山东阳谷、河北邯郸和河南安阳的田间高抗种群,江苏徐州、湖北武汉的田间中等抗性水平种群和新疆沙湾的田间敏感种群,采用酶标板酶动力学法测定了各品系（种群）的3龄幼虫个体酯酶活性频率分布和平均酯酶活性。结果表明,偃师敏感品系、湖北敏感品系和新疆沙湾田间敏感种群的酯酶活性个体频率分布相似,三个品系（种群）的平均酯酶活性相近,分别为991、1 138、1 055 mOD·min^-1·larva^-1。室内选育的PCP20抗性品系、YG45抗性品系及山东阳谷、河北邯郸 、河南安阳田间高抗种群的高酯酶活性（活性在1 800 mOD·min^-1·larva^-1以上）个体频率明显高于三个敏感品系（种群）,平均酯酶活性在1 510～2 482 mOD·min^-1·larva^-1之间。江苏徐州、湖北武汉的田间中抗水平种群高酯酶活性个体频率及平均酯酶活性都介于敏感和高抗品系（种群）之间,平均酯酶活性为1 258～1.404 mOD·min^-1·larva^-1。棉铃虫各品系（种群）平均酯酶活性与对拟除虫菊酯类杀虫剂抗性个体频率的相关性要比对有机磷类的高,相关系数分别为0.82和0.42。分析各品系（种群）高酯酶活性个体频率与棉铃虫对拟除虫菊酯类、有机磷类杀虫剂抗性个体频率的相关性,得到相似的结果。考虑到酯酶并不是棉铃虫对拟除虫菊酯抗性的主要机理,建议酯酶活性可作为棉铃虫抗药性生化检测的一个参考指标。本文还讨论了酯酶与棉铃虫对拟除虫菊酯类杀虫剂及有机磷类杀虫剂抗性的关系。     

Chemotaxonomic Profiling Through NMR1

A metabolite screening of cyanobacteria was performed by nuclear magnetic resonance (NMR) analysis of the soluble material obtained through sequential extraction of the biomass with three different extractive ability solvents (hexane, ethyl acetate, and methanol). Twenty-five strains from the Coimbra Collection of Algae (ACOI) belonging to different orders in the botanical code that represent three subsections of the Stainer-Rippka classification were used. The ¹H NMR spectra of hexane extracts showed that only two strains of Nostoc genus accumulated triacylglycerols. Monogalactosyldiacylglycerols and digalactosyldiacylglycerols were the major components of the ethyl acetate extracts in a mono- to digalactosyldiacylglycerols ratio of 4.5 estimated by integration of the signals at δ 3.99 and 3.94 ppm (sn3 glycerol methylene). Oligosaccharides of sucrose and mycosporine-like amino acids, among other polar metabolites, were detected in the methanolic extracts. Strains of Nostocales order contained heterocyst glycolipids, whereas sulphoquinovosyldiacylglycerols were absent in one of the studied strains (Microchaete tenera ACOI 1451). Phosphathidylglycerol was identified as the major phospholipid in the methanolic extracts together with minor amounts of phosphatidylcholine based on ¹H, ³¹P 2D correlation experiments. Chemotaxonomic information could be easily obtained through the analysis of the δ 3.0–0.5 ppm (fatty acid distribution) and δ 1.2–1.1 ppm (terminal methyl groups of the aglycons in heterocyst glycolipids) regions of the ¹H NMR spectra of the ethyl acetate and methanol extracts, respectively.     

Trichoderma asperelloides antagonism to nine Sclerotinia sclerotiorum strains and biological control of white mold disease in soybean plants

Sclerotinia sclerotiorum is an important plant pathogen with worldwide distribution that causes severe economic losses of various agricultural crops such as soybean. This fungus is normally controlled with synthetic chemical fungicides that pose risks to the environment, and can be harmful to human health, and they can also induce resistance in pests. The aim of this study was to investigate the potential of Trichoderma asperelloides as a biocontrol agent towards white mold disease on soybeans crops. The antagonism of two strains of T. asperelloides (T25 and T42) isolated from soil samples was determined in-vitro by dual-culture confrontation testing on nine S. sclerotiorum strains obtained from sclerotia collected on diseased soybean plants. The mycelial growth and inhibition of carpogenic and ascospore germination by T. asperelloides extracts, as well as the efficacy of these on white mold control in soybeans were evaluated. Both strains of T. asperelloides exhibited high potential of antagonism. Methanolic and ethyl acetate extracts of the two T. asperelloides strains showed excellent growth inhibition (60–100%) on all of the pathogens tested. The ethyl acetate extracts of both T. asperelloides strains exhibited the highest efficacy against carpogenic germination, decreasing by 20–30% the number of ascospores per apothecium. Strains of T. asperelloides tested were more efficient in controlling white mold than two commercial products made from Trichoderma harzianum. The new strains of T. asperelloides have potential for successful biological control of white mold disease of soybean crops in the field.     

