February, 1924
INDUSTRIAL A N D ENGINEERING CHEMISTRY
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The Rapid Analysis of Sugars’ Purification and Concentration of Enzyme Solutions By F. W. Reynolds CARBOHYDRATB LABORATORY, B U ~ A OF W CXEXISTRY, WASHINGTON, D. C.
T
Yeast extracts may be freed of color and substances causing The writer has found in turbidity by dialyzing, or by washing on an ultra-filer. and subthe case of invertase and enzymes as hydrosequently acidifying with acetic acid, whereby the substances causmelibiase that efficient clarilytic reagents in the ing turbidity are flocculated and can be removed by filtration on fication of the autolyzed analysis of PolYsaccharides has long been recognized. yeast extract3 can be accomTheir use, however, has Highly active preparations of invertase and melibiase may be Plished in 12 simple manner obtained by concentrating crude or purijied yeast extracts by ultra-that is, by dialyzing the been limited by the fact that, with the dilute Sohcrude extract in collodion filtration. A simple ultra-fitter may be constructed of materials dialyzing sacks for 24 hours tions available the time rereadily Ultra-filtration should be useful as a means of concentrating O r longer against running quired for hydrolysis was in other enzymes for use as analytical reagents and also in the study tap water and then acidifymany cases too long for ing with glacial acetic acid satisfactory work and the of enzymic action of plant and fruitjuices. results were somewhat unin the proportion of about certain This is especially four drops per 100 cc. of true of some of the so-called weak enzymes, such as the dialyzed extract. This treatment causes flocculation melibiase, maltase, and lactase. Enzymes are no more of a considerable amount of material, and after standing difficult to use than other reagents, provided certain funda- over night the precipitate may be filtened off on fluted mental conditions are sufficiently controlled. By far the paper. This treatment causes no inactivation or loss of greater portion of these difficulties and uncertainties dis- either invertase or melibiase, as was shown by a number appear when enzyme solutions of sufficient activity are used. of activity tests made before and after clarification. The acIt is quite practicable to prepare invertase solutions which, tivity of solutions clarified in this manner is practically the when used in the proportion of 10 cc. of the enzyme solution same as that of solutions prepared by the lead acetate and to 100 cc. of the clarified sucrose solution, will complete the dialysis method. Neutral lead acetate causes no precipitate hydrolysis of the sucrose in less than 15 minutes a t room in yeast extracts clarified as described above. temperature. The advantage of using concentrated enzyme During the course of dialysis the enzyme solution is consolutions is not limited to a saving of time. The end point siderably diluted owing to the passage of water to the inside of the hydrolysis is sharp and distinct, instead of being some- of the dialyzing bag. This added water, together with most what uncertain as is the case when dilute enzyme solutions are of the water originally present, may be removed by the process used. The enzyme has the advantage over an acid in that no of ultra-filtration described below. Dilution during dialysis destruction of sugar is possible and the hydrolysis isspecific. may, however, be largely prevented by fitting the collodion When used as an analytical reagent, it is desirable that the dialyzing sack with a cork stopper and cementing it thereto enzyme solution be free from inactivating substances, color, with collodion. An upright glass tube of small diameter turbidiiy, and optically active substances the optical activity should penetrate the stopper and reach to a height of about of which may change during the hydrolysis. The present 4 feet above the stopper. The bag is completely filled with paper describes a method for preparing highly active solutions the enzyme solution to be dialyzed and is then immersed in of the enzymes invertase and melibiase which meet these running water. Osmotic pressure forces the liquid inside the requirements. bag up into the upright tube to a height of 3 or 4 feet. As PURIFICATION OF ENZYME SOLUTIONS the dialysis proceeds the height of the liquid gradually falls, reaching a minimum of 2 or 3 inches. It should be possible Enzymes have frequently been purified by precipitation of actually to concentrate the enzyme during dialysis by adthe e n z P e from solution by means of alcohol, acetone, and mitting fresh enzyme solution through the upright tube as other reagents, the precipitated material being redissolved in fast as the pressure falls. This has no advantage Over water* Some investigators have emPb’ed selective ad- ultra-filtration, however, and has therefore not been further sorption on aluminium hydroxide, fuller’s earth, finely divided investigated, carbon, etc. The most important work along this line is OF ENZYME SOLUTIONS BY ULTRACONCENTRATION probably that of Willstatter.2 Such methods are usually atFILTRATION tended Sy a rather large loss of enzymes unless the work is done with extreme care by an experienced investigator. The means available for concentrating enzyme solutions Xeutral lead acetate has been frequently used as a reagent are rather limited. Concentration in vacuo a t low temperafor precipitating impurities from autolyzed yeast extract. ture may be used, but in this case there is also more or less Lead saks have an inactivating effect on enzymes, and excess destruction of enzymes, especially in cases where the temperaof such salts must therefore be carefully removed by pre- ture is not carefully controlled. The fact that collodion memcipitatian, hydrogen sulfide or potassium oxalate being branes of scitable composition are impermeable to enzymes satisfactory deleading agents. The acetic acid or potassium but are permeable to water and to highly dispersed substances acetate remaining in the solutions may be removed by dialy- present in the enzyme solution, renders ultra-filtration sis. Enzyme solutions prepared in this way are usually too through such a membrane an ideal method for concentrating dilute to be used in rapid analytical work, especially in the enzymes. Enzyme solutions purified by any method may case of the so-called weaker enzymes, such as melibiase. be readily concentrated by filtering off the required amount
HE value of certain
1 Received August 22, 1923. * A n n . , 426, 1 (1921).
8 Bakers’ or top yeast contains invertase, and brewers’ or bottom yeast contains both invertase and melibiase.
INDUSTRIAL A N D ENGINEERING CHEMISTRY
170
of water; the enzyme, being retained by the filter, is thereby concentrated. Any suitable type of ultra-filter may be used. The one employed in this laboratory was improvised from materials which are generally available, and on account of its simplicity and adaptability to this purpose will be described in detail. CONSTRUCTION OF A COLLODION ULTRA-FILTER For the preparation of a suitable type of collodion membrane 5 grams of Anthony's snowy cotton4 are dissolved in a mixture of 50 cc. 95 per cent alcohol, 50 cc. U. S. P. ether, and 5 cc. glacial acetic acid. About 100 cc. of this solution are poured into a cylinder of about 2000 cc. capacity and the open end o,€ the cylinder is covered immediately with the hand. The cylinder is then inclined and rotated, keeping the open end covered to prevent evaporation, until the inside surface of the cylinder is uniformly coated with the collodion. The cylinder is then inverted and uncovered and allowed to drain and dry for 10 minutes. At the end of this time it is placed in an upright position and filled with water. I n 10 or 15 minutes the collodion membrane will have loosened from the cylinder and can be removed after pouring out the water. The sack should be filled with water and wiped dry on the outside and examined for leaks. It is then emptied and cut open longitudinally and the flat membrane which is obtained is spread out on a wet piece of filter paper. The membrane should never be allowed to dry, as it then becomes impermeable. A circular piece about 7 or 8 inches in diameter is then cut out with a pair of scissors. The bottom is cut from a large wide-mouth bottle or 2-liter Erlenmeyer flask and the edge ground flat and smooth with emery powder on a flat piece of metal or g l ~ .The bottle or flask is placed concentrically on the circular piece of collodion, and the edges of the membrane are folded up to the sides of the
iaa/F/n o r
U
Fro. DIAGRAM
OF
Vaseline
ULTRA-FILTER
bottle and cemented thereto with collodion. This cementing collodion should contain an increased percentage of ether (about 4 parts of ether to 1 of alcohol), as a collodion of this composition dries quickly to a hard, impermeable film. It is well to coat most of the outside surface of the 1 All types of nitrocellulose do not give the same results, It is therefore recommended that Anthony's snowy cotton be used as this was found to yield the most satisfactory results of any variety readily obtainable in the United States.
Vol. 16, No. 2
bottle with this cementing collodion, making it continuous with the filtering membrane; a small varnish brush is conveniently used. Three or four thicknesses of wet filter paper are placed in a Buchner funnel having an outside diameter of about 8 inches. The bottle, fitted with the collodion membrane, is then placed in the Buchner funnel with the collodion membrane resting on the filter paper. Melted vaseline is poured between the edge of the bottle or flask and the side of the funnel to a depth of an inch or so in order to make an air-tight seal. The Buchner funnel is then used in the customary manner, with vacuum, and a suction flask as a receiver. Water filters through this membrane a t the rate of about 500 cc. per hour, depending on the vacuum. With yeast extracts the rate of filtration is slower, varying from 100 cc. per hour to 25 cc. per hour, depending upon the character of the solution. When not in use the membrane should be kept covered with water to which a little camphor or chloroform has been added as a preservative. Iltra-filtration may be kept going continuously, day and night, with very little attention, by using a constant-level siphon for supplying the enzyme solution, thus compensating for the relatively slow rate of filtration. Fig. 1 shows the construction of the complete filter. It is necessary to keep the enzyme solution thoroughly agitated during the ultra-filtration, as the enzyme is apparently precipitated-possibly owing to the high concentration at the surface of the membrane-unless this precaution is taken.
SIMULTANEOUS CONCENTRATION AND PURIFICATION BY ULTRA-FILTRATIO N A crude, autolyzed yeast extract may be concentrated to small volume and then washed with distilled water. This can be very conveniently accomplished by means of a constant level siphon arrangement shown in Fig. 2. This process accomplishes the same results as dialysis so far as purification is concerned, and is much more convenient owing to the fact that smaller volumes are handled. About 5 liters of the crude extract may be concentrated to approximately onefifth this volume and washed with 1 Liter of water. If the extract is then removed from the funnel and diluted to 5 liters and 10 cc. of glacial acetic acid are added, flocculation occurs to the same extent as if the extract had been dialyzed. After standing over night the flocculated material may' be filtered off and the enzyme solution returned to the ultrafilter and concentrated to the desired volume. It may then be washed with distilled water to remove any remaining color. It has been found preferable to bring about the flocculation of impurities after a relatively small amount of washing, inasmuch as the filtration after flocculation is much more rapid than before. The rate of filtration of the crude extract through a membrane 6 inches in diameter is usually about 50 to 75 cc. per hour a t the start, and gradually decreases to about 20 to 25 cc., owing probably to an increase in the viscosity of the extract, The rate of filtration during washing continues practically unchanged. After flocculation the rate of filtration is about 75 cc. per hour. Agiven membrane may be used continuously for months and maintains practically the same permeability. When not in use it should be kept COVered with water to which some pregervative has been added. A 1 : 2000 solution of chinosol (potassium oxyquinoline sulfonate) is an excellent preservative. If the membrane is to be allowed to stand for some time without use, some of the chinosol solution may be filtered through the membrane to disinfect the apparatus. Even when the apparatus is in continuous use, it is well to wash it out occasionally with pure water to which some of the preservative may be added. Toluene and chloroform are satisfactory preservatives when the filter is not idle for any great length of time.
February, 1924
INDUSTRIAL A N D ENGINEERING CHEMISTRY
ACTIVITYO F ENZYME SOLUTIONS The method of expressing the activity of the enzymes invertase and melibiase employed by the writer consists in using 21s an index the hydrolysis velocity constant k in the 1 a where t is unimolecular reaction formula k = t log I
until it gave a reading of 35 seconds per I; I1 100 revolutions on the Stormer a t 39" C.; jl ;I the specific gravity was 1.03 (the viscosity of gelatin solutions reaches a point of equIIibrium very quickly a t temperatures above 38" C., hence it is advisable to work a t temperatures above 38" C.);,glycerol diluted with water tested 35 seconds per 100 revolutions a t 26.53 0.; the specific gravity was 1.19.
