Introduction Hrl
In nature, the vast majority of enzyme-catalyzed reactions are carried out in water. Traditionally, enzymic catalysis in organic synthesis is carried out in aqueous solution. In some cases, water-miscible organic cosolvents are added to improve the solubility of the hydrophobic substrate in the reaction solution. With the addition of an organic cosolvent, the equilibrium constant of the hydrolytic enzyme-catalyzed reaction can change and subsequently affect the yields of the product 1 . Recent...
Regioselective Carbohydrate Acylation
In one of our research programs aimed at preparing new trimodular nonionic surfactants made of a sugar with a lipophilic tail linked by an aminoacid residue 30 , several esterifications of suitable sugar derivatives have been attempted using different lipases. The potential of lipases as catalysts for the acylation of carbohydrates is currently of considerable interest, as the reaction occurs selectively at the primary hydroxyl group of the sugar. The need for protection and deprotection steps...
Conversion and Enantiomeric Excess Determination
1. Ten microliters of reactants were withdrawn at scheduled times see Note 3 , diluted in a 1-mL tube with 100 L of acetone and centrifuged for a few seconds to clear the solution from enzyme powder and molecular sieves. 2. Ten microliters of the acetone solution were analyzed by gas chromatography GC under the following conditions oven temperature 130 C for 10 min, then to 160 C with a heating rate of 0.5 C min. The running time was approx 30 min see Note 4 . 3. The enantiomeric ratio was...
Proteases
Proteases have been intensively studied in water-poor organic solvents using transesterification reactions of N-protected amino acid esters 1 . Also, peptide bond formation reactions have been catalyzed by proteases in nonaqueous media, which are obviously of greater practical importance. In order to minimize hydrolysis, both types of enzymatic reactions have to be performed under water-poor conditions. This has the drawback that the protease activity is generally low and, therefore,...
What to PreEquilibrate
Thought must be given to the initial hydration state of every component of the reaction mixture. If only some are pre-equilibrated, then water redistribution after they are combined will give a final water activity perhaps quite different from the intention. For example, if a biocatalyst brought to a desired water activity is suspended in a dried solvent, it will probably rapidly lose water. The equilibrium water activity eventually reached may be significantly reduced. Such combination of a...
Nocardia rhodocrous Cells 6
Thawed cells of N. rhodocrous 1 g wet cells , suspended in 2.0 mL of a water-saturated mixture of benzene and n-heptane 1 1, v v under mild soni- Fig. 3. Schematic model of lipid-coated lipase entrapped in hydrophobic photo-crosslinked resin gel matrix. Fig. 3. Schematic model of lipid-coated lipase entrapped in hydrophobic photo-crosslinked resin gel matrix. cation 10 kHz for several seconds , were mixed with 1.0 mL of the same solvent containing 1.0 g of ENTP or ENTB and 10 mg of benzoin...
General Comments on Producing PropanolRinsed Enzyme Preparations
Figure 1 illustrates the process involved in the production of propanol-rinsed enzyme preparations PREPs . The initial step is to immobilize the enzyme on a support material in aqueous buffered solution. This could be via covalent attachment or by an adsorption process. With subtilisin Carlsberg and a-chymotrypsin, we have adopted a very simple method of adsorbing the protein onto chromatog-raphy-grade silica gel. For other enzymes, this may not be the best support and materials such as...
Control of Water Activity by Means of Celite R640 Adsorption Isotherms in
The following diagrams show how aw varies as different amounts of water are added to Celite rods in dry toluene. The adsorption isotherms are obtained by mixing 1 mL of dry toluene, three Celite rods 95 mg , and water into 5 mL vials with screw caps and Teflon-lined septa, after which the system is equilibrated for at least 24 h in an air-bath-type thermostatted orbital shaker. After equilibration, water activity is measured by sealing the humidity sensor into the open end of the thermostatted...
References Bzc
1. Livingston, A. G. 1991 Biodegradation of 3,4-dichloroaniline in a fluidized bed bioreactor and a steady-state biofilm kinetic model. Biotechnol. Bioeng. 38,260-272. 2. Ebert, C., Gardossi, L., and Linda, P. 1998 Activity of immobilised penicillin ami-dase in toluene at controlled water activity. J. Mol. Catal. B Enzymatic 5, 241-244. 3. De Martin, L., Ebert, C., Garau, G., Gardossi, L., and Linda, P. 1999 Penicillin G amidase in low-water media immobilisation and control of water activity by...
Celite as a Support for Biocatalysts
Celite is a solid support widely used for the adsorption of biocatalysts. Many examples of improved enzyme performances by means of adsorption onto From Methods in Biotechnology, Vol. 15 Enzymes in Nonaqueous Solvents Methods and Protocols Edited by E. N. Vulfson, P. J. Halling, and H. L. Holland Humana Press Inc., Totowa, NJ Celite powder are reported in the literature 5-16 . Porous Celite beads and rods are used primarily as supports for the immobilization of whole cells 17-19 and fungi...
Solvents For Poly Allylamine Hydrochloride
Fig. 1. Preparation and use of microencapsulated biocatalysts in organic solvents. A Aqueous buffer containing biocatalyst and a water-soluble amine is dispersed in an organic solvent e.g., decane, see Notes 1 and 2 in the presence of an emulsifier B an organic-soluble crosslinker is added to the solvent to initiate interfacial polymerization and stirring is continued to harden the microcapsules see Note 4 C the microcapsules are allowed to sediment D the polymerization solvent is decanted and...
Activation of Bakers Yeast
1. Add 1.25 g of dried Baker's yeast to 25 mL of sterile distilled water containing 10 glucose and 0.1 w v of yeast extract in a 100-mL conical flask. 2. The culture is incubated at 25 C in a Luckham R300 shaker-incubator at 150 rpm for 30 min. 3. The activated yeast is agitated for 2 min using a laboratory rotamixer Spinmix, Gallenkamp to provide a homogenous suspension of cells. 4. A 1.5-mL aliquot of the homogenous suspension of the yeast cells is spun in a lab bench centrifuge at 4000g for...
Activity Determination of Subtilisin
3. One of the following organic solvents 1,4-dioxane, carbon tetrachloride, benzene, toluene. Transesterification, Hydrolytic, and Total Transesterification Plus Hydrolytic Activity of Subtilisin and Subtilisin PEG in 1,4-Dioxane, and of Lipase PC and Lipase PC PEG in 1,4-Dioxane and Carbon Tetrachloride Transesterification, Hydrolytic, and Total Transesterification Plus Hydrolytic Activity of Subtilisin and Subtilisin PEG in 1,4-Dioxane, and of Lipase PC and Lipase PC PEG in 1,4-Dioxane and...
HPLC Analyses
All HPLC analyses were performed using a Waters liquid chromatography system purchased from Waters Scientific Mississauga, Ontario, Canada . For the measurements of enzyme activity and enantioselectivity in the resolution of ibuprofen presented in this chapter, various types of column were used A reverse-phase column CSC-S, Spherisorb ODS-2, 5 m 25 cm x 4.6 mm , CSC, Montr al, Qu bec, Canada , allowing the separation of ibuprofen, alcohol, and ester. Two chiral columns Chiralcel OD 25 cm x 4.6...
Immobilization of Lipases on Hydrogels
Abu Bakar Salleh, Norhaizan M. Esa, Mahiran Basri, Che Nyonya A. Razak, Wan Md Zin W. Yunus, and Mansor Ahmad Immobilization of enzymes can be achieved by methods of varied complexity and efficiency 1 on a variety of supports. For example, enzymes can be adsorbed onto insoluble materials, copolymerized with a reactive monomer, encapsulated in gels, crosslinked with a bifunctional reagent, covalently bound to an insoluble carrier 2 or entrapped within an insoluble gel matrix of natural or...
