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164,643-654 (1987)
0FEBS 1987
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 1.1.3.4); peroxidase(EC 1.11.1.7);
a-amylase (EC 3.2.1 .l); 0-amylase (EC 3.2.1.2); glucoamylase (EC
3.2.1.3); a-glucosidase (EC 3.2.1.20); pullulanase (EC 3.2.1.41);
cyclodextnnase (EC 3.2.1.54); isoamylase (EC 3.2.1.68).
Note. Parts of this paper, including Materials and Methods, are
presented in miniprint at the end of the paper.
644
1
Mr
94
67
43 -
30 20.1 14.4
3 4
pl
- 10.65
- 9.45
- 8.3
- 7.3
- 5.9
- 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
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
Proportion
Residues
a-amylase glucoamylase
a-amylase glucoamylase
mo1/100 mol
Asx
Thr
Ser
Glx
Pro
GIY
Ala
CYS
Val
Met
Ile
Leu
TYr
Phe
LYS
His
Arg
TrP
Total number
of residues
11.7
8.36
8.73
7.02
5.38
8.82
6.07
1.64
6.99
0.22
5.00
6.24
5.29
3.25
2.21
8.12
2.35
2.44
10.9
7.30
9.01
7.18
3.22
8.18
12.8
0.95
6.92
0.45
2.30
5.58
3.17
3.94
1.88
5.98
3.07
7.22
55
39
41
33
25
41
29
8
33
1
23
29
25
15
10
38
12
11
44
29
36
29
13
33
52
468
404
28
2
9
23
13
16
8
24
12
29
645
Table 2. Estimation of the apparent relative molecular mass of the
extracellular C. antarctica amylases
Experimental conditions are described in Materials and Methods
Procedure
Apparent M ,
a-amylase
glucoamylase
Gel filtration
Sephadex (3-75 sf
Ultrogel AcA 54
Bio-Gel P-100 f
Superose 12
Superose 12"
37000
29 000
49 000
25 000
48 ooo
16000
34000
46000
25000
48 ooo
SDS-PAGE
51 000
48000
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.
10
PH
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
100
75 o\"
..
-*295
315
335
L1
._
>
._
U
50
8
.-?
U
0
-
25
45
65
85
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-')
646
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
647
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
a-Amylase
Glucoamylase
~~
Km
Glucose
p-Nitrophenyl a-D-glucoside
Panose
Maltose
Maltotriose
Maltopentaose
Maltoheptaose
Short-chain aniylose
a-Cyclodextrin
j-Cyclodextrin
y-Cyclodextrin
Maldex 15
Soluble starch (Difco)
Soluble starch (UCB)
Soluble starch (Merck)
Soluble starch (Zulkowsky type)
Amylose (potato)
Amylopectin (potato)
Glycogen (rat liver)
Glycogen (oyster)
Pullulan
Dextran
Wheat starch
Corn starch
Waxy corn starch
Rice starch
Potato starch
Acarbose
Trestatin A
Trestatin C
Bay e 4609
1-Deoxynojirimycin
Maltitol
Tris
2-Amino-2-ethyl-I ,3-propanediol
Methyl a-D-glucoside
Phenyl a-D-glucoside
k,
A,
Ki
YO
mM
K,
k,
A,
K,
S-'
mM
13.3(C)
-
2140
n.d.
n. d.
0.02
0.37
23.0
34.2
43.8
0.08
1.9
32.2
55.8
60.1
97.1
94.0
98.0
84.5
86.2
4.5
7.1
n. d.
n. d.
43.9 { 100:
0.22 (86.3)
0.22 i73.2)
1.8 {83.8)
44.6 (98.7)
0.27
0.18
2.6
4.8
10.9
13.8
14.8
n.d.
n.d.
n.d.
