Crystallisation of α-amylase from Bacillus subtilus

347

BIOCHIMICA ET BIOf'KYSICAACTA

Short Communications sc11010 Crystallisation The crystallisation

of wamylase

of a-amylase

(a-1,4-glucan

Bacillus subtilis has been reported The purification require

several

for preparing

crystalline

BABBAR

crude

obtained

amylase

used

et al.’ by submerged

fermentation

strain

of B. subtilis. The filtered

about

one-sixth

of its volume

for large-scale

by this method

as a starting

by these methods

to contain

B. subtilis a-amylase

The entire procedure up to crystallisation

enzyme

EC 3.2.1.1)from

of colored material

enzyme was reported

ried out within a few hours and is suitable The

4glucanohydrolase,

of the enzyme and the removal

this communication.

Bacillus subtilus

by MEYER et al.l, HAGIHARA~ and FISCHER et a1.3-s.

steps. The recrystallised

as an impurity6. ,4 simple procedure

The crystalline

from

preparation

of the enzyme.

was obtained

of wheat bran-peanut

fermentation

is described in

of the enzyme can be car-

is free from proteolytic

material broth

and precipitated

a peptidase

to

meal medium by a

was evaporated

with ethanol.

enzymes.

according

in vactlo to

The precipitate

was

then filtered and dried. Activated >zoo

charcoal

was prepared

by refluxing

IOO g of charcoal

(Merck

mesh B.S. sieve) with 400 ml of 6 N HCl for 3 h. It was then filtered,

free of chloride of charcoal

with glass-distilled

varied

greatly

in their

water and dried for 4 h at 160’. Different effectiveness

for removal

of colored

2183,

washed brands

impurities

from the amylase. Amylase was assayed according to BERNFELD~ with Merck soluble starch as substrate. A unit of amylase represents the formation of reducing substances equivalent activity

to I pmole

maltose

is expressed

determined

according

per min under

as units

of amylase

the conditions activity

of assay

at 30”. Specitic

per mg of protein.

Protein

was

to LOWRY et al.g.

Crude amylase powder (8 g) was stirred with IZO ml water for 30 min and filtered. The filtrate was made upto x50 ml with water, mixed with 0.75 g NaCl and o.zzg calcium acetate and adjusted to pH 7.0. The solution was then heated at 70” for I h, filtered and made up to 175 ml. The brown liquid was cooled to room temperature

(about

27”), stirred with 8 g of activated charcoal for 20 min and then filtered. Colored impurities were removed at this stage and the filtrate was water clear. The charcoal was washed twice with about 20 ml water and the filtrates combined. All subsequent operations were carried out at o-5”. An equal volume of acetone was added to the enzyme solution and the precipitate containing amylase was collected by centrifugation. A gentle stream of cold air was blown over the precipitate to remove the acetone and the minimum amount of 0.01 M calcium acetate required to dissolve the precipitate (about 0.5 ml) was then added. The pH of the solution was 7.0 and the protein concentration about 15%. On keeping at o” needle-shaped crystals appeared within 24 h. The yield of crystals reached a maximum in 10-15 days. Toluene mav be added to prevent microbial growth. Biochim.

Biophys.

.-1cta, Or; (1962)

347-348

SHORTCOMMUNICATIONS

348

The enzyme may be recrystallised by the following procedure which is mostly similar to that of STEIN ANDFISCHER~; the crystals were centrifuged, washed thrice with small amounts of 0.01 M calcium acetate by centrifugation and redissolved by the cautious addition of the minimum amount of 0.1 iV NH,OH to give a final pH of 9.5-10.0. Any undissolved residue was removed by centrifugation and the pH of the supernatant was adjusted to 6.8 by the addition of 0.1 N acetic acid. Recrystallisation was completed within two weeks and may be hastened by seeding. TABLE 1 PURIFICATION OF B. subtilis Cl-AMYLASE

Crude extract Heat treatment at 70’ Charcoal treatment First crystals

j.1

5.0 4.5 2.5

287 287 .5o9 1452

(100) 98 88 49

STEIN ANDFISCHER~reported that crystalline a-amylase of B. subtilis contained a peptidase as impurity and was protected against proteolytic action by calcium which was also bound to the enzyme. In the presence of EDTA which combined with metal ions, they observed progressive loss of amylase (activity even in presence of DFP). Three different batches of recrystallised enzyme prepared by our procedure were tested for their stability in the presence of EDTA essentially under the conditions described by STEIN ANDFISCHER~ using 0.006 M EDTA, with a molar ratio of EDTA to amylase of 400 : I, in 0.02 M phosphate buffer (pH 7.0) at 25’. There was no appreciable loss of amylase activity even after 5 h, indicating the absence of proteolytic enzymes inactivating amylase in these preparations. The peptidases in the crude amylase were found to be completely destroyed by the heat treatment at 70~ since tests for peptidase activity with casein were negative. National Chemical Laboratory, Poona (India)

I. J. BABBAR V. K. POWAR V. JAGANNATHAN

I K. MEYER, M. FULD AND P. BERNFELD, Experientia, 3 (1947) 411. 2 B. HAGIHARA, Ann. Rept. Sci. Works Fat. Sci. Osaka Univ.. 2 (1954) 35. 8 E. H. FISCHER AND E. A. STEIN, Arch. sci. (Geneva), 7 (1954) 131. 4 J. FELLIG, E.A.STEIN AND E.H.FISCHER, Helv.Chim. Acta, 40 (1947) 529. 5 E. 4. STEIN AND E. H. FISCHER, in C. S. VESTLING, Biochemical Preparations, Vol. 8, John Wiley and Sons, Inc., New York, 1961, p. 34. B E.A.STEIN AND E.H.FIscHER,J.B~o~. Chem., 232 (1958) 867. 7 I.J. BABBAR, R.M. BEHKI AND M.C.SRINIVASAN, Indian Patent No.66096 (1958). BP. BERNFELD. in S. P. COLOWICK AND N. 0. KAPLAN, Methods in Enzvmolorv, __ Vol. I, Academic Press,Inc.,Nm York, 1955, p. 149. @ O.H.LOWRY,N.J. ROSENBROUGH, A.L.FARRAND R.J.RANDAI.L, J.Biol.Chem., I93 (1951) 265. IDM. KUNITZ, J. Gen. Physiol., 30 (1947) 291.

Received April 24th, 1962 Biochim. Biophys. Acta, 65 (1962) 347-348