A thermostable β-1,6-endoglucanase or pustulanase (E.C. 3.2.1.75) which catalyses the cleavage of β-1,6 bonds in pustulan and other beta-glucans containing 1,6 linked glucose units. The enzyme is now a component in a standard assay for beta-glucan from Baker's yeast as documented in US Pharmacopeia Food and Chemicals Index 7th Edition, 3rd Supplement p. 1686-1689. Available as 100 units or 500 units. Shipping within a week, shipping and handling charges additional to price listed below. Product : Cel136 | Pustulanase 100 Units | Price : 310.00 EUR Product : Cel136L | Pustulanase 500 units | Price : 990.00 EUR Read more below or download the product sheet Storage and Shelf Life The product is freeze-dried and is stored in a frozen condition by Prokazyme. Upon receipt, customers shall store the product at 1–4°C in a dark place. When stored under these conditions, the product has a shelf life of 12 months. - Enzyme activity: Cel136 Beta-glucanase (glucan endo-1,6-β-glucosidase) hydrolyses Beta-glucan polysaccharides with β-(1,6) linkages - see further below for substrate specificity Activity determination: The standard assay for activity was done by incubating the enzyme at 80°C for 10 min. with 1% (w/v) pustulan as substrate in 0,1 M sodium phosphate buffer at pH 5.5. The reducing sugars released were detected by the dinitrosalisylic acid method using glucose as standard. Unit definition: One unit (U) of enzyme activity is the amount that leads to the release of 1 μmol reducing sugars per minute. Synonyms: glycoside hydrolase, pustulanase, glucan endo-1,6-β-glucosidase, 6-β-D-glucan glucanohydrolase, endo-1,6-β-glucanase; β-1→6)-β-D-glucanase; β-1,6-glucanase-pustulanase; β-1,6-glucan hydrolase; β-1,6-glucan 6-glucanohydrolase; 1,6-β-D-glucan glucanohydrolase Protein family: Glycosyl hydrolase family 30 ( GH30 ) - CAZy database family 30 Enzyme classification: EC 3.2.1.75 Source: Proprietary metagenome environmental DNA Beta-Glucan Assay: Beta-glucanase (Pustulanase) Cel136 is recommended for the determination of yeast Beta-Glucan. The United States Pharmacopeia (USP) and the USA-based company Biothera have developed the first official monograph for beta glucan from bakers yeast (Saccharomyces Cerevisiae), recognized for its immune health benefits and used as dietary supplement in food and beverages. The monograph was issued in the Third Supplement of the Seventh Edition of the Food Chemicals Codex (compendium of standards for the purity and identity of food ingredients) and includes a protocol for beta-glucan assay using the Cel136 enzyme. See "Beta Glucan from Baker’s Yeast (Saccharomyces cerevisiae)". Food Chemical Index, 7th Edition, Third supplement, p. 1686-1689. (2011) Properties of Cel136 Beta-glucanase (Pustulanase): Substrate specificity: Cel136 Beta-glucanase (Pustulanase) Cel136 hydrolyses β-glucan polysaccharides with β-(1,6) linkages such as Pustulan ( β-(1,6)-D-glucan ) and Gentiobiose ( β-(1,6)-linked disaccharide) or β-glucans with mixed linkages, including β-(1,6) linkages, such as Laminarin ( β-(1,3/1,6)-D-glucan ) and Yeast β-glucan ( β-(1,3/1,6)-D-glucan ). Substrate specificity analysis (below) indicates that the enzyme does not cleave β-(1,3) or β-(1,4) linkages as it does not show activity on the β-glucans Lichenan ( β-(1,3/1,4)-D-glucan ) or CM-Cellulose ( β-(1,4)-D-glucan). Temperature Optimum: The enzyme is relatively active in a rather broad temperature range (65-90°C)with optimum around 80°C pH Optimum: suitable pH range is about 5-8 with optimum around 5.5 References Beta Glucan from Baker’s Yeast (Saccharomyces cerevisiae). Food Chemical Index, 7th Edition, Third supplement, p. 1686-1689. (2011) - inlcudes assay for beta-glucan using the Cel136 product. M. Danielson et al.: Enzymatic Method to Measure β-1,3-β-1,6-Glucan Content in Extracts and Formulated Products (GEM Assay), J. Agric. Food Chem. 58: 10305-10308 (2010).
A thermostable β-1,6-endoglucanase developed from environmental DNA
Tsc DNA ligase from Thermus scotoductus is a thermostable NAD+ dependent double-stranded DNA ligase with activity at temperatures between 15 and 75°C. The enzyme has no activity on blunt-end DNA fragments. Thermostable Tsc DNA ligase and Rma DNA ligase (Dlig120) have shown to have favorable properties in comparison to other DNA ligases. For reference please consider the following papers: Housby & Southern 2002: Thermus scotoductus and Rhodothermus marinus DNA ligases have higher ligation efficiencies than Thermus thermophilus DNA ligase. Anal. Biochem. 302:88-94 - link to article Housby JN et al. 2000. Optimised ligation of oligonucleotides by thermal ligases: comparison of Thermus scotoductus andRhodothermus marinus DNA ligases to other thermophilic ligases. Nucl. Acid Res. 28:e10 - link to article Thorbjarnardottir SH et et al. 1995: Cloning and sequence analysis of the DNA ligase-encoding gene of Rhodothermus marinus, and overproduction, purification and characterization of two thermophilic DNA ligases. Gene 161:1-6 - link to article Jonsson Zo et al . 1994: Sequence of the DNA ligase-encoding gene from Thermus scotoductus and conserved motifs in DNA ligases. Gene 151: 177-180 - link to article Product Dlig119 Tsc DNA ligase is available as 500 units and 2000 units together with 10x reaction buffers. - Shipping within a week, shipping and handling charges additional to price listed below. Product : Dlig119S | DNA Ligase 500 Units | Price : 310.00 EUR Product : Dlig119L | DNA Ligase 2000 Units | Price : 450.00 EUR For further information read below or download the product sheet Product description Introduction: Tsc DNA ligase catalyzes the NAD-dependent ligation of adjacent 3´-hydroxyl and 5´-phosphate termini in duplex DNA structures. In contrast to T4 DNA ligase, Tsc DNA ligase has no detectable activity on blunt end DNA fragments. Unlike T4 DNA ligase, Tsc DNA ligase shows only minimal ligation activity under optimal temperature conditions for 4 bp as well as 2 bp of cohesive ends. Tsc DNA ligase has no activity on RNA targets. Tsc DNA ligase is isolated and purified from an E.coli strain carrying a plasmid with the cloned DNA ligase gene from the thermophilic bacteria Thermus scotoduc-tus isolated in Iceland (1, 2). The half-life of Tsc ligase is 26 min at 91°C (3). The enzyme has a broad range of reaction temperatures with the lower limit around 15°C and the upper limit determined by the melting temperature (Tm) of the DNA substrate. The enzyme is also active in various DNA polymerase buffers within the pH range of 7-9. Under optimal conditions the rate and extent of oligonucleotide ligation is much higher for Tsc DNA ligase compared to other commonly available thermostable ligases (4,5). Applications: Tsc DNA ligase is an ideal enzyme for applications re-quiring high temperature, high-stringency ligations of double-stranded DNA.Tsc DNA ligase may be applied to: Ligase Chain Reaction (LCR) (6-8) for amplification of DNA targets. Oligonucleotide Ligation Assay (OLA) (9-10) for mutational analysis. Repeat Expansion Detection (11) for determining genetic anomalies, such as trinucleotide repeats, research only, not for diagnostic purposes (for e.g. Fragile X, Huntington disease). Gene Synthesis (12) from overlapping oligonucleo-tides. Storage: Storage and dilution buffer: 20 mM Tris-HCl, 50 mM KCl, 0,1 mM EDTA, 0,1% Triton X-100 (v/v), 1 mM dithiothreitol (DTT), 50% glycerol (v/v), pH 7,6 (25°C). Tsc Ligase is stable when stored at –15°C to -25°C. Reaction conditions: 1 x reaction buffer (10 x supplied) 20 mM Tris-HCl, 20 mM KCl, 10 mM MgCl2, 0,1% Nonidet P40 (v/v), 0,5 mM NAD, 1 mM DTT, pH 7,5 (25°C). Concentration and unit definition Concentration 10 U/μl. One unit of Tsc DNA Ligase catalyzes the ligation of 50% of the cos sites of 1μg BstEII digested λDNA in 1 min at 45°C. Application Protocol: Reaction protocol for oligonucleotide ligation: Thaw the components listed below and place them on ice. Vortex briefly and centrifuge all reagents before setting up the reactions. Set up the reaction components in a microfuge tube placed on ice: Component Volume Final conc. Reaction buffer (10x) 2,0 μl 1 x Oligo 1 X μl 1-30nM Oligo 2 X μl 1-30nM Template DNA X μl 0,1 ng Tsc DNA Ligase 0,5 μl 5 U Add sterile H2O Up to 20,0 μl TOTAL 20,0 μl A typical temperature profile is: 94°C 2 min, 94°C 30 sec, 45-65°C 3 min and repeat last two temperatures for 30 cycles. 