Soc. [5] Sohn was set to voice the Marvel Comics character Ganke Lee in the Sony Pictures Animation film Spider-Man: Into the Spider-Verse, though his lines were ultimately deleted from the final film. Means with standard deviations are shown from triplicate reactions. doi: 10.1007/s40089-017-0215-1. For this reason, we selected a biocatalyst based on amino-modified MCF, pH 6.7, for further research. Geburtstag. Initial tested conditions for the TaGalU reaction and comparison of observed enzyme activities. Relative specific TaGalU activity of the free and immobilized enzyme depending on the reaction temperature. By just varying the concentration of dissolved oxygen the expression level of human CYPs can be 10‐fold lower compared to the expression level under optimal conditions (Vail et al., 2005). Both variants of TaGalU showed a strong substrate inhibition at concentrations above 2 mM UTP (Figure 8A), whereas changes in the G1P concentration (Figure 8B) did not show any inhibition and were fitted to the Michaelis–Menten model. 1, 167–176. Number of times cited according to CrossRef: Two‐step Screening for Identification of Drug‐metabolizing Bacterial Cytochromes P450 with Diversified Selectivity. Gustafson, G. L., and Gander, J. E. (1972). But promising approaches for engineering of bacterial CYPs were published (Li et al., 2008; Otey et al., 2006). This may be caused by the supported steric hindrance of the active site, by the change of enzyme flexibility after immobilization, or by diffusional resistance to the substrate transport. 100% relative specific activity corresponds to 1568 U mg–1. The technical demand and the expense for insect cell cultures are usually quite high, but extraordinary high expression levels can be achieved with those systems. Hundred μL of the clear supernatant was used for HPLC separation combined with UV/Vis-detection of the product UDP-glucose (see section “HPLC Measurement and Determination of the Specific TaGalU Enzyme Activity”). (ed.) Res. (2019). Blood Sub. Protein Expr. However, further temperature increase resulted in a significant decrease of the activity of the free enzyme, whereas immobilized TaGalU still showed higher activities. Covalent immobilization of trypsin on glutaraldehyde-activated silica for protein fragmentation. Utilization of lignocellulosic biomass by oleaginous yeast and bacteria for production of biodiesel and renewable diesel. For this reason, the activity was measured after 1-month of storage at 4°C. Enhanced production of β-glucosides by in-situ UDP-glucose regeneration. IOP Conf. Delignification of vineyard pruning residues by alkaline peroxide treatment. AK wrote the first draft of the manuscript and visualized the data. Peter Nidetzky prägte die Entwicklungsjahre des österreichischen Reitsports wie kein Zweiter, veranstaltete legendäre Turniere, die bis heute atmosphärisch unerreicht sind, und hat ein riesengroßes Herz für Kinder. Cytochromes P450 as useful biocatalysts: addressing the limitations. Am. Herein, we report on the identification, gene expression, biochemical characterization as well as immobilization of the uridine-5′-diphosphate-glucose (UDP-glucose) pyrophosphorylase originating from the thermostable soil actinobacterium Thermocrispum agreste DSM 44070 (TaGalU). There are some biomimetic oxidations described in literature, for example, Udenfriend reaction (Udenfriend et al., 1954) or CH oxidation by a [Fe(S,S‐PDP)(CH3CN)2](SbF6)2 catalyst (Chen and White, 2007), which are useful strategies for the synthesis of some human drug metabolites. (2017). Alternative Solvents for Natural Products Extraction. Differential activity of lytic polysaccharide monooxygenases on celluloses of different crystallinity. Use of synthetic fusion gene to produce biodiesel from lignocellulosic biomass. doi: 10.1016/j.molcatb.2007.09.018, Moffatt, J. G., and Khorana, H. G. (1958). Enzyme Microb. Usually the whole cell biotransformation process is also carried out in a wave bag reactor. The remaining specific activity of this enzyme was 66.03 U mg–1 and demonstrates how stable this enzyme is even without immobilization. As the optimal reaction and immobilization conditions for TaGalU were established, a deeper look inside the performance of the free and immobilized enzyme was made. There are several examples where active metabolites were discovered during the drug development process and marketed as drugs (Fura et al., 2004). Appl. doi: 10.1093/jxb/erx190, Honda, Y., Zang, Q., Shimizu, Y., Dadashipour, M., Zhang, Z., and Kawarabayasi, Y. Indeed, these processes catalyzed by enzymes are often referred to as “white biotechnology,” which underlines their positive impact on the environment (Grunwald, 2015; Sheldon