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Eur. J. Biochem.

164,643-654 (1987)
0FEBS 1987

Purification and characterization of extracellular a-amylase and glucoamylase

from the yeast Candida antarctica CBS 6678
Laboratory of Industrial Microbiology and Biochemistry, University of Leuven, Heverlee
(Received November 6, 1986) - EJB 86 1177

An a-amylase and a glucoamylase were purified to homogeneity from the culture fluid of /3-cyclodextringrown Candida antarctica CBS 6678 by protamine sulfate treatment, ammonium sulfate precipitation, gel filtration
(Sephadex G-75 sf, Ultrogel AcA 54), DEAE-Sephacel chromatography, hydroxyapatite chromatography and
affinity chomatography on acarbose - AH-Sepharose 4B.
Both enzymes were monomeric glycoproteins with fairly different amino acid compositions. Their apparent
relative molecular mass, sedimentation coefficient (s&, ,), isoelectric point, absorption coefficient (280 nm), pH
and temperature optima were estimated as 48500, 4.7 S, 10.1, 1.74 cm2 mg-', 4.2 and 57"C, respectively, for
glucoamylase and as 50000, 4.9 S, 10.3, 1.53 cm2 mg-l, 4.2 and 62"C, respectively, for a-amylase.
Kinetic analyses indicated that both enzymes preferentially hydrolyzed high-molecular-mass substrates, including some raw starches. a-Amylase was active on cyclodextrins, whereas debranching activity was demonstrated
for glucoamylase. Trestatins were potent inhibitors of both a-amylase (Ki< 1 pM) and glucoamylase (Ki
< 0.1 pM), being more effective than Bay e 4609 (Ki < 10 pM). Glucoamylase was selectivity and strongly
inhibited by acarbose (Ki < 0.1 pM). Activity of the latter enzyme was also affected by 1-deoxynojirimycin (Ki
< 1 mM), maltitol and amino alcohols (Ki < 10 mM).
Unlike a-amylase, glucoamylase adsorbed strongly onto raw starch, the adsorption site being non-identical
with the active site.
In recent years the capability of some yeast species to
degrade starch has aroused the interest of several researchers,
as the potential value of these microorganisms for certain
biotechnological applications, such as the production of
single-cell protein or ethanol from a starchy biomass, was
recognized [l]. In addition there are current attempts to introduce foreign starch-degrading activity into the nonamylolytic yeast Saccharomyces cerevisiae by recombinant
DNA technology [l].
Most research dealing with microbial amylolytic enzymes
has been focused on the enzymes from bacteria and
filamentous fungi, several of which have found industrial
applications [2]. However, far fewer data are available on the
amylases from yeasts, although the starch-degrading enzymes
of some promising species, such as Lipomyces kononenkoae,
Saccharomycopsis Jibuligera, Schwanniomyces spp. and Filobasidium capsuligenum have been partially characterized [3,
41. Recently we observed that Candida antarctica CBS 6678
secretes with /3-cyclodextrinas sole carbon source significantly
higher levels of amylolytic activity than any of the currently
recognized, active starch-degrading yeast species [5]. This observation urged us to investigate further the extracellular
Correspondence to H . Verachtert, Laboratorium voor Industrielc
Microbiologie en Biochemie, Katholieke Universiteit Leuven, Kardinaal Mercierlaan 92, B-3030 Heverlee/Lcuvcn, Belgium
Abbreviation. AH-Sepharose 4B, 1-aminohexyl-Sepharose 4B.
Enzymes. Glucose oxidase (EC; peroxidase(EC;
a-amylase (EC 3.2.1 .l); 0-amylase (EC; glucoamylase (EC; a-glucosidase (EC; pullulanase (EC;
cyclodextnnase (EC; isoamylase (EC
Note. Parts of this paper, including Materials and Methods, are
presented in miniprint at the end of the paper.

amylolytic system of this strain, which was originally isolated

from rice and described as Trichosporon oryzae by Ito et al.
[6]. According to recent taxonomic studies strain CBS 6678 is
currently accomodated within the genus Candida [7, 81.
In the present paper the purification and characterization
of an a-amylase and a glucoamylase from C. antarcticu are
reported with special reference to some interesting properties
which have been assigned previously to mould amylases but
rarely to the enzymes from yeast species.
Enzyme purification
C. antarctica CBS 6678 was grown in a medium with
P-cyclodextrin as sole carbon source to induce enhanced secretion of amylolytic enzymes IS]. The culture supernatant was
treated with protamine sulfate, which reduced its viscosity
and thereby facilitated concentration of the fluid by salt
precipitation. Through Sephadex G-75 sf gel filtration two
peaks with amylolytic activity, corresponding to a-amylase
and glucoamylase respectively, were separated (Supplement
Fig. 1). a-Amylase was further purified by Ultrogel AcA 54,
DEAE-Sephacel and hydroxyapatite chromatography.
@-Amylasewas eluted from the hydroxyapatite column between 40mM and 50mM potassium phosphate buffer
(Supplement Fig. 2A). A similar procedure was employed for
glucoamylase, however, with a final purification by
acarbose - AH-Sepharose 4B affinity chromatography
(Supplement Fig.2B). The results of a typical purification are
summarized in Supplement Table 1. Both enzymes appeared
homogeneous when analyzed by cathodic PAGE, SDS-PAGE




43 -

30 20.1 14.4

3 4


- 10.65

- 9.45

- 8.3
- 7.3
- 5.9

Table 1. Amino acid composition of C. antarctica amylases

Cysteine was determined as cysteic acid [17] and tryptophan was
measured spectrophotometrically [16]. Values for serine and threonine
were extrapolated to zero time of hydrolysis, whereas those for
isoleucine and valine were obtained after hydrolysis for 72 h. The
other values are averages from 24-h, 48-h and 72-h hydrolyses
Amino acid

- 5.34
- 4.7

- 4.4

- 3.5

Fig. 1. Determination of M, and p l o j C . antarctica amylases. SDSPAGE (lanes 1, 2) and isoelectric focusing (lanes 3, 4) of purified
a-amylase (lanes 1, 3) and glucoamylase (lanes 2, 4) were carried out
as described in Materials and Methods. The positions of markers
for isoelectric point (PI) and relative molecular mass (multiples of
x M,) are indicated

and isoelectric focusing (Fig. I), analytical ultracentrifugation

and FPLC on Superose 12 in 6 M guanidine hydrochloride.
Apart from the a-amylase and glucoamylase, zymograms
of the concentrated culture fluid from C. antarctica, grown in
P-cyclodextrin or starch media, revealed no additional
amylolytic enzymes. Furthermore, column effluents showed
no other pullulanase or cyclodextrinase activity than the one
associated with glucoamylase and a-amylase, respectively (see
Substrate specificity), whereas no isoamylase was detectable.
Known starch-degrading yeasts also secrete a-amylase and/or
glucoamylase, but no P-amylase or true pullulanase [3, 4, 11,
271. Secretion of an isoamylase and a specific cyclodextrinase
has been reported for L. kononenkoae only [28,29].