62.3
60.8
92.2
94.5
81.1
50.7
90.6
79.4
70.6
0.40
0.05
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
402
59.6
42.2
27.0
-
0.087h (C)
63.1
0.066' (C)
32.2
0.037 (C)
24.7
8 S b (C)
21.8
0.45(C)
532
5.3 (C)
500
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
648
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
20
Ratio: units.mg
PH
649
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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21. Ansari, A. A. & Mage, R. G. (1976) Anal. Biochem. 74, 118125.
22. Fohlman, J., Eaker, D., Karlsson, E. & Thesleff, S. (1976) Eur.
J . Biochem. 68,457 - 469.
23. McKenzie, H. A. & Dawson, R. M. C. (1969) in Data for biochemical research (Dawson, R. M. C., Elliott, D. C., Elliott,
W. H. & Jones, K. M., eds) 2nd edn, pp.475-508, Oxford
University Press, London.
24. Medda, S., Saha, B. C. & Ueda, S. (1982) J . Ferment. Technol.
60,261 -264.
25. Saha, B. C. & Ueda, S. (1983) J . Ferment. Technol. 61, 67-72.
26. Takahashi, T., Kato, K., Ikegami, Y .&hie, M. (1985) J. Biochem.
(Tokyo) 98,663 - 671.
27. De Mot, R., Van Oudendijck, E. & Verachtert, H. (1985) Antonie
van Leeuwenhoek. J. Microbiol. 51,275-287.
28. Spencer-Martins, I. (1982) Appl. Environ. Microbiol. 44, 12531257.
29. Spencer-Martins, I. (1984) Znt. J . Microbiol. 2, 31 -38.
30. Svensson, B., Pedersen, T. G., Svendsen, I., Sakai, T. & Ottesen,
M. (1982) Carlsberg Res. Commun. 47, 55-69.
31. Manjunath, P., Shenoy, B. C. & Raghavendra Rao, M. R. (1983)
J . Appl. Biochem. 5,235 - 260.
32. Tamaki, H. (1980) Doshisha Joshi Daigaku Gakujutsu Kenkyu
Nempo 31,210 - 286.
33. Erratt, J. A. (1980) Ph. D. Thesis, University of Western Ontario,
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34. Van Iersel, J., Jzn, J. F. & Duine, J. A. (1985) Anal. Biochem. 151,
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35. Tucker, M., Grohmann, K. & Himmel, M. (1984) Biotechnol.
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36. Spencer-Martins, I. & van Uden, N. (1979) Eur. J . Appl.
Microbiol. Biotechnol. 6,241 -250.
37. Bhella, R. S. & Altosaar, I. (1985) Can. J. Microbiol. 31, 149-153.
38. Malamud, D. & Drysdale, J. W. (1978) Anal. Biochem. 86,620647.
39. Takahashi, T., Tsuchida, Y. & Irie, M. (1978) J . Biuchem.
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Blakebrough, W. & Parker, K., eds) vol. 3, pp. 50-80, Applied
Science Publishers, London.
41. Takagi, T., Toda, H. & Isemura, T. (1971) in The enzymes (Boyer,
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48,1611-1616.
44. Ueda, S. & Saha, B. C. (1983) Enzyme Microb. Technol. 5, 196198.
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90,311 -314.
46. Modena, D., Vanoni, M., Englard, S. & Marmur, J. (1986) Arch.
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48. Nanmori, T., Shinke, R., Aoki, K. & Nishira, H. (1983) Agric.
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49. Taniguchi, H., Chung, M. J., Yoshigi, N. & Maruyama, Y. (1983)
Agric. Biol. Chem. 47, 511 -519.
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55. Oteng-Gyang, K., Moulin, G. & Galzy, P. (1981) Z . Allg.
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44,301 -307.
57. Simdes-Mendes, B. (1984) Can. J. Microbiol. 30, 1163- 1170.
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Biotechnol. 18,271 -278.
59. Pestana, F. & Castillo, F. J. (1985) MZCREN J . Appl. Microbiol.
Biotechnol. 1, 225 -237.
(ryclamaituhexdusr),
material to
Supplemeniary
y-cyrlodrxlrin
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
rxtrarellular
R-cyclodextrin
(cyclamaltahrptaose),
( 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,
a-amylase
r h a i n a m y l o s r (DP = 7 5 ) , m d l t i t o l ,
shoi't
AiP,
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.
grl
Verdchtert
METHODS
S O I U I ~ I ?S L d P C h ,
platrd
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
Aalst,
Belgium.
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
Mullrn,
i l u l l k r and F.
UK.
1-deuxynojirimycin
.ant1
H n l i r r . P I n o S c was
D i o - C ~ l HTI'
(Irydronyapatite)
were p u r c h r l s t ~ d r r o m B i u - R a d ,
topentdos?
l,lannheim,
from Difrn,
f r o m RDH C h e m i c a l s ,
and B i o - G e l
Rirhmnnd,
M1, USA.
Cutrndnn
and P.
flric
ABTS.
mdl-
F.
c i l i m i c d , nrt.rse,
WCI-P
~ p ~ o d u c tO sf
was obtdiriell
Phadrbds
tloli
MeTik,
Ddrmstddt,
FRC.
from Plidrmdcid I i n s C h e m i c a l s ,
l,rblrls,
5rl.liadrx
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
Sweden:
Uppsdld,
DtAE-Scphacri,
Supcrosr
Hoffrnann-LaRoche,
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
pulliil,iii,
drid
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
Webcr,
1'-100
CA, USA.
I'nolr,
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
Drlruil,
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??
c
obtainrd
FRC.
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 .
A.
Ziilkowsky-type
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
-
Cliemicdls
__-
s t d r c l i were o h t d i n e d
and U l t r o -
AcA
Sweden.
IMATFRIALS AND
_-
Colnbrook, UK,
from Kocii-Lighr,
dcxtran
n-D-qlucosidi.
. ~ n dw l i c a t
i>oldlr,
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
idki.
3apan.
Clycoqen
t o S t e l L e n e l di.(9!.
rice
f r o m UCB,
~ - g - ~ i r ~ n ~ i -
s t d r c l l , corn
nrusseis,
k i t w a s f r o m Wako C h e m i c a l s ,
WYDS
140,
1-O-mrthyl-o-D-qluco-
7 1 2 0 0 ) . PGn-enLymes,
,mid A H - S e p h d r O S e
qiurn.
St.Louis,
: 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
Co.,
porifird
Brl-
Amaga-
from r a t l i v e r according
65 1
Yeast
%rp
-____
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 .
Delft,
Ultro"r.1
'
C
30
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 %
D-cyclodextrin
u ~ e df o r
29
amylases.
t h e p r o d u c t i o n of
Erlenmeyer f l a s k i ,
in ILL
OC
a d d e d a s zole c a r b o n
a C C t d t l l brifft-r
[lo),
s o l e c a r b o n s o u r c e , was u s e d f o r
inoculation.
'C,
and t h e ~ u p e c n a t a n tA
S
c
(2.5
5.8
of
method),
m?ihnii$l.
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 -
addition.
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
method,
an3
[3,117.
b u f f e r pH 7.6
at
10
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
rquilihratrd
OC.
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 ,
solved
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
wd5
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 !
glucoamylase
(PCO-4BTS
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
~ i i ef r a c -
S
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
WCTC
(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
Tnr
r a t e of
5 days,
After
(ON5 m e t h o d ] ,
azide.
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
as
t o a column
% 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
of
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)
with
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
culture,
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
(2.6
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
qrdphy
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
with
4 . U I t r o q e i A c 4 54 gel
i l.
t r.
a.
ti.
o n. .
o f. a_
- a m.
y_
l a s e_
.
T_
he _____~~
_______......._.....
. .f .
the
dis-
and exhails-
buffer.
f"?
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
phy
280 nm
d l
RiO-Gel
were
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
UBCd.
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 .
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
rlrrldient
amylases
of
p o t d s 5 1 ~ mp h o s p h a t e
buffer
fate
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
solution
cipitalion,
a n d s t o r r d a t -20 'C
550-mi
pH 6 . 8
t h e same
fractions
linear
(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 ,
S
t.
e.
p.
I.
P.
r.
o.
t.
a.
r.
n.
i.
n.
e.
s.
u.
l.
f.
a.
t.
e.
p.
r.
e.
c.
i.
pitation.
..
..
...
.
..
.
.
.
.
n h ~ l e3 . 9 - m i
i n 25 mM s o d i u m
phosphdle
p H 4.21
biiffpr
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
O C
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
w.15
jo
fur
OC.
mill 0
farmed.
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
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-
5.8
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
the
f o r 45 m i n a t
3.6
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
S
.L_
r p ..
6.
. .D
.E
..
hE
..
- S.
c.
Q.
___._____.._..
h.
acri ch'0matoyr?e?i-of-q!u~oa~i!asp.
Ion
3 0 .in
a-emylasr.,
pH
same b u f f e r a t
0.07
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 .
rrdCtiDrls
b y U\J
the
r a t s of
6.1
rni.h-l,
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
dr'd
giucoamylaSe
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
~r
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
respec-
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 .
The
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
was
3.5-ml
tivclv,
drtrated.
contdininq
aCli"ity,T"TTrS,~"IIdiny
roam"lasC1.
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).
WPL-P
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
usinq
Like
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
Irre.!-.sre?~d~~.c,!!_if.sE!.fIllf~i~o?-~f.Efude.e?rYn?e.
ihr c o r t c e n t r d l r d
concentrated
~ 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 .
ylucuaioylase
were p o o l e d ,
tion5
5,
<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 .
of
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 -
,affinity
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
(5.6
ml),
sample
from
w i t h 0.1
M NaCl.
After
M sodium
applica-
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
tely
48 a s d e s c r i b e d b y
m a t r i x 1 0 s e q ~ ~ i l i b r d t eadt 2 0 'C
areLdle buffer
liuiis.
(1.1
frac-
w i t h 50 rnl o f
The e l u a t e w a z
immediaand then
roncrrilrdlrd
a t -20
'C
(151.
,achieved
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
IH
7.6
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 .
652
aria*
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
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 ,
O C
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
Bencze
Se d i m e n t d t i o n
on o f
sllrrific
Guanidine
m1.q"
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
and
method
SolvCrlt d r n s i t y and
a n d 0.711
m1.9-l
as substrate.
X in i4cilvaine
(2
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
plots.
for
Fn/vrn?
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 -
samples
WCFP
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 )
vuium
were
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
in
in
nlir
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
in
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
(19,201
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
partial
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 )
viscosity.
A Siiperose 1 2 FPLC c o l u m n
l31.
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
8-cyrlodcxtrin
FPLC
a Pharmacia
iii
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
5tdndilrd
d5
The suqacs p r o d u c e d by l h y d r o l y s i s o f
(18).
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"
6.5
5YStFrn.
The netitrdl
T C S ~ P ~ ~ I Y AF S
~ i~
i p.r i o s r
ml.l>-'.
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.?,
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
Rio-Gel
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 " ,
ire,
focusing
(DNS mcthodl.
vity
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
was
inactivation
Tibermdl
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 .
fPr
Per gel
inac-
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
pa5
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
described
[ 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 .
pH
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
SDS-PAGC
i d s
5 % polyacryidmidr
as IUhStrati..
4.)
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
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
buffrr
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
M
55
O C
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
fCrs
gel,
5 0 mM g i y c i n ? / H C i
12.5-25
W P ~ Pa
at
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
fo?
5 7: 2 - m e r c a p t o e t h a n o i .
