Substrate Sucrose Research Articles

Educational activity of enzyme kinetics in an undergraduate biochemistry course: invertase enzyme as a model.

This article aims to simplify and facilitate the process of practical teaching of enzyme kinetics by utilizing minimal teaching laboratory requirements. Simultaneously, it ensures that students comprehend the enzyme kinetics experiment effectively. The focus is on teaching students how to estimate the maximum velocity (Vmax) and Michaelis constant (Km) of β-fructofuranosidase enzyme (also known as invertase) isolated from dry yeast. The invertase enzyme catalyzes the hydrolysis of sucrose substrate into glucose and fructose, employing the Michaelis-Menten approach of evaluating invertase enzyme kinetics as well as Lineweaver-Burk linear graphic approach of evaluating the Michaelis-Menten enzyme kinetics. The practical experiment seeks to reinforce the concepts of initial velocity dependence on substrate concentration. The data presented in the work were generated from a genuine practical biochemistry course enrolled by second-year undergraduate students in the Department of Pharmacy and the Department of Medical Laboratory Science. While there were minor variations in the invertase enzyme kinetic parameters among students, they successfully carried out the experiment. The students accurately estimated the Vmax and Km of the invertase enzyme in the sucrose hydrolysis chemical reaction. Moreover, they demonstrated an understanding of the meanings of the kinetic parameters (Km and Vmax) and the utility of the Lineweaver-Burk plot.

Blood Coagulation-Inspired Fibrin Hydrogel for Portable Detection of Thrombin Based on Personal Glucometer.

As one of the biomarkers of coagulation system-related diseases, the detection of thrombin is of practical importance. Thus, this study developed a portable biosensor based on a personal glucometer for rapid detection of thrombin activity. Fibrinogen was used for the detection of thrombin, and the assay principle was inspired by the blood coagulation process, where thrombin hydrolyzes fibrinogen to produce a fibrin hydrogel, and the amount of invertase encapsulated in the fibrin hydrogel fluctuates in accordance with the activity of thrombin in the sample solution. The quantitative assay is conducted by measuring the amount of unencapsulated invertase available to hydrolyze the substrate sucrose, and the signal readout is recorded using a personal glucometer. A linear detection range of 0-0.8 U/mL of thrombin with a limit of detection of 0.04 U/mL was obtained based on the personal glucometer sensing platform. The results of the selectivity and interference experiments showed that the developed personal glucometer sensing platform is highly selective and accurate for thrombin activity. Finally, the reliability of the portable glucometer method for rapid thrombin detection in serum samples was investigated by measuring the recovery rate, which ranged from 92.8% to 107.7%. In summary, the fibrin hydrogel sensing platform proposed in this study offers a portable and versatile means for detecting thrombin using a personal glucometer. This approach not only simplifies the detection process, but also eliminates the need for large instruments and skilled operators, and substantially reduces detection costs.

A Critical Review on Immobilized Sucrose Isomerase and Cells for Producing Isomaltulose.

Isomaltulose is a novel sweetener and is considered healthier than the common sugars, such as sucrose or glucose. It has been internationally recognized as a safe food product and holds vast potential in pharmaceutical and food industries. Sucrose isomerase is commonly used to produce isomaltulose from the substrate sucrose in vitro and in vivo. However, free cells/enzymes were often mixed with the product, making recycling difficult and leading to a significant increase in production costs. Immobilized cells/enzymes have the following advantages including easy separation from products, high stability, and reusability, which can significantly reduce production costs. They are more suitable than free ones for industrial production. Recently, immobilized cells/enzymes have been encapsulated using composite materials to enhance their mechanical strength and reusability and reduce leakage. This review summarizes the advancements made in immobilized cells/enzymes for isomaltulose production in terms of refining traditional approaches and innovating in materials and methods. Moreover, innovations in immobilized enzyme methods include cross-linked enzyme aggregates, nanoflowers, inclusion bodies, and directed affinity immobilization. Material innovations involve nanomaterials, graphene oxide, and so on. These innovations circumvent challenges like the utilization of toxic cross-linking agents and enzyme leakage encountered in traditional methods, thus contributing to enhanced enzyme stability.

Activity of hydrolytic lipase enzyme synthesised by Aspergillus terreus isolated from petroleum-contaminated soil

Lipase enzyme-mediated processes have biological significance and tremendous potential in various industrial and technological areas due to the enzymatic properties and broad specificity for a wide spectrum of substrates. This research involved the production of hydrolytic lipase enzyme by Aspergillus terreus extracted from petroleum-contaminated soil and optimising the environmental parameters that may affect lipase production. Hydrolytic lipase enzyme activity of the isolated Aspergillus terreus from petroleum-contaminated soil was undertaken in submerged fermentation. Dry cell weight, lipase enzyme pellets, and lipase enzyme concentration by measuring the percentage transmission by spectrophotometry were measured to determine the effects of various abiotic factors (pH, temperature, carbon and nitrogen sources, lipid substances and vegetable oils) affecting cell growth and lipase enzyme production by Aspergillus terreus on modified lipase production media. The results showed a precipitate on Tween 20 and a bright pink-fluorescent halo on the rhodamine B agar plate. After temperature adjustment to 35 °C, statistical analysis showed that Aspergillus terreus produced a dry cell weight of 3.14 g/L and a lipase enzyme pellet of 0.33 g with a spectrophotometric transmission of 0.03%. The sucrose substrate displayed a dry cell weight of 3.11 g/L and a lipase enzyme pellet of 1.15 g. Spectrophotometric analysis showed that sucrose, glucose and peptone had a similarity in transmission at p = 0.321. The addition of Neem oil improved enzyme yield as a dry cell weight of 3.02 g/L and a lipase enzyme pellet of 0.95 g was obtained. Statistically, sunflower and tributyrin similarly affected enzyme production by showing a spectrophotometric transmission at p = 0.024. This study has indicated that Aspergillus terreus could be employed as a reliable source of lipase enzymes for industrial processes due to its stability across different abiotic factors.

From Milk Kefir to Water Kefir: Assessment of Fermentation Processes, Microbial Changes and Evaluation of the Produced Beverages

The aim of the present study was to investigate the feasibly of using traditional milk kefir grains for the production of water kefir-like beverages and assess the changes in the physicochemical characteristics and the microbial populations of the fermented beverages. To this end, experiments of milk fermentation were primarily conducted at different temperatures and upon selection of the optimal, a gradual substitution of the substrate was performed by replacing milk from a sucrose-based solution. After the successful fermentation of the sucrose substrate, fruit juices were used as fermentation substrates. Sensory evaluation of the sugar-based beverages was also performed in order to access their acceptability for consumption. According to the results, the transition from milk to water kefir is indeed feasible, leading to the production of beverages with relatively higher ethanol concentrations (up to 2.14 ± 0.12% w/v) than milk kefir and much lower lactic acid concentrations (up to 0.16 ± 0.01% w/v). During the fermentation of the sugary substrates, yeasts seemed to be dominant over lactic acid bacteria, in contrast to what was observed in the case of milk kefir, where LAB dominated. The sensory evaluation revealed that all sugar-based beverages were acceptable for consumption, with the fruit-based ones obtaining, though, a better score in all attributes.

ENVIRONMENTAL PERFORMANCE OF MICROBIAL FUEL CELL BASED LIVE SLUDGE FOR VOLTAGE PRODUCTION AND CONGO RED DYE REMOVAL

In this work, Single chamber Microbial fuel cells (SCMFCs) are a versatile technology is depending on the interaction mechanisms of bacteria, to produce bioelectricity simultaneously and treat Congo red (CR) dye from aqueous solution at different pH (6.5-8). Electricity generation from the biodegradable organic substrate (sucrose) accompanied by decolorization of azo dye was investigated in the batch test results showed that more than 99% decolorization demonstrated at UV-Visible Spectrophotometer (500 nm) was achieved within 20 days and maximum output voltage (889 mv) had been obtained in an open circuit at a pH value of 7.5. Microbial community analysis showed that species in live sludge and the impact of bacteria grown on removal and voltage.

The role of the C domain in the thermostability of GH70 enzymes investigated by domain swapping

Abstract Sucrose-active enzymes belonging to the glycoside hydrolase (GH) family 70 are attractive tools for the synthesis of oligosaccharides, polysaccharides or glycoconjugates. However, their thermostability is an important issue for the development of robust and cost-effective enzyme-based processes. Indeed, GH70 enzymes are mesophilic and no thermophilic representatives have been described so far. Furthermore, structurally guided engineering is a challenge given the size of these proteins (120 to 250 kDa) and their organization in five domains. Herein, we have investigated the possible role of the domain C in the stability of GH70 enzymes. The alternansucrase (ASR) is the most stable enzyme of the GH70 family. Structural comparison of ASR to other GH70 enzymes highlighted the compactness of its domain C. We assumed that this atypical structure might be involved in the stability of this enzyme and decided to introduce this domain in another much less stable GH70 enzyme of known three-dimensional structure, the branching sucrase GBD-CD2. The chimeric GBD-CD2 exhibited a lower specific activity on sucrose substrate but its specificity was unchanged with the enzyme remaining specific for the branching of dextran via α-1,2 linkage formation. Interestingly, the chimera showed a higher melting temperature and residual activity than the wild-type enzyme after 10 min incubation at 30 °C showing that the domain C can affect GH70 enzyme stability and could be a potential target of both random or rational mutagenesis to further improve their stability.

Novel carotenogenic gene combinations from red yeasts enhanced lycopene and beta-carotene production in Saccharomyces cerevisiae from the low-cost substrate sucrose.

Carotenoids (C40H56) including lycopene and beta-carotene are relatively strong antioxidants that provide benefits to human health. Here, we screened highly efficient crt variants from red yeasts to improve lycopene and beta-carotene production in Saccharomyces cerevisiae. We identified that crt variants from Sporidiobolus pararoseus TBRC-BCC 63403 isolated from rice leaf in Thailand exhibited the highest activity in term of lycopene and beta-carotene production in the context of yeast. Specifically, the phytoene desaturase SpCrtI possessed up to 4-fold higher in vivo activity based on lycopene content than the benchmark enzyme BtCrtI from Blakeslea trispora in our engineered WWY005 strain. Also, the geranylgeranyl pyrophosphate (GGPP) synthase SpCrtE, the bifunctional phytoene synthase-lycopene cyclase SpCrtYB, and SpCrtI when combined led to 7-fold improvement in beta-carotene content over the benchmark enzymes from Xanthophyllomyces dendrorhous in the laboratory strain CEN.PK2-1C. Sucrose as an alternative to glucose was found to enhance lycopene production in cells lacking GAL80. Lastly, we demonstrated a step-wise improvement in lycopene production from shake-flasks to a 5-L fermenter using the strain with GAL80 intact. Altogether, our study represents novel findings on more effective crt genes from Sp. pararoseus over the previously reported benchmark genes and their potential applications in scale-up lycopene production.

Study on the thermoelectric properties of Al-ZnO thin-film stack fabricated by physical vapour deposition process for temperature sensing

In this work, Al-ZnO thin-film stacks were fabricated by the physical vapour deposition (PVD) process. ZnO films with different oxygen compositions were deposited by reactive DC magnetron sputtering. According to the results, it was observed that the deposition parameters influenced the thin film’s thermoelectric properties. The thermoemf response of the Al-ZnO stacks was studied through experiments to calibrate the same as a thin-film thermocouple (TFTC). The best linear response was observed for the stack with ZnO having an oxygen composition of 80.45% for temperatures between 360 K and 400 K. The stack showed a sensitivity of 5.22 mV/K and 0.78 mV/K, in the same temperature range, on the glass substrate and the sucrose substrate, respectively. A comparative study of the thermoemf response was carried out between Al-ZnO and Al-Ag stacks on glass and sucrose substrates, and wired thermocouples.

Biotechnological purification of a β-fructofuranosidase (β-FFase) from Aspergillus tamarii kita: Aqueous two-phase system (PEG/Citrate) and biochemical characterization

β-fructofuranosidases (EC3.2.1.26) are members of the GH32 family of glycoside hydrolases, which include more than 390 enzymes of vegetable and microbial origins, used in several biotechnological applications. Thus, this research aimed to produce a β-fructofuranosidase obtained by Aspergillus tamarii through solid state fermentation, and to purify by Aqueous Two-Phase System (ATPS). Summary results presented the optimal parameters to produce the β-fructofuranosidase used wheat bran as a substrate at 30 °C for 48 h, and purification process using ATPS with polyethylene glycol and sodium citrate (PEG/sodium citrate), where the β-fructofuranosidase preferably partitioned to the salt-rich phase, the best run (24% of PEG 400, 20% sodium citrate, pH 8) which presented a higher purification factor 6.42 with 12.39 U/mL activity and 352% yield. Optimum parameter was pH 5.15 and temperature of 55 °C, respectively. The purified enzyme showed excellent thermal stability and exhibited a half-life of 60 min at 65 °C. Kinetics results for enzyme showed for Sucrose substrate the enzyme showed Km of 42.9 ± 2.21 mM and Vmax of 180.2 ± 2.8 μM min−1 mg−1 of protein. Although Vmax was the highest for 1-Kestose (219.4 ± 2.7 μM min−1 mg−1 of protein) the preferred substrate of Aspergillus tamarii β-fructofuranosidase (β-FFase) was Nystose (Km of 3.8 ± 0.15 mM). SDS-PAGE revealed a single band of protein at ~66 kDa. Finally, this study demonstrated the potential of ATPS to purify a β-fructofuranosidase with application in biotechnological field aiming to functional foods.

Bacterial α-Glucan and Branching Sucrases from GH70 Family: Discovery, Structure-Function Relationship Studies and Engineering.

Glucansucrases and branching sucrases are classified in the family 70 of glycoside hydrolases. They are produced by lactic acid bacteria occupying very diverse ecological niches (soil, buccal cavity, sourdough, intestine, dairy products, etc.). Usually secreted by their producer organisms, they are involved in the synthesis of α-glucans from sucrose substrate. They contribute to cell protection while promoting adhesion and colonization of different biotopes. Dextran, an α-1,6 linked linear α-glucan, was the first microbial polysaccharide commercialized for medical applications. Advances in the discovery and characterization of these enzymes have remarkably enriched the available diversity with new catalysts. Research into their molecular mechanisms has highlighted important features governing their peculiarities thus opening up many opportunities for engineering these catalysts to provide new routes for the transformation of sucrose into value-added molecules. This article reviews these different aspects with the ambition to show how they constitute the basis for promising future developments.

Synthesis and characterization of Bifidogenic raffinose-derived oligosaccharides via acceptor reactions of glucansucrase E81

Glucansucrases can perform acceptor reactions with different sugars to produce distinct oligosaccharides which can potentially act as prebiotics depending on their characteristics. Using cheap substrate sucrose and under mild and environmentally friendly conditions, glucansucrase-derived oligosaccharides can be produced with high yields. In this study, raffinose-derived oligosaccharides up to degree of polymerization (DP) 7 produced by glucansucrase E81 were characterized and their prebiotic functions were evaluated. The main product was a DP 4 oligosaccharide containing two products formed by the transfer of the glucose to raffinose at either the 3- (81%) or 4-O (19%) position of its galactose residue confirmed by NMR spectroscopy and LC-MS-LTQ analysis. The purified DP 4 raffinose-derived oligosaccharides were subjected to in vitro human gastric fluid and α-amylase digestion tests and no digestion was observed under both cases. Finally, prebiotic potential of DP 4 raffinose-derived oligosaccharides was tested with probiotic and pathogenic strains and only growth of Bifidobacteria promoted, thus supported the Bifidogenic activity of DP 4 raffinose-derived oligosaccharides.

High and ultra low concentrations of Mercuric chloride initiate their specific action on binding sites of invertase and modify its interaction with sucrose

Background: Mercuric chloride is known to inhibit the activity of enzymes. It is used in homeopathy at ultra low concentration (ULC) and is known as Mercurius corrosivus (Merc cor). ULCs of Merc cor are reported to promote enzyme activity. Objective: To see whether the mother tincture (?) of Merc cor and its ULCs interact with an enzyme invertase at its binding sites and influence enzyme’s action on its substrate sucrose. Methods: Merc cor ? (0.15 M HgCl2) was diluted with deionized and distilled (DD) water 1:100 and succussed 10 times to prepare Merc cor 1 cH or 1st potency. This potency was further diluted and succussed in 200 and 1000 steps to prepare 200cH and 1000cH potencies, respectively. Merc cor 200 cH and 1000cH were prepared in 90% ethanol. The two potencies and blank 90% EtOH were diluted with DD water 1:1000 to minimize ethanol content to a negligible amount 0.09%. The control was DD water (55mM). The drugs, EtOH and water control were mixed separately with 0.037 mM invertase in DD water. Using an isothermal calorimetry (ITC) instrument the substrate sucrose (65mM) was injected at 2 µl every 2 min into 300 µl invertase solution 20 times at 25 0C. Molecular modeling study was done to predict possible binding sites and nature of binding between the enzyme and HgCl2 and between the enzyme and water. Potencies after dilution are virtually water. Fluorescence spectra of invertase (4µM) mixed with drug/control solutions were also obtained to see the effect of drugs on protein folding. Results: Thermodynamic parameters like binding constant, change in enthalpy, entropy and Gibbs free energy showed marked variation in treatment effects on the enzyme. Molecular modeling study also shows variation in binding between invertase and HgCl2 and between invertase and water. Fluorescence spectra show variation in quenching related to different treatments. Conclusion: Merc cor mother tincture and its potencies interact at different binding sites of invertase and modify the enzyme’s action on sucrose. Drug solutions induce conformational changes in the enzyme.

Kinetics of Saccharomyces cerevisiae Fermentation under Metal Ions Stress during Ethanol Production

This research investigated the effects of inorganic compounds or metal ions (calcium ion, Ca2+; potassium ion, K+; magnesium ion, Mg2+) on ethanol production efficiency invertase, an enzyme produced by Saccharomyces cerevisiae, in sucrose solution, which was the substrate for yeast fermentation. The results showed that all metal ions (concentration 0.20 and 0.60 % (w/v)) acted as inhibitors on invertase activity in the order Ca2+ > K+ > Mg2+. Subsequently, these ions inhibited sugar conversion, reducing sucrose utilization and less glucose and fructose consumption based on the high content of remaining sugars in the culture medium. The reduction of the substrate was due to the consumption and an increased growth rate of S. cerevisiae, which all resulted in low efficiency of ethanol production and an increase in glycerol content. The glycerol content was increased due to yeast cells' developed mechanism or adaptation to enhance cell survival following metal ion contamination, especially from Ca2+ and K+; furthermore, the glycerol content significantly increased during the changed conditions, such as when the sugars were nearly all consumed. The kinetic parameters such as specific growth rate (µ-1), substrate consumption rate (Qs), and ethanol production of the research work were also undertaken. In conclusion, metal ion contamination in the sucrose substrate of yeast fermentation resulted in low efficiency of ethanol production, specific growth rate, and substrate consumption rate decrease with the Ca2+ ion (concentration 0.20 - 0.60 % (w/v)) acting more harshly as an inhibitor of ethanol production than the other ions, particularly where there was a high concentration of contamination.

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.

Recombinant Expression and Biochemical Characterization of Levansucrase from Halophilic Bacteria <i>Bacillus licheniformis</i> BK1 and BK2

Levansucrase was an extracellular polysacharride (EPS) which has a role in synthesizing levans by transferring fructose moiety from sucrose to acceptor molecules. In the previous study, we have successfully cloned the levansucarese gene from two Bacillus licheniformis strains of BK1 and BK2 labeled as lsbl-bk1 and lsbl-bk2. The present study aims to optimize the expression level of both genes in E. coli expression system and also to obtain the optimum conditions for the recombinant enzymes activity by applying the response surface methodology (RSM). The optimization result found that the highest Lsbl-bk1 production in E. coli expression system was occurred when the recombinant cells grown in the medium containing 0.6% (w/v) NaCl at 42°C, and induced by 0.6 mM IPTG. Different optimum conditions were found for Lsbl-bk2 production. It was achieved when 1.1% (w/v) NaCl added to the production medium and induced by 0.7 mM IPTG at 40°C. RSM optimization result for biochemical characterization of Lsbl-bk1 levansucrase showed the highest specific activity achieved at 56°C and pH 7.5, whereas for the Lsbl-bk2 levansucrase reached the highest specific activity at 50°C and pH 7.5. The addition of Co2+, Ti2+, Mg2+, Ba2+, Zn2+, Fe3+, Ca2+ metal ion to both levansucrases solution did not significantly altered their specific activity, indicating that both levansucrases are not metalo enzymes. Furthermore, the specific activity of levansucrase was also not affected by the addition of 1-25% (w/v) NaCl, suggesting that the variation of ionic strength did not alter the native state of both enzymes. The plot results of levansucrase specific activities toward sucrose concentration showed that both levansucrases follow Michaelis-Menten profile with kcat/KM values ​​about 3.8 and 3.6 s-1/mM respectively. These data indicated that the recombinant levansucrases from halophilic bacteria B. licheniformis BK1 and BK2 are a non metaloenzyme with high affinity and binding rate to sucrose substrate, in which the catalytic efficiency on hydrolysis reactions is relatively low.

Purification and biochemical characterization of an extracellular fructosyltransferase enzyme from Aspergillus niger sp. XOBP48: implication in fructooligosaccharide production.

An extracellular fructosyltransferase (Ftase) enzyme with a molar mass of ≈70kDa from a newly isolated indigenous coprophilous fungus Aspergillus niger sp. XOBP48 is purified to homogeneity and characterized in this study. The enzyme was purified to 4.66-fold with a total yield of 15.53% and specific activity of 1219.17 Umg-1 of protein after a three-step procedure involving (NH4)2SO4 fractionation, dialysis and anion exchange chromatography. Ftase showed optimum activity at pH 6.0 and temperature 50°C. Ftase exhibited over 80% residual activity at pH range of 4.0-10.0 and ≈90% residual activity at temperature range of 40-60°C for 6h. Metal ion inhibitors Hg2+ and Ag+ significantly inhibited Ftase activity at 1mmol concentration. Ftase showed K m, v max and k cat values of 79.51mmol, 45.04µmolmin-1 and 31.5min-1, respectively, with a catalytic efficiency (k cat/K m) of 396µmol-1min-1 for the substrate sucrose. HPLC-RI experiments identified the end products of fructosyltransferase activity as monomeric glucose, 1-kestose (GF2), and 1,1-kestotetraose (GF3). This study evaluates the feasibility of using this purified extracellular Ftasefor the enzymatic synthesis of biofunctional fructooligosaccharides.

Investigation of Optimal Conditions for Production, Characterization, and Immobilization of Fructosyltransferase and β-Fructofuranosidase by Filamentous Fungi

This work's objective was the extracellular production, partial characterization, and immobilization of the enzymes fructosyltransferase (Ftase) and β-fructofuranosidadase (Ffase) by filamentous fungi. Aspergillus niger ATCC 9642 and Penicillium brasilianum were evaluated for the production of fructosyltransferase (Ftase) and β-fructofuranosidadase (FASE) enzymes. The A. niger presented the highest activity of FTase (24.86 µmol/min.mL) and FFase (28.68 µmol/min.mL) in medium composed of 20% sucrose, 0.5% yeast extract, 1% NaNO3, 0.05% MgSO4.7 H2O, 0.25% KH2PO4, 0.5% NH4Cl and 0.25% NaCl inoculated using 5x107spores/mL and incubated at 25°C, pH 5.5, 150 rpm for 48 h. Presenting optimum pH and temperature of 2.39 and 60°C. Thermal stability has shown that the enzyme FFase is more thermally stable when compared to FTase. Stability against different pHs showed similar behavior for FTase and FFase; the optimum pH being between 2.0 and 3.0. FTase and FFase showed storage stability in freezing and refrigeration temperature for approximately 400 h. The kinetic parameters, Km and Vmax, for the sucrose substrate were 24.60mM and 104.16 μmol/min.mL for FTase and 3.91mM and 20.24 μmol/min.mL for FFase. The immobilization process displayed a yield of 6744.66% for FFase and 3928.90% for FTase, with enzymatic activities of 364.79 U/g and 220.34 U/g, and 4 and 3 times reuse, respectively.

Features of isolation of the anthrax pathogen depending on the type of nutrient medium

The purpose of the work is to develop a nutrient medium for differentiation of bacillus from soil aerobic bacilli. In order to achieve the set goal, we used the method of introduction into the environment of cultivation of microorganisms separated from animals and objects of external environment (water, soil, feed, air, scrapes from different surfaces suspected of contamination by their bacillus ) of nutrient substrate sucrose, used by bacteria of Bacillus genus for synthesis of product of their metabolism, a sign absent in bacillus . This feature is essential for identification and differentiation of bacillus from closely related saprophytes. To identify and differentiate the bacillus , the microbes isolated from the external environment were cultivated in a nutrient medium consisting of agar (MPA) and () synthesis of sucrose in the amount of 10% to 100 ml of melted agar. The proposed nutrient media was prepared as follows. agar (500 ml) was melted at 1000C, 10 g of sucrose was added per 100 ml of medium and after the complete dissolution of sucrose, the nutrient medium was poured into dishes and used for sowing the studied material for identification and differentiation of grown crops. The efficiency of the method has been tested in production experiments with positive evaluation. For this purpose, soil samples taken from the territory of old cattle cemeteries were fractionally sown on MPA and for 16-18 hours at 370C and examined crops for the presence of matte and rough (R-form) colonies.

Insights into the pH-dependent, extracellular sucrose utilization and concomitant levan formation by Gluconobacter albidus TMW 2.1191

Many bacteria and archaea produce the polydisperse fructose polymer levan from sucrose upon biofilm formation via extracellular levansucrases (EC 2.4.1.10). We have investigated levansucrase-release and -activities as well as molecular size of the levan formed by the acetic acid bacterium Gluconobacter albidus TMW 2.1191 at varying environmental pH conditions to obtain insight in the ecological role of its constitutively expressed levansucrase and the produced levan. A buffer system was established enabling the recovery of levansucrase-containing supernatants from preincubated cell suspensions at pH 4.3–pH 5.7. The enzyme solutions were used to produce levans at different pH values and sucrose concentrations. Finally, the amounts and size distributions of the produced levans as well as the corresponding levansucrase activities were determined and correlated with each other. The data revealed that the levansucrase was released into the environment independently of its substrate sucrose, and that more levansucrase was released at pH ≥ 5.0. The glucose release and formation of high molecular weight levans (> 3.5 kDa) from 0.1 M initial sucrose was comparable between pH ~ 4.3–5.7 using equal amounts of released levansucrase. Hence, this type of levansucrase appears to be structurally adapted to changes in the extracellular pH and to exhibit a similar total activity over a wide acidic pH range, while it produced higher amounts of larger levan molecules at higher production pH and sucrose concentrations. These findings indicate the physiological adaptation of G. albidus TMW 2.1191 to efficient colonisation of sucrose-rich habitats via released levansucrases despite changing extracellular pH conditions in course of acid formation.