Physicochemical characteristics
The sedimentation coefficients, corrected at 20 "C in water
and at zero concentration ( s & , ~ ) , were 4.9 S for a-amylase
and 4.7 S for glucoamylase (Supplement Fig. 3), corresponding well to the ones estimated for several fungal
amylases [30 - 321. However, significantly higher values of
about 7 S have been reported for the DEX-encoded
glucoamylases of Saccharomyces diastaticus by Erratt [33].
Ultraviolet absorption at pH 7 displayed a simple spectrum with a peak at 277 nm for a-amylase and at 280 nm for
glucoamylase, and A2so/A260values of 2.88 and 1.93 respectively (Supplement Fig. 4A). The absorption coefficients at
280 nm were determined as 1.53 cm2 mg-' for a-amylase and
1.74 cm2 mg- ' for glucoamylase by the methods of Scopes
and of Van Iersel et al. [34]. The alkaline spectra (Supplement
Fig. 4B) revealed a considerably higher tyrosine/tryptophan
ratio for a-amylase (2.17) than for glucoamylase (0.44) [16].
By analytical isoelectric focusing PI values of 10.3 and 10.1
were estimated for a-amylase and glucoamylase respectively
(Fig. 1). A minor, diffuse band (PI % 10) was present in the
glucoamylase sample. Such microheterogeneity is often displayed in isoelectric focusing by glycoproteins judged homogeneous by other techniques [20]. The basic nature of both
amylases was confirmed by their lack of binding to DEAESephacel at pH 7.6 (see Purification) and the absence of



a-amylase glucoamylase

a-amylase glucoamylase

mo1/100 mol
Total number
of residues








anodic electrophoretic mobility at pH 8.3. As compared to

most mould and yeast amylases with isoelectric points below
7 [31, 35 - 371, the PI values of the C. antarctica amylases are
unusually high. However, a few mammalian a-amylases [38]
and three glucoamylases from a Rhizopus species [39] have PI
values around 9.
The amino acid compositions of the C. antarctica amylases
are shown in Table 1. As compared to the composition of the
S. diastaticus glucoamylases [33 - 351 and of several mould
amylases [30, 31, 40 -421, the C. antarctica enzymes contain
an unusually high amount of histidine. However, a common
feature is the presence of a relatively large number of
hydroxyamino acid residues (threonine, serine). The latter are
major potential glycosylation sites [31, 401. Indeed, neutral
carbohydrate (as mannose) was present in both the C. antarctica a-amylase (2.4%) and glucoamylase (7.4%0),which was
confirmed by their staining with the periodic acid/Schiff s
base reagent after electrophoresis. The glycoprotein nature
of extracellular yeast amylases has been reported by several
authors [3, 11, 27, 32, 33, 43-45]. A 300-kDa glucoamylase
from S . diastaticus with an extremely high carbohydrate
content (8O0/,) was recently characterized by Modena et al.
A discrepancy was observed between the values for the
apparent relative molecular mass determined by SDS-PAGE
and gel filtration (Table 2). When using agarose or dextranbased media (Sephadex, Ultrogel, Superose) M , estimates
were significantly lower than for SDS-PAGE. However, M ,
values obtained with a polyacrylamide gel (Bio-Gel P), or
an agarose-containing medium (Superose) in 6 M guanidine

Table 2. Estimation of the apparent relative molecular mass of the
extracellular C. antarctica amylases
Experimental conditions are described in Materials and Methods

Apparent M ,


Gel filtration
Sephadex (3-75 sf
Ultrogel AcA 54
Bio-Gel P-100 f
Superose 12
Superose 12"

29 000
49 000
25 000
48 ooo

48 ooo


51 000


In 6 M guanidine hydrochloride.

hydrochloride were consistent with the results from SDSPAGE, and close to the values calculated from carbohydrate
contents and amino acid compositions, i.e. 48500 for
glucoamylase and 50000 for a-amylase. Evidently the substrate similarity of the agarose or dextran-containing gels
leads to interaction with the matrices, resulting in significant
underestimates of M , for the yeast amylolytic enzymes. Such
anomalous behaviour has been reported for some bacterial
[47-491 and fungal [50, 511 amylases as well. The different
affinities of the C. antarctica enzymes for Sephadex and BioGel were exploited for their purification as described above.
Apparently no such interactions occurred in the presence of
6 M guanidine hydrochloride, which enables subunit M ,
estimation [21], or with a polyacrylamide gel. The above data
indicate that a-amylase and glucoamylase are monomeric proteins with M , of 50000 and 48500 respectively, which are
values in the range of most amylases from yeast [3,4,27] and
fungi [2, 31, 40,411.


Fig.2. Effects o f p H on the C. antarctica amylases. Relative stability
(A) and activity (B) of a-amylase ( W , V, 0 )and glucoamylase (0,
V , 0)in 50 mM glycine/HCl ( 0 ,O ) ,McIlvaine buffer (m, 0),and
Clark/Lubbs buffer (V,V). Experimental conditions are described
in Materials and Methods


Effects of temperature and p H

Both a-amylase and glucoamylase from C. antarctica were

optimally active at pH 4.2 (Fig.2B). As compared to a-amylase, the pH/activity curve of glucoamylase was shifted to
slightly lower pH values. Glucoamylase was also more acidstable but, unlike a-amylase, its stability rapidly decreased at
alkaline pH (Fig.2A). Mould and other yeast amylases usually also have pH optima between 4 and 6 [3, 4, 27, 31, 39411.
The effect of temperature on the catalytic activity is shown
in Fig.3A. Between 40C and 45C these Arrhenius plots
displayed a discontinuity of slope and, above 60-65"C,
thermal inactivation. Possible explanations for such biphasic
Arrhenius curves have been discussed by Dixon and Webb
[52]. From the linear parts the apparent activation energies
for hydrolysis of soluble starch were calculated to be 59 kJ
mol-' and 35 kJ mo1-l for glucoamylase and a-amylase,
respectively, between 25C and 40"C, and 33 kJ mol-' and
5.8 kJ mol- ',respectively, in the 45 - 60C range. Clearly the
values for a-amylase were distinctly lower than for
glucoamylase, as reported also for the Aspergillus niger
enzymes [53, 541.
The apparent activation energies for thermal inactivation
in the absence of substrate were estimated to be 281 kJ mol-'
for glucoamylase and 212 kJ mol-' for a-amylase (Fig.3B).

75 o\"










Temperature : "C
Fig. 3. Effects of temperature on the C. antarctica amylases. Arrhenius
plots for (A) the hydrolysis of soluble starch by ct-amylase ( 0 )or
glucoamylase (0)and (B) the thermal inactivation of a-amylase ( O ) ,
a-amylase with soluble starch (W), glucoamylase (0)or glucoamylase
with soluble starch (0).
(C) Overall effect of temperature on the
activity of a-amylase ( 0 )or glucoamylase (0)under standard assay
conditions. Experimental conditions are given in Materials and Methods. )t = initial reaction rate (pmol min-I); vi = thermal inactivation
rate (h-')

