10 mL Hexokinase (420 U/mL) + G6P-DH (210 U/mL)
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|Content:||10 mL Hexokinase (420 U/mL) + G6P-DH (210 U/mL)|
|Formulation:||In 3.2 M ammonium sulphate|
|Stability:||> 4 years at 4oC|
Hexokinase: Hexokinase; ATP:D-hexose 6-phosphotransferase
G6P-DH: glucose-6-phosphate dehydrogenase (NADP+); D-glucose-6-phosphate:NADP+ 1-oxidoreductase
Hexokinase: ~ 420 U/mL
G6P-DH: ~ 210 U/mL
Hexokinase: Recombinant from yeast
G6P-DH: Recombinant from Leuconostoc mesenteroides
Hexokinase: Catalyses the reaction:
ATP + D-hexose = ADP + D-hexose 6-phosphate Phopshorylates D-glucose, D-mannose, D-fructose, sorbitol and D-glucosamine.
Glucose-6-phosphate dehydrogenase: Catalyses the reaction: D-glucose 6-phosphate + NADP+ = 6-phospho-D-glucono-1,5-lactone + NADPH + H+
Hexokinase: One Unit of hexokinase activity is defined as the amount of enzyme required to produce one µmole of NADH from NAD+ in the presence of D-glucose and glucose-6-phosphate dehydrogenase at pH 7.4 and 25oC.
G6P-DH: One Unit of Glucose-6-phosphate dehydrogenase activity is the amount of enzyme required to convert one μmole of glucose-6-phosphate to 6-phosphogluconate per minute, in the presence of NADP+ at pH 7.4 and 25oC.
|Application examples:||Applications for the measurement of glucose and other hexoses in carbohydrate research and in the food and feeds, fermentation, wine, beverage and dairy industries.|
High purity Hexokinase (yeast) / Glucose-6-phosphate dehydrogenase (G6P-DH) (Leuconostoc mesenteroides) for use in research, biochemical enzyme assays and in vitro diagnostic analysis.
Find out more of our analytical enzymes.
(Escherichia coli) E-GMPK - Guanylate kinase (prokaryote) E-PKRM - Pyruvate kinase (rabbit muscle) E-TMPK - Thymidylate kinase (prokaryote) E-UMPK - Uridylate kinase (prokaryote) E-ADHEC - Alcohol dehydrogenase (Escherichia coli) E-FDHCB - Formate dehydrogenase (Candida boidinii) E-GALDH - Galactose dehydrogenase (soil prokaryote) E-GALMUT - Galactose dehydrogenase - Galactose mutarotase E-GPDH5 - Glucose-6-phosphate dehydrogenase (Leuconostoc mesenteroides) E-GPDHEC - Glucose-6-phosphate dehydrogenase
(Escherichia coli) E-HBDH - 3-Hydroxybutyrate dehydrogenase (prokaryote) E-INDHBS - myo-Inositol dehydrogenase (Bacillus subtilis) E-ICDHBS - Isocitrate dehydrogenase (Bacillus subtilis) E-DLDHLM - D-Lactate dehydrogenase
(Leuconostoc mesenteroides) E-LLDHP - L-Lactate dehydrogenase (Porcine) E-LMDHEC - L-Malate dehydrogenase (Escherichia coli) E-MNHPF - Mannitol dehydrogenase
(Pseudomona fluorescens) E-PGDHEC - 6-Phosphogluconate dehydrogenase
(Escherichia coli) E-XYLMUT - Xylose dehydrogenase + Xylose mutarotase
A novel enzymatic method for the measurement of lactose in lactose‐free products.
Mangan, D., McCleary, B. V., Culleton, H., Cornaggia, C., Ivory, R., McKie, V. A., Delaney, E. & Kargelis, T. (2018). Journal of the Science of Food and Agriculture, 99, 947-956.
