504 GLYCOSIDASES [76]

[76] ~ - D - F r u c t o f u r a n o s i d e F r u c t o h y d r o l a s e f r o m Y e a s t

B y AIDA GOLDSTEIN and J. OLIVER LAMPEN

R-B-Fructofuronoside q- H20 -~ ROH q- fructose

fl-D-Fructofuranoside fructohydrolase (EC 3.2.1.26) of yeast, more commonly known as invertase, was shown to occur as two distinct forms. External invertase, the larger and predominant form of the enzyme, is found pr imari ly outside of the cell membrane and contains up to 50% carbohydra te depending on the species of yeast and purification method. 1 In ternal invertase, the smaller enzyme, is found entirely within the cell. I t constitutes only a small percentage of the total invertase act ivi ty of the cell and contains little or no carbohydrate. 2-4 Although two separate purification schemes are presented, a way is suggested for purifying both enzymes from one batch of cells.

I. External Invertase

Assay Method

Principle. The most common colorimetric methods use glucose oxidase in combination with a chromogen to estimate the amount of glucose re- leased from the enzymic hydrolysis of sucrose. In the two-step method 4 described below, the first step is the enzymic hydrolysis of sucrose to glucose and fructose followed by heat inactivation of the enzyme. The second step is the determination of the liberated glucose using glucose oxidase, peroxidase, and o-dianisidine. The reaction is stopped with acid, and the developed color is read at 540 nm.

Reagents

Sodium acetate buffer, 0.2 M, pH 4.9 Sucrose 0.5 M Potassium phosphate buffer, 0.5 M, p H 7.0 Solution A consists of 10 ml of glucose oxidase (84 mg),~ 10 mg of

1N. P. Neumann and J. O. Lampen, Biochemistry 6, 468 (1967). 2 j. Friis and P. Ottolenghi, C. R. Trav. Lab. Carlsberg 31, 259 (1959). 3 S. Gascon and P. Ottolenghi, C. R. Tray. Lab. Carlsberg 36, 85 (1967).

S. Gascon and J. O. Lampen, J. Biol. Chem. 243, 1567 (1968). 5 Miles Laboratories, Inc., Kankakee, Illinois. When glucose oxidase was obtained

from Nutritional Biochemical Corp., Cleveland, Ohio, Solution A contained 100 mg of glucose oxidase, 10 mg of peroxidase, and 100 ml of 0.1 M phosphate buffer, pH 7. Specific activity of Miles glucose oxidase is 160 ~tmole units/mg, and NBC enzyme is 134 ~mole units/mg.

[76] ~-D-FRUCTOFURANOSIDE FRUCTOHYDROLASE FROM YEAST 505

peroxidase, and 90 ml of potassium phosphate buffer, 0.1 M, pH 7.0

Solution B consists of 300 mg o-dianisidine in 50 ml of distilled water. Store refrigerated in dark bottle.

Solution C consists of 1.0 ml of solution A, 0.5 ml of solution B, and 8.5 ml of 45% glycerol (v/v).~ This solution can be stored frozen up to a month.

Hydrochloric acid, 6 N Glucose, 2 mM used as standard Enzyme solution, which is diluted to give 0.2-0.5 unit invertase per

ml (0.05-0.1 t~g/ml)

Procedure. The reaction is carried out in two steps as follows: (a) Combine in a 12 X 100 mm test tube 50 ~l of acetate buffer and 5-20 ~l of enzyme solution. Place tube in a 30 ° bath and start the reaction by the addition of 25/~l of sucrose solution. After incubating for 5-10 min at 30 °, stop the reaction by adding 0.1 ml of phosphate buffer, pH 7, and immedi- ately heat for 3 min in a boiling water bath. The addition of the phos- phate buffer before heating slows down the reaction and also renders the enzyme more sensitive to the heat treatment.

(b) After the tube cools down to 30 °, add 1.0 ml of solution C to the total reaction mixture and incubate 20 min at 30 °. Add 1.5 ml 6 M HC1 to stop the reaction and read the developed red color at 540 nm. This step measures the glucose released during the enzymic hydrolysis of su- crose in the first step.

