Purify Enzyme Amylase From Candida

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Eur. J. Biochem. 164,643 -654 (1987)0 EBS 1987

Purification and characterization of extracellular a-am ylase and glucoamylasefrom the yeast Candida antarctica CBS 6678

Rene DE MOT and Hubert VERACHTERTLaboratory 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-cyclodextrin-grown 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 andaffinity chomatography on acarbose AH-Sepharose 4B.

Both enzymes were monomeric glycoproteins with fairly different amino acid compositions. Their apparentrelative molecular mass, sedimentation coefficient (s&, ,), isoelectric point, absorption coefficient (280 nm), pHand 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, in-cluding some raw starches. a-Amylase was active on cyclodextrins, whereas debranching activity was demonstratedfor glucoamylase. Trestatins were potent inhibitors of both a-amylase K i 1 pM) and glucoamylase K i< 0.1 pM), being more effective than Bay e 4609 (Ki < 10 pM). Glucoamylase was selectivity and stronglyinhibited 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-identicalwith the active site.

In recent years the capability of some yeast species todegrade starch has aroused the interest of several researchers,as the potential value of these microorganisms for certainbiotechnological applications, such as the production ofsingle-cell protein or ethanol from a starchy biomass, wasrecognized [l]. In addition there are current attempts to in-troduce foreign starch-degrading activity into the non-amylolytic yeast Saccharomyces cerevisiae by recombinantDNA technology [ l ] .

Most research dealing with microbial amylolytic enzymeshas been focused on the enzymes from bacteria andfilamentous fungi, several of which have found industrialapplications [2]. However, far fewer data are available on theamylases from yeasts, although the starch-degrading enzymesof some promising species, such as Lipomyces kononenkoae,Saccharomycopsis Jibuligera, Schwanniomycesspp. and Filo-

basidium capsuligenum have been partially characterized [3,41. Recently we observed that Candida antarctica CBS 6678secretes with /3-cyclodextrin as sole carbon source significantlyhigher levels of amylolytic activity than any of the currentlyrecognized, active starch-degrading yeast species [5]. This ob-servation urged us to investigate further the extracellular

Correspondence to H . Verachtert, Labora torium voor IndustrielcMicrobiologie en Biochemie, Katholieke Universiteit Leuven, Kar-dinaal 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 (EC3.2.1.3); 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, arepresented in miniprint at the end of the paper.

amylolytic system of this strain, which was originally isolatedfrom rice and described as Trichosporon o ryzae by Ito et al.[6]. According t o recent taxonomic studies strain CBS 6678 iscurrently accomodated within the genus Candida [7, 81.

In the present paper the purification and characterizationof an a-amylase and a glucoamylase from C . antarcticu arereported with special reference to some interesting propertieswhich have been assigned previously to mould amylases butrarely to the enzymes from yeast species.

RESULTS AND DISCUSSION

Enzyme purification

C. antarctica CBS 6678 was grown in a medium withP-cyclodextrin as sole carbon source t o induce enhanced secre-

tion of amylolytic enzymes IS]. The culture supernatant wastreated with protamine sulfate, which reduced its viscosityand thereby facilitated concentration of the fluid by saltprecipitation. Through Sephadex G-75 sf gel filtration twopeaks with amylolytic activity, corresponding to a-amylaseand glucoamylase respectively, were separated (SupplementFig. 1). a-Amylase was further purified by Ultrogel AcA 54,DEAE-Sephacel and hydroxyapatite chromatography.@-Amylase was eluted from the hydroxyapatite column be-tween 40mM and 50mM potassium phosphate buffer(Supplement Fig. 2A). A similar procedure was employed forglucoamylase, however, with a final purification byacarbose AH-Sepharose 4B affinity chromatography(Supplement Fig.2B). The results of a typical purification are

summarized in Supplement Table 1. Both enzymes appearedhomogeneous when analyzed by cathodic PAGE, SDS-PAGE


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Mr

9 4 -67 -

43 -

3 -

20.1

14.4-

1 2 3 4 pl

0.65

- .45- 8.3- .3

.9

- .34- .7

.4

- .5

Fig. 1. Determination o M, and p l o j C . antarctica amylases. SDS-PAGE (lanes 1, 2) and isoelectric focusing (lanes3, 4) of purifieda-amylase (lanes 1, 3) and glucoamylase (lanes2, 4) were carried ou tas described in Materials and Methods. The positions of markersfor isoelectric point (PI) and relative molecular mass (multiplesof

x M,) are indicated

and isoelectric focusing (Fig. I), analytical ultracentrifugationand FPLC on Superose 12 in 6 M guanidine hydrochloride.

