Dextransucrase Activity Research Articles

Interactions of dextransucrase purified from Streptococcus mutans 890 with plant polyphenols

Plant polyphenols have been extensively studied for their chemopreventive properties for human health. Dextransucrase plays an essential role in synthesizing exopolysaccharides from its exclusive substrate sucrose in Streptococcus mutans. In the present study, the effect of polyphenols gallic acid and tannic acid was investigated on the dextransucrase activity. The enzyme was purified by ethanol precipitation followed by column chromatography by Sephadex G-200 gel chromatography, followed by PEG-400 treatment. The purified enzyme exhibited 52 fold enrichment with 17.5% yield and specific activity of 3.54 Units/mg protein. On SDS-PAGE enzyme protein gave a single band with a molecular weight of 160 kDa. Dextransucrase activity was inhibited 80–90% by 0.04 mM tannic acid (TA) or 0.4 mM gallic acid (GA) suggesting that tannic acid has 10- fold more inhibitory potential than gallic acid on the activity of dextransucrase. CD/ORD studies revealed modifications in the tertiary structure of enzyme protein in presence of tannic acid and gallic acid, which were further confirmed by fluorescence spectra of the protein in presence of tannic acid. These results suggest that inhibition of dextransucrase activity in S. mutans by polyphenols may have potential applications in the prevention and control of dental caries.

Insights into extracellular dextran formation by Liquorilactobacillus nagelii TMW 1.1827 using secretomes obtained in the presence or absence of sucrose

Dextrans are α-(1,6)-linked glucose polymers, which are exclusively produced by lactic acid bacteria from sucrose via extracellular dextransucrases. Previous studies suggested that the environmental pH and the presence of sucrose can impact the release and activity of these enzymes. To get deeper insight into this phenomenon, the dextransucrase expressed by water kefir borne Liquorilactobacillus (L.) nagelii TMW 1.1827 (formerly Lactobacillus nagelii) was recovered in supernatants of buffered cell suspensions that had been incubated with or without sucrose and at different pH. The obtained secretomes were used to time-dependently produce and recover dextrans, whose molecular and macromolecular structures were determined by methylation analysis and AF4-MALS-UV measurements, respectively. The initial pH of the buffered cell suspensions had solely a minor influence on the released dextransucrase activity. When sucrose was present during incubation, the secretomes contained significantly higher dextransucrase activities, although the amounts of totally released proteins obtained with or without sucrose were comparable. However, the dextransucrase appeared to be released in lower amounts into the environment if sucrose was not present. The amount of isolable dextran increased up to 24 h of production, although the total sucrose was consumed within the first 10 min of incubation. Furthermore, the sucrose isomer leucrose had been formed after 10 min, while its concentrations decreased over time and the portions of longer isomaltooligosaccharides (IMOs) increased. This indicated that leucrose can be used by L. nagelii TMW 1.1827 to produce more elongated and branched dextran molecules from presynthesized IMOs, while disproportionation reactions on short IMOs may appear additionally. This leads to increasing amounts of high molecular weight dextran in a state of sucrose depletion. These findings reveal new insights into the pH- and sucrose-dependent kinetics of extracellular dextran formation and may be useful for optimization of fermentative and enzymatic dextran production processes.

Chemical kinetics, thermodynamics and inactivation kinetics of dextransucrase activity by ultrasound treatment

In this work, the impact of ultrasound on dextransucrase activity was investigated, and a biphasic model was introduced to investigate the inactivation kinetics of the dextransucrase catalytic reaction. In addition, a chemical kinetic model, the Michaelis–Menten equation, Eyring transition state theory and the Arrhenius equation were applied. Short periods of exposure to low-intensity ultrasound significantly increased the activity of dextransucrase. The maximum activity was 1.25 ± 0.01 u/mL at an ultrasound intensity of 1.8 W/cm3 for 3 min, which was 27.6% higher than the activity of the untreated enzyme (0.98 ± 0.038 U/mL). In addition, after the ultrasound treatment, the kinetic model of the dextransucrase catalytic reaction was fitted to a first-order kinetic model at low sucrose concentrations. At low sucrose concentrations, the Ea, ΔH, ΔS and ΔG of ultrasound-treated dextransucrase were reduced by 33.7, 35.7, 28.8 and 0.38%, respectively. Furthermore, compared with the parameters of the untreated sample, the value of Vmax the treated sample increased, and Km decreased. The thermal inactivation model of the dextransucrase catalytic reaction in a shaker was fitted to a biphasic model, and the percentage of the thermally stable fraction of ultrasound-treated dextransucrase was increased, while the rate of deactivation was decreased. Moreover, the degree of thermal inactivation was inversely proportional to the dextransucrase concentration. The fluorescence spectra showed a decrease in the amount of tryptophan on the surface of dextransucrase treated with ultrasound. In conclusion, short-term treatment with low ultrasound intensity increases the activity, thermal stability and reaction rate of dextransucrase.

Antibodies generated against dextransucrase exhibit potential anticariostatic properties in Streptococcus mutans.

Streptococcus mutans is a common principal causative agent of dental caries. In this communication, we describe that the antibodies raised against purified dextransucrase effectively inhibited the growth of S. mutans. The purified enzyme showed 58-fold enrichment, 17.5% yield and a specific activity of 3.96 units/mg protein. Purified IgG fraction of the antibody showed significant affinity with the antigenic protein. Immunotritation of the enzyme with dextransucrase antibody showed a gradual increase in inhibition of dextransucrase activity. The growth of S. mutans was also inhibited by 85% in the presence of 28 μg of IgG fraction of the antibody. Antibodies also impaired glucosyltransferase activity (72.8%) and biofilm formation by 92.6% in S. mutans. Western blot analysis revealed no cross reactivity with the various tissues of mice, rat, rabbit and humans. Dot blot analysis showed little reactivity with Lactobacillus acidophilus and Staphylococcus aureus and there was no reactivity with other bacterial strains like Enterococcus faecalis, Escherichia coli and Salmonella typhimurium. These findings suggest that antibody raised against dextransucrase exhibit inhibitory effects on the growth of S. mutans and biofilm formation with no reactivity with various mammalian tissues, thus it could be an effective anticariogenic agent.

PH dependent effects of sodium ions on dextransucrase activity in Streptococcus mutans

Dextransuccrase (E.C 2.4.1.5) is a key enzyme in S. mutans for the metabolism of sucrose which helps in the adherence and accumulation of bacteria on tooth surface leading to the formation of dental caries. Dextransuccrase resembles in its catalytic properties with the brush boarder sucrase and exhibits pH dependent inhibitory and stimulatory effects in response to Na+. In this communication we studied the effect of monovalent cations on the activity of dextransuccrase from S. mutans. The percentage inhibition of dextransuccrase was 65% at 0.5 mM NaCl which enhanced to 90% at 20 mM sodium concentration. However there was no effect on dextransucrase activity in presence of other monovalent cations (Rb+, Cs+, and K+) tested. Enzyme activity was enhanced 20–24% in acidic pH but was strongly inhibited (59–89%) around neutral and alkaline pH by 0.5–2.0 mM sodium chloride. Upon dialysis, 86% of enzyme activity was restored to control values. There was no effect of 2 mM NaCl on glucosyltransferase activity of the enzyme. Kinetic studies revealed that enzyme showed biphasic effects in response to Na+ ions. At acidic pH the enzyme exhibited mixed type of activation affecting both Vmax and Km, while in alkaline pH, the enzyme showed V- type effect reducing Vmax by 74% without affecting Km. The effects of sodium ions on dextransuccrase activity were specific, thus it can be useful to block its catalytic activity, and reducing the cariogenic potential of S. mutans.

Karakteristik Isolat Bakteri Penghasil Dekstran dari Batang Tebu (Saccharum officinarum L.)

Dextran is a long chain of carbohydrate polymers synthesized by dextransucrase enzyme with sucrose as a substrate. The physical, chemical conditions, temperature, and the concentration of sucrose in the isolate bacterial growth media play an important role in dextransucrase production. This study aims to isolate and characterize the dextransucrase producing bacteria. The bacteria were isolated from sugar cane. TBT 3.2 Isolate which had the highest viscosity (1.48 cP) and crude dextran (7.87 g/L) production was selected. TBT 3.2 Isolate was identified as Paenibacillus polymyxa with 99% similar identity. The Paenibacillus polymyxa TBT 3.2 was characterized based on their ability to produce dextran and cell number. The bacteria had the optimum growth conditions on the media for producing dextran at pH 7, temperature 30°C, and concentration of 20% sucrose. The highest dextransucrase activity and the highest specific activity were obtained after 25 minutes of incubation, with the activities of 29.41 mU/mL and 0.46 U/mg. Based on the results of this study, the Paenibacillus polymyxa TBT 3.2 can be used as a dextran producing bacteria .

Entrapment of purified novel dextransucrase obtained from newly isolated Acetobacter tropicalis and its comparative study of kinetic parameters with free enzyme

Purified Acetobacter tropicalis dextransucrase was immobilized in different matrices viz. calcium-alginate, κ-carrageenan, agar, agarose and polyacrylamide. Calcium-alginate was proved to be superior to the other matrices for immobilization of dextransucrase enzyme. Standardization of immobilization conditions in calcium-alginate resulted in 99.5% relative activity of dextransucrase. This is the first report with such a large amount of relative activity as compared to the previous reports. The immobilized enzyme retained activity for 11 batch reactions without a decrease in activity which suggested that enzyme can be used repetitively for 11 cycles. The dextransucrase was also characterized, which revealed that enzyme worked best at pH 5.5 and 37 °C for 30 min in both the free as well as immobilized state. Calcium-alginate immobilized dextransucrase of A. tropicalis showed the Km and Vmax values of 29 mM and 5000 U/mg, respectively. Free and immobilized enzyme produced 5.7 mg/mL and 2.6 mg/mL of dextran in 2 L bench scale fermenter under optimum reaction conditions. This immobilization method is very unconventional for purified large molecular weight dextran-free dextransucrase of A. tropicalis as this method is used usually for cells. Such reports on entrapment of purified enzyme are rarely documented.

Production of short-chain fructooligosaccharides with a recombinant produced and immobilized fructosyltransferase

Twelve bacterial strains were isolated from beehives of Yemen, Nigerian and Libyan honey bee. All the isolates showed dextransucrase activity with degrees of variation. Esawy-1 recorded the highest activity (10.53) and was identified morphologically and based on 16S rRNA as Enterococcus faecalis. Some isolate safety was assessed such as hemolytic activity and resistance to antibiotic. The isolate showed sensitivity to most tested antibiotic such as levofloxacin, ofloxacin, ciprofloxacin and vancomycin. Also, the hemolytic activity was negative. A 2- level Plackett–Burman design was used to determine the bioprocess parameters that affected dextransucrase productivity. Central composite design was consequently showed 6.70-fold improvement in dextransucrase productivity in compared to the basal medium. Also, the results showed a 96% correlation between sucrose concentration and the amount of dextran yield. FTIR and 1HNMR spectrum confirmed that the formed polysaccharide was dextran comparing to a standard dextran. Enterococcus faecalis Esawy KR758759 dextran showed a fibrinolytic activity matching the standard Hemoclar preparation (40%) while the anticoagulation activity was negative. Also, it recorded a promising prebiotic against Lactobacillus casie (1.82). All previous results recommended the Enterococcus faecalis dextran to be used in food and pharmaceutical fields and paid attention to the honey as a source of potential bacterial dextransucrase.

Optimization of Enterococcus faecalis Esawy KR758759 dextransucrase and evaluation of some dextran bioactivities

Twelve bacterial strains were isolated from beehives of Yemen, Nigerian and Libyan honey bee. All the isolates showed dextransucrase activity with degrees of variation. Esawy-1 recorded the highest activity (10.53) and was identified morphologically and based on 16S rRNA as Enterococcus faecalis. Some isolate safety was assessed such as hemolytic activity and resistance to antibiotic. The isolate showed sensitivity to most tested antibiotic such as levofloxacin, ofloxacin, ciprofloxacin and vancomycin. Also, the hemolytic activity was negative. A 2- level Plackett–Burman design was used to determine the bioprocess parameters that affected dextransucrase productivity. Central composite design was consequently showed 6.70-fold improvement in dextransucrase productivity in compared to the basal medium. Also, the results showed a 96% correlation between sucrose concentration and the amount of dextran yield. FTIR and 1HNMR spectrum confirmed that the formed polysaccharide was dextran comparing to a standard dextran. Enterococcus faecalis Esawy KR758759 dextran showed a fibrinolytic activity matching the standard Hemoclar preparation (40%) while the anticoagulation activity was negative. Also, it recorded a promising prebiotic against Lactobacillus casie (1.82). All previous results recommended the Enterococcus faecalis dextran to be used in food and pharmaceutical fields and paid attention to the honey as a source of potential bacterial dextransucrase.