a-glucosidase [60,68]. Glucose was the sole hydrolysis product
of the glucoamylase from starch or pullulan as indicated by
TLC analysis (results not shown). Further evidence for the exo
type of hydrolysis was obtained with endo-specific, artificial
substrates, namely Amylase B-AR [12], Alphachrome [69] and
Phadebas [70], which were not susceptible to the enzyme.
The above substrates were readily hydrolyzed by the
Candida a-amylase. The hydrolysis products from starch were
maltose (major product), small maltooligosaccharides and a
minor amount of glucose (results not shown). Like a few
other a-amylases [3,11,71- 731, the C. antarctica enzyme was
capable of hydrolyzing cyclic, non-reducing dextrins
(Table 3), which further confirms its endo mode of action.
TLC analysis revealed that P-cyclodextrin wdS first linearized
to maltoheptaose, which was further hydrolyzed to maltose
and glucose (results not shown). The effect of the degree of
polymerization of the cyclic dextrins on the a-amylase activity,
also evident from the kinetic parameters (Table 3), was much
more pronounced than with the linear maltooligosaccharides.
In fact, only y-cyclodextrin was hydrolyzed at a comparable
rate. This typical kinetic pattern, also described for the
Aspergillus [71, 721 and the pancreas and saliva [73]
Substrate specificity
a-amylases, is quite different from the one observed for true
The purified a-amylase and glucoamylase from C. antarc- cyclodextrinases [74]. As far as yeasts are concerned, such a
ticu were characterized by their substrate specificity (Table 3). specific enzyme has only been reported for L. kononenkoae
In addition, kinetic constants (K,,,, k,) were determined with ~ 9 1 .
The observation that the C. antarctica a-amylase and
several soluble substrates (Table 3).
In general, high-molecular-mass substrates containing glucoamylase, respectively, hydrolyzed P-cyclodextrin and
a-1,4 linkages were the better substrates for both enzymes. pullulan slowly and selectively was exploited routinely to
The enhanced activity with increasing degree of polymeriza- estimate their activities in column effluents during purification was also evident within the homologous series of maltose tion, employing the simple 3,s-dinitrosalicylate method. Thus,
up to short-chain amylose, with decreasing K,,, and increasing extensive dilutions and the use of the more laborious
k , values (Table 3). Similar kinetic results have been obtained Alphachrome and peroxidase/glucose oxidase/2,2'-azinobismethods was avoided.
with Aspergillus and Rhizopus glucoamylases [42, 61 - 631. (3-ethylbenzthiazoline-6-sulphonate)
Raw starches, especially those from wheat and potato,
The relative activity of purified S. diastaticus glucoamylases
also increased with increasing degree of polymerization of were hydrolyzed by the C. antarctica a-amylase and glucomaltooligosaccharides but, as compared to the Candida amylase, the latter enzyme displaying the highest activity. The
enzyme, better activity on small substrates was shown [35, ability of some amylases, especially from filamentous fungi,
461. As compared to cr-amylase, K , values with polysac- to degrade raw starch has been established for some time [75,
charides were lowest for glucoamylase, resembling the proper- 761. However, apart from the glucoamylases of Saccharomyties of the Succharomycopsis,fibuligera enzymes [64] but quite copsis fibdigera [44] and C . antarctica (this paper), this phedifferent from the L. kononenkoae [36] and the nomenon has not yet been reported for yeast amylases. The
potential of yeast species for digesting raw starch merits furSchwanniomyces [45, 56, 571 amylases.
Only the glucoamylase of C . antarctica slowly hydrolyzed ther investigation as it has been suggested that this property
a-1,6 linkages in panose, dextran and pullulan (Table 3). Fur- could be advantageously used for the alcoholic fermentation
thermore, glycogen with its relatively high number of a-1,6 of starch without precooking [76].
branch points was a good substrate for glucoamylase, but not
for a-amylase. As specific extracellular debranching enzymes, Inhihition
such as pullulanase or isoamylase, are usually not produced
The effect of several potential amylase inhibitors on the
by moulds or yeasts [2], the glucoamylase debranching activity
is essential to enable extensive starch hydrolysis by these C. antarctica enzymes was characterized as shown in Table 3.
Glucoamylase was competitively inhibited by cyclic
microorganisms. Indeed, the most active amylolytic yeasts
display such activity [3, 11, 27, 44, 45, 56, 651. However, dextrins and a-D-glucosides (Ki between 15mM and 60 mM)
the debranching activity of S. diustaticus glucoamylases is and, somewhat more strongly, by glucose, amino alcohols
and maltitol (Ki 5 extremely low, if detectable [33, 35, 461. The C. antarctica (Tris, 2-amino-2-ethyl-l,3-propanediol
glucoamylase also released, albeit very slowly, glucose from 13 mM). However, a-amylase was only very weakly affected
cross-linked dextran (Sephadex), without affecting its chro- by maltitol and not at all by the amino alcohols. The inhibition
matographic properties, however. This affinity may account of a-amylase by a and P-cyclodextrin was similar to their
for the observed, pronounced retardation of the enzyme effects on glucoamylase. Maltose was a non-competitive induring Sephadex G-75 sf gel filtration. A low exodextranase hibitor (Ki = 8.8 mM) of a-amylase. The inhibitory effect of
activity has equally been assigned to Rhizopus and Aspergillus maltose and glucose on a-amylase and glucoamylase, respecglucoamylases [66, 671 but it has not yet been reported for tively, has previously been observed for the L. kononenkoae
another yeast glucoamylase. The negligible activity of the enzymes [36], but further kinetic data on yeast amylase inhibiCandida glucoamylase on a-glucosides further indicates it to tion are scarcely available, if at all. On the other hand, the
be a true glucoamylase rather than a starch-degrading mould enzymes have been intensively studied. Inhibition of

Intrinsically glucoamylase is more thermally stable than

a-amylase, which is also the case for the Schwanniomyces
enzymes [45, 55, 561. The much lower energies previously
reported for the amylases from L. kononenkoae ( < 100 kJ
mol-') [36] and Schwanniomyces alluvius (< 170 kJ mol-l)
1571 indicate the C . antarctica enzymes to be less susceptible
to heat inactivation. As demonstrated by the parallel shifts
in the Arrhenius plots (Fig. 3 B), the enzymes, especially aamylase, were stabilized by added substrate. This phenomenon has also been described for glucoamylases from
Aspergillus spp. [58, 591.
Under standard assay conditions the temperature optima
were 62C for a-amylase and 57C for glucoamylase, with
glucoamylase activity being much more temperature-dependent (Fig. 3 C). The optimum for glucoamylase is within the
usual range for yeast glucoamylases (SO - 60C), but the value
for a-amylase is considerably higher than for most other yeasts
(not higher than 50C) [3,4,27]. However, an a-amylase with
optimum at 70C is secreted by L. starkeyi [60].

Table 3. Substrate specificity and kinetic constants of the C. antarctica amylases
Relative activities 1/13 were determined under standard assay conditions (3,5-dinitrosalicylate method) with oligosaccharides, a-glucosides
(I0 mM) and polysdccharides (I"/.). No activity of n-amylase and glucoamylase was found with melibiose, trehalose, saccharose, methyl
a-D-glucoside or phenyl a-wglucoside (n. d. = not detectable). Values between { } correspond to activities on boiled starches (previously
heated for 45 min at 100C in substrate buffer). Michaelis constants (K,) and reaction rates at infinite substrate concentrations (V) were
obtained from Lineweaver-Burk plots. The molar activity (k,) was calculated from V and the respective molar enzyme concentrations.
Inhibition constants (Ki) and the type of inhibition (C = competitive, N = non-competitive; indicated in parentheses) were determined from
Dixon plots. No inhibitory effect on a-amylase was observed for Tris, 2-amino-2-ethyl-l,3-propanediol
and 1-deoxynojirimycin. An average
M , of 3750 was assumed for the homologous inhibitor mixture Bay e 4609 [84]. Activities were determined with the 3,5-dinitrosalicylate
method (40C; pH 4.2). Concentrations causing 50% inhibition of glucoamylase adsorption onto raw starch (Zs0) were determined at 4C in
0.1 M sodium citrate buffer pH 5 according to Takahashi et al. [26]
Substrate or inhibitor




p-Nitrophenyl a-D-glucoside
Short-chain aniylose
Maldex 15
Soluble starch (Difco)
Soluble starch (UCB)
Soluble starch (Merck)
Soluble starch (Zulkowsky type)
Amylose (potato)
Amylopectin (potato)
Glycogen (rat liver)
Glycogen (oyster)
Wheat starch
Corn starch
Waxy corn starch
Rice starch
Potato starch
Trestatin A
Trestatin C
Bay e 4609
2-Amino-2-ethyl-I ,3-propanediol
Methyl a-D-glucoside
Phenyl a-D-glucoside













n. d.
n. d.
n. d.
43.9 { 100:
0.22 (86.3)
0.22 i73.2)
1.8 {83.8)
44.6 (98.7)

