SPRIN Ketoreductase Kit

Transcription

1 SPRIN Ketoreductase Kit SPRIN Ketoreductase Kit Normal (12 preparations) Product code: SKKN Covalently immobilised preparations of different Ketoreductases on Acrylic Resin Kit Description: The kit contains 12 different Ketoreductases immobilised on acrylic resin, and cofactors, SPRIN imibond GD (Glucose dehydrogenase immobilised on acrylic resin) and glucose for cofactor regeneration. Content Immobilised KREDs SPRIN imibond GD Glucose 12 vials with different enzymatic preparations, 250 mg wet each 1 vial, 50 mg 1 vial, 30 mg 1 vial, 500 mg wet 1 vial, 1 g Storage: Store at 2 8 C. 1

2 Specifications SPRIN immobilised Ketoreductases Covalently immobilised preparations of recombinant Ketoreductases from Escherichia coli Enzyme Recommended Name: Alcohol dehydrogenase EC numbers for the 12 different Ketoreductases: SPRINzymes EC number K K K K K K K K K K K K Properties Activity of immobilised Ketoreductases is reported at page 5 Characteristics Matrix: acrylic polymer Particle size: μm Water content: 60 70% SPRIN imibond GD Covalently immobilised preparation of recombinant Glucose dehydrogenase from Escherichia coli Enzyme Recommended Name: Glucose 1-dehydrogenase EC number: Properties Activity: 7 U/g wet (A.M. S042, page 4) Characteristics Matrix: acrylic polymer Particle size: μm Water content: 60 70% Recommendations: for a proper use of SPRIN Ketoreductase Kit, magnetic stirring should be avoided. We suggest, when working in batch, the following stirring methods: blood-rotator, orbital shaker, mechanical stirrer (recommended 120 rpm). 2

3 General overview of substrate specificity for the different SPRIN immobilised Ketoreductases: SPRINzymes Cofactor Substrates Linear Ketone Aromatic Ketone α-ketoester β-ketoester Linear Aldehyde 2-Butanone Acetophenone Ethyl pyruvate ECAA DL-Glyceraldehyde K01 K02 K03 K04 K05 K06 K07 K08 K09 K10 K11 K12 ECAA: Ethyl-4-chloroacetoacetate Legend: Symbol Activity (U/g wet ) <

4 Ketoreductase activity assay (A.M. S041) Enzyme activity is determined monitoring the reduction in absorbance at 340 nm of NAD(P) that is consumed during the reaction. Enzyme activity is expressed as U/g, where one Unit corresponds to the amount of enzyme that reduces 1 µmol of substrate per minute at p 7.0 at 30 C. Selected substrates Glucose dehydrogenase activity assay (A.M. S042) The assay is based on the oxidation of glucose to gluconolactone using NAD(P) + as a cofactor. The reaction is monitored by following the increase of absorbance at 340 nm. Enzyme activity is expressed as U/g, where one Unit corresponds to the amount of enzyme that reduces 1 µmol of glucose per minute at p 8.0 at 30 C. 4

5 Activity assays and relative activity values for the different SPRIN immobilised Ketoreductases: SPRINzymes Cofactor Activity assay Activity (U/g wet ) 1 K01 K02 K03 K04 K05 K06 K07 K08 K09 K10 K11 Acetophenone (8.80 mm, ml), K12 >0.15 (7.50 mm, 0.30 ml), K12 (35-40 mg) 1 Activity (U/g wet ): one Unit correspond to the amount of enzyme that reduces 1 µmol of substrate per minute at p 7.0 at 30 C. Ethyl-4-chloroacetoacetate (3.87 mm, ml), (7.50 mm, 0.30 ml), K01 (20-25 mg) Ethyl-4-chloroacetoacetate (3.87 mm, ml), (7.50 mm, 0.30 ml), K02 (30-35 mg) Ethyl pyruvate (8.80 mm, ml), (7.50 mm, 0.30 ml), K03 (30-35 mg) 2-Butanone (8.80 mm, ml), (7.50 mm, 0.30 ml), K04 (30-35 mg) Ethyl-4-chloroacetoacetate (3.87 mm, ml), (7.50 mm, 0.30 ml), K05 (25-30 mg) DL-Glyceraldehyde (8.80 mm, ml), (7.50 mm, 0.30 ml), K06 (30-35 mg) Ethyl pyruvate (8.80 mm, ml), (7.50 mm, 0.30 ml), K07 (30-35 mg) Ethyl pyruvate (8.80 mm, ml), (7.50 mm, 0.30 ml), K08 (25-30 mg) DL-Glyceraldehyde (8.80 mm, ml), (7.50 mm, 0.30 ml), K09 (75-80 mg) Ethyl-4-chloroacetoacetate (3.87 mm, ml), (7.50 mm, 0.30 ml), K10 (35-40 mg) Ethyl pyruvate (8.80 mm, ml), (7.50 mm, 0.30 ml), K11 (30-35 mg) 5 >1.50 >0.80 >1.50 >1.50 >0.15 >0.15