99°C for 10 min. Activity assay: The enzyme assay for unit definition was ligation of cos sites of λDNA digested with BstII. Component Volume Final conc. Reaction buffer (10x) 2,0 μl 1 x λDNA (BstEII digested) X μl 1 μg Tsc DNA Ligase Dilution serie Add sterile H2O Up to 20,0 μl TOTAL 20,0 μl Incubate at 45°C for 1-15 min. Stop reaction in dry ice/ethanol bath. Incubate for 10 min at 65°C before analysis on agarose gel (melting of not ligated cos sites) Results are assayed by agarose gel electrophoresis and ethidium bromide staining. Quality Control: Quality Control: Each lot of ThermoPhageTM ssDNA ligase is assayed for activity and for contaminating activities as stated be-low. Absence of DNA endonuclease: 0,25 μg supercoiled pBR322 DNA is incubated with increasing amounts of Tsc DNA ligase in 25 μl reactions at 37°C for 16 h. >100 U of Tsc DNA ligase show no relaxation of the supercoiled structure of pBR322 DNA. 0,25 μg of λ-DNA Eco RI/HindIII fragments is incu-bated with Tsc DNA ligase in 25 μl reactions at 37°C and 64°C for 16 h. 100 U of Tsc DNA ligase show no alteration of the banding pattern. Absence of exonuclease: Increasing amounts of Tsc DNA ligase are incubated in 50 μl test buffer containing [3H]-labelled DNA at 37°C and 64°C for 4 h. The amount of enzyme, which shows no exonuclease activity is >100 U. Absence of Rnases: RNaseAlertTM Lab Test Kit (cat no. 1964) from Ambion was used to detect RNase activity according to the manufacturer protocol. No RNase activity was detected after incubating X U of Tsc DNA ligase at 37°C after 1 hour. References: Jonsson ZO, et al. 1994. Gene 151: 177-80 Kristjansson JK et al. 1995. System. Appl. Microbiol. 17: 44-50 Thorbjarnardottir SH et al. 1995. Gene 161: 1-6 Housby JN, et al. 2000. Nucleic Acids Research. 28: e10. Housby JN, et al. 2001. Anal. Biochem. 302: 88-94 Landegren U, et al 1988. Science 241: 1077-1080 Landegren U, et al 1988. Science 242: 229-237 Barany F, 1991. PCR Meth. and Appl.. 1: 5-16 Nickerson DA, et al. 1990. Proc. Natl. Acad. Sci. USA 87: 8923-8927 Baron H, et al. 1996. Nature Biotech 14: 1279-1282 Schalling M, et al. 1993. Nature Genetics 4: 135 Sutton JR, et al. 1992. Transgenic Research 1: 228
A thermostable NAD+ dependent double-stranded DNA ligase with activity at temperatures between 15 and 75°C.
DNA ligase from Rhodothermus marinus is a thermostable NAD+ dependent double-stranded DNA ligase with broad temperatures range between 5 and 65°C. The enzyme catalyzes ligation of DNA with high fidelity and is suitable for various applications including gene synthesis. Product Dlig120 Rma DNA ligase is available as 500 units and 2000 units together with 10x reaction buffers. - Shipping within a week, shipping and handling charges additional to price listed below. Product : Dlig120S | DNA ligase 500 Units | Price : 310.00 EUR Product : Dlig120L | DNA Ligase 2000 Units | Price : 450.00 EUR For further information read below or download the product sheet Product description Introduction: Rma DNA ligase catalyzes the NAD-dependent ligation of adjacent 3´-hydroxyl and 5´-phosphate termini in duplex DNA structures. In contrast to T4 DNA ligase, Rma DNA ligase has no detectable activity on blunt end DNA fragments. Unlike T4 DNA ligase, Rma DNA ligase shows only minimal ligation activity under optimal temperature conditions for 4 bp as well as 2 bp of co-hesive ends. Rma DNA ligase has no activity on RNA targets. Rma DNA ligase is isolated and purified from an E.coli strain carrying a plasmid with the cloned DNA ligase gene from the thermophilic bacteria Rhodother-mus marinus isolated in Iceland (1, 2). The half-life of Rma ligase is 7 min at 91°C (2). The enzyme has a broad range of reaction temperatures with the optimal activity about 55°C. Under optimal conditions the rate and extent of oligonucleotide ligation is higher for Rma DNA ligase compared to Tth and Taq DNA ligases (3,4). Applications: Tsc DNA ligase is an ideal enzyme for applications requiring high temperature, high-stringency ligations of double-stranded DNA.Tsc DNA ligase may be applied to: Gene Synthesis (12) from overlapping oligonucleo-tides. Storage: Storage and dilution buffer: 20 mM Tris-HCl, 50 mM KCl, 0,1 mM EDTA, 0,1% Triton X-100 (v/v), 1 mM dithiothreitol (DTT), 50% glycerol (v/v), pH 7,6 (25°C). Rma DNA ligase is stable when stored at –15°C to -25°C. Reaction conditions: 1 x reaction buffer (10 x supplied) 20 mM Tris-HCl, 20 mM KCl, 10 mM MgCl2, 0,1% Nonidet P40 (v/v), 0,5 mM NAD, 1 mM DTT, pH 7,5 (25°C). Concentration and unit definition Concentration 10 U/μl. One unit of Rma DNA ligase catalyzes the ligation of 50% of the cos sites of 1μg BstEII digested λDNA in 1 min at 45°C. Application Protocol: Activity assay: The enzyme assay for unit definition was ligation of cos sites of λDNA digested with BstII. Component Volume Final conc. Reaction buffer (10x) 2,0 μl 1 x λDNA (BstEII digested) X μl 1 μg Tsc DNA Ligase Dilution serie Add sterile H2O Up to 20,0 μl TOTAL 20,0 μl Incubate at 45°C for 1-15 min.Stop reaction in dry ice/ethanol bath. Incubate for 10 min at 65°C before analysis on agarose gel (melting of not ligated cos sites) Results are assayed by agarose gel electrophore-sis and ethidium bromide staining. Quality Control: Quality Control: Each lot of Tsc DNA Ligase is assayed for activity and for contaminating activities as stated below. Absence of DNA endonuclease: 0,25 μg supercoiled pBR322 DNA is incubated with increasing amounts of Tsc DNA ligase in 25 μl reactions at 37°C for 16 h. >100 U of Tsc DNA ligase show no relaxation of the supercoiled structure of pBR322 DNA. 0,25 μg of λ-DNA Eco RI/HindIII fragments is incu-bated with Tsc DNA ligase in 25 μl reactions at 37°C and 64°C for 16 h. 100 U of Tsc DNA ligase show no alteration of the banding pattern. Absence of exonuclease: Increasing amounts of Tsc DNA ligase are incubated in 50 μl test buffer containing [3H]-labelled DNA at 37°C and 64°C for 4 h. The amount of enzyme, which shows no exonuclease activity is >100 U. Absence of Rnases: RNaseAlertTM Lab Test Kit (cat no. 1964) from Ambion was used to detect RNase activity according to the manufacturer protocol. No RNase activity was detected after incubating X U of Tsc DNA ligase at 37°C after 1 hours. References: Alfredsson GA, et al. 1988. J. Gen. Microbiol. 134:299-306 Torbjarnardottir SH et al. 1995. Gene 161: 1-6 Housby JN, et al. 2000. Nucleic Acids Research. 28: e10. Housby JN, et al. 2001. Anal. Biochem. 302: 88-94 Sutton JR, et al. 1992. Transgenic Research 1: 228
A thermostable NAD+ dependent double-stranded DNA ligase with broad temperatures range between 5 and 65°C.
ThermoPhage™ Polynucleotide Kinase is a novel thermoactive kinase catalysing 5’ phosphorylation of DNA at elevated temperatures and is the only available thermostable enzyme of this kind. The enzyme has very low 3' phosphatase activity. Thermophage™ Polynucleotide Kinase is well suited for various applications including radioactive labelling of nucleic acids (Blondal et al 2005, J Biol Chem 280:5188-94 ) Available as 500 units and 1000 units (select product above) together with 10x reaction buffers. Shipping within a week, shipping and handling charges additional to price listed below. Product : Pnk162S | Polynucleotide kinase 500 Units | Price : 310.00 EUR Product : Pnk162L | Polynucleotide kinase 1000 Units | Price : 450.00 EUR For further information read more below or download the product sheet Introduction: ThermoPhageTM Polynucleotide Kinase (PNK) catalyses the transfer of phosphate (Pi) from the γ- position of ATP to the 5’ end of a hydroxylated nucleic acid (RNA or DNA, single or double stranded) or an exchange of the Pi to a mono-phosphorylated nucleic acid (1). ThermoPhageTM PNK is derived from bacteriophage RM378 that infects Rhodothermus marinus (2). The recombinant PNK protein was expressed and purified from E. coli. ThermoPhageTM PNK has a temperature optimum between 60-70°C. For short incubation time (1 hour or less) temperature optimum of the enzyme is about 70°C but for longer incubation protocols we recommend 60-65°C. ThermoPhage PNK has Mn2+ -dependent 3’ phosphohydrolase activity that does not interfere with its 5’ kinase activity. Applications: Labeling of nucleic acids using 32P-γ-ATP for probes and DNA sequencing. Phosphorylation of nucleic acids for subsequent ligation for cloning. Phosphorylation of oligonucleotides for ligase reaction like the Ligase Chain Reaction and similar procedures (3-4). Phosphorylation of nucleic acids with modified phosphates (i.e. thiol-phosphates) for subsequent modifications and/or labeling. Notes: ThermoPhage PNK is inhibited by high (>50 mM) salt concentrations (KCl and NaCl), and also strongly inhibited by phosphate ions and ammonium ions. Labeling of 5’phosphorylated nucleic acids (exchange reaction) will work in the same buffer as the forward reaction but more time and enzyme is needed. If using protocols including RNA and RNAse inhibitors, make sure they are active at high temperature, for example SUPERase InTM (Ambion) or RNAse-Free Ribonuclease inhibitor (CHIMERx). Storage: Storage and dilution buffer: 10 mM Tris (pH 8), 50 mM KCl, 0.1 mM EDTA, 0.1 μM ATP, 1 mM DTT and 50 % glycerol. ThermoPhageTM PNK is stable for one year when stored at –25 to -15 °C. Reaction conditions for unit definition: 1 x reaction buffer (50 mM MOPS (pH 8.5), 1 mM DTT, 10 mM MgCl2, 10 mM KCl), with 2 nmol ATP, 25 μg/ml BSA, 5% PEG6000, 1 nmol ssDNA oligonucleotides and ThermoPhageTM PNK enzyme incubated at 70°C for 15 minutes in 20 μl volume. Concentration and unit definition: Concentration 25 U/μl One unit of ThermoPhageTM Polynucleotide Kinase catalyses the transfer of 1 nmol of γ-phosphate from ATP to 5’ hydroxylated end of oligonucleotides at 70°C in 30 minutes (5). Application Protocol: Reaction protocol: For optimized phosphorylation reaction protocol use 10-20 units of enzyme in a 25-50 μl reaction volume with 1x reaction buffer, 5 % PEG6000, 25 μg/ml BSA, 10-100 μM ATP and substrate. Incubate at 60-70°C for 1-2 hours. For labeling reaction using 32P, 33P-γ-labeled ATP use 10 pmol of the labeled ATP, at least 10 pmol substrate and 10 units PNK in its standard buffer as described above. Incubate at 60-70°C for 1-2 hours. If doing an exchange 5’ kinase reaction, increase the incubation time and/or the amount of enzyme used. Activity assay: 1x ThermoPhageTM PNK buffer, 25 μg/ml BSA, 2 nmol ATP (mixture of ATP and 32P-γ-ATP) and 1 nmol ssDNA 5’ hydroxylated oligonucletide oligomer substrate and 0.1-0.5 unit PNK in 20 μl reaction volume. After incubation at 70°C for 15 minutes, the reactions were terminated by heating it at 95°C for 5 minutes, and the samples captured on DE81 filters and washed twice in 250 mM phosphate buffer. The filter was then dried and counted for radioactivity in a liquid scintillation counter and the incorporation of phosphate determined. Characterization: Temperature Stability: ThermoPhageTM Polynucleotide kinase is stable at 60°C but starts to loose activity at extended time at 65- 70°C. The standard assay was done at 50, 60, 65 and 70°C and samples taken at time 0, 30, 60 and 120 min (Fig 1). Fig 1: Thermostability over time at 50-70°C. The enzyme is stable at 50-60°C but starts to loose activity at extended time at 65-70°C. Temperature Optimum: The temperature optimum of the ThermoPhage™ polynucleotide kinase is between 60°C and 70°C. We recommend 70°C for short incubation times (1 hour). For longer incubation times we recommend incubating at 60°C. However, the enzyme shows relatively high activity up to 90°C for shorter incubation times as shown in Figure 2. Fig 2: Activity of ThermoPhageTM Polynucleotide kinase incubated at given temperature for 1 hour under standard assay conditions. 5' Kinase Activity: ThermoPhage Polynucleotide kinase was assayed with 10 μM ATP (mixture of 32P-γ- and unlabeled ATP) and 20 μM dA20 or rA20 oligomers under standard conditions and 5% PEG6000. Reaction time was 30 min at 70°C in 10 μl reaction volumes. Reactions were terminated by adding stop solution (95% formamide 20 mM EDTA, 0.05% bromophenol blue and 0.05% Xylene Cyanol FF). Samples were run on 20% UREA-PAGE gels, dried and autoradiographed. Fig 3: Phosphorylation of ssDNA (dA20) and RNA (rA20) under standard condition. The PAGE gel shows complete depletion of ATP in the labeling reation using 5 units ThermoPhage PNK enzyme. 3' Phosphohydrolase Activity: ThermoPhageTM PNK has optimal 3’ phosphohydrolase activity at pH 5-7 and mainly in the presence of Mn2+ as seen in figure 4. The main substrate is 2’-3’ cAMP as seen in figure 5. The 3’ hydrolase activity should not interfere with the 5’ kinase reaction of the ThermoPhageTM PNK. Fig 4: pH profile of ThermoPhageTM PNK 3’ phosphohydrolase activity on 2’-3’ cAMP. Fig 5: Comparison of 3’ phosphohydrolase activity of ThermoPhage PNK and T4 PNK on 100 μM 2’-3’cAMP, 3’TMP and dA15-PO4- substrates. Quality Control: Each lot of ThermoPhageTM Polynucleotide kinase is assayed for activity and for contaminating activities as stated below. Absence of DNA endonuclease: 0,25 μg supercoiled pBR322 DNA is incubated with increasing amounts of ThermoPhageTM PNK in 25 μl reactions at 37°C and 64°C for 4 h. 100 U of ThermoPhageTM PNK show no relaxation of the supercoiled structure of pBR322 DNA. 0,25 μg of λ-DNA Eco RI/HindIII fragments is incubated with ThermoPhageTM PNK in 25 μl reactions at 37°C and 64°C for 4 h. 100 U of ThermoPhageTM PNK show no alteration of the banding pattern. Absence of exonuclease: Increasing amounts of ThermoPhageTM PNK are incubated in 50 μl test buffer containing [3H]-labelled DNA at 37°C and 64°C for 4 h. The amount of enzyme, which shows no exonuclease activity is at least 200 units. Absence of Rnases: RNaseAlertTM Lab Test Kit (cat no. 1964) from Ambion was used to detect RNase activity according to the manufacturer protocol. No RNase activity was detected after 1 hour incubation of 100 units of ThermoPhageTM PNK. References: Sambrook, J., Fritsch, E. F., and T., M. (1989) Molecular cloning, a laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY. Hjörleifsdottir, S. et al. (2002) Bacteriophage RM 378 of a thermophilic host organism - US Patent No. 6,492,161 Blondal et al. (2005) Characterization of a 5′-Polynucleotide Kinase/3′-Phosphatase from Bacteriophage RM378 - J Biol Chem 280:5188-94. Wu, D. Y. & Wallace, R. B. (1989) Genomics 4, 560-569. Barringer, K., et al. (1990) Gene 89, 117-122. Richardson, C. C. (1965) Proc Natl Acad Sci U S A 54, 158-165.
ThermoPhage™ Polynucleotide Kinase is a novel thermoactive kinase catalysing 5’ phosphorylation of DNA at elevated temperatures and is the only available thermostable enzyme of this kind.
A moderately thermostable Glucansucrase (4,6-Alpha-Glucanotransferase, reuteransucrase). The enzyme transfers glucose units from sucrose to make a highly branched, high molecular weight alpha-D-Glucan with α(1→4) glucosidic linkages and also some α(1→6) linked glucosyl units. The enzyme is available for purchase as 100 or 500 microliter solution (1 mg/ml). Product Gtfa163S | 100 microliter | EUR 310 Product Gtfa163L | 500 microliter | EUR 700 For purchase select size and add to cart above. Shipping within a week, shipping and handling charges additional to price listed above. For further information read more below or download the product sheet Enzyme activity: Gtfa163 Glucansucrase (Reuteransucrase) catalyses the transfer of glucose from sucrose to make a highly branched, high-molecular-weight α-D-glucan with α(1→4) glucosidic linkages but also some α(1→6)-linked glucosyl units and 4,6-disubstituted glucosyl units at the branching points. As an example, glucose transferase activity was determined at 50°C for 60 hours in a 25 mM sodium acetate buffer at pH 4.7, containing 1 mM CaCl2 and 100 mM sucrose. This gave complete utilization of the sucrose with about 75% going into α-D-glucan. Activity determination: Glucansucrase activity was determined at 50°C in a 25 mM sodium acetate buffer at pH 4.7, containing 10 mM CaCl2 and 30 nM enzyme. Unit definition: One unit (U) of enzyme activity is the amount that leads to the release of 1 μmol of fructose from sucrose per minute. Synonyms: glycoside hydrolase, glucosyltransferase, 4,6-alpha-Glucanotransferase, Reuteransucrase Protein family: Glycosyl hydrolase family 70 ( GH70 ) - GH-H clan - CAZy database GH70 family Enzyme classification: EC 2.4.1.- Source: The lactic acid bacteria Lactobacillus reuteri strain 121 Protein sequence: NBCI protein entry Structural information: The crystal structure of Gtfa163 Glucansucrase (Reuteransucrase) from Lactobacillus reuteri121 has been determined and shown here in Figure 1 (Pijning et al. 2012). - PDB entry 4AMC Figure 1: Crystal structure of Gtfa163 Glucansucrase (truncated form comprising residues 745-1763 including catalytic domain) Applications: Exopolysaccharide synthesis: The enzyme produced a α-D-glucan polymer (MW 3.5x106 Da) with a composite structure, that includes all identified structural elements, as shown in Fig. 2 below (van Leeuwen et al 2008) Figure 2. Composite model of a alpha-D-glucan product made by Gtfa163 Glucansucrase Oligosaccharides from sucrose: With only sucrose present, and before branching starts, the enzyme activity will yield a linear oligosaccharides via alternating α(1→4) and α(1→6) linkages. Malto-oligosaccharides (DP2-DP6) in the absence of sucrose are slow substrates. With high concentration of sucrose the formation of low-molecular-mass oligosaccharides was favoured over high-molecular-mass polysaccharides. Properties of Gtfa163 Glucansucrase: Figure 3. Temperature optimum: the enzyme has optimum activity around 50°C (Kralj et al. 2004). Figure 4. pH profile: Solid line, transferase activity; dashed line, hydrolysis activity (Kralj et al. 2004). References: Van Leeuwen SS, Kralj S, van Geel-Schutten IH, Gerwig GJ, Dijkhuizen L, Kamerling JP (2008) Structural analysis of the α-D-glucan (EPS35-5) produced by the Lactobacillus reuteri strain 35-5 glucansucrase GTFA enzyme. Carbohydr. Res. 343: 1251–1265. Kralj S, van Geel-Schutten GH, van der Maarel MJEC & Dijkhuizen L (2004) Biochemical and molecular characterization of Lactobacillus reuteri 121 reuteransucrase. Microbiology 150: 2099–2112. Leemhuis H, Pijning T, Dobruchowska JM, van Leeuwen SS, Kralj S, Dijkstra BW, Dijkhuizen L. (2013) Glucansucrases: Three-dimensional structures, reactions, mechanism, α-glucan analysis and their implications in biotechnology and food applications. J. Biotechnol. 163:250-272. Pijning T, Vujicic-Zagar A, Kralj S, Dijkhuizen L & Dijkstra BW (2012) Structure of the α-1,6/α-1,4-specific glucansucrase GTFA fromLactobacillus reuteri 121. Acta Cryst. F68: 1448-1454.
A moderately thermostable Glucansucrase (4,6-Alpha-Glucanotransferase, reuteransucrase) from Lactobacillus reuteri strain 121
A thermostable 4-α-glucanotransferase that elongates linear alpha-glucan chains in starch and amylose by catalyzing the transfer of one glucose unit from the non-reducing end to a new position in an acceptor, which may be triose or larger. The enzyme can be used various applications, such as for modifications of alpha-polysaccharides and is ideal for making clear size ladders of oligosaccharides larger than maltose. The enzyme was developed from metagenome DNA obtained from environmental sample from a geothermal environment. Available for purchase as 1000 units. For purchase select product and add to cart above. We accept payments with credit cards via PayPal or direct bank transfer. Shipping within a week, shipping and handling charges additional to price listed above. Product : Elo127 | Glucan elongation enzyme 1000 Units | Price : 330.00 EURFor further product information read more below or download the product sheet (pdf format). Mode of action: The enzyme catalyzes the formation of the alpha-1,4-glucosidic linkages. The enzyme transfers a segment of a (1->4)-alpha-D-glucan to a new position in an acceptor, which may be glucose or a (1->4)-alpha-D-glucan. Unit Definition: Half unit (U) is defined as the amount of enzyme which lowers the OD at 650 nm from 1.2 to 0.7 in 30minutes. Unit Assay Conditions: Branching enzyme activity was routinely determined in a 50mM KPO4 pH 8.0 buffer for 10 minutes, with 0.01% (w/v) final conc. of amylose. Source: Bacterial Environmental DNA Enzyme properties: Temperature Optimum: The enzyme in relatively active in a rather broad temperature range with optimum around 70°C (Figure 1) pH Optimum: pH optimum is around 7 (Figure 2)
A thermostable 4-α-glucanotransferase that elongates linear alpha-glucan chains in starch and amylose.
A thermostable Alpha-L-Rhamnosidase (Naringinase, RhamA) that catalyzes the cleavage of the bond between terminal L(+)-rhamnose and the aglycone of rhamnose-containing glycosides. The enzyme is very active on naringin but has also substantial activity with hesperidin as substrate. Available as 100 units. For purchase select product and add to cart above. We accept payments with credit cards via PayPal or direct bank transfer. Shipping within a week, shipping and handling charges additional to price listed. Product : Rham142 | Naringinase 100 Units | Price : 390.00 EUR For further information read more below or download the product sheet (pdf format). Enzyme activity: Rham142 Rhamnosidase A (naringinase, alpha-L-rhamnosidase) catalyzes the cleavage of the bond between terminal L(+)-rhamnose and the aglycone of rhamnose-containing glycosides. Hydrolysis of terminal non-reducing α-L-rhamnose residues in α-L-rhamnosides: naringin, hesperidin and rutin Activity determination: Rhaminosidase activity was routinely determined in a 100mM KPO4 pH 7.5 buffer for 10 minutes with 2.0mM final conc. of p-nitro-phenyl-α-L-rhamnopyranoside. Unit definition: One unit (U) of enzyme activity is the amount that leads to the release of 1 µmol of p-nitro-phenyl-α-L-rhamnopyranoside (pnpR) per minute Synonyms: glycoside hydrolase; RhamA; naringinase; hesperidinase; α-L-rhamnosidase A; α-L-rhamnosidase N; α-L-rhamnoside rhamnohydrolase Protein family: Glycosyl hydrolase family 78 ( GH78 ) - GH-H clan - CAZy database GH78 family Enzyme classification: EC 3.2.1.40 Source: Bacteria; Thermomicrobia strain PRI-1686 Protein sequence: NBCI protein entry Structural information: The crystal structure of alpha-L-rhamnosidase from Bacillus sp. GL1, sharing 52% sequence identity with ThermoactiveTM Rhamnosidase A, has been determined to a resolution of 1.9 Å ( Cui et al. 2007 ). -Protein Data Bank entry 2OKX Substrates: Alpha-L-rhamnosidasaes catalyse the release of terminal rhamnose residues from polysaccharides and glycosides. Of the many natural compounds that contain terminal alpha-L-rhamnose, the flavonoids naringin, hesperidin, rutin and quercitrin have been the main natural test-substrates for alpha-L-rhamnosidases. Of these compounds, ThermoactiveTM Rhamnosidase A was found to be most active on Naringin as shown in Figure 1 (Birgisson et al 2004). The structure of naringin (4′,5,7-trihydroxyflavanone-7-α-L-rhamnopyranoside-(1,2)-β-D-glucopyranoside) and the hydrolysis by rhamnosidase is shown in Figure 2. Figure 1. Substrate specificity: hydrolysis of terminal non-reducing α-L-rhamnose residues in the flovonoids naringin, hesperdin, rutin and quercitrin Figure 2. Structure of naringin, with rhamnose 1,2 linked to a glucose residue linked to the flavonoid skeleton, and the hydrolysis of the terminal α-L-rhamnose residues by rhamnosidase Applications: Naringin is a source of bitter flavor in fruit juice and rhamnosidases with naringinase activity are frequently used for debittering citrus juice. Other biotechnological applications include manufacture of prunin; manufacture of alpha-L-rhamnosidese fom natural glycosides; clarification of juices; enhancement of wine aromas by hydrolysis of terpenyl glycosides; conversion of chloropolysporin B to chloropolysporin C and production of pharmaceutically important compounds by removal of rhamnose residues from steriods such as diosgene, desglucoruscin and ginsenosides-Rg2 (Yadav et al. 2010). Beta-glucosidases may be used in combination with alpha-L-rhamnosidases for removal of glucose from the flavonoid skeleton. ThermoactiveTM Rhamnosidase A has been successfully demonstrated for use in production of rhamnose from narigin in a bioreactor containing immobilized E. coli cells expressing the gene for the enzyme (Birgisson et al 2007). L-Rhamnose or its derivatives are suitable chiral structural component and can be used for the synthesis of pharmaceutical products, plant protection agents and the preparation of fragrances in the foodstuffs and perfume industries. Properties of ThermoActive™ Rhamnosidase A: Figure 3. Temperature spetrum: The enzyme in relatively active in a rather broad temperature range (45-75°C)with optimum around 65°C Figure 4. pH spectrum: pH range is about 4.5-9 with optimum about pH 7.5 References: Birgisson H, Hreggvidsson GO, Fridjónsson OH, Mort A, Kristjánsson JK, Mattiasson B (2004) Two new thermostable alpha-L-rhamnosidases from a novel thermophilic bacterium. Enzyme Microb. Technol. 34: 561-571 Birgisson H, Wheat JO, Hreggvidsson GO, Kristjánsson JK, Mattiasson B. (2007) Immobilization of a recombinant Escherichia coli producing a thermostable α-l-rhamnosidase: Creation of a bioreactor for hydrolyses of naringin. Enzyme and Microbial Technology 40:1181-1187 Cui Z, Maruyama Y, Mikami B, Hashimoto W and Murata K (2007) Crystal Structure of Glycoside Hydrolase Family 78 α-L-Rhamnosidase from Bacillus sp. GL. J Mol Biol 374: 384–398 Yadav V, Yadav PK, Yadav S & Yadav KDS (2010) α-L-Rhamnosidase: A review. Process Biochem 45:1226–1235
A thermostable Alpha-L-Rhamnosidase (Naringinase, RhamA) from Thermomicrobia sp.
A Thermostable α-L-rhamnosidase that catalyzes the cleavage of the bond between terminal L(+)-rhamnose and the aglycone of rhamnose-containing glycosides. The enzyme is also very active on naringin. L-Rhamnose or its derivatives is a suitable chiral structural component and can be used for the synthesis of pharmaceutical products, plant protection agents and the preparation of fragrances in the foodstuffs and perfume industries. Available as 100 units. Shipping within a week, shipping and handling charges additional to price listed below. For purchase select product and add to cart above. We accept payments with credit cards via PayPal or direct bank transfer. Shipping within a week, shipping and handling charges additional to price listed. Product : Rham143 | Hesperedinase 100 Units | Price : 390.00 EUR For further information read more below or download the product sheet (pdf format). Mode of action: The enzyme catalyzes the cleavage of the bond between terminal L(+)-rhamnose and the aglycone of rhamnose-containing glycosides. Hydrolysis of terminal non-reducing α-L-rhamnose residues in α-L-rhamnosides, Naringin, Hesperdin and Rutin. Unit Definition: One unit (U) of enzyme activity is the amount that leads to the release of 1 µmol of p-nitro-phenyl-α-L-rhamnopyranoside (pnpR) per minute. Temperature Optimum: The enzyme in relatively active in a rather broad temperature range (45-75°C)with optimum around 65°C (Figure 1) pH Optimum: pH range is about 4.5-9 with optimum about pH 7.5 Substrate specificity: Hydrolysis of terminal non-reducing α-L-rhamnose residues in α-L-rhamnosides, Naringin, Hesperdin and Rutin. Unit Assay Conditions: Rhaminosidase activity was routinely determined in a 100mM KPO4 pH 7.5 buffer for 10 minutes with 2.0mM final conc. of pnpR. References: 1) Birgisson, H., Hreggvidsson, G. O., Fridjónsson, O. H., Mort, A., Kristjánsson, J. K., Mattiasson, B., Two new thermostable α-L-rhmnosidases from a novel thermophilic bacterium. Enzyme and Microbial Technology 2004;34:561-571. 2) Birgisson, H., Wheat, J. O., Hreggvidsson, G. O., Kristjánsson, J. K., Mattiasson, B. Immobilization of a recombinant Escherichia coli producing a thermostable α-l-rhamnosidase: Creation of a bioreactor for hydrolyses of naringin. Enzyme and Microbial Technology 2007;40:1181-1187
A Thermostable α-L-rhamnosidase from Thermomicrobia sp.