References Bjm
1. Collins, A. N., Sheldrake, G. N., and Cosby, J. eds. Chirality in Industry II. Wiley, New York. 2. Faber, K., Ottlina, G., and Riva, S. 1993 Selectivity-enhancements of hydrolases reactions. Biocatalysis 8, 91-123. 3. Klibanov, A. M. 1989 Enzymatic catalysis in anhydrous organic solvents. Trends Biochem. Sci. 14, 141-144. 4. Wescott, C. R. and Klibanov, A. M. 1994 The solvent dependence of enzyme specificity. Biochim. Biophys. Acta 1206, 1-9. 5. Carrea, G., Ottlina, G., and Riva, S. 1995...
Microencapsulation of Enzymes and Cells for Nonaqueous Biotransformations
Jeffrey A. Khan and Evgeny N. Vulfson 1. Introduction The use of biocatalysts in nonaqueous organic solvents is now a well-established method for the preparation of pharmaceutical products 1-2 , food ingredients 3-5 , and intermediates used in the fine chemicals industry 6-10 . One of the advantages of this approach is that it enables the bioprocess operation at much higher concentrations of poorly water-soluble substrates, thus making both the synthesis and product recovery more attractive...
References Xqd
1. Yang, Z. and Russell, A. J. 1995 Fundamentals of non-aqueous enzymology, in Enzymatic Reactions in Organic Media Koskinen, A. M. P. and Klibanov, A. M., eds. , Blackie Academic amp Professional, Glasgow, pp. 43-69. 2. Dordick, J. S. 1988 Biocatalysis in nonaqueous media. Appl. Biochem. Biotechnol. 19, 103-112. 3. Halling, P. J. 1994 Thermodynamic predictions for biocatalysis in non-conventional media theory, tests and recommendations for experimental design and analysis. Enzyme Microb....
Weiss Formerly Philips LiCl Humidity Sensor
We currently use this sensor for gas-phase humidity measurements. The sensor has an electrically heated LiCl solution that comes into equilibrium for water exchange with the gas phase. The steady-state temperature at which this occurs depends on the partial pressure of water vapor in the gas and is measured by a Pt resistance thermometer. The element temperature and the water partial pressure are related by a calibration curve. A simplified theoretical picture suggests that the calibration...
Water Concentrations by Coulometric Karl Fischer
For measurement of low water concentrations, the coulometric Karl Fischer KF method is generally the best. This is based on the electrolytic generation of the iodine involved in the Karl Fischer reaction, rather than a volumetric titration. The resulting electrical measurements are more sensitive at low levels. We have used the Metrohm 684KF and Fisher Instruments Accumet KF instruments. A variety of reagents and recommendations for their use are available from both Riedel de Haen Hydranal and...
References Xzw
1. Anderson, E. M., Larsson, K. M., and Kirk, O. 1998 One biocatalyst many applications the use of Candida antarctica B-lipase in organic synthesis. Biocatal. Biotransform. 16, 181-204. 2. Stead, P., Marley, H., Mahmoudian, M., Webb, G., Noble, D., To Ip, Y., et al. 1996 Efficient procedures for the large-scale preparation of 1S,2S -trans-2-methoxycyclohexanol, a key chiral intermediate in the synthesis of tricyclic p-lactam antibiotics. Tetrahedron Asymmetry 7, 2247-2250. 3. Kazlauskas, R. J....
GasPhase Sensor Response and Equilibration Times
In discussing the response time of sensors in the gas phase, it is important to distinguish the true response time of the sensor from the equilibration time of the system. The sensor response time may be only a minute or two, as shown by for example removing it from a water-saturated vessel to the laboratory air. However, the reading will take 1 h or more to reach a final value after the sensor is sealed into an unstirred headspace above water or a salt solution this reflects the time for...
Notes Bwo
1. Analysis of the mixture composition can be performed by taking samples at intervals. After a 20-fold dilution in water, filter to eliminate the enzyme and analyze by high-performance liquid chromatography HPLC on a reverse-phase column Nucleosil C18, 6 m, 250 mm x 4 mm inside diameter I.C.S., France . Eluent 50 50 methanol water flow rate 0.5 mL min temperature 33 C injection volume 10 L detection refractometer RI-Detector 8110, I.C.S. . 2. The yield is defined as the molar ratio of...