6 Background Ketoreductases (KREDs) are a large protein super-family mainly of NAD(P)-dependent oxidoreductases involved in carbonyl metabolism. Catalysis is promoted by a conserved tetrad of active site residues (Tyr, Lys, Asp and ys). Ketoreductases typically catalyse reduction of carbonyl compounds to corresponding alcohols or the reverse oxidation reaction, finding applications in pharmaceuticals, flavouring, aroma, agricultural and fine chemicals production processes. SPRIN Ketoreductases are covalently immobilised on acrylic polymers and are conceived as a screening tool. All the preparation can be further optimised for each specific process of interest, in terms of activity, stability and recyclability. 6

7 Ketoreductases Ketoreductases (KREDs) are enzymes capable of reducing ketones to the corresponding chiral alcohols. KRED immobilised R R' R * R' NAD(P) NAD(P) + SPRIN immobilised Ketoreductases are either (nicotinamide adenine dinucleotide) or (nicotinamide adenine dinucleotide phosphate) dependent. The reduced form of these cofactors delivers the hydride-equivalent necessary for the reduction of carbonyl groups to the corresponding alcohol. Chiral alcohols are frequently required as important and valuable intermediates in the synthesis of numerous pharmaceuticals and other fine chemicals. ydrogen source for cofactor regeneration Cofactors like and are expensive, therefore using them in stoichiometric amounts is hardly ever applicable, except in small scale screening applications. In commercial processes the cofactors are recycled and used in catalytic amounts, significantly reducing the production costs. Alcohols or Sugar with Glucose dehydrogenase can be used to carry out the regeneration of the cofactor, parallel to the conversion of the substrate to product. Alcohols Alcohols such as ethanol and 2-propanol have been widely used as co-substrate for cofactor recycling. 7

8 Sugars and Glucose dehydrogenase xidised cofactors can be regenerated by coupling the ketone reduction to glucose oxidation catalysed by a second immobilised enzyme preparation, SPRIN imibond GD, immobilised Glucose dehydrogenase. The enzyme reduces NAD + or NADP + using glucose as a hydride donor, the glucose is oxidised to gluconolactone that hydrolyses spontaneously to the corresponding acid. This last reaction is irreversible. R R' KRED immobilised R * R' NAD(P) NAD(P) + + GD immobilised Gluconolactone Glucose Substrate specificities Ketoreductases react with a wide range of substrates, such as Aliphatic or Aromatic Ketones, Aldehydes or ɑ/β Ketoesters (page 3). 8

9 Application KREDs catalyse reduction of carbonyl compounds to the corresponding alcohol or the reverse oxidation reaction: Reduction of Aliphatic and Aromatic Aldehydes; Reduction of Aliphatic and Aromatic Ketones; Reduction alpha/beta-ketoesters; xidation of Monosaccharides; Reduction Ketosteroides. Reaction Media Non-aqueous solvents; rganic solvent (which facilitate hydrophobic substrates solubilisation); Biphasic systems (to reduce concentration of toxic substrate and product around enzymes in aqueous layer, and to simplify work-up procedure). Literature R. Kratzer et al., Life, 2006, 58 (9), 499. F. ildebrand et al., Tetrahedron: Asymmetry 2006, 17, K. Goldberg et al., Appl. Microbiol. Biotechnol. 2007, 76, 237. K. Goldberg et al., Appl. Microbiol. Biotechnol. 2007, 76, 249. J.M. Woodley Trends Biotechnol. 2008, 26, 321. T. Matsuda et al., Tetrahedron: Asymmetry 2009, 20, 513. S. Leuchs et al., Chem. Biochem. Eng. Q. 2011, 2, 267. Y. Ni et al., Biotechnol. Adv. 2011, article in press. G.A. Petkova et al., Biochim. Biophys. Acta 2012, 1824,