Effect of temperature on production of oligosaccharides and dextran by Weissella cibaria 10 M

The formation of HoPS and oligosaccharides in sourdough fermentation improves bread quality but is dependent on the expression of glycansucrases by lactic acid bacteria. Data on the expression of dextransucrases by Weissella spp., however, are limited. This study therefore aimed to assess dextansucrase expression in W. cibaria 10 M, focusing on the effect of temperature. The effect of temperature on growth, oligosaccharide and dextran synthesis by W. cibaria 10 M was determined and the expression and activity of cell-associated dextransucrase from W. cibaria 10 M were investigated. The oligosaccharides profiles were measured by thin layer chromatography and high performance anion exchange chromatography coupled to pulsed amperometric detection. Dextran formation was quantified by size exclusion chromatography. W. cibaria grew fastest at 30 °C but oligosaccharide formation was highest at 20 °C or less. Dextransucrase expression as measured by reverse transcription quantitative PCR, SDS-PAGE, and activity of cell-associated dextransucrase were maximal at 15 °C. Cold shift incubation, characterized by incubation at 30 °C to obtain biomass, followed by shift to 6 °C to induce dextransucrase expression, supported high dextransucrase activity in laboratory media. Cold shift fermentation of wheat and sorghum sourdoughs supplemented with 15 or 30% sucrose increased the yields of oligosaccharides, and resulted in formation of 16 and 12 g/kg dextran in wheat and sorghum sourdoughs, respectively. Dextran formation was decreased in favour of oligosaccharide formation when doughs were supplemented with maltose. In conclusion, cold shift fermentation of sourdough with W. cibaria supports high dextran yields or formation of oligosaccharides without excess acidification.

Production, purification and biochemical characterization of dextransucrase from Lactic acid bacteria isolated from sauerkraut

In this study Lactobacillus sp. has been isolated form Sauerkraut for the production of dextransucrase. The fermentation process is continued by the mixed activity of lactic acid bacteria (LAB) present in the medium. Morphological, physiological and biochemical analysis deciphered the presence of acid producing bacteria belongs to the genera Lactobacilli. Characteristically the isolates were catalase producing but oxidase negative significant to the Lactobacilli genera. The isolated bacterium was tested to be positive in the production of dextransucrase in Tsuchiya medium. Maximum protein production containing dextransucrase was achieved to be 400g/l which is significantly high in terms of concentration. Partially purified enzyme exhibited Vmax=2.9±0.2µmole/min, Km=1.1±0.2mg/ml and Kcat=2.75s-1 and the enzyme followed first order rate kinetics. Better exploration strategy for higher dextransucrase activity from Lactobacillus sp. by inducing operon to higher dextran production can be used later as potent nutraceuticals.

The thermoduric effects of site-directed mutagenesis of proline and lysine on dextransucrase from Leuconostoc mesenteroides 0326

Dextransucrase (DSR, EC2.4.5.1) from Leuconostoc mesenteroides 0326 which catalyzes sucrose to produce dextran, are popularly used in food and medicine industries. However, its poor kinetic stability especially at higher temperatures is a limiting factor for developing industrial applications. The aim of this study is to improve the enzyme activity and thermal stability of dextransucrase by single, double and triple mutations of proline and lysine. In this work, Pro-473, Pro-678, and Pro-856 were selected as engineering targets and individually replaced with serine. Lys-378, Lys-725, and Lys-955 were replaced with threonine, respectively. Mutant P473S/P856S (MW: 170kDa) was selected as the highest enzymatic activity mutant from fourteen mutants. Specifically, the mutant P473S/P856S showed a significant increase in thermal inactivation with a 7.4-fold increase in half-life at 35°C and a 2-fold increase in catalytic efficiency compared with the wild-type. The results of structural simulation are demonstrated that the new intramolecular hydrogen bonds in mutated enzymes increased structural stability, thereby increasing the thermal stability. The thermal stable mutants have an enormous application potential in food and pharmaceutical industry.

Improvement of Dextransucrase Activity by Glucose Acclimation Cultivation and Kinetics of Sucrose Consumption

Dextransucrase is the enzyme that catalyzes the synthesis of dextran from sucrose. Though dextran is an unwanted deterioration product in the sugar industry, it is being widely used in medicine, cosmetics, and several food areas. Currently, the natural enzyme activity of dextransucrase is low. Thus, it is of great interest to understand how to improve the dextransucrase activity. In the study, Leuconostoc mesenteroides (L.M.CICC-20724) were cultured in a glucose-based solid medium (glucose was the sole carbon source) and then transferred to a sucrose-based solid medium (sucrose was the sole carbon source) for rejuvenation. It was observed that the dextransucrase production was inhibited when L.M.CICC-20724 using the glucose-based solid medium. After 3 months of acclimation, the activity of dextransucrase produced by the acclimated L. mesenteroides (Glu-L.M.) achieved a stable level at approximately 4.20 U/mL. The dextransucrase activity (approximately 1–2 U/mL) increased more than two times with L.M.CICC-20724 in comparision with original one. Dextran products generated from Glu-L.M. and L.M.CICC-20724 were studied using Fourier transform infrared spectroscopy and results showed that both dextrans had a consistent structural composition. The kinetics of sucrose consumption was studied. Under the condition of 0.5 U/mL dextransucrase activity, the consumption of relative low initial sucrose concentrations (0.1, 0.2 and 0.4 M) more fitted to the first-order reaction kinetics, while the consumption of relative high initial sucrose concentrations (0.6 and 0.8 M) was fitted better to the second-order reaction kinetics.

A novel approach of integrated bioprocessing of cane molasses for production of prebiotic and functional bioproducts

In this work, the sugar industry by-product cane molasses was investigated as feedstock for acceptor reactions by dextransucrase from Leuconostoc mesenteroides MTCC 10508, leading to the biosynthesis of oligosaccharides. The starch industry corn fiber residue was used as a source for acceptor molecules, maltose, in the reaction. Production of approximately 124g oligosaccharides (DP3-DP6) per kg of fresh molasses was achieved. Further, cane molasses based medium was demonstrated as a sole carbon source for L. mesenteroides growth and dextransucrase production. d-Fructose released by dextransucrase activity as processing by-product was transformed into the functional monosaccharide with zero caloric value, d-psicose, by inducing its epimerization. Quantitative analysis approximated 37g d-psicose per kg of fresh molasses. Thus, the study established a novel approach of integrated bioprocessing of cane molasses into prebiotic and functional food additives.

Mutation of AN39-1 for production and characterization of constitutive, thermostable and pH-resistant dextransucrase / Konstitutif, sıcaklığa ve pH’ya dayanıklı dekstransukrazların üretimi ve karakterizasyonu için AN39-1’in mutasyonu

Abstract Objective: Leuconostoc mesenteroides AN39-1 has recently been isolated from Crataegus orientalis var. Orientalis. It produces inducible extracellular dextransucrase (EC 2.4.1.5) forming dextran from sucrose. The aim of this study was (1) to obtain constitutive, pH-resistant and thermostable dextransucrase, (2) to characterization of these dextransucrase. Methods: Mutagenesis was carried out on the parent strain (AN39-1) using UV, ethyl methane sulfonate, and N- methyl- N´-nitro-N-nitrosoguanidine. Dextransucrases from wild type (AN39-1) and the mutant strain (A26-2/11) were purified by polyethylene glycol (PEG) precipitation and characterized. Results: Mutants (A26, A26-2, and A26-2/11) hyper producing and constitutive for dextransucrase were isolated. The mutants (A26, A26-2, A26-2/11) produced 7.2, 8.1, and 2.0 times more dextransucrase activity as compared to parent strain on sucrose medium, respectively. In addition, the mutants produced dextransucrase on glucose medium with higher activities (3.0-5.8 times) than what the parental strain produced on sucrose medium. The mutant enzyme (A26-2/11) was much more thermostable than the native enzyme and resistant to pH more than dextransucrase of AN39-1. The dextransucrase from mutant strain was stable up to 35°C and pH of 7.5 for 3 hr. Conclusion: The structures of dextrans produced by wild type and mutant enzymes were similar to commercially produced B-512 F dextran. Thus, the newly dextransucrases produced by mutant strain could find industrial applications at higher temperature and pH.