Relationship between macrotetrolide production and specific activity of some hydrolases in a high and a low producing strain of Streptomyces griseus

The activities of five hydrolytic enzymes in the culture filtrate and in cell-free extracts from strains of Streptomyces griseus, differing in macrotetrolide production, have been determined over a fermentation period of 200 h. The specific activities of phosphatase, phosphodiesterase, and adenosine triphosphatase in the medium, and phosphatase and phosphodiesterase in the cell-free extract were lower in the low than in the high producing strain. No significant difference was found between the strains, for adenosine triphosphatase and protease activity in the cell-free extract or protease activity in the medium. The specific activity of esterase was higher in the low than in the high producing strain.     

The study of antagonistic interactions among pelagic bacteria: a promising way to coin environmental friendly antifouling compounds

Ten strains of marine bacteria (SCH0401–SCH0410) were isolated from Ayajin, the east coast of South Korea. In spectrophotometer based chemotaxis assay the ethyl acetate extract (300 μg) of SCH0402 decreased the optical density (OD) of the motile target strains SCH0401, SCH0402, SCH0407 and SCH0408 by two to six times when compared to control. Tributyltin oxide (TBTO) decreased the OD of all target strains by only two times. The most active strain SCH0402 was identified as Shewanella oneidensis by using 16S rDNA gene sequence analysis. Similarly, the target motile strains SCH0401, SCH0402, SCH0407 and SCH0408 were identified as Alteromonas marina, Shewanella oneidensis, Roseobacter gallaeciensis and Bacillus atrophaeus, respectively. The growth inhibition zone produced by the test bacterial extracts against the target strains were three to eight times smaller when compared to that of TBTO. Even though, SCH0402 showed six times weaker antibacterial activity, the repellent activity was three times stronger than TBTO. Therefore, the higher negative chemotactic activity would be better to select eco-friendly antifouling compounds than the other antibacterial activities.     

Heterologous expression of a thermophilic esterase in <Emphasis Type="Italic">Kluyveromyces</Emphasis> yeasts

In the present work, a thermophilic esterase from Thermus thermophilus HB27 was cloned into Kluyveromyces marxianus and into Kluyveromyces lactis using two different expression systems, yielding four recombinant strains. K. lactis showed the highest esterase expression levels (294 units per gram dry cell weight, with 65% of cell-bound enzyme) using an episomal system with the PGK promoter and terminator from Saccharomyces cerevisiae combined with the K. lactis k1 secretion signal. K. marxianus showed higher secretion efficiency of the heterologous esterase (56.9 units per gram dry cell weight, with 34% of cell-bound enzyme) than K. lactis. Hydrolytic activities for the heterologous esterases were maximum at pH values between 8.0 and 9.0 for both yeast species and at temperatures of 50 °C and 45 °C for K. marxianus and K. lactis, respectively. When compared to previously published data on this same esterase produced in the original host or in S. cerevisiae, our results indicate that Kluyveromyces yeasts can be considered good hosts for the heterologous secretion of thermophilic esterases, which have a potential application in biodiesel production or in resolving racemates.     

Biochemical characterisation of esterases active in hydrolysing xenobiotics in wheat and competing weeds

Esterase activities toward model xenobiotic substrates ( p -nitrophenyl acetate, naphthyl acetate) and pesticide esters (diclofop methyl, bromoxynil octanoate, binapacryl) have been compared in crude extracts from wheat (Triticum aestivum L.) and Triticum progenitors of wheat. Esterase activities were also determined in the weeds, wild oat ( Avena fatua ) and two populations of black-grass ( Alopecurus myosuroides ), one of which (Rothamsted) is susceptible to herbicides, while the other (Peldon) shows cross-resistance to multiple classes of herbicides. Esterase activity toward the model substrates was highest in wheat, while the weeds were more active in hydrolysing the pesticides. Using isoelectric focussing (pH 4–8), 13 proteins with esterase activity toward α -naphthyl acetate could be resolved in hexaploid wheat (genome AABBDD). The pattern of these activities was most similar to that of the diploid progenitor T. tauschii (DD), excepting a major acidic esterase (pI 4.6), which originated from T. urartu (AA). Resolved esterase activities in the weeds were distinct from those observed in the Tritcum species. However, unlike the case with other classes of xenobiotic-metabolising enzymes, the complement of esterases in the Peldon and Rothamsted populations of black-grass appeared to be identical. In all species, the more basic esterases (>pI 5.0) were sensitive to inhibition by organophosphate and carbamate insecticides, suggesting that they were B-class esterases. In contrast, the acidic wheat esterase (pI 4.6) with the greatest activity toward α -naphthyl acetate was insensitive to insecticides. This wheat-specific esterase was purified 7000-fold by a combination of hydrophobic interaction chromatography, gel filtration and anion-exchange chromatography. The purified esterase behaved as a monomeric 45-kDa protein showing high activity toward p -nitrophenyl acetate and α -naphthyl acetate, but limited activity toward the pesticides.     