With each set of measurements there should be included a sucrose blank, an enzyme blank, and three glucose standards (20-80 ~moles) which were carried through both steps of the reaction.

Definition of Unit and Specific Activity. One unit of invertase act ivi ty is defined as the amount of enzyme at pH 4.9 which hydrolyzes sucrose to produce 1 t~mole of glucose per minute at 30 °. Specific act ivi ty is expressed as units pei milligram protein. In impure preparations, protein was determined by the method of Lowry et al. 7 using bovine serum albumin as standard. For purified enzyme preparations, the protein content was est imated spectrophotometr ical ly at 280 nm using either lysozyme as a s tandard or a specific extinction coefficient t~,/~l%lcmj~ of 23.0.1

Separation o] External and Internal Invertases ]or Assay Purposes. Crude extracts from yeast cells usually contain both external and internal invertase so that assaying the extract gives total invertase activity. To

M. E. Wasko and E. W. Rice, Clin. Chem. 7, 542 (1961). 70. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem.

193, 265 (1951). See also this series, Vol. 3 [73].

506 GLYCOSIDASES [76]

determine the contribution of each form of the enzyme, it is necessary to separate the activities by ion-exchange chromatography. This is ac- eomplished by placing a 50-t~l sample, diluted to ca. 2 ml with 0.05 M Tris-HC1 buffer, pH 7.3, on a 1 X 10 cm DEAE-Sephadex A-50 column equilibrated with 50 mM Tris .HC1 buffer, pH 7.3. First wash the column with 10 ml of equilibrating buffer and then elute the external enzyme with ca. 25 ml, 0.2 M NaC1, and the internal enzyme with ca. 25 ml, 0.4 M NaC1 in 50 mM Tris. HC1 buffer, pH 7.3, collecting 2-ml fractions. Pool the active tubes of each form of invertase, determine volumes and reassay for activity.

Purif ication Procedure s

Unless otherwise specified, procedures are carried out at 0-5 °, eentrif- ugations are performed on a Sorvall RC2-B centrifuge at maximum oper- ating speeds for the rotors used (14,000 g for large volumes and 40,000 g for small volumes), and Buffer refers to 10 mM potassium phosphate buffer, pH 6.5.

Step 1. Preparation o] Crude Extract. The yeast paste, 500 g of mutant strain FH4C is suspended in 500 ml of Buffer containing 2 mM PMSF, 9 and the cells are broken by passage two times through a pre- chilled laboratory Sub-Micron Disperser TM at a continuous operating pressure of 9000 psi. The homogenate is centrifuged for 60 min, and the pellet is discarded. Both the external and internal invertases are con- tained in the supernatant liquid or crude extract.

Step 2. Ammonium Sulfate Fractionation. To the crude extract, add slowly with constant stirring solid ammonium sulfate to 85% saturation [560 g (NH4)2S04 per liter extract]. The solution is stirred an additional 4 hr and then centrifuged for 40 min. This step separates the external and internal invertases with the external invertase remaining in the 85% (NH4)2SO4 soluble fraction. The 0-85% (NH4)~SO4 fraction can be stored frozen and used later for the purification of internal invertase (see Section II) .

Step 3. Batch Adsorption on DEAE-Sephadex. Dialyze the 85% (NH~)~SO~ soluble fraction overnight in the cold room against running tap water. The dialysis bags are only half filled to allow for a 2-fold

s j. S. Tkacz, Ph.D. Dissertation, 1971. The procedure described is modified to shorten the purification time. PMSF or phenylmethylsulfonylfluoride is added as a protease inhibitor. It is pre- pared immediately before use as a 40 mM solution in 95% ethanol and added to the buffer in a 1:20 ratio to give a final concentration of 2 mM PMSF.

10 Model 15 M~STA, Manton-Gaulin Manufacturing Co., Inc., Everett, Massachusetts.

[76] ~-D-FRUCTOFURANOSIDE FRUCTOHYDROLASE FROM YEAST 507

increase in volume during dialysis. To the dialyzed solution, add slowly with stirring 10 g of DEAE-Sephadex A-50 previously equilibrated with Buffer, and continue stirring for 2 hr. Allow the resin to settle and assay the liquid above the resin for activity. If more than 10% remains in the liquid, add more resin. When most of the enzyme is adsorbed, decant the liquid and wash the resin three times with l-liter volumes each of Buffer containing 50 mM NaC1. Pack the resin into a colunm 4 cm in diameter and wash the resin with 1.5 column volumes of Buffer containing 0.10 M NaC1. The enzyme is then eluted with buffer containing 0.20 M NaC1, collecting 10 ml per tube. All the tubes containing substantial ac- tivity are pooled.

Step ~. DEAE-Sephadex Column. Dialyze the pooled tubes from the batch step overnight against running tap water. Place the dialyzed solu- tion on a 4 X 27 cm (350 ml) DEAE-Sephadex A-50 column previously equilibrated with Buffer. Wash the column successively with 300 ml each of 50 mM NaC1 and 80 mM NaC1 in Buffer. The enzyme is then eluted from the column with 0.16 M NaC1 in Buffer collecting 5 ml per tube. Pool the tubes containing activity.

Step 5. SE-Sephadex Column. The pooled fraction from above is dialyzed 24 hr against 2 changes, 6 liters each of 10 mM sodium citrate buffer, pH 3.7. If a precipitate forms, centrifuge the dialyzed solution before placing on a 4 X 27 cm (350 ml) SE-Sephadex A-50 column which was equilibrated with 10 mM sodium citrate buffer, pH 3.7. Wash the column with one column volume of the equilibrating buffer before eluting

T A B L E I PURIFICATION OF EXTERNAL INVERTASE FROM YEAST STRAIN FH4C

Total Total Specific Re- Volume protein act ivi ty act ivi ty covory

Fract ion (ml) (rag) (units) (uni ts /mg) (%)

Homogenate a 1220 66,500 1,250,000 19 - - Crude extract 1290 28,000 865,000 31 100 85 % Ammonium 1520 3,420 685,000 200 79

sulfate supe rna t an t Ba tch DEAE- 276 323 587,00O 1820 68

Sephadex I )EAE-Sephadex 220 152 b 524,000 3450 60

chromatography SE-Sephadex and 174 96 b 466,000 4800 54

water dialysis

a Homogenate from 500 g of yeast paste (FH4C). b Protein was determined at 280 nm using h:l% of 23. Other protein values were ~280

determined by the method of Lowry using lysozyme as s tandard .

508 GLYCOSIDASES [76]

the enzyme with a 900-ml linear gradient from 0 to 0.3 M NaC1 in 10 m M citrate buffer, pH 3.7. Collect 5-ml fractions and pool the tubes with activity. At this stage the enzyme should be pure.

Step 6. Dialysis and Lyophilization. The pooled tubes are dialyzed 36 hr against 4 changes, 6 liters each, of distilled water and then lyophi- lized. Store the enzyme desiccated below 0% From Table I it can be seen that the purified enzyme has a specific activity of 4800 with about 54% recovery.

Properties

Substrate Specificity. Invertase will hydrolyze any compound with an unsubstituted fl-D-fructofuranosyl residue, such as sucrose, raffinose, and methyl-fl-D-fructofuranoside. Maltose is not a substrate for the enzyme.

At high substrate concentrations (1 M), invertase exhibits transferase activity, transferring the fl-D-fructofuranosyl residue to pr imary alcohols, such as methanol, ethanol, and n-propanol. Isopropanol was the only sec- ondary alcohol showing acceptor activity. 11

Some amines, such as 2-amino-2-hydroxymethyl propane-l,3-diol (Tris) 1~ and aniline, will reversibly inhibit invertase. Iodine will also inhibit the enzyme, and in some instances the inhibition can be reversed with mercaptoethanol. 13