and Woodley, 2018). Mater. Cytochrome P450 monooxygenases (CYPs) are important enzymes in the metabolism of xenobiotics. Bioeng. (2015). Moreover, it exhibited improved thermal long-term stability and reusability. © 2010 Wiley Periodicals, Inc. A review on delignification of lignocellulosic biomass for enhancement of ethanol production potential. Biotechnol. Kinetic constants for free TaGalU calculated by the fits. 100% relative specific activity corresponds to 1430 U mg–1. ISRN Biochem. Mesopor. (2009). To test the cofactor specificity, we also used manganese chloride (MnCl2), nickel chloride (NiCl2), cobalt chloride (CoCl2), calcium chloride (CaCl2) and zinc chloride (ZnCl2) instead of MgCl2 with an improved activity assay with 2 mM UTP and 30 mM G1P. Expression of Piromyces rhizinflata cellulase in marine Aspergillus niger to enhance halostable cellulase activity by adjusting enzyme-composition. A new functionally based kinetic model for enzymatic hydrolysis of pure cellulose by the Trichoderma cellulase system is presented. UDP-glucose formation was monitored at a wavelength of 260 nm with an isocratic method with 70 % acetonitrile and 30 % ammonium acetate buffer (134 mM, pH 5.35). Biochimica et Biophysica Acta (BBA) - Biomembranes. 29, 75–83. 42, 4289–4299. View the profiles of people named Peter Nidetzky. With respect to the biocatalytic activity for large‐scale applications E. coli expression systems are generally superior to S. cerevisiae or other yeasts regarding both CYP expression levels and CYP yields. Mini-scaffoldin enhanced mini-cellulosome hydrolysis performance on low-accessibility cellulose (Avicel) more than on high-accessibility amorphous cellulose. Additionally, UTP, as a reagent with a higher amount of phosphate groups, could strongly interact with the functional groups of the silica carrier or silica itself, limiting their accessibility to the enzyme (Keerti et al., 2014; Rahimizadeh et al., 2016). Sohn directed The Good Dinosaur, which was released in November 2015. Interactions between CYP3A4 and Dietary Polyphenols. For G1P, the concentration was adjusted from 0.01 to 45 mM, holding UTP concentration at a value of 2 mM. From our experience the E. coli strains developed in Dundee are highly suitable whole cell biocatalysts for the performance of human CYP‐catalyzed reactions. The Effect of Alkaline Pretreatment Methods on Cellulose Structure and Accessibility. Herein, we report the biochemical characterization of the first thermostable and highly active UDP-glucose pyrophosphorylase from the thermophilic soil actinobacterium Thermocrispum agreste DSM 44070 (TaGalU). 247, 1387–1397. For purification, the cell suspensions were thawed as fast as possible in a warm water bath and the following solutions were added per portion: 1 mM magnesium chloride (MgCl2), 240 U DNase I and 12 mg Lysozyme. On the ancestral UDP-glucose pyrophosphorylase activity of GalF from Escherichia coli. Various authors have suggested that it could be caused by the changes in physical and chemical properties of an immobilized enzyme. In the Expertise Bioreactions of Novartis Pharma AG (Basel, Switzerland), 14 different recombinant human CYP isoenzymes functionally coexpressed with human CYP reductase in E. coli are established as whole cell biotransformation systems for the synthesis of phase I metabolites. From AAG561 four different metabolites were synthesized yielding between 1.8 and 13.6 mg after purification (Fig. DK immobilized the enzyme and did the majority of the experiments with the immobilized enzyme. Unbound protein was removed by washing with 100 mM phosphate buffer pH 7.0 (1x), 100 mM phosphate buffer pH 7.0 with 500 mM NaCl (2x), 100 mM acetate buffer pH 4.5 (1x) and several times with distilled water to remove the acid. Improvement of enzyme activity, stability and selectivity via immobilization techniques. a: Hydroxylation and oxidative ring formation. The temperature and the protein concentration were increased as follows: 10, 22, 30, 50, and 80°C; 0.013 μg mL–1, 0.04 μg mL–1, 0.08 μg mL–1, and 8 μg mL–1, respectively. Identification of a fungus able to secrete enzymes that degrade regenerated cellulose films and analyses of its extracellular hydrolases. Cellulose Hydrolysis by Cellobiohydrolase Cel7A Shows Mixed Hyperbolic Product Inhibition. In the last decades the time span allotted for drug discovery and development activities has been shortened significantly. Valorization of exhausted sugar beet cossettes by successive hydrolysis and two fermentations for the production of bio-products. Positive and negative signs indicate advantageous or disadvantageous properties. In Dundee a cloning strategy was developed where various human CYP