Apart from the a-amylase and glucoamylase, zymogramsof the concentrated culture fluid from C. antarctica, grown inP-cyclodextrin or starch media, revealed no additionalamylolytic enzymes. Furthermore, column effluents showedno other pullulanase or cyclodextrinase activity than the oneassociated with glucoamylase and a-amylase, respectively (seeSubstrate specificity), whereas no isoamylase was detectable.Known starch-degrading yeasts also secrete a-amylase and/orglucoamylase, but no P-amylase or true pullulanase [3, 4, 11,271. Secretion of an isoamylase and a specific cyclodextrinase

has been reported for L . kononenkoaeonly [28,29].

Physicochemical characteristics

The sedimentation coefficients, corrected a t 20 C in waterand at zero concentration ( s , ~ ) ,were 4.9 S for a-amylaseand 4.7 S for glucoamylase (Supplement Fig. 3), cor-responding well to the ones estimated for several fungalamylases [30 21. However, significantly higher values ofabout 7 S have been reported for the DEX-encodedglucoamylases of Saccharomyces diastaticus by Erratt [33].

Ultraviolet absorption at pH 7 displayed a simple spec-trum with a peak at 277 nm for a-amylase and at

280nm for

glucoamylase, and A2so /A260 alues of 2.88 and 1.93 respec-tively (Supplement Fig. 4A). The absorption coefficients at280 nm were determined as 1.53 cm2 mg-' for a-amylase and1.74 cm2 mg- ' for glucoamylase by the methods of Scopesand of Van Iersel et al. [34]. The alkaline spectra (SupplementFig. 4B) revealed a considerably higher tyrosine/tryptophanratio for a-amylase (2.17) than for glucoamylase (0.44) [16].

By analytical isoelectric focusing PI values of 10.3 and 10.1were estimated for a-amylase and glucoamylase respectively(Fig. 1). A minor, diffuse band (PI % 10) was present in theglucoamylase sample. Such microheterogeneity is often dis-played in isoelectric focusing by glycoproteins judged homo-geneous by other techniques [20]. The basic nature of both

amylases was confirmed by their lack of binding to DEAE-Sephacel at pH 7.6 (see Purification) and the absence of

Table 1. Amino acid composition o C . antarctica amylasesCysteine was determined as cysteic acid [17] and tryptophan wasmeasured spectrophotometrically [16]. Values for serine and threoninewere extrapolated to zero time of hydrolysis, whereas those forisoleucine and valine were obtained after hydrolysis for 72 h. Theother values a re averages from 24-h, 48-h and 72-h hydrolyses

Amino acid Proportion Residues

a-amylase gluco- a-amylase gluco-

amylase amylase

mo1/100 mol

AsxThrSerGlxProGIYAlaCYSValMetIleLeuTYrPheLYSHisArgTrP

11.7 10.9 558.36 7.30 398.73 9.01 417.02 7.18 335.38 3.22 258.82 8.18 416.07 12.8 291.64 0.95 86.99 6.92 330.22 0.45 15.00 2.30 23

6.24 5.58 295.29 3.17 253.25 3.94 152.21 1.88 108.12 5.98 382.35 3.07 122.44 7.22 11

44293629133352

428

29

2313168

241229

Total numberof residues 468 404

anodic electrophoretic mobility at pH 8.3. As compared to

most mould and yeast amylases with isoelectric points below7 [31, 35 71, the PI values of the C. antarctica amylases areunusually high. However, a few mammalian a-amylases [38]and three glucoamylases from a Rhizopusspecies [39] have PIvalues around 9.

The amino acid compositions of the C .antarctica amylasesare shown in Table 1. As compared t o the composition of theS . diastaticus glucoamylases [33 51 and of several mouldamylases [30, 31, 40 -421, the C. antarctica enzymes containan unusually high amount of histidine. However, a commonfeature is the presence of a relatively large number ofhydroxyamino acid residues (threonine, serine). The latter aremajor potential glycosylation sites [31, 401. Indeed, neutralcarbohydrate (as mannose) was present in both the C. antarc-tica a-amylase (2.4%) and glucoamylase (7.4%0), which wasconfirmed by their staining with the periodic acid/Schiff sbase reagent after electrophoresis. The glycoprotein natureof extracellular yeast amylases has been reported by severalauthors [3, 11, 27, 32, 33, 43-45]. A 300-kDