Powder enzymes

Harkonen, H., Koskinen, M., Linko, R, Siika-aho, M. and Poutanen, K., Granulation of enzyme powders in a fluidized bed spray granulator, Lebensmittel Wissenschaft Technologie, 26 (1993) 235-241. 

Figure 9.2 A schematic presentation of different types of enzyme preparations used in non conventional media, a enzyme powder, b enzyme crystals c enzyme on a porous support d covalently modified enzyme dissolved in the solvent e enzyme solubilised by surfactant ...

The most straightforward way of using sohd enzymes in organic media is to suspend the solid enzyme directly in the solvent. If one wants to get quick results from a bioconversion and does not want to optimise the efficiency of the enzyme, this method is the obvious first choice. There are many example in the hterature where enzymes have been used successfully in synthesis just as powders directly from the enzyme manufacturer. Sometimes there is a need to dissolve the enzyme powder and re-lyophilise it from a buffer with a more suitable composition, see the section 9.6 pH control in non-conventional media . 

Most enzyme powders are prepared by lyophilisation (freeze drying). However, the lyophilization procedure might inactivate the enzyme to some extent. To avoid this and thereby increase the activity of lyophilized enzymes in dry organic solvents, the lyophilization can be carried out in the presence of lyoprotectants such as sorbitol (Dabulis and Klibanov, 1993). The inactivation is believed to be caused at least partly by a reversible conformational change in the enzyme. This process can be reversed and the enzyme reactivated by the addition of small amoimts of water (Dabulis and Klibanov, 1993). 

In many applications of enzymes in non-conventional media, the enzymes are nsed in the immobilised form on support materials which often are porous. These immobilised preparations usually express considerably higher specific activity (moles snbstrate converted per unit enzyme and time) than enzyme powders. The reason can be facilitation of mass transfer either by the spreading of the enzyme on a large surface area or by better suspension of the catalyst particles (enzyme powders often tend to aggregate). An alternative explanation is that the support might protect the enzyme from inactivation dnring the drying of the preparation. 

Furthermore, the type of enzyme formulation (free enzyme, immobilized enzyme, or whole cells) plays a key role in determining the progress of the overall reaction. For most applications, lyophilized enzyme powders have been used with good results presumably they dissolve into the liquid phase. When poorly soluble products are formed, the enzyme can be recovered by washing with water [52]. For co-factor-dependent reactions permeabilized cells may be used [44]. When using immobilized enzymes, it has been demonstrated that the chemical nature and the pore size of the support are very important parameters to consider [8, 41]. 

Lyophilization employs sub-zero temperatures in combination with very low pressure to withdraw water from the protein solution typical values would be -80 °C and 1-3 mbar. In these conditions, as the water phase diagram reveals, water sublimes, leaving a fluffy porous enzyme powder. As typical run times are on the order of a day, lyophilization is not the method of choice for large-scale enzyme drying operations. On a laboratory scale, however, lyophilization is a very effective method. 

Although freeze drying has often been used to prepare stable dry enzyme preparations we found that when preparing enzyme powders and electrodes it was preferable to vacuum dry the enzyme in the presence of stabilizers (Fig. 8). However this was not true in all cases. Cholesterol oxidase was found to be stabilized best by freeze drying in stabilizers and buffer at pH 5.5 well below the pH for optimal activity (Gibson and Woodward unpublished results). 

Plot the numerical data you recorded in the four columns on graph paper. Note that somewhere between 3 and 5 min. your graphs are linear. Obtain the slopes of these linear portions and record them on your Report Sheet. Calculate the activities of your enzyme first as (a) units per mL sample solution and second as (b) units per mg enzyme powder. 

The enzyme powder obtained was assayed to determine the optimum pH and temperature for fatty acids production (7). A 20% butterfat emulsion was used as the enzyme substrate instead of an olive oil emulsion. The optimum pH for fatty acid production was 7.7. The optimum temperature was 37°C. 

Immobilized enzymes are currently the object of considerable interest. This is due to the expected benefits over soluble enzymes or alternative technologies. The number of applications of immobilized enzymes is increasing steadily [5]. Occasionally, however, experimental investigations have produced unexpected results such as a significant reduction or even an increase in activity compared with soluble enzymes. Thus, cross-linked crystals of subtihsin showed 27 times less activity in the aqueous hydrolysis of an amino acid ester compared to equal amounts of soluble enzyme [6]. On the other hand, in the application of hpo-protein lipase in the solvent-mediated synthesis of esters there was a 40-fold increase in activity using immobihzed or otherwise modified enzyme preparations as compared to enzyme powder [7]. 

For reactions in water-immiscible organic solvents the simplest method of immobihzation is to use dried enzyme powders and to suspend them in the solvent [104]. They can be removed by filtration or centrifugation and reused afterwards. However, even this simple method can cause trouble. 

A powder of hpoprotein hpase (LPL) esterified an organic substrate in toluene at a rather poor reaction rate (Table 4), which was to some extent explained by adhesion of the sticky enzyme powder to the surface of the reaction vessel [7]. When polyethylene glycol (PEG) was bound covalently to LPL and this modified enzyme was dissolved in toluene, approximately 3.5 U mg of enzyme protein were assayed. After simple addition of PEG to the reaction mixture together with LPL powder, the same poor reaction rate of the enzyme powder alone was observed. On the other hand, when LPL powder was lyophilized together with PEG the resultant preparation had an activity of 1.8 U mg L In this case, the enzyme... 

In the case of enzyme powders, proper formulation and storage conditions will ensure reasonable activities. This is achieved by the addition of various compoimds prior to the drying process to improve distribution. Such compounds can also be quite useful as stabilizers and as protective agents. Suitable preparations are usually provided by enzyme manufacturers for dedicated enzymes. 

For patients who are unable to swallow capsules, the contents may be emptied into applesauce, jelly, or some other nonalkatine vehicle, provided that the patient does not chew the microencapsulated beads. Side effects of pancreatic enzyme products are uncommon. Perianal irritation resembling diaper rash may occur in infants fed excess quantities of enzyme powders. Hyperuricosuria also has been reported to occur secondary to pancreatic enzyme use, apparently related to their high purine content. Proximal colonic stricture (fibrosing colonopathy) is a dose-related side effect associated with lipase doses in excess of 24,000 units/kg per day. ... 

Immobilization, dehned as the physical confinement or localization of an enzyme into a specihc micro-environment, has been a very common approach to prepare enzymes for aqueous as well as nonaqueous applications. For nonaqueous enzymol-ogy, immobilization improves storage and thermal stability, facilitates enzyme recovery, and enhances enzyme dispersion. In addition, immobilized enzymes are readily incorporated in packed bed bioreactors, allowing for continuous operation of reactions. Moreover, lyophilized enzyme powders often aggregate and attach to reactor walls, particularly when the water activity is moderately high. The major disadvantage of immobilization is low activity, induced by pore diffusion mass transfer limitations and by alteration of protein stmcture. For enzymes in nonaqueous media, the following broad categories of immobilization exist ... 

A simple way of applying enzymes in a non-aqueous reaction is through addition of lyophilized enzyme powder. With a hydrophobic solvent, this will create an enzyme suspension and, if other conditions (substrate, temperature, etc.) are acceptable, it will probably work. For a quick screening of a range of enzymes it may even be the most optimal setup. It is, however, well known that many enzymes lose activity upon lyophilization (which to some degree can be prevented by the use of lyoprotectants). Another concern is that enzyme dust is potentially allergenic if inhaled. 

Figure 2. Distribution of protein and enzymic activities after gel filtration of a partly purified commercial enzyme powder (Cellulose 36, Rohm Haas Co., Philadelphia) on two connected Sephadex G-75 columns. The void volume of the system was 1170 ml. The fraction volume was 27 ml. (2)...

To facilitate lipase-reactions in supercritical CO2, Bruns et al (32) impregnated a lipase within amphiphilic co-networks that comprised a fluorophilic phase. This resulted in substantially higher enzyme activity relative to the enzyme powder. It may be noted that the bio-artificial materials reported by Silvestri et al (7) can also be considered a form of enzyme immobilization. 

Kasei Kogyo Co., Ltd. Since the enzyme powder suppli contains sdts (12% sodium acetate and 24% calcium acetate) as stabiluer, it was purified by re-precipitation at pH 7.0 and the precipitate was freeze-dried at -40 C before use. TTie other chemicals used were of reagent grade. 

Effect of Lactose Addition on Production of Stabilized Enzyme Powders... 

Effect of Lactose Addition on Production of Stabilized Enzyme Powders by Spray Drying with an Exhaust Air Temperature Variation from 60°C to 70°C... 

H. A. Herrmann, I. Good, and A. Laufer, Manufacturing and downstream processing of detergent enzymes. Powdered Detergents (M.S. ShoweU, ed.) Marcel Dekker, New York, p. 251,1997. 

Figure 2.9 Enzymatic synthesis of jj-nitrobenzyl-D-glucopyranoside (pNBG) by reverse hydrolysis in a monophasic dioxane-water system. The reaction was performed in 1.0 mL of dioxane-buffer medium (Na2HP04-KH2PO4, 70 mM, pH 6.0) by shaking a mixture of 0.25 mmol glucose, 1.0 mmol pNBA and 5 mg of enzyme powder at 50 °C and 160 rpm. Symbols ( ) initial rate ( ) final conversion.

Special precautions. There are no reported risks associated with the glycosidase however, many enzymes are immunogenic, so skin contact and inhalation of enzyme powders should be avoided. Safety glasses and gloves should be worn during this experiment and work should be carried out in a fume hood. 

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