cDNAs were fused with a modified ompA leader sequence in order to achieve higher CYP expression levels by minimizing the formation of mRNA secondary structures. Chem. Phosphite‐driven Self‐sufficient Cytochrome P450. Synthesis of cyclophosphamide metabolites by a peroxygenase from Marasmius rotula for toxicological studies on human cancer cells. The enzyme activity was only measured in the direction of UDP-glucose formation in a reaction volume of 1 mL for the free enzyme and of 3 mL for the immobilized enzyme, with a standard test of a buffer containing 2 mM UTP, 2 mM G1P, 4 mM MgCl2, 50 mM Hepes, pH 7.0 and an appropriate amount of TaGalU, if not otherwise indicated. Insights on How the Activity of an Endoglucanase Is Affected by Physical Properties of Insoluble Celluloses. A broad‐range yeast expression system reveals rxula adeninivorans expressing a fungal self‐sufficient cytochrome P450 monooxygenase as an excellent whole‐cell biocatalyst. Therefore, the recyclability of TaGalU immobilized on MCF in a batch system was investigated for five consecutive reactions (Figure 9). Enzyme in der chemischen und pharmazeutischen Industrie. SuSy representatives are stable and can be employed as whole cell biocatalysts (Schmölzer et al., 2017), but the reaction toward UDP-glucose is not favored with respect to the equilibrium at certain pH-ranges and lower UDP concentrations (Gutmann and Nidetzky, 2016), and the enzymes show activities in a range of 14–53 μmol min–1 mg–1. When mammalian cells are applied as expression hosts for recombinant human CYPs (Crespi et al., 1991; Friedberg et al., 1999a), there is no need for the coexpression of reductase since mammalian cell lines contain endogenous reductase. Koo, H. M., Yim, S.-W., Lee, C.-S., Pyun, Y. R., and Kim, Y. S. (2014). This could explain why the concentration of UTP needs to be at least 10 times higher to detect a very small TaGalU activity, than the concentration of G1P (see Figure 8). Mat. Before the enzyme assays were carried out, the carriers were centrifuged (8,667 × g, 20 min, 5°C) and washed with 100 mM phosphate buffer pH 7.0. For the immobilized TaGalU the situation is vice versa with a lower affinity (higher Km) for G1P. Metabolites of AAG561 prepared by biotransformation with E. coli JM109 expressing rhCYP 3A4. It was generally observed that TaGalU immobilized on amino-functionalized carriers showed much better activities than when immobilized on epoxy-modified silica. Microbiol. The recombinant expression of human CYPs in insect cell, realized by infection with baculovirus DNA, has been reported several times (Buters et al., 1994; Mao et al., 2008; Rushmore et al., 2000). It consisted of a backbone (5740 bp) coding for a resistance against Ampicillin and the codon usage optimized UDP-glucose pyrophosphorylase (galU) gene from Thermocrispum agreste DSM 44070 as insert (911 bp, NCBI accession of the protein sequence: WP_028847555; GenBank accession of the codon usage optimized nucleotide sequence: MT321102; see Supplementary Material) under the control of the lactose or isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible lac-promoter. Also other mammalian CYPs were expressed in E. coli and applied as whole cell biocatalysts (Uno et al., 2008). To deeply illustrate the impact of immobilization on enzyme activity, the specific activity of TaGalU before and after immobilization on amino modified MCF at pH 6.7 was measured (Figure 4). They turned out to be technically robust and to express the recombinant human CYP reliably to sufficient expression levels enabling preparative syntheses in the scale of up to several hundred milligrams of drug metabolites. Pre-incubation of the reaction mixture was carried out at 50°C for 15 min, enzyme was added after storage on ice to start the reaction. Learn about our remote access options, Novartis Institutes for Biomedical Research, GDC/PSB, WSJ‐508.2.02A, CH‐4056 Basel, Switzerland; telephone: +41‐61‐324‐0460; fax: +41‐61‐324‐2103, E-mail address: stephan.luetz@novartis.com. After 2 h only activities between 50 and 200 U mg–1 (about 6 and 29% of the activities before incubation) were observed. Enantioenriched Compounds via Enzyme-Catalyzed Redox Reactions. Efficient Screening of Cytochrome P450 BM3 Mutants for Their Metabolic Activity and Diversity toward a Wide Set of Drug-Like Molecules in Chemical Space. It is desirable to identify such metabolites early in drug development. We also tested this hypothesis with both substrates and the free TaGalU, but the activity was not increased. Therefore, immobilization of enzymes is usually required in industrial applications (Mateo et al., 2007; Zhang et al., 2015).
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