Stability. External invertase can be stored in the lyophilized state below 0 ° in a desiccator for at least a year without appreciable loss of activity. I t also can be stored frozen in solution for a few months as long as the pH is around 4.5-5.0.

pH Optimum. The enzyme has a broad pH optimum, between pH 3.5 and 5.0, which drops off rapidly on the acid side and more slowly on the alkaline side of the optimum range. At pH 7, only a few percent of the maximum activity remains.

pH and Heat Stability. When incubated at 30 °, the external enzyme is stable for at least 2 hr between pH 3 and 7.1~ At 56 ° the enzyme is stable for 15 rain at pH 4.9, but becomes very unstable above pH 6. ~

Kinetic Properties. The Kn, value for sucrose is 25-26 mM, and for raffinose 150 mM. 14 These values may vary sharply depending on the source of invertase and the pH.

21A. Baseer and S. Shall, Biochim. Biophys. Acta 250, 192 (1971). 12 K. Myrb~ck, Ark. Kemi 25, 315 (1965). 13 A. Waheed and S. Shall, Biochim. Biophys. Acta 242, 172 (1971). 14S. Gascon, N. P. Neumann, and J. O. Lampen, J. Biol. Chem. 243, 1573 (1968). i~ Unpublished data.

[761 ,~.D-FRUCTOFURANOSIDE FRUCTOHYDROLASE FROM YEAST 509

II. Internal Invertase

Assay Method

The assay procedure is the same as that described for external inver- tase except that the 0.2 M sodium acetate buffer, pH 4.9, 'contains 0.5 mg bovine serum albumin per milliliter.

Purification Procedure 16

Conditions used are the same as those described for external invertase except that buffer A refers to 50 mM Tris.HC1, pH 7.3 and buffer B to 50 mM sodium acetate, pH 4.9. To use the 0-85% (NH4)2S04 fraction from step 2 under external invertase for purification of the internal inver- tase, it is dissolved in a minimum volume of buffer A, centrifuged to remove insoluble material and dialyzed overnight against buffer A. The purification is continued with step 3 below.

Step 1. Preparation o] Crude Extract. The yeast paste, 2 kg from mutant strain FH4C, is suspended in 2 liters of buffer B containing 2 mM PMSF. The cells are broken and centrifuged as described under step 1 for external invertase. The supernatant solution or crude extract con- tains both the internal and external invertases.

Step 2. pH 4.9 Step. With rapid stirring, the crude extract is brought to pH 4.9 by the addition of 2 M acetic acid. Stir for an additional 2 hr and then allow the solution to stand overnight in the cold. Centrifuge and discard the inactive precipitate.

Step 3. Batch Adsorption on DEAE-Sephadex. The pH of the super- natant solution is adjusted with 1 M Tris to pH 7.3 and the conductivity is adjusted with 2 M NaC1 until it is equivalent to buffer A containing 0.16 M NaC1. Now add with stirring, 25 g DEAE-Sephadex previously equilibrated with buffer A containing 0.16 M NaC1. Stir for an additional 2 hr and allow the resin to settle. This step separates the internal and external invertases, the internal binding to the resin and the external re- maining in solution. 17 In order to cheek the liquid for loss of internal inx, ertase, it is necessary to separate the two enzyme activities by ion exchange chromatography (see under external invertase assay for the method). If more than 25% remains in solution, add more resin. When

1, Modified from that of S. Gascon and J. O. Lampen, J. Biol. Chem. 243, 1567 (1968).

l~The external invertase can be purified from the solution by first dialyzing the solution overnight against running tap water and then following the purification scheme for external invertase, starting at step 3.

510 GLYCOSIDASES [75 ]

most of the internal invertase is bound, decant the liquid and wash the resin 2 times with 1 liter each of 0.16 M NaC1 in buffer A. Pack the resin in a 4-cm diameter column and wash with two column volumes of 0.17 M NaC1 in buffer A. The enzyme is eluted with 0.3 M NaC1 in buffer A collecting 10-ml fractions. Pool all tubes with enzyme activity.