Background: In recent years there has been a surge in the number of commercially available lactose‐free variants of a wide variety of products. This presents an analytical challenge for the measurement of the residual lactose content in the presence of high levels of mono‐, di‐, and oligosaccharides. Results: In the current work, we describe the development of a novel enzymatic low‐lactose determination method termed LOLAC (low lactose), which is based on an optimized glucose removal pre‐treatment step followed by a sequential enzymatic assay that measures residual glucose and lactose in a single cuvette. Sensitivity was improved over existing enzymatic lactose assays through the extension of the typical glucose detection biochemical pathway to amplify the signal response. Selectivity for lactose in the presence of structurally similar oligosaccharides was provided by using a β-galactosidase with much improved selectivity over the analytical industry standards from Aspergillus oryzae and Escherichia coli (EcLacZ), coupled with a ‘creep’ calculation adjustment to account for any overestimation. The resulting enzymatic method was fully characterized in terms of its linear range (2.3-113 mg per 100 g), limit of detection (LOD) (0.13 mg per 100 g), limit of quantification (LOQ) (0.44 mg per 100 g) and reproducibility (≤ 3.2% coefficient of variation (CV)). A range of commercially available lactose‐free samples were analyzed with spiking experiments and excellent recoveries were obtained. Lactose quantitation in lactose‐free infant formula, a particularly challenging matrix, was carried out using the LOLAC method and the results compared favorably with those obtained from a United Kingdom Accreditation Service (UKAS) accredited laboratory employing quantitative high performance anion exchange chromatography - pulsed amperometric detection (HPAEC‐PAD) analysis. Conclusion: The LOLAC assay is the first reported enzymatic method that accurately quantitates lactose in lactose‐free samples.Hide Abstract
Microbial diversity analysis and screening for novel xylanase enzymes from the sediment of the Lobios Hot Spring in Spain.
Knapik, K., Becerra, M. & González-Siso, M. I. (2019). Scientific Reports, 9(1), 1-12.
Here, we describe the metagenome composition of a microbial community in a hot spring sediment as well as a sequence-based and function-based screening of the metagenome for identification of novel xylanases. The sediment was collected from the Lobios Hot Spring located in the province of Ourense (Spain). Environmental DNA was extracted and sequenced using Illumina technology, and a total of 3.6 Gbp of clean paired reads was produced. A taxonomic classification that was obtained by comparison to the NCBI protein nr database revealed a dominance of Bacteria (93%), followed by Archaea (6%). The most abundant bacterial phylum was Acidobacteria (25%), while Thaumarchaeota (5%) was the main archaeal phylum. Reads were assembled into contigs. Open reading frames (ORFs) predicted on these contigs were searched by BLAST against the CAZy database to retrieve xylanase encoding ORFs. A metagenomic fosmid library of approximately 150,000 clones was constructed to identify functional genes encoding thermostable xylanase enzymes. Function-based screening revealed a novel xylanase-encoding gene (XynA3), which was successfully expressed in E. coli BL21. The resulting protein (41 kDa), a member of glycoside hydrolase family 11 was purified and biochemically characterized. The highest activity was measured at 80°C and pH 6.5. The protein was extremely thermostable and showed 94% remaining activity after incubation at 60°C for 24 h and over 70% remaining activity after incubation at 70°C for 24 h. Xylanolytic activity of the XynA3 enzyme was stimulated in the presence of β-mercaptoethanol, dithiothreitol and Fe3+ ions. HPLC analysis showed that XynA3 hydrolyzes xylan forming xylobiose with lower proportion of xylotriose and xylose. Specific activity of the enzyme was 9080 U/mg for oat arabinoxylan and 5080 U/mg for beechwood xylan, respectively, without cellulase activity.Hide Abstract
Response to sulfur dioxide addition by two commercial saccharomyces cerevisiae strains.
Morgan, S. C., Haggerty, J. J., Johnston, B., Jiranek, V. & Durall, D. M. (2019). Fermentation, 5(3), 69.
Sulfur dioxide (SO2) is an antioxidant and antimicrobial agent used in winemaking. Its effects on spoilage microorganisms has been studied extensively, but its effects on commercial Saccharomyces cerevisiae strains, the dominant yeast in winemaking, require further investigation. To our knowledge, no previous studies have investigated both the potential SO2 resistance mechanisms of commercial yeasts as well as their production of aroma-active volatile compounds in response to SO2. To study this, fermentations of two commercial yeast strains were conducted in the presence (50 mg/L) and absence (0 mg/L) of SO2. Strain QA23 was more sensitive to SO2 than Strain BRL97, resulting in delayed cell growth and slower fermentation. BRL97 exhibited a more rapid decrease in free SO2, a higher initial production of hydrogen sulfide, and a higher production of acetaldehyde, suggesting that each strain may utilize different mechanisms of sulfite resistance. SO2 addition did not affect the production of aroma-active volatile compounds in QA23, but significantly altered the volatile profiles of the wines fermented by BRL97.Hide Abstract
Stressler, T., Tanzer, C., Ewert, J., Claaßen, W. & Fischer, L. (2017). Protein Expression and Purification, 131, 7-15.