92.7 {IOO)
6.0 (96.3)
1.2 C97.3)
23.4 (99.8)
43.8 {99.5}

47.5 (C)
26.3 (C)
15.1 (C)

I5 0


0.087h (C)
0.066' (C)
0.037 (C)
8 S b (C)
5.3 (C)
9.4 (C)
8.5 (C)
56.2 (C)
34.8 (C)

the glucoamylases from Aspergillus and Rhizopus species by inhibited by these cyclic oligosaccharides (a-amylase, P-amyglucose and a-glucosides has been demonstrated [61, 771, lase, pullulanase) [79 - 821, the relatively high Ki values
whereas maltose is known to inhibit the Aspergillus awamori (> 10 mM) for both the C. untarctica glucoamylase and aa-amylase [37]. Recently Iwama et al. [78] noticed that several amylase indicate these ligands not to be suitable for effective
amino alcohols, including Tris and 2-amino-2-ethyl-l,3-pro- affinity chromatography of these yeast amylases. During the
panediol and maltitol affect the Aspergillus and Rhizopus last decade several amylase inhibitors of microbial origin have
glucoamylases, reporting Kivalues close to the one we found been isolated, including acarbose (Bay g 5421) and Bay e
for the C. antarctica glucoamylase. Although immobilized a 4609 [83, 841, 1-deoxynojirimycin [85], amylostatins [86] and
and P-cyclodextrin have been adopted to purify, from various trestatins [87], which are all characterized by a monosources, different types of amylolytic enzymes, which were saccharide or oligosaccharide composition. A limited amount

of literature data, almost exclusively dealing with enzymes
other than yeast amylases, are available on the characterization of these substances. Therefore, several of these
compounds were included in our study (Table3). 1Deoxynojirimycin selectively inhibited glucoamylase (Ki =
0.45 mM). Bay e 4609 displayed considerably lower Ki values
(< 10 pM) for both amylases, with a-amylase being slightly
more affected. An even stronger effect on a-amylase (Ki <
1 pM) and, especially, on glucoamylase (Ki < 0.1 pM) was
obtained with trestatins, trestatin C being most effective.
Acarbose also strongly inhibited glucoamylase (Ki < 0.1 pM)
but it had only a moderate effect on a-amylase activity (Kl =
1.5 mM).
Apparently, 1 -deoxynojirimycin, which is essentially a
strong a-glucosidase inhibitor [85, 881, also inhibits the
Candida glucoamylase, in a manner similar to its effect on a
Rhizopus glucoamylase [89]. The very strong inhibition of
the C. antarctica amylases by trestatins is consistent with a
preliminary report of their effects on a-amylase (pancreas,
Bacillus subtilis, Aspergillus oryzae) and the A. niger
glucoamylase [87]. Although a-amylase was slightly more
affected than glucoamylase, Bay e 4609 [84, 881 proved not to
be a selective a-amylase inhibitor in the case of C. antarctica.
On the other hand, its low-molecular-mass homologue,
acarbose, was a potent selective inhibitor of the Candida
glucoamylase. A pronounced inhibitory effect of acarbose has
also been reported for a few other glucoamylases [3, 27, 88,
901. According to Clarke and Svensson [91], two specific
tryptophanyl residues were involved in the strong binding of
acarbose to the glucoamylase of A. niger resulting in a typical
ultraviolet difference spectrum between 260 nm and 320 nm.
We also recorded such a characteristic difference spectrum for
the C. antarctica glucoamylase in the presence of this inhibitor,
which suggests a similar interaction with the yeast enzyme
(Supplement Fig. 5).
We used the high and specific affinity of acarbose for
the Candida glucoamylase during purification of the enzyme,
employing acarbose coupled to AH-Sepharose 4B. a-Amylase, characterized by a 17000-fold higher Ki for acarbose
(Table 3), was not bound. A high concentration of the competitive inhibitor Tris (1.7 M) released the enzyme from the
matrix. Recently acarbose affinity chromatography was also
successfully applied for the purification of the A. niger
glucoamylase [15]. As compared to other described affinity
matrices for amylases, based on immobilized starch,
amylopectin of glycogen [92 -941, the use of acarbose allows
the efficient separation of a mixture of amylolytic enzymes
while ligand degradation is avoided. Based on the inhibitory
action of P-thiomaltosides, affinity chromatography
employingp-aminophenyl 1-thio-P-D-maltopyranosidelinked
to Sepharose has been proposed for the purification of
pancreatic a-amylase [95], but its specificity with regard to
other types of amylases has not yet been investigated.
Raw-starch adsorption of glucoamylase
Unlike a-amylase, glucoamylase from C. antarctica was
strongly adsorbed onto raw starches. When applying the same
amount of enzyme (4 units/mg starch) more than 90% was
retained on corn, waxy corn and wheat starches, whereas
only 70% and 60% was bound to potato and rice starches
respectively (results not shown). When the enzyme/starch
ratio was increased, wheat starch proved to have a higher
adsorption capacity than corn starch (Fig. 4A). In addition,
glucoamylase adhesion to corn starch was somewhat more




Fig. 4. Adsorption of the C . antarctica glucoamylase onto raw starches.

(A) Adsorption capacity at pH 5 of corn starch (0)and wheat starch
( 0 )at different enzyme/starch ratios. (B) Effect of pH on the adsorption of glucoamylase (4 units added/mg starch) onto corn (0)or
wheat ( 0 )starch. Experimental conditions are described in Materials
and Methods

pH-dependent, although a similar pH optimum between pH

5 and 5.5 was obtained (Fig.4B). For other glucoamylases,
adsorption also preferentially occurred between pH 2 and 6
without a very pronounced pH optimum [24, 25, 441.
Significant desorption could not be achieved unless fairly
concentrated, alkaline washing buffers were employed, such
as 1.2 M sodium borate pH 9.5 or 3 .S M Tris/HCl pH 9,
which indicates a strong adhesion of the Candida glucoamylase to the raw starch [44]. However, recoveries did not
exceed 50%. One should bear in mind the fact that the stability
of this glucoamylase is strongly reduced at alkaline pH.
Several lines of evidence indicate that the active sites of
glucoamylases are not identical with their raw-starch adsorption sites [26, 76, 96-98], which is consistent with our results
obtained with the Candida glucoamylase. Indeed, Table 3
demonstrates that the affinity of several inhibitors for the
enzyme (Ki) is not correlated with their capability to interfere
with raw-starch adsorption (Zs,,). However, as can be seen
also for a homologous series of maltooligosaccharides, the
increasing size of the linear pseudooligosaccharide inhibitors
(1-deoxynojirimycin < maltitol < acarbose < trestatin A
< trestatin C < Bay e 4609) appears to enhance interference
with starch adsorption.
In general the phenomenon of raw-starch adsorption of
an enzyme appears to be associated with its capability to digest
it, which is the case for a number of mould glucoamylases [75,
761. However, as far as yeasts are concerned, these properties
have been assigned only to the glucoamylases of Saccharomycopsisfibuligera [44] and of C. antarctica (this paper).
Clearly the extracellular amylolytic system of C. antarctica, consisting of an a-amylase and a glucoamylase with
debranching activity, contains the necessary enzymes for extensive starch degradation. The yeast enzymes have a number
of properties in common with the mould enzymes, such as
glycosylation, substrate spectrum and inhibition pattern. In

addition, the a-amylase has a high optimum temperature and
displays some raw-starch digestion. The latter is more pronounced for glucoamylase, which also strongly adsorbs onto
raw starch. It would be of interest to investigate whether these
favourable properties are more widely distributed amongst
amylolytic yeasts. Acarbose affinity chromatography, as
applied for the C. antarctica glucoamylase, should be a
powerful technique for purification of other yeast and mould
glucoamylases because of the potent and selective action of
the inhibitor. Such selectivity is not exhibited by other, highly
effective microbial pseudooligosaccharide inhibitors of the
Candida amylases.
C. antarctica is not suitable for direct alcoholic fermentation ofstarch, since this species lacks glucose fermentation [7,
81. However, several interesting properties of its amylases,
discussed above, render this yeast a potentially valuable
source of amylase genes for introduction into proven industrial Saccharomyces strains [I].
The authors wish to thank W. Broekaert, B. Cammue (Laboratory of Plant Biochemistry), J. Knaepen (Laboratory of Industrial
Microbiology and Biochemistry) and G. PrCaux, R. Witters (Laboratory of Biochemistry) of the University of Leuven for their assistance
with several analyses.