Inhibitors of sensillar esterase reversibly block the responses of moth pheromone receptor cells

Three volatile alkyl-thio-trifluoro propanones inhibiting the esterase in olfactory sensilla of the silkmoths Antheraea polyphemus and A. pernyi were used to test the hypothesis that enzymatic pheromone degradation is responsible for the decline of the receptor potential after pheromone stimulation. Test stimuli were the pheromone components (E,Z)-6,11-hexadecadienyl acetate, a substrate for the sensillar esterase, and (E,Z)-6,11-hexadecadienal, not degraded by the esterase. Each compound acts on a separate type of receptor cell. In both receptor cell types the trifluoro propanones caused a partially reversible reduction of sensitivity as indicated by smaller receptor potential amplitudes and lower nerve impulse frequencies. Since application of the esterase inhibitors did not prolong the decline of the receptor potential of the acetate cell, the esterase is not responsible for the rapid pheromone deactivation. When the trifluoro propanones were applied after the pheromone at high concentrations, they rapidly inhibited (repolarized) both receptor cell types. Experiments with local application of trifluoro propanones revealed that the inhibitory effect spreads within seconds along the length of the sensillum. The inhibition of the electrophysiological responses might be due to an antagonistic action of the trifluoro propanones at the pheromone-binding sites, either at the receptor molecules or at the pheromone-binding protein. Accepted: 4 February 1998     

Amino acid and glucose fermentation by Treponema denticola

Summary Treponema denticola was grown in serum-containing media to which ¹⁴C-labelled compounds were added. Determinations of radioactivity in the products formed indicated that the organism fermented alanine, cysteine, glycine, serine, and glucose. Fermentation products included acetate, lactate, succinate, formate, pyruvate, ethanol, CO₂, H₂S, and NH₃. The products formed from glucose constituted a small portion of the total products. Assays of enzymatic activities in cell extracts indicated that the organism degraded glucose via the Embden-Meyerhof pathway. T. denticola possessed a coenzyme A-dependent CO₂-pyruvate exchange activity associated with a clostridial-type clastic system for pyruvate metabolism. Phosphotransacetylase and acetate kinase activities were present in cell extracts. Acetyl phosphate formation and benzyl viologen reduction were detected when cell extracts were incubated with pyruvate, serine or cysteine. The data indicate that T. denticola is an amino acid fermenter and that it possesses the enzymes needed for the fermentation of glucose. However, glucose does not serve as the primary substrate when the organism grows in media including both this carbohydrate and amino acids.     

Purification and characterization of antibacterial substances produced by a marine bacterium Pseudoalteromonas haloplanktis strain

Aims: To purify and characterize compounds with antimicrobial activity from Pseudoalteromonas haloplanktis inhibition (INH) strain. Methods and Results: The P. haloplanktis isolated from a scallop hatchery was used to analyse antibacterial activities. Crude extracts were obtained with ethyl acetate of the cultured broth, after separation of bacterial cells, and assays against six strains of marine bacteria and nine clinically important pathogenic bacteria. The active compounds were purified from ethyl acetate extracts, by a combination of SiO₂ column and thin layer chromatography. Two active fractions were isolated, and chemical structures of two products from the major one were unambiguously identified as isovaleric acid (3-methylbutanoic acid) and 2-methylbutyric acid (2-methylbutanoic acid), by comparing their mass spectra and ¹H- and ¹³C-nuclear magnetic resonance spectra to those of authentic compounds. Conclusions: In the antibacterial activity of P. haloplanktis INH strain, extra cell compounds are involucred, mainly isovaleric and 2-methylbutyric acids. Significance and Impact of the Study: Production of antimicrobial compounds by marine micro-organisms has been widely reported; however, the efforts not always are conducted to purification and applications of these active compounds. This study is a significant contribution to the knowledge of compounds unique from marine bacteria as potential sources of new drugs in the pharmacological industry.     

Expression and transcriptional activity of alternative splice variants of Mitf exon 6

Heme proteins––hemoglobin and myoglobin possess esterase activities. Studies with purified hemoglobin from normal individuals and diabetic patients revealed that the esterase activity as measured from hydrolysis of p-nitrophenyl acetate (p-NPA) was higher in diabetic condition and increased progressively with extent of the disease. HbA_1c, the major glycated hemoglobin, which increases proportionately with blood glucose level in diabetes mellitus, exhibited more esterase activity than the non-glycated hemoglobin fraction, HbA₀, as demonstrated spectrophotometrically as well as by activity staining. Glycation influenced esterase activity of hemoglobin by increasing the affinity for the substrate and the rate of the reaction. Both HbA₀ and HbA_1c-mediated catalysis of p-NPA hydrolysis was pH-dependent. Esterase activity of in vitro-glycated myoglobin (GMb) was also higher than that of its non-glycated analog (Mb). The amplified esterase activities of hemoglobin and myoglobin might be associated with glycation-induced structural modifications of the proteins.