Step 4. DEAE-Sephadex Column, pH 7.3. The pooled solution is di- luted with distilled water until the conductivity is equivalent to 0.16 M NaC1 in buffer A and is then placed on a 2 ;K 33 cm (100 ml) D E A E - Sephadex A-50 column equilibrated with 0.16 M NaC1 in buffer A. The column is washed with one column volume of equilibrating buffer and the enzyme is eluted with 1 liter linear gradient from 0.16-0.50 M NaC1 in buffer A, collecting 5-ml fractions. Pool the fractions with enzyme activity.

Step 5. First DEAE-Sephadex Column, pH 4.9. Dialyze the pooled fractions overnight against 4 liters of buffer B containing 50 m M NaCI and remove the inactive precipitate by centrifugation. Place the super- na tan t solution on a 2 X 18 cm (55 ml) DEAE-Sephadex A-50 column equilibrated with 50 m M NaC1 in buffer B, wash with one column volume of equilibrating buffer and elute with a 600 ml of linear NaC1 gradient from 0.05-0.35 M in buffer B. Collect 4-ml fractions and pool the tubes with activity.

Step 6. Second DEAE-Sephadex Column, pH 4.9. Add distilled water to the pooled tubes until the conductivity is equivalent to buffer B con- taining 0.1 M NaC1. Place the solution on a 2 )< 13 cm column (40 ml)

TABLE I I PURIFICATION OF INTERNAL INVERTASE FROM YEAST STRAIN FH4C

Total Total Specific Re- Volume protein b activity activity covery

Fraction (ml) (mg) (units) (units/rag) (%)

Crude extract a 3630 54,500 181,000 3 100 pH 4.9 step 4075 61,100 178,000 3 100 Batch DEAE-Sephadex 112 677 91,500 135 50 DEAE-Sephadex 113 148 87,100 590 48

column, pH 7.3 1st DEAE-Sephadex 158 36 80,000 2200 44

column, pH 4.9 2nd DEAE-Sephadex 32 12 56,600 4700 32

column, pH 4.9

a From 2 kg yeast paste. b Protein determined by method of O. H. Lowry, N. J. Rosebrough, A. L. Farr,

and R. J. Randall, J. Biol. Chem. 19@, 265 (1951), using bovine serum albumin as a standard.

[76] ~-D-FRUCTOFURANOSIDE FRUCTOHYDROLASE FROM YEAST 511

equilibrated with 50 mM NaC1 in buffer B, wash successively with 70 ml of 0.1 M NaCI, 70 ml of 0.13 M NaC1, and 40 ml of 0.15 M NaC1, all salts in buffer B. Elute the enzyme with 0.18 M NaC1 in buffer B collecting 2-ml fractions, and pool the tubes with activity. Dialyze the pooled fractions overnight against 4 liters 20 mM potassium phosphate buffer, pH 7.0, and store frozen. The overall recovery is 32% and the specific activity of the purified enzyme is 4700. Table II summarizes the purification of internal invertase.

Properties

Substrate Specificity. Same as for the external invertase. [nhibitors and Activators. The internal invertase, in addition to being

inhibited by the same amines as the external invertase, is also reversibly inhibited by some salts at high concentrations. Ammonium sulfate at 0.1 M concentration inhibits the enzyme 100%, while sodium sulfate and sodium chloride partially inhibit the enzyme. 11

Bovine serum albumin when added to the assay mixture stimulates the activity of the purified internal invertase.

Stability. Internal invertase can be stored frozen in solution at pH 7-7.5 for a few months without loss of activity.

pH Optimum. Same as for the external invertase. pH and Heat Stability. The purified internal invertase is stable for

at least 2 hr at 30 ° between pH 6.5 and 9. TM In the presence of bovine serum albumin, the enzyme becomes stable down to pH 4.9.15

At 56 °, the purified enzyme is unstable at all pH's. But in the presence of BSA, mannan, or external invertase, it exhibits similar heat stability properties as external invertase, being stable at pH 4.9, but not above pH 6 when heated 10 rain at 56°. ~

Kinetic Properties. Same as for external invertase.