Synthesis of Imidoesters
Synthesis of imidoesters are according to the procedure adapted from Hunter and Ludwig 4 as described by CH3OH, 0 C, 1.5 h OCH nitrile OCH3 Imidoesters with different hydrophobicity could be synthesized easily from their corresponding nitriles or alcohols in relatively high yield. Methyl acetimidate imidoester I methyl benzimidate imidoester II , methyl 3-phenylpropionimidate imidoester V , and methyl 4-phenylbutyrimidate imidoester VI are very soluble in aqueous 0.1 M borate buffer, pH 8.5....
Synthesis of 2Heptanone
1. Encapsulated spores 200 L containing 4.84 x 1010 cells are added to 1.8 mL of n-decane containing 500 mM methyl octanoate in a sealed 10-mL Wheaton vial. 2. The bioconversion is performed at 25 C in a Luckham R300 shaker-incubator at 150 rpm. 3. The reaction is monitored by removal of 50- L aliquots of the organic layer at regular intervals over a period of 1-14 d. These samples are diluted 10-fold with dichloromethane containing 3-decanone as an internal standard and analyzed by gas...
Regiospecific Interesterification of Triglyceride by Rhizopus delemar Lipase 10
The reaction was carried out at 40 C with shaking 120 strokes min . The reaction mixture was composed of entrapped preparations of free lipase or C-lipase corresponding to 5 mg of free lipase , 0.25 g of olive oil, and 0.25 g of stearic acid in 10 mL of water-saturated -hexane. After development by thin-layer chromatography TLC , the reformed triglyceride was scraped off from a thin-layer plate and extracted with ethyl ether. After hydrolysis of triglyceride, liberated fatty acids were...
Endress Hauser Aluminum Oxide Humidity Sensor
We used this sensor extensively in the past, as it is the only one we know of that can be immersed directly in organic liquids although only the more nonpolar ones . It is also unaffected by most organic vapors when used in the gas phase, but its lower sensitivity and tendency to drift make it no longer our choice for this, compared with the LiCl sensor. The sensor and corresponding hygrometer are manufactured by the German division of Endress amp Hauser Maulburg . In our studies, we have used...
References Mnc
1. Whitesides, G. M. and Wong, C.-H. 1985 Enzymes as catalysts in synthetic organic chemistry. Angew. Chem. Int. Ed. Engl. 24, 617-638. 2. Klibanov, A. M. 1983 Immobilized enzymes and cells as practical catalysts. Science 219, 722-729. 3. Klibanov, A. M. 1986 Enzymes that work in organic solvents. CHEMTECH 16, 354-359. 4. Klibanov, A. M. 1990 Asymmetric transformations catalyzed by enzymes in organic solvents. Acc. Chem. Res. 23, 114-120. 5. Jones, J. B. 1986 Enzymes in organic synthesis....
References 1
1. Inada, Y., Yoshimoto, T., Matsushima, A., and Saito, Y. 1986 Engineering physicochemical and biological properties of proteins by chemical modification. Trends Biotechnol. 4, 68-73. 2. Inada, Y., Matsushima, A., Kodera, Y., and Nishimura, H. 1990 Polyethylene glycol PEG -protein conjugates application to biomedical and biotechnological processes. J. Bioact. Compat. Polym. 5, 343-364. 3. Inada, Y., Matsushima, A., Hiroto, M., Nishimura, H., and Kodera, Y. 1994 Modification of proteins with...
Introduction
This section of Enzymes in Nonaqueous Solvents Methods and Protocols covers methods for the control of general conditions affecting the behavior of enzymes in nonaqueous solvents. Just as in aqueous media, the activity, specificity, and stability of enzymes in nonaqueous media are rather strongly dependent on reaction conditions. Some of the important factors, e.g., temperature and substrate concentration, are the same as in conventional aqueous media, although the dependence may be...
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