The aminopeptidase A (PepA; EC 22.214.171.124) is an intracellular exopeptidase present in lactic acid bacteria. The PepA cleaves glutamyl/aspartyl residues from the N-terminal end of peptides and can, therefore, be applied for the production of protein hydrolysates with an increased amount of these amino acids, which results in a savory taste (umami). The first PepA from a lactobacilli strain was recombinantly expressed in Escherichia coli in a recently published study and harbored a C-terminal His6-tag for easier purification. Due to the fact that a His-tag might influence the properties of an enzyme, a simple purification method for the non-His-tagged PepA was required. Surprisingly, the PepA precipitated at a very low ammonium sulfate concentration of 5%. Unusual for a precipitating step, the purity of PepA was over 95% and the obtained activity yield was 110%. The high purity allows biochemical characterization and kinetic investigation. As a result, the optimum pH (6.0–6.5) and temperature (60–65°C) were comparable to the His6-tag harboring PepA; the KM value was at 0.79 mM slightly lower compared to 1.21 mM, respectively. Since PepA is a homo dodecamer, it has a high molecular mass of approximately 480 kDa. Therefore, a subsequent preparative size-exclusion chromatography (SEC) step seemed promising. The PepA after SEC was purified to homogeneity. In summary, the simple two-step purification method presented can be applied to purify high amounts of PepA that will allow the performance of experiments in the future to crystalize PepA for the first time.Hide Abstract
Orlandi, I., Stamerra, G., Strippoli, M. & Vai, M. (2017). Redox Biology, 12, 745-754.
Resveratrol (RSV) is a naturally occurring polyphenolic compound endowed with interesting biological properties/functions amongst which are its activity as an antioxidant and as Sirtuin activating compound towards SIRT1 in mammals. Sirtuins comprise a family of NAD+-dependent protein deacetylases that are involved in many physiological and pathological processes including aging and age-related diseases. These enzymes are conserved across species and SIRT1 is the closest mammalian orthologue of Sir2 of Saccharomyces cerevisiae. In the field of aging researches, it is well known that Sir2 is a positive regulator of replicative lifespan and, in this context, the RSV effects have been already examined. Here, we analyzed RSV effects during chronological aging, in which Sir2 acts as a negative regulator of chronological lifespan (CLS). Chronological aging refers to quiescent cells in stationary phase; these cells display a survival-based metabolism characterized by an increase in oxidative stress. We found that RSV supplementation at the onset of chronological aging, namely at the diauxic shift, increases oxidative stress and significantly reduces CLS. CLS reduction is dependent on Sir2 presence both in expired medium and in extreme Calorie Restriction. In addition, all data point to an enhancement of Sir2 activity, in particular Sir2-mediated deacetylation of the key gluconeogenic enzyme phosphoenolpyruvate carboxykinase (Pck1). This leads to a reduction in the amount of the acetylated active form of Pck1, whose enzymatic activity is essential for gluconeogenesis and CLS extension.Hide Abstract
Ewert, J., Glück, C., Strasdeit, H., Fischer, L. & Stressler, T. (2017). Enzyme and Microbial Technology, 10, 69-78.
The aminopeptidase A (PepA; EC 126.96.36.199) belongs to the group of metallopeptidases with two bound metal ions per subunit (M1M2(PepA)) and is specific for the cleavage of N-terminal glutamic (Glu) and aspartic acid (Asp) and, in low amounts, serine (Ser) residues. Our group recently characterized the first PepA from a Lactobacillus strain. However, the characterization was performed using synthetic para-nitroaniline substrates and not original peptide substrates, as was done in the current study. Prior to the characterization using original peptide substrates, the PepA purified was converted to its inactive apo-form and eight different metal ions were tested to restore its activity. It was found that five of the metal ions were able to reactivate apo-PepA: Co2+, Cu2+, Mn2+, Ni2+ and Zn2+. Interestingly, depending on the metal ion used for reactivation, the activity and the pH and temperature profile differed. Exemplarily, MnMn(PepA), NiNi(PepA) and ZnZn(PepA) had an activity optimum using MES buffer (50 mM, pH 6.0) and 60°C, whereas the activity optimum changed to Na/K-phosphate-buffer (50 mM, pH 7.0) and 55°C for CuCu(PepA). However, more important than the changes in optimum pH and temperature, the kinetic properties of PepA were affected by the metal ion used. While all PepA variants could release N-terminal Glu or Asp, only CoCo(PepA), NiNi(PepA) and CuCu(PepA) could release Ser from the particular peptide substrate. In addition, it was found that the enzyme efficiency (Vmax/KM) and catalytic mechanism (positive cooperative binding (Hill coefficent; n), substrate inhibition (KIS)) were influenced by the metal ion. Exemplarily, a high cooperativity (n > 2), KIS value >20 mM and preference for N-terminal Glu were detected for CuCu(PepA). In summary, the results suggested that an exchange of the metal ion can be used for tailoring the properties of PepA for specific hydrolysis requirements.Hide Abstract
Stressler, T., Ewert, J., Merz, M., Funk, J., Claaßen, W., Lutz-Wahl, S., Schmidt, H., Kuhn, A. & Fischer, L. (2016). PloS one, 11(3), e0152139.
Lactic acid bacteria (LAB) are auxotrophic for a number of amino acids. Thus, LAB have one of the strongest proteolytic systems to acquit their amino acid requirements. One of the intracellular exopeptidases present in LAB is the glutamyl (aspartyl) specific aminopeptidase (PepA; EC 188.8.131.52). Most of the PepA enzymes characterized yet, belonged to Lactococcus lactis sp., but no PepA from a Lactobacillus sp. has been characterized so far. In this study, we cloned a putative pepA gene from Lb. delbrueckii ssp. lactis DSM 20072 and characterized it after purification. For comparison, we also cloned, purified and characterized PepA from Lc. lactis ssp. lactis DSM 20481. Due to the low homology between both enzymes (30%), differences between the biochemical characteristics were very likely. This was confirmed, for example, by the more acidic optimum pH value of 6.0 for Lc-PepA compared to pH 8.0 for Lc-PepA. In addition, although the optimum temperature is quite similar for both enzymes (Lc-PepA: 60 °C; Lc-PepA: 65 °C), the temperature stability after three days, 20 °C below the optimum temperature, was higher for Lc-PepA (60% residual activity) than for Lc-PepA (2% residual activity). EDTA inhibited both enzymes and the strongest activation was found for CoCl2, indicating that both enzymes are metallopeptidases. In contrast to Lc-PepA, disulfide bond-reducing agents such as dithiothreitol did not inhibit Lc-PepA. Finally, Lc-PepA was not product-inhibited by L-Glu, whereas Lc-PepA showed an inhibition.Hide Abstract
Stressler, T., Pfahler, N., Merz, M., Hubschneider, L., Lutz-Wahl, S. Claaßen, W., & Fischer, L. (2016). Applied Microbiology and Biotechnology, 1-17, 7499-7515.
Nowadays, general and specific aminopeptidases are of great interest, especially for protein hydrolysis in the food industry. As shown previously, it is confirmed that the general aminopeptidase N (PepN; EC 184.108.40.206) and the proline-specific peptidase PepX (EC 220.127.116.11) from Lactobacillus helveticus ATCC 12046 show a synergistic effect during protein hydrolysis which results in high degrees of hydrolysis and reduced bitterness. To combine both activities, the enzymes were linked and a fusion protein called PepN-L1-PepX (FUS-PepN-PepX) was created. After production and purification, the fusion protein was characterized. Some of its biochemical characteristics were altered in favor for an application compared to the single enzymes. As an example, the optimum temperature for the PepN activity increased from 30°C for the single enzyme to 35°C for FUS-PepN. In addition, the temperature stability of PepX was higher for FUS-PepX than for the single enzyme (50 % compared to 40 % residual activity at 50°C after 14 days, respectively). In addition, the disulfide bridge-reducing reagent β-mercaptoethanol did not longer inactivate the FUS-PepN activity. Furthermore, the KM values decreased for both enzyme activities in the fusion protein. Finally, it was found that the synergistic hydrolysis performance in a casein hydrolysis was not reduced for the fusion protein. The increase of the relative degree of hydrolysis of a prehydrolyzed casein solution was the same as it was for the single enzymes. As a benefit, the resulting hydrolysate showed a strong antioxidative capacity (ABTS-IC50 value: 5.81 µg mL-1).Hide Abstract