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P u i ' i f i c s t i o n a n d C h d r a C t P r i z d t i o n of




( c y c l o m d l t u u c i a o s e ) , q u a n i d i n e hydrochluridr,

r h a i n a m y l o s r (DP = 7 5 ) , m d l t i t o l ,



and melibioae.

a n d q l u c o a m y l a s e F r o m t h e y e a s t C a n d i d a a n t a r c t i c a CBS 6678
Alphachrome *as

R . Oe M o t a n d H.




S O I U I ~ I ?S L d P C h ,


naldrx I S (a-dmylase d e x t r i n ,

F r o m Amylurn,

DE = 1 5 ) a n d w a x y c o r n



l o w i n q r n m p o u n d s were q e n e r u u s l y s u p p l i e d


i l u l l k r and F.




H n l i r r . P I n o S c was

D i o - C ~ l HTI'


were p u r c h r l s t ~ d r r o m B i u - R a d ,

from Difrn,

f r o m RDH C h e m i c a l s ,
and B i o - G e l


M1, USA.


and P.





c i l i m i c d , nrt.rse,


~ p ~ o d u c tO sf

was obtdiriell





from Plidrmdcid I i n s C h e m i c a l s ,



G-75 sr,

a n d ~ i e ~ t r ~ p I ) o r e Ts h~e s p. l

~ r i d r l t i r r q , I iic.

Thc f o l l o w i n g





w e r ~p u r c h a s e d

nrisiun1 : a - c y c ~ o d r x t r i n

markers n e r f f r o m S r r v a ,

i r o m Sigma Chemical

US,\, t h r f o I l n n i r 8 r i p r o d i i c t s w e r e u s e d

ma1 t o t r i o s e ,

p o t a t o starches,

from Borhringer,

T r e s t d t i n s A d n d C were k i n d l y

Basel, S w i t z r r l d n d . The f o l l o s i n q product:

f r o " , J~~~~~~




YedsL N i t r o q c n B a s e d n i s o l u b l e s t d r c h were


s u p p l i ~ db y H .

soluble starch,

i z , I L u w M o l c r u l a r P ~ l q h tC n l i h r a t i o n K i t s f o r g r i f i i l r d -

L e l l ~ r h o fand

a n d Hay e 4 6 0 9 b y W.R.

a n d m d I l c i l i e p l d ~ ~wc??


The F o l -

b y O a y e r AG,

W ~ p p r r l d l , FRG : a r a r b n s e ( H a y q 5421) by K .


p - n i t r o p h c n y l - o - D - q l u c o s l d r , t r e h a l ~ s e a n d S i l i c a G e l 60


s t d r c l i were o h t d i n e d

and U l t r o -

5'1 ,and A m p h o l i n e c a r r i e r a m p h o l y t c s f r o m LKB, B r o m n a ,




Colnbrook, UK,

from Kocii-Lighr,


. ~ n dw l i c a t


4H. S o l u b l ?

s t i l ~ c h ci ~c r e p u r r h a s r d

T l i e h m y l a s o R-An




t o S t e l L e n e l di.(9!.


f r o m UCB,

~ - g - ~ i r ~ n ~ i -

s t d r c l l , corn


k i t w a s f r o m Wako C h e m i c a l s ,



7 1 2 0 0 ) . PGn-enLymes,

,mid A H - S e p h d r O S e



: p r ~ l d m i n es u l f a t r ,

d m y l o s e arid d r n y l o p r c l i r i ,

O ) S ~ C Ty l y r o g r n ,

( B ~ C P D ~+Ci r-i





from r a t l i v e r according

65 1


s t r a i n and growth conditions


C a n d i d a a n t a r c t i c a CBS 6 6 7 8 w a s o b t a i n e d f r o m

C e n t r a a i b u r P d u voor S c h i m m e i c u i t u r e s ,

Yeast D i v i s i o n ,

S t o c k c u l t u r e s *ere p r o p a g a t e d a t

The N e t h e r l a n d s .





4 "C.

R l r f f ~ r e d Y e a s t N i t r o q e n B a s e m e d i u m pH 6.5

I %


u ~ e df o r



t h e p r o d u c t i o n of

Erlenmeyer f l a s k i ,

in ILL


a d d e d a s zole c a r b o n

a C C t d t l l brifft-r


s o l e c a r b o n s o u r c e , was u s e d f o r



and t h e ~ u p e c n a t a n tA







i s ~ d m y l d ~ ep, u l l u i a n a s r a r i d c y c l u d e x t r i r i d s e a o t i -


n - d m y l a s ~ ( A l p h a c h r o m e and Phadebas

t h ? Arnylrls? R - A R




b u f f e r pH 7.6



o f t h ? sam? b u f f e r

w h i l ? 3.2-ml

mi . h - '

(2.5 m l l h a s a p p l i e d


The c o l u m n

f l o w r a t ? of

a t

The m d j o r

r r a c t i o n z were c a l i e c l e d .

200 m l

t h e s d m ? b u f f e r c o n t a i n i n g 0 . 5 I4 N a C l a t a f l o w

1 8 rn1.h".

T h r d r n y l a ~ ~ - p ~ s i t i v f. r- r r t i o n s

employing carhoxympthyl-

t i v e l y dialyzed aqainst t h i s

1 1 ? \ , w a s u s e d i n some e x p e r i m e n t s

were p o o l e d ,

i n 2 mM s o d i u m p h o s p h a t e b u f f c r pH 6 . 8 ,


r a t e of

s i i l f a t ~(80 % saturation),

p r ~ ~ i p i l d wl i~r hd a m m o n i u m

I n


amylase a c t i v i t y e l u t e d a t t h e v o i d voiurnc.

A d s o r b P d m a t e r i a l w a s t h e n r ~ m n v r db y w a s h i n q w i l l !



amylns? a s s i i h s t i a t r

The p r e c i p i -

same b u f f e r .

dgairist the

X 23 r m l o f DEAE-Srphacel

140 m l

p l ~ kw i t h a l l

and p r o t e i n d e t e r m i n a t i o n

dctcrminrd as described previously

~ i i ef r a c -

t r p 5 .^ _ _D. .l . .A. .r. .-. .S. .e
p h a c ~ l r h r o m l o q r a p h y o f ~ ~ - d m y l a ~ Ttlr
c .

ellitrd wilh


(80 % saturationl.