A thermostable β-glucosidase. The enzyme catalyses the transfer of D-glucose residues in disaccharides, alkyl glycosides or other aglycone-linked glycosides to water (hydrolysis) or to alcohols depending on reaction conditions. The enzyme is available for purchase as 100 units size For purchase select product and add to cart above. We accept payments with credit cards via PayPal or direct bank transfer. Shipping within a week, shipping and handling charges additional to price listed. Product : Bgl162 | Beta-glucosidase 100 units | Price : 310.00 EUR For further information read more below or download the product sheet Enzyme activity: Bgl162 β-glucosidase catalyses deglycosylations and transglycosylation reactions with retaining mechanism. The enzyme can both catalyse hydrolysis of various glycosides and transglycosylations with alcohols. Activity determination: Glycosidase activity was determined at 85°C in a 20 mM citrate phosphate buffer at pH 5.6 and incubated for 5 minutes with 2.92 mM final concentration of p-nitrophenyl-β-D-glucanopyranoside. Unit definition: One unit (U) of enzyme activity is the amount that leads to the release of 1 μmol of p-nitrophenyl from p-nitrophenyl-β-D-glucanopyranoside (pNPG) per minute. Synonyms: β-glucosidase, glycoside hydrolase, β-D-glucoside glucohydrolase Protein family: Glycosyl hydrolase family 3 ( GH3 ) - CAZy database GH3 family Enzyme classification: EC 3.2.1.6 Source: The thermophilic bacteria Thermotoga neopolitana Protein sequence: NBCI protein entry Structural information: The crystal structure of β-glucosidase from Thermotoga neopolitana has been determined to 2.05 Å resolution (Pozzo et al. 2010). - PDB entry 2X41 Figure 1: Crystal structure of β-glucosidase 3B from Thermotoga neopolitana Substrates and Applications: Polyphenol glycoside hydrolysis: The enzyme may be used for hydrolysis of various glycosides such as polyphenol glycosides including naturally occurring antioxidants such as quercetin-glycosides found in various vegetables (Fig. 2). For complete hydrolysis of 1 μmol of quercetin-4-glycoside in 5 minutes at 80°C and pH 5.5, about 28 pmol (~25 μg) enzyme was needed (Turner et al 2006) Alkyl glycoside synthesis: The enzyme may be used for transglycolysation with alcohols as acceptors such as in synthesis of alkyl glycosides. At 60°C, the ratio of alcoholysis to hydrolysis is 5.1 with a pNPG conversion rate of 153U/mg whereas at 90°C the ratio of alcoholysis to hydrolysis is 4.5 and the pNPG conversion rate of is 831U/mg. (Turner et al. 2007) Properties of ThermoActive™ β-glucosidase: Figure 2. Temperature optimum: the enzyme has optimum activity around 90°C Figure 3. Example of substrate specificity: quercetin References: Turner C, Turner P, Jacobson G, Waldebäck M, Sjöberg P, Nordberg Karlsson E & Markides K. (2006). Subcritical water extraction and β-glucosidase catalyzed hydrolysis of quercetin in onion waste. Green Chem. 8: 949–959. Turner P, Svensson D, Adlercreutz P & Karlsson EN (2007). A novel variant of Thermotoga neapolitana β-glucosidase B is an efficient catalyst for the synthesis of alkyl glucosides by transglycosylation. J. Biotechnol.130: 67–74. Pozzo T, Pasten JL, Karlssoon EN and Logan DT 2010. Structural and functional analyses of beta-glucosidase 3B fromThermotoga neopolitana: a thermostable three-domain representative of glycosidase hydrolase 3. J Mol Biol 397: 724-39
A thermostable β-glucosidase transferring D-glucose residues
Fuc123 Alpha-L-Fucosidase is thermostable enzyme that cleaves alpha-glucan residues from various alpha-fucosylated oligosaccharides including hydrolysis of alpha-L-fucose from algal fucosidan (Ascophyllum nodosum). The enzyme is available for purchase as 30 units of enzyme. Shipping within a week, shipping and handling charges additional to price listed below. Product : Fucl123 | Fucosidase 30 units | Price : 310.00 EUR For further information read more below or download the product sheet Enzyme activity: Fuc123 Alpha-Fucosidase is a glycosyl hydrolase that cleaves alpha-glucan residues from various alpha-fucosylated oligosaccharides including hydrolysis of alpha-L-fucose from algal fucosidan (Ascophyllum nodosum). Activity determination: The standard assay is performed by mixing 10 μL enzyme sample to 80 μL of 125mM phosphate buffer at pH 6,5 with 20 μL PNP-alpha-fucopyranoside, incubated at 65°C. Reaction is stopped with 200μL 0,5M sodium borat and OD measured at 405 nm. Unit definition: One unit of activity is defined as the amount of enzyme, which liberates 1 μmol of p-nitrophenol per minute under the given assay conditions. Synonyms: glycoside hydrolase, alpha-L-fucoside fucohydrolase, alpha-fucosidase Protein family: Glycosyl hydrolase family Enzyme classification: EC 3.2.1.51 Source: Proprietary environmental DNA Substrates: Fucoidan is a group of sulfated polysaccharides containing fucose and includes molecules with a backbone built of (1→3)-linked alpha-L-fucopyranosyl or alternating (1→3)- and (1→4)-linked alpha-L-fucopyranosyl residues, but also includes sulfated galactofucans with backbones built of (1→6)-beta-D-galacto- and/or (1→2)-beta-D-mannopyranosyl units and may include branching and various substitutions of sugar residues. Fucoidan from seaweed has found use as dietary supplement and has been implicated as having potential bioactive functions in humans ( Ale et al 2011 ). Properties of ThermoActive™ Alpha-Fucosidase: Temperature Optimum: The enzyme in relatively active in a temperature range (60-70°C)with optimum around 65°C pH Optimum: suitable pH range is about 6-7 with optimum around 6.5 References: Ale MT, Mikkelsen JD & Meyer AS (2011). Important Determinants for Fucoidan Bioactivity: A Critical Review of Structure-Function Relations and Extraction Methods for Fucose-Containing Sulfated Polysaccharides from Brown Seaweeds. Marine Drugs 9: 2106–2130
Thermostable Alpha-L-Fucosidase that cleaves alpha-glucan residues from various alpha-fucosylated oligosaccharides.
A thermostable α-Galactosidase catalyzing the hydrolyzis of -1.6 linked -galactose residues from oligosaccharides such as raffinose, melibiose, and stachyose or from polymeric substrates such as galactomannans. Available as 1000 units. Shipping within a week, shipping and handling charges additional to price below. Product : Agal104 | Alpha-Galactosidase 1000 Units | Price : 310.00 EUR For further information read more below or download the product sheet Mode of action: The enzyme catalyses the hydrolyzis of a-1.6 linked a-galactose residues from oligosaccharides Unit Definition: One unit (U) is defined as the amount of enzyme which liberates 1 μmol of p-nitrophenol per min from pNP-α-galactoside, under the given assay conditions. Unit Assay Conditions: One unit (U) is defined as the amount of enzyme which liberates 1 µmol of p-nitrophenol per min from pNP-α-galactoside, under the given assay conditions. Enzyme properties: Temperature Optimum: The enzyme has best activity in a temperature range (55-65°C) with optimum around 60°C pH Optimum: optimal range is pH 5-7,5. Relative thermostability of Agal104
A thermostable α-Galactosidase catalyzing the hydrolyzis of -1.6 linked -galactose residues from oligosaccharides such as raffinose, melibiose, and stachyose or from polymeric substrates such as galactomannans.