14 h
d t
35
'C.
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
iisrd
: phosphoryiase b
(940001.
b o v i n e s e ~ u r na l b u m i n
T h r procedure
(670001,
ovalbumin
modified
soybean tcypsin
i
iaiinoi
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),
d5
foliaws.
Ciucuamyldse
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,
B-lactoqlobulin
nUClrdsP
(9.45)
2nd
Urtrrmindtion of
I$
values
(8.31,
ribu-
WAS
same
buffer.
(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 ) .
chromdtoqrdphy
t r y p s i n o g P n A (25000).
5f
for
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
O C
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 )
rxcliisionidiffusion
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
bovine
t1,e
(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
(4.71.
was
( 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 ) ,
s.irnpies
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
i24.251
ovalbumin
(43000). chymo-
a n d C i b n n u C i P d S e A (137001. Columns
(1.6
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
thl'ee
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
buffers
(0.5-1.5
or w i t h Sodium b u r d t r b u f f p r s (0.5-1.2
8.5-7.51.
d"",,rill
14:
14;
pH
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
"lC
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
"f
rslr.3srd r n 7 y m e .
653
RESU
TS
1.8
1900
0.09 I
"
",
100
20
40
60
80
Elution volume
ml
F i n a l p u r i f i ca t i o n s t e p s f o r__
thr
dmyldses.
A.
---I
CrddicnL
rlution
o-amylase
of
F l u t i o n o f q l i i r n n m y l a s ~ froni
(drrow).
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.
670
402
1 . 7 M l r i s I l i C 1 pH 7.6
40 w i t h
536
268
s i u m p h o s p h n t r pH 6 . 8 ;
t i t ? . R.
,170
134
Eupplrinrnt Fig.2.
-~
C. d n t n r r l i c d
'
iii
(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
0.2
4.2
8 1 0
Concentrot ion
Supplement
Fig.3.
coefficients
( 0 )W
240
280
: 9. I-'
(so,w)t h ? C.
for
metitation ~ o r f f i c i e n l s
coamylase
~ C Cd
d n t a r c t i c a amylases.
(520,w)
of
elerminrd for
r e n t r a t i n n s a s drsrrihrd
e-amylase (*I
Sedi-
and g l u -
different protein
con-
i n M a t e r i a l s and M e t h o d s
260
280
300
320
Wovelength : nm
Siipplemenl I iq.5.
C.
(a)
Ultrdviolrl
d ~ t d r r t i c dg l r l c u a m y l a s ~ i
iri
drdrbosr
(ildsrlinr).
5 0 mM s o d i u m a c r t . i t r
of
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 .
the
Giucodmyiase
? 7 . 3 u M a c d r h u s e arid ( b ) w i L h u u l
Thr rrtismr
biiffrr
was u s e d .
( 7 . 6 6 I.M)
p H 4.2
iiC 25
The v e r t i c a l
bar
dissolved i n
wd5
"C.
scan spced
represents 0.01
320 240
280
320
654
P u r l f i r a t i n n st?p
Fn!y.i?
Vnluiil~T o t a l
pl"tein
mi
Sephadrx 6-75
,,?I
t i l t T A t i " "
sf
U l t i ' o q e i X r A 54
<,PI f i l t r a t i o n
mq
Total
Specific
aPtiYity a c ' t i * i t y
units
~rn
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
ih
-faid
ii1150
33.6
LOO
44500
147.4
ion
10140
30.3
55.9
0.9
44380
132.5
59.7
0.9
9680
55.3
95.4
1.7
47050
240.3
94.5
1.6
8500
524.7
83.7
15.6
383611
384.4
86.2
2.6
7720
704.4
76.1
22.8
~ 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
factnr
5i10.0
72.R
3.4
1173.9
53.2
35.0
?i?70
779.1
47.8
5.3
3500
1296.3
3+.5
38.6
13130
887.2
29.5
6.0
5400