~ ~ n ~ c n t ar f at et r ~U l t r o q ~ l f i l t r d t t u o

w i t h 1 0 mM T r i s I H C l

v i t i r i


r a t e of

5 days,


ollected for purification of

(ON5 m e t h o d ] ,


a t a flow

w e r e p o o l e d a n d c o n c e n t r a t e d b y ammo-

extensively d i a l y z e d


t o a column

Total amyioiytic activity

% sodium

t a t e w n s r e d i s s o l v e d i n 10 m M T r i s i H C l b u f f e r pH 7 . 6

the e x t r a c e l l u l a r a m y l a s e s .

Enzyme a s s a y s


50 mi4 s u d i u ~

f r a c t i o n s rere c o i i e c t e d .

a n d 3.4-mi

niiim s i i l f d t e p r r c i p i t d t i o n

9 f o r 30 m i " a t

t h r c u l t u r e s were c e n t r i f u g e d a t 14000 X

X 6 5 cm)


r n n t a i n i n q 0.U7

pH 5.8

t i o n s w i t h a-amylase


q r o w n i n t h e same medium w i t h 1 % s o l u b l e s t a r c h ( M e r c k )

8.5 m1.h-l

source, was

e a c h c o n t a i n i n g 170 ml o f

A 2-day


e q i r i l i b r a t e d .at 10 'C

AcA 54,

p r o t ~ i n sw e r e e l i r t e d w i t h t h e l a m e b u f f e r

T h e y e a s t was g r o w n a t

t h i s m ~ d i u r n , on a r e c i p r o c a l s h a k e r .

Sephadrx 6-75 c h r o m a t o -

( 3 m l J was a p p l i e d t o a c o l u m n


on s l a n t s c o n t a i n i n g s o l u b l e S t a r c h (10) and m a i n t a i n e d a t


4 . U I t r o q e i A c 4 54 gel
i l.
t r.
o n. .
o f. a_
- a m.
l a s e_
he _____~~
. .f .

c o n r r n t r d t e d a-amylase sample After



and exhails-


the rStirnaLi0" o f a-amy1ase.


I T E e - 6 : . C L d f " l Y " C " T 1 T ' E - c ~ E ~ ~ ~ ~ ~ ~ ~ ~ e ~ y - ~ ~ - ~ T " Y ! " T' Eh .e

P r o t e i n c n n r r n t r . ~ t i o nw a s e s t i m a t e d b y t h e m e t h o d o f

( 1 3 1 u s i n q b o v i n e serum a l b u m i n a s

Lowry rt a l .

F a r Lhe p u r i f i e d e n z y m e s ,

a b s o r p t i o n measurements

( 3 . 4 m l ) ua5 l o a d e d o n t o a ralumn (2.5


280 nm

d l



a f t e r i o n exchange chromdtogrd-

CoilCentrdte w i t h a - a s y l a s r

~ t a n d a ~ d .

i n 2 mM

HTP e q u i l i b r a t e d

X 25 cml o f

Sodium phosphate

b u f f e r pH


6.8 a t 10

T h e c o l u m n was w a a h r d w i t h 1 2 0 m i o f

O C .

buffcr a t a flaw rat? of

2.8 rn1.h-l

was e l u t e d by a

wcrr c ~ l l r c l e d . .-Amylase

Purification o f e*trace11uiar




p o t d s 5 1 ~ mp h o s p h a t e



Protarnine sul-

(2.5 % i n 50 mM s o d i u m a c e t a t e b u f f e r



a n d s t o r r d a t -20 'C


pH 6 . 8

t h e same


(0-70 m M ) .

c o n c e n t r a t e d b y ~ a l pt r e -

Th? a c t i v r f r a c t i o n s w r r e p o o l e d ,

n h ~ l e3 . 9 - m i

i n 25 mM s o d i u m


p H 4.21


D H 5.8.

- a s d d d f d u n d c r s t i r r i n g t o t h e c u l t u r e ~ u p e ~ n a t d n( itU 8 0
rnl) a t 0


u n t i l na more f i b r o u s p r e c i p i t a t e





mill 0

S . T ' e . ! r - ? " ? ~ ~ f ~ ' - ~ ~ ! f ? T ~ - e ~ e c l p l ' d i f ~ ? .C r o u n d

monium s u l f a t e
n a t a n t of


a t 14000 X g

ThC p r e c i p i t a t ? w a s r e m o v e d b y c e n t r i f u g a t i o n

was s l o w i y d d d r d cinder s t i r r i n g

the previous step a t

Stirred for

s o l u l i r i r i was

"C t o 75 % s a t u r a t i o n .

30 m i n .

aft?^ a f u r t h e r

w i t h o u t s t i r r i n g t h e m i x t u r e was c e n t r i f u g e d
1 7 0 0 0 X 9 dnrl 0 'C

s o l i d am-

t o t h e super-


t o c o l l e c t the precipitate,

and d i a i y s e d c k h a u s t i v e l y a q a i n s t


?xrhdnrlP Chromatoqrdphy was c a r r i e d

f o r 45 m i n a t


nhieh was

then r e d i s s o l v e d i n ~ u l d5 0 m M s a d r u m r i t r a t ? b u f f e r

4 "C

r p ..
. .D
- S.
acri ch'0matoyr?e?i-of-q!u~oa~i!asp.

3 0 .in



same b u f f e r a t


S e p h a d e x 6-75 s f

w i t h 50 m M s o d i u m c i t r d t e
At a flow

% soilium a z i d e .

b y U\J


r a t s of



p r o t e i n was r

T h e s e f r a c t i o n s \*err s e p d r d t e l y



a n d r o n c ~ n t r i l t ~ bdy a m m o n i u m s u l f a t e p r e c i p i t a t i o n


by c e n t r i f u q a t i o n

(25000 X g;

t h e r p u r i f i e d by steps 4-6


p o o i ~ d

(80 %
30 m i " ;

t h e p r ~ c i p i t d t ea n d d i a l y s i s a g a i n s t

50 m M s o d i u m r i t r a t r b u f f e r

pH 5 . 8 .


c o v a l r r i t l y roiipled to AH-Sejiharose

114). A column

O i l r r i i l rL a l .

Tau ~ e p d r d t ? p e a k s w i t h a m y l a s e

t o ol-amyldse

sat~rationl. follonrd





1 4 ~ e _ ' - _ A L ' ~ ? f ~ ~ _ ~ ? 1 ~ n ? ~ T o ~ ~ a i ~ ~ ~ . ~ f . 1 !A"c oa r"b~o !s ~e ? ~ .




extensjrrly dialyzed against

same h u f f p r .

a n d eluted a t

b u r r e r p H 5.8

r ~ i i e ~ t ~ ~d i ., , t i ~ onf

a h i o r h a n c r (280 nm).


The a c t i v p f r a c

b y s d i t p r r c i p i t d t ~ a n( H O %

m r y m r s o l u t i o n ( 1 1 m i l was t h e n a p p l i e d l o

a c c i i u m n (2.6 X 110 r m l o f

10 " C



i n 0 . 1 M s o d i u m acetate b u f f e r pH 4.2

M NaCl, and

c o n t d i n i n q 0.5

ihr c o r t c e n t r d l r d


~ a t o r a t i o n l ,dissolved

t n o h t a i n a c o n c e n t r a t e d enzyme s o l u t i o n .

*1~bsn e t a d s o r b e d .


were p o o l e d ,



<out a 5 i n s t e p

U l t r o g e l c o n c ~ n t r a t ew i t h q l u r o a m y l a s e .


m l

~ n i y r n ew~e r e f u r -

( ~ ~ - d m y l 3 5 a~n1d 5 t e p S 7 - 9 ( g i u -


p H 4.7

r o n t a i n i n q 0.5

t i o n c ~ f t h e giucaamyidse
rndloqraphy s t e p
w i t h 50 rnl o f





w i t h 0.1

M NaCl.