A thermostable β-galactosidase that hydrolyses terminal, non-reducing β-D-galactose residues in β-D-galactosides. From the thermophilic bacterium Thermus brockianus. Available as 1000 units. Shipping within a week, shipping and handling charges additional to price below. Product : Bgal112 | Beta-Galactosidase 100 Units | Price : 310.00 EUR Bgal112 | Beta-Galactosidase 500 Units | Price : 990.00 EUR For further information read more below or download the product sheet Mode of action: The enzyme hydrolyses terminal, non-reducing b-D-galactose residues in b-D-galactosides. Unit Definition: One unit (U) is defined as the amount of enzyme which liberates 1 mmol of p-nitrophenol per min from pNP-b-galactoside, under the given assay conditions. Unit Assay Conditions: The standard assay for cellulase activity was done by incubating the enzyme at 80°C for 10 min, with 1 % (w/v) pustulan, as a substrate in 0.1M sodium phosphate buffer, pH 5,5. The reducing sugars released were detected by the dinitrosalicylic acid method using glucose as standard. Enzyme properties: Temperature Optimum: The enzyme activity is limited below 65°C but increases with temperature up to 100°C pH Optimum: pH range is about 4-7 with optimum around 6.5 Thermostability: The ß-galactosidase heat stability at 70,80 and 91°C
Thermostable Beta-Galactosidase from Thermus brockianus
A highly thermostable Laminarinase (Beta-glucanase). The enzyme is active on β-1-3 linkages in polysaccharides of D-glucose units with β-1-3 linkages including β-glucans with mixed linkages such as β-1-3 and β-1-4 or β-1-3 and β-1-6 linkages includinglaminarin, lichenan, barley beta-glucan and scleroglucan. Uniquely active on recalcitrant beta-glucans such as scleroglucanand curdlan. The enzyme is available for purchase as 200 units size (see unit definition below). Shipping within a week, shipping and handling charges additional to price listed below. Product : Bglu110 | Laminarinase 200 Units | Price : 310.00 EUR For further information read more below or download the Product sheet Enzyme activity: ThermoActive™ Laminarinase catalyses thehydrolysis of β-1-3 linkages in polysaccharides of D-glucose residues connected by β-1-3 linkages or mixture of β-1-3 linkages and either β-1-4 linkages or β-1-6 linkages . This includes barley beta-glucan, laminarin, lichenin, scleroglucan and curdlan. The activity of the enzyme on barley β-glucan indicates that the enzyme is also able to hydrolyse β-1-4 linkages. The enzyme does not appear to hydrolyze β1-6 linkages and the extent of hydrolysis of Laminarin is dependent on the source of the substrate, i.e. proportion of β1-3 vs β1-6 linkages. Activity determination: The standard assay for activity was done by incubating the enzyme at 75°C for 15 min. with 1% (w/v) lichenan as substrate in 0,1 M sodium phosphate buffer at pH 7.0. The reducing sugars released were detected by the dinitrosalisylic acid method using glucose as standard. Unit definition: One unit (U) of enzyme activity is the amount that leads to the release of 1 μmol reducing sugars per minute. Synonyms: glycoside hydrolase, 1,3-[1,3;1,4]-β-D-Glucan-3(4)-glucanohydrolase, Endo-1,3(4)-β-glucanase,laminaranase, lichenase, licheninase. Protein family: Glycosyl hydrolase family 16 ( GH16 ) - CAZy database Enzyme classification: EC 3.2.1.6 Source: The thermophilic bacteria Rhodothermus marinus Protein sequence: NBCI protein entry Structural information: The crystal structure of Laminarinase from Rhodothermus marinus (96% sequence identity with Bglu110 Laminarinase/Lichenase) has been determined to 1.9 Å resolution. - PDB entry 3ILN Crystal structure of Laminarinase from Rhodothermus marinus Substrates: The molecule laminarin (also known as laminaran) is a storage glucan produced in brown algae through photosynthesis. The polysaccharide is made up of glucose residues withβ-1,3-linkages and β-1,6-linkages. It is a linear polysaccharide, with a β(1→3):β(1→6) ratio typically 3:1 but the ratio may vary with the source of the polysaccharide. β-1-3 and β-1-6 linkages found in laminarin Lichenin, also known as lichenan or moss starch, is a complex glucan occurring in certain species of lichensandconsists of repeating glucose units linked by β-1,3 and β-1,4 glycosidic bonds. It can be extracted from Cetraria islandica(Iceland moss). Scleroglucan, is formed by the fungus Sclerotium rolfsii. A chemically analogous polysaccharide, Schizophyllan (Sizofiran, Sonifilan, SPG) is a neutral extracellular polysaccharide produced by the fungus Schizophyllum commune. Schizophyllan is a β-1,3 beta-glucan with β-1,6 branching. Both polysaccharides share the chemical structure of the backbone with curdlan. Properties of Bglu110 Laminarinase/Lichenase (β-glucanase): Temperature optimum: the enzyme has optimum activity around 80°C pH optimum: the enzyme has optimum activity around pH 7 Thermostability: Activity of the enzyme as a function of time at different temperature - ( open circles: 80°C; closed circles: 85°C; open squares: 94°C ) References: Spillaert, R. Hreggvidsson, G.O., Kristjansson, J.K., Eggertsson, G.and Palsdottir, 1994. Cloning and sequencing of aRhodothermus marinus gene, bglA, coding for a thermostable β-glucanase in Eschericha coli. Eur. J. Biochem. 224:923-930
A highly thermostable Beta-glucanase active on β-1-3 linkages in polysaccharides of D-glucose units with β-1-3 linkages
A thermostable alcohol dehydrogenase. Most active on n-butanol but low activity on ethanol. Available as 100 units. Shipping within a week, shipping and handling charges additional to price listed below. Product : Adh131 | Alcohol dehydrogenase 100 Units | Price : 310.00 EUR For further information read more below or download the product sheet Mode of action: The enzyme is an alcohol dehydrogenase most active on n-butanol but with low activity on ethanol. Unit Definition: One unit (U) of enzyme activity is defined as the absorbance measured after incubation for 10 min at 340nm (NAD+ turning into NADH) units (U) are defined as mmol/min of product released. Temperature Optimum: The enzyme in relatively active at broad temperature range (50-65°C)with optimum around 60°C. pH Optimum: pH range is about 8-9 with optimum around pH 8,0. Thermostability: The enzyme is stable over 400 minutes at temperature at 65°C. Unit Assay Conditions: 100 µl sample: 100 µl 1-butanol (110mM) 200 µl NAD+ (50mM) 100 µl KPO4 pH 8 (500mM) 10 µl zinksalt (100mM) 390 µl dH2O Incubated at 60°C for 10min. Reaction stopped on ice. Spin shortly at 4°C before measuring the absorbance at 340nm.
A thermostable alcohol dehydrogenase. Most active on n-butanol but low activity on ethanol.
A highly thermostable Lysozyme developed from a unique thermophilic bacteriophage. The enzyme breaks down the cell wall of specific bacteria and is especially active against cells of thermophilic bacterial strains of the genus Thermus including T. thermophilus and T. aquaticus. The enzyme is available for purchase in 0.1 mg size. Shipping within a week, shipping and handling charges additional to price listed below. Product : Lys164 | ThermoPhage Lysozyme | Price : 310.00 EUR For further information read more below or download the product sheet Enzyme activity: ThermoPhage Ts Lysozyme promotes lysis of certain bacteria probably through hydrolysis of linkages between sugar residues and peptides of the cell wall peptidoglycan. Synonyms: muramidase, N-acetylmuramide glycanhydrolase, glycoside hydrolase Enzyme classification: EC 3.5.1.28 Source: Thermophilic bacteriophage Ph2119 Description: ThermoPhage™ Lysozyme originates from a thermophilic bacteriophage known to infect thermophilic bacteria of the genus Thermus. The enzyme promotes lysis of certain bacterial species but has activity spectrum very different from the commonly used Hen egg white lysozyme (HEWL). The enzyme has high activity against bacteria of the genus Thermus including Thermusthermophilus and Thermus aquaticus as shown in the Table: Bacterial species Ts Lysozyme HEWL Thermus thermophilus HB8 100 43 Thermus aquaticus 100 41 Deinococcus radiodurans 25 21 Echerichia coli 34 100 Salmonella panama 10 35 Pseudomonas fluorescence 13 40 Serratia marcescens 0 35 Enterococcus faecalis 4 0 Bacillus subtilis 2 0 Bacillus cereus 15 75 Staphylococcus aureus 0 0 Staphylococcus intermedius 0 5 Staphylococcus epidermis 0 0 Sarcina lutea 2 48 Streptococcus pyogenes 0 11 Lactococcus lactis 0 11 Relative activity (%): ThermoPhage™ Lysozyme vs. Hen Egg White Lysozyme Temperature profile: The enzyme has >80% activity between 40°and 100°C pH optimum: the enzyme has optimum activity around pH 7.5 - 8 Thermostability: Relative activity of ThermoPhage™ Lysozyme at different temperature vs. the activity of Hen Egg White Lysozyme Applications: The enzyme is suitable for efficient lysis of Thermus bacteria such as for increasing total DNA or plasmid DNA isolation (See figure below). Reactions and enzyme dilutions can be done in a 10 mM K/PO4 buffer at pH 8.0. The use of EDTA is not recommended. DNA isolation: ThermoPhage™ Lysozyme improves plasmid preparation from Thermus thermophilus HB8 References: Plotka M, Stefanska A, Kaczorowska A-K, Morzywolek A, Fridjonsson OF, Dunin-Horkawicz S, Hreggvidsson GO, Kristjansson JK, Dabrowski S, Bujnicki JM and Kaczorowski T. 2014 Purification and characterization of a novel highly thermostable lysozyme fromThermus scotoductus MAT2119 bacteriophage Ph2119 that shows amino-acid sequence similarity to eukaryotic peptidoglycan recognition proteins (PGRPs). Appl. Environ. Microbiol.
A highly thermostable Lysozyme developed from a unique thermophilic bacteriophage.