Simple synthesis of isomaltooligosaccharides during Sauerkraut fermentation by addition of Leuconostoc starter and sugars

Isomaltooligosaccharides (IMOs), prebiotic compounds stimulating the growth of intestinal bacteria, were synthesized in sauerkraut by inoculating psychrotrophic Leuconostoc citreum KACC 91035 strain with high dextransucrase activity. For the glucose transferring reaction of dextransucrase, sucrose and maltose were added in sauerkraut (29 and 28 mM, respectively, w/v) as glucosyl donor and acceptor molecule, respectively. For 12 days of fermentation at 10℃, IMOs were gradually produced by consuming sucrose and maltose, and the synthesis rate and maximal concentration of IMOs were 2.04 and 20.2 mM, respectively. This result demonstrates a simple method to manufacture a synbiotic sauerkraut product by adding probiotic lactic acid bacterium and sugars as ingredients.

Effect of sucrose on the growth and dextransucrase activity of Leuconostoc mesenteroides

Effect of sucrose on the growth and dextransucrase activity of Leuconostoc mesenteroides

Comparison of inhibitory activity of bioactive molecules on the dextransucrase from Streptococcus mutans.

The effect of chitosan with different molecular weights and other natural substances on dextransucrase (DSase) activity from a representative oral pathogen Streptococcus mutans was elucidated. Among other bioactive substances, amino-monosaccharides such as glucosamine, mannosamine, and galactosamine exerted the enzyme inhibitory activity over 95% of DSase. The specified hydrolysates derived from the hydrolysis of high molecular weight chitosan (HMWC) designated to CTSN, CTSN-P, CTSN-B, and CTSN-S with different molecular weights ranging from 3 to 8 kDa showed the similar inhibitory activity toward DSase. Also, the hyaluronic acid (MW 8.9 kDa), sulfated chitin, and amino-monosaccharides demonstrated the significant activity, CTSN, CTSN-P, CTSN-B, and CTSN-S are of potent bioactive substances that can be prepared in the cheapest way compared with other molecules tested available for antibacterial agent useful for human oral health.

Influence of terpenoids and acarbose on glycosyltransferases produced by strain Leuconostoc mesenteroides URE 13

A study of the influence of different plant terpenoids and amino sugar derivate acarbose on the activity of glycosyltransferase complex and purified dextransucrase from Leuconostoc mesenteroides URE 13 strain was carried out. All the tested terpenoids showed an inhibitory effect on glycosyltransferases from strain URE 13 at concentration 0.34 mmol. Out of all studied diterpenoids splendidin showed the strongest inhibitory effect decreasing the activity of both glycosyltransferase complex and dextransucrase with 70% and 90%, respectively. The triterpenoid ursolic showed the second strongest inhibitory effect as the enzyme complex and dextransucrase from strain URE 13 retain 27% and 13% of their initial enzyme activity. Despite the higher degree of inhibition of purified dextransucrase, compared to the enzyme complex, a complete inhibition of the enzyme was not observed at the highest used terpenoid concentration (3.42 mmol). When acarbose was used as an inhibitor, a complete inhibition of dextransucrase was observed at concentration of 6.9 mmol, while the enzyme complex retained 8% of its enzyme activity. Ki values of 0.28 mmol for splendidin, 0.37 mmol for ursolic acid and 0.29 mmol for acarbose were determined from the kinetic studies of purified dextransucrase.

Biotechnological Valorization of Cashew Apple: A Review

Cashew apple, the peduncle of cashew fruit, is an agricultural waste byproduct from harvesting cashew nuts. Cashew apple juice contains about 10% reducing sugar. Its bagasse contains about 20% of cellulose. The byproducts can be used as a substrate for several microbial fermentation processes. Wine and bioethanol were produced by Saccharomyces cerevisiae. Probiotic beverage and lactic acid were produced by Lactobacillus casei. Biosurfactants-rhamnolipids, emulsan and surfactin were synthesized by Pseudomonas aeruginosa, Acineto- bacter calcoaceticus and Bacillus subtilis, respectively. Tannase and pectinase were produced during solid-state fermentation of Aspergillus spp. Prebiotic oligosaccharides were synthesized by the activity of dextransucrase produced by Leuconostoc spp. Cashew apple is a potential substrate for producing a variety of products, depending on the type of microorganisms used.