M sodium


t h e i o n exchange c h r o -

i n a r t i v e m a t e r i a l was washed n u t

t h e above b u f f e r ,

Llutiuri or

bvuriii eii/ymr.

i.7 M T r i s l l l C l l l u f f e r

48 a s d e s c r i b e d b y

X 4 cml containlnq this

m a t r i x 1 0 s e q ~ ~ i l i b r d t eadt 2 0 'C

areLdle buffer




w i t h 50 rnl o f

The e l u a t e w a z

immediaand then


a t -20




d i d l y r r d a g a i n s t 5 0 m M s o d i u m a c e t a t e pH 5 . 8



whil? collrctinq I-mi

w . 3 ~

by bdI1 prc, ipit.itiun.

The e n z y n c w a s s t o r e d

i n 25 mM s o d i t i n p h u ~ . p h . ~l bt u~ f ~f r r p H 5 . 8 .


Amino a r i d c o m p o s i t i o n and c a r b o h y d r a t e

60 c m ) ,

T h e d m i i i o a c i d c o m p o ~ i t i o no ~f t h e p u r i f i e d e n z y m e s w e r e
HC1 a t

drtrrmined after hydrolysis with ronstant-boiling


4 8 a n d 72 h o u r s w i t h

and u n d r r vacuum f o r 2 4 ,


C a r l o f r b a A u t o m a t i c Arninoacid Andlyier.
c y s t e i n e were d e t e r m i n e d


Se d i m e n t d t i o n

on o f

511940 ~ p m . C o r r r c t i o n s were made f o r




T h e ~ n i t i a i r r a c t i u n r d t r s O f h y d r o l y s i s were d e t e r m i n e d

a t ~ c v e r d l t m p ~ r a t u r e v s l u ~ s ( 2 5 'C-75



SolvCrlt d r n s i t y and

a n d 0.711


as substrate.

X in i4cilvaine


Thc a p p a r e n t a c t i v a t i o n

e n r r q i r s f o r ~ t d r c h h y d r o l y s i s werc o b t a i n e d

from Arrhenius




g l u c o a m y l a s ~ . T h r s ~ d i m r n t a t i o nC o p f f i r i e n t s a t z e r o < ' o n -



i n c u b a t e d i n M c I i v a i n ? h i i f f r r pH 4.2

a t s r ~ r r d lt e m p r r a t u P ~ v a l u e s ( 4 5 * C - 7 0 ' r l

c e n t r a t i o n were d ~ t c r m i n e db y l i n e a r c x t r d p o l d t i o n .

thrn rapidly

e l e c t r o p h o r e s i s and i s o e l e c t r i c

OC) b y t h e O N 5

~ m p l o y i n ql u l k o w s k y s n l < i h l s s t d r c h

b u f i e r pH 4 . 2 )

rs c a i w i d t e d from

f o r a-amylase

t e m p e r a t u r ? a n d pH

E f f e c t s of



t h c a t i n a a c i d ~ a r n p o s i t i a n s and r d r b o h y d r a t c C o n t e n t S

Polyarrylamide qel


the p u r i -

S p i n r o m o d ~ i F a n a l y t i c a l u l t r a c e n t r i f u g e a t 2 0 'C

d S 0.72

( H H 1 0 1 3 0 ) was e q u i l i b r a t e d

w i t h t h i s s o i v r n t a t 2 0 rn1.h.'.

pH 7 a n d c l u t e d

2 5 m M s o d i u m r i t r a t e b u f f e r pH 5 . 8 w a s c d r l . i e d


gudnidine hydro-

h y d r o c h l o r i d e w a s p u r i f i e d d c c o r d i n q t o Fohlrnan e t a1.1??1.

Spdim~ntationvelocity ultracentriruqot


presence o f

6 M i q u d n i d i n r h y d r o c h l o r i d e b u f f e r e d w i t h 10 m M T r i s i H C l

rl n d 1y Ii 5

f i e d enzymes a t d i f f e r e n t c o n c e n t r a t i o n s (1-9 g . 1 - l )


A Siiperose 1 2 FPLC c o l u m n


a n a l y i e d b y TLC a s d e s c r i b e d p r e v i o u s l y

i n the

r e d i i r r d a n d d i k y l a t e d a s d e s c r i b e d b y A n s d r i a n d Mag? I Z I } .

solyble StdrCh,

o r p u l l u l a n w i t h p u r i f i e d d m y l a s e s , were



a Pharmacia


c h l o r i d ~ ,t h e a m y l a s r s a n d t h e m a r k e r p r o t e i n s w e r e f i r s t



The suqacs p r o d u c e d by l h y d r o l y s i s o f


For g e l f i l t r a t i o n

c a r b o h y d r a t e c o n t e n t was e s t i m a t e d b y t h e

p h ~ n o l - s x i l f u r i r a c i d m v t h o d i i s l n g mannose

% Sodium

a n d 5.0

I ? c v l i i r n n (HIS i O I 3 0 1 * a s

f l n n r d t r o i 70 m1.h"



arid Schmid ( 1 6 ) a n d o f Muore ( 1 7 ) , r e s p e , " t i \ e l y .

The netitrdl

T C S ~ P ~ ~ I Y AF S
~ i~
i p.r i o s r


opcr,itcd a I

c o n t a i n i n g 0.02

M N a C l a t f l o w r a t e s o f 5.?,

azidr and 0.i

T r y p t o p h a n arid

a c c o r d i n g Lu t h e n i ~ t h o d s o f

P - 1 0 0 f i n e ( 1 . 6 X 4 3 ern) w e r e e l u t P d w i t h


50 mM s o d i u m c i t r d t e b u f f e r p H 5 . 8

c o n l ~ l io n

f u r 5 t a 60 m i " ,

and assayed f o r residual a c t i -


(DNS mcthodl.


F o r some e x p e r i m e n t s 1 . 6 7 % L u i k o a s k y

C a t h u d i c ~ i e c t r o p h o ~ r s iu sn d e r n o n d c n d t u r i n g c o n d i t i o n s
~ o l u b l rs t , i r r h w a s i n r l u d e d i n t h e i n c u b a t i o n m i x t u r e .
p e r f o r m e d i n 6.5


X piilydcryiamide g e l rods (gel buffer



0 . 0 6 M KOHI0.375

M a c e t i c a c i d pti 4 . 3 ;

r a t e s w ~ r cc a l c u l a t e d f r o m p l o t s o f

e i e c t r o p h o r r s i s buf-

r c s i d u a l a r t i v i l y v e r s u s i n c u b d t i o n t i m e . From R r r h e n i u s
: 0 . 3 5 M 6 - a l a n i n ~ l 0 . 1 4 M a c e t i c a c i d pH 4 . 5 1 .