The Ice and Fire™ Phosphorylation kit includes optimized reagents for the 5’ end labeling of nucleic acids. The novel protocol provides easy "one-step" handling by combining the low temperature activity of thermolabile Shrimp Alkaline Phosphatase (SAP) and the high temperature activity of the thermostable ThermoPhage™ polynucleotide kinase (PNK). Shrimp alkaline phosphatase digests mono-, di- and triphosphates from a 5’ end of nucleic acids leaving a 5’ hydroxyl group. PNK catalyzes the transfer of the gamma phosphate from ATP to the 5'-OH of a nucleic acid molecule. The combination of the two enzymes in a simplified temperature-controlled procedure allows for a rapid and efficient 5’ end labeling of phosphorylated nucleic acids. Available as kit for 50 reactions. Shipping within a week, shipping and handling charges additional to price listed below. Product : IaF170S | Ice and Fire kit 50 reactions | Price : 310.00 EUR For further information read more below or download the product sheet Background: The Ice and Fire Phosphorylation kit provides optimized reagents for the 5' end labeling of nucleic acids. The simple protocol utilizes low and high temperature to control the activity of thermolabile Shrimp Alkaline Phosphatase (SAP) and thermostable ThermoPhage™ polynucleotide kinase (PNK) in one solution and optimized reaction buffer (patent pending). The kit is designed to label 5' ends of DNA or RNA molecules with terminal mono-, di- or triphosphates. Other nucleic acids such as PCR products, oligonucleotides,restrictiondigested plasmid DNA, or complex sample RNA or DNA can also be labeled using the kit. The substrate can be of virtually any length. Nucleic acids with a 5'-phosphate needs to be dephosphorylated before the kinase reaction. The kit includes Shrimp Alkaline Phosphatase and thermostable polynucleotide kinase enzyme mixture. This allows for dephosphorylation of the nucleic acids at lower temperature followed by heat inactivation of the phosphatase prior to addition of radiolabeled [γ-32 P]ATP and labeling by the novel thermostable polynucleotide kinase (1). The SAP denaturation and the PNK reaction are done at 70°C. No SAP activity can be detected after the heating step, followed by the PNK reaction. Nucleic acids with a 5'-hydroxyl (-OH) group can also be labeled using this kit. In this case the phosphatase is directly inactivated by heating followed by addition of gamma radiolabeled ATP and incubation at 70°C. Benefits: Efficient 5’ end labeling of phosphorylated nucleic acids, single- or double-stranded Simple temperature control of enzymatic activity No extra cleanup steps like phenol/chloroform or spin columns are needed between the reactions, only heat denaturation of the SAP Single tube format, simplified procedure Applications: 5’ - end label of phosphorylated DNA and RNA for: Nuclease protection assays Hybridization blots Quantitive PCR Product Components: 10X reaction buffer SAP/PNK enzyme mixture Phosphorylated ssDNA oligomer (10 pmol/μL) for use as positive control Components not supplied with the kit: Radiolabeled γ-ATP [32P] ATP Nuclease free water Protocol I: Protocol for 5’ radio-labeling of nucleic acids: The Ice and Fire phosphorylation kit provides controls and optimized reagents for quick and easy end-labeling of DNA or RNA 1) Dephosphorylation of target nucleic acid (SAP reaction): 10x SAP/PNK buffer 2.5 μL SAP/PNK enzyme mix 1 μL Target DNA/RNA 10-100 pmol Nuclease free water up to 24 μL Incubate at 37°C for 60 minutes 2) Phosphorylation of target nucleic acid (PNK reaction): After incubation at 37°C the sample is heated to 70°C. After 15 minutes at 70°C add X μL radiolabeled [γ-32 P]ATP to the reaction equivalent to a 2-5 molar excess ATP over ends to be labeled (for example using 7,000 Ci/mmol, 150 mCi/ml [γ-32 P]ATP, 1 μL is about 25 pmol). Incubate at 70°C for additional 30-60 minutes. Stop the reaction by cooling down to +4°C or put on ice. Optional: Add 1 mM EDTA and/or heat the reaction at 95°C for 5 minutes to terminate the reaction. Optional: Clean the probe by spin column (for example Qiagen nucleotide removal kit, Cat. No. 28304 or equivalent spin columns) or PAGE gel purification as described by Sambrook et al. (2). The radiolabeled nucleic acids are now ready for use Protocol II: Protocol for 5’ labeling of 5’ hydroxylated nucleic acids: Prokazyme Ltd provides ThermoPhageTM PNK also for labeling of 5’ hydroxylated nucleic acids but the Ice and Fire phosphorylation kit can also be used for this kind of applications. 1) Reaction mixture 10x SAP/PNK buffer 2.5 μL SAP/PNK enzyme mix 1 μL Target DNA/RNA 10-100 pmol Nuclease free water up to 24 μL Incubate at 70°C for 15 minutes to inactivate the SAP enzyme. 2) Polynucleotide kinase 5’ labeling of target nucleic acid: Add X μL radiolabeled [γ-32 P]ATP to the reaction equivalent to 2-5 molar excess ATP over ends to be labeled (for example using 7,000 Ci/mmol, 150 mCi/ml [γ-32 P]ATP, 1 μL is about 25 pmol). Incubate at 70°C for 60 minutes. Stop the reaction by cooling down to +4°C or put on ice. Optional: Add 1 mM EDTA and/or heat the reaction at 95°C for 5 minutes to terminate the reaction. Optional: Clean the probe by spin column (for example Qiagen nucleotide removal kit, Cat. No. 28304 or equivalent spin columns) or PAGE gel purifications as described by Sambrook et al. (2). The radiolabeled nucleic acids are now ready for use Protocol III: Phosphorylation without radioactive ATP: This kit can also be used for phosphorylation of 5’ hydroxylated nucleic acids prior to cloning procedure etc. 1) Reaction mixture 10x SAP/PNK buffer 2.5 μL SAP/PNK enzyme mix 1 μL Target DNA/RNA 1-500 pmol Nuclease free water up to 24 μL Incubate at 70°C for 15 minutes to inactivate the SAP enzyme. 2) Polynucleotide kinase 5’ phosphorylation of target nucleic acid: Add ATP to the reaction to a final concentration of 10-100 uM (at least twice the concentration of nucleic acids ends to be phosphorylated). Incubate at 70°C for 60 minutes. Stop the reaction by cooling down to +4°C or put on ice. Optional: Clean the probe by spin column (for example Qiagen nucleotide removal kit, Cat. No. 28304 or equivalent spin columns) or PAGE gel purifications as described by Sambrook et al. (2). The nucleic acids are now ready for cloning procedure Evaluation Protocol: If specific radioactivity needs to be determined or if the researcher wants to evaluate the efficiency of the reaction, the following evaluation protocol can be used. Note that scintillation counter, scintillation liquid and DE81 filters or equivalent instruments and components are required for this evaluation. Dilute 1 μL of the reaction 1/10 and spot 1μL to a Whatman Inc. DE81 filter (Cat no. 3658023) or its equivalent. As a positive control, same amount is put on one filter that does not undergo the washing steps. Reaction solution without enzyme mix can be used as negative control and washed with the samples. The sample and negative control filters are washed twice in 400 ml sodium phosphate buffer pH 7 for 30 minutes, dried and put into scintillation counter vials, scintillation liquid added and counted for radioactivity in an liquid scintillation counter. To determine the specific activity of the labeled nucleic acid, calculate the total cpm incorporated in the reaction and divide it by the amount of nucleic acid labeled in pmol. (#total cpm incorporateddilution factorvolume) / (#total pmol nucleic acid) Expected specific activity: When [γ-32P]ATP with a specific activity of 7000 Ci/mmol is used for the kinase reaction, the nucleic acid should be labeled to at least 1 x 106 cpm/pmol on the reference date. Decay of 32P from the reference day stated on the vial of radiolabeled ATP, can be calculated as: (original cpm/μl) / (2(days/14.3)) References: Blondal, T., Hjorleifsdottir, S., Aevarsson, A., Fridjonsson, O.H., Skirnisdottir, S., Wheat, J.O., Hermannsdottir, A.G.,Hreggvidsson, G.O., Smith, A.V. and Kristjansson, J.K. (2005) J Biol Chem, 280, 5188-5194. Sambrook, J., Fritsch, E.F. and T., M. (1989) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor NY.
The Ice and Fire™ Phosphorylation kit includes optimized reagents for the 5’ end labeling of nucleic acids.