Per gel

p l o t s the dppdrent a c t i v a t i o n energies f o r thermal


rod 5 m A was a p p l i e d f o r d b o u t 1 . 5 h . A n o d i c e l e c t r o p h o r e s i s
1 i " 4 i i 0 n we r e c* I c I I 1a t e d .

crndpr n o n d r n a t u r i n g c o n d i t i o n s

c a r r i e d o u t 8 s previously


T h e o v ~ r a l lt e m p r r a t u r e d e p e n d e n c e o f c a t a l y t i c a c t i v i t y


[ 3 1 . P r o t e i n s were s t a i n e d w i t h Coomassie h r i l l i a n l
under s t a n d a r d

b l u e R 250.

s e p a r a t e g e l s were

In addition,

a s s a y c o n d i t i o n s was d e t e r m i n e d

by m ~ a s u r i i i q

stained for
d c t i v i t i v s i n t h e ranqe

35 OC-90

"C u s ~ n yt h e

DNS m c t h o d

amylase and q l y c o p r o t r i n 1 3 , i l l .

w i t h 2 % l u l k o w s k y s o l u b l e ~ t a r c hi n M c I l v a i n e b u f f c r

i d s

cnrrivd out using

5 % polyacryidmidr

as IUhStrati..


b u f f e r pti 7.6

s t a c k i n q q ~ il n 0 . 1 7 M T r i s I H C I

with O.i

T h r s o m e s ~ b s t r a t cw a s d i s s o l v e d i n

SO5 a n d

(pH < 3.81,

M T r i s / H C l b u i f r r p H 8.7

i n 0.37

c o n t a i n i n g 0.1

Mcilvdine buffer

(pH 3.4-1.6)

or ClarkILubbs

X 505. Thc
1231 (pH > 8 ) t o e s t i m a t e r e l a t i v e a c t i v i t i e s a t

e l e c t r o p h o r ~ s i sb u f f e r c o n s i s t e d o f 0.05 M T r i s l 0 . 3 8 4



w i t h t h ? DNS m e t h o d

i n t h e r a n g e pH 2 - 9 .

These b u f -

1 SDS. P r i o r t o a p p l i c a t i o n t o t h e

g l y c i n e pH 8 . 3 w i t h 0 . 1


5 0 mM g i y c i n ? / H C i

% polydrrylamide sepdrating gradient gel


t h e s a m p l e s were b o i l e d f o r 5 rnin w i t h t h e sarnplc

W P ~ Pa


l r o used t o d r t e r r n i n ? t h e r e l a t i v e s t a h i i i t y

bufd i f f e r r n t pH v a l u e s ( 2 - 1 0 ) o f d m y l a s e s a m p l e s w h e n i n c u b a t e d

f s r , 1 0 m M T r i s I H C I pH 8 c o n t a i n i n g I m M E D T A , i % SDS a n d

5 7: 2 - m e r c a p t o e t h a n o i .

14 h

d t



For t h e estimation o f t h e apparent

r e l a t i v e m o l e c u l a r mass,

t h e f o i l o w i n q blr m a r k e r s

W ? ~ P

Haw starch . i d s o r p t i o n

: phosphoryiase b


b o v i n e s e ~ u r na l b u m i n
T h r procedure



soybean tcypsin


1used b y

llrda a n d c o - w o r k e r s

~ n h i h i t o r(20100),




u n i t s 1 s ~ r rm i x r ~ d w i t h 2 0 0 m y v f
sodium < : i t r d t e

I s o c l e c t r i c f o c u s i r i q was c a r r i r d o u t i n 0 . 5
a t 8 *C.

(pii 3.5-10.51

a c r y l a m i d e qel p l a t e s

b u f f ? = (ptl 3-6.2)

f e r r i t i n (4.41,





Urtrrmindtion of







(5000 X

4: Ill m i n ;

4 'C)

and washed w i t h

R p s i d u d l a c t i v i t y i n t h e combined super-

~ n d t a n t sw a s d e t r r m i n e d t o c a l c u l a t e t h e r i m o u n t o f a d s o r b e d
r n i y m r . When s t i i d y i n q a d s o r p t i o n

i n t h e p r ~ s c n c eo f

hy ye1 f i l t r a t i o n

c o d m y l d ~ ew d s e s t i m d t p d b y U V a b s o r h a n c e a t 2811 n m 1 2 6 ) .


employing spvcral media.

calibrated using the foliorinq markers :

t r y p s i n o g P n A (25000).


5 U h s t r d t c S or i n h i h i l o r s

h o v i n p scrum a l b i i n l i n ( 6 7 0 0 0 1 ,

o f S e p h d d c x 6-75


S t a r c h was t h e n p r e c i p i t a t e d

cvtnchromr c (10.65).

f o r t h e p u r i f i e d a m y l d s e ~w e r e r s t i m a t e d b )

f a r h column

(5.341, c - i i r i a l h u m i n

whale rnyogiohin

ml of O.i M


(5.3). h o r s e n i y n q l o b i n ( 7 . j I .

r a w ~ t d r r hi n 4

and i n c u b d t e d a t 4

Thc p l mdrkcrs
by r r n t r i f u q a t i o n



( u p t o 8000 U N S

mm p o l y ? O rnin w i t h r r c l ~ l d rm i x i n q .

were : d r n y l u g l u c ~ s i d d s e ( 3 . 5 ) ,


dnd e - l a c t a l l J u m i n

5 ~ r u m. i l h i i m i n


(43000). cdrbunic anhydrase (30000),


(43000). chymo-

a n d C i b n n u C i P d S e A (137001. Columns


X 95 c m l ,

l l l t r o g e l AcA 5 4 ( 2 . 6 X

D c r o r p t i o n was a t t e m p t e d


times for

pH 8-91

b y r a s h i n q t i l e s t a r c h a t 4 'C

? O rnin w i t t i T r i s I H C l



or w i t h Sodium b u r d t r b u f f p r s (0.5-1.2






T h e c o m b i n e d v r s h i n q s were d i d l y r e d a t 4 " C

i i q a i n s t 0.1

th? amount of non-adac~rbcd glu-

M s o d i u m r i t r a t r b u f f e r pH 5 h r f o r e e s t i m a t i n g

rslr.3srd r n 7 y m e .





0.09 I







Elution volume


F i n a l p u r i f i ca t i o n s t e p s f o r__








F l u t i o n o f q l i i r n n m y l a s ~ froni

q l u c o d m y l a s e wcce o n l y d e l e c l r d
o f A a n d B.



1 . 7 M l r i s I l i C 1 pH 7.6

40 w i t h



s i u m p h o s p h n t r pH 6 . 8 ;
t i t ? . R.



Eupplrinrnt Fig.2.

C. d n t n r r l i c d



(0-70 m l l pcitds-

from h y d r o x y a p a -

d r d ~ h o 5 ~ - A t i - S r p l i d ~ " ~ ~

o-Amylase and

the m d j o r

p r o t e i n pcaks

~ ~ ~ p e r t i v ~ l y



8 1 0

Concentrot ion




( 0 )W



: 9. I-'

Determination of the sedimentation

(so,w)t h ? C.

metitation ~ o r f f i c i e n l s


~ C Cd

d n t a r c t i c a amylases.


elerminrd for

r e n t r a t i n n s a s drsrrihrd

e-amylase (*I


and g l u -

different protein


i n M a t e r i a l s and M e t h o d s





Wovelength : nm
Siipplemenl I iq.5.




d ~ t d r r t i c dg l r l c u a m y l a s ~ i

the presence rif



5 0 mM s o d i u m a c r t . i t r

1 5 nm.rnin-'

d i f f p r p n r ? s p ~ r t r u mn f

drrced b y a c d r b u s e .



? 7 . 3 u M a c d r h u s e arid ( b ) w i L h u u l

Thr rrtismr


was u s e d .

dbsorpt inn tunits

( 7 . 6 6 I.M)

p H 4.2

iiC 25

The v e r t i c a l


dissolved i n



scan spced

represents 0.01

320 240




P u r l f i r a t i n n st?p


Vnluiil~T o t a l


Sephadrx 6-75
t i l t T A t i " "


U l t i ' o q e i X r A 54
<,PI f i l t r a t i o n


aPtiYity a c ' t i * i t y


it s . r n g -

Hydro x y n pa t i t P
r hr o m d t oq P a ph y

f f in i t y
<, h rtlrnd t " 4 P d I, h v
h r d

rbo SP





































~ Z ~ O O

DCAl - S r p h a c ? l
i.11 r nm d t o qr a p h y

R P C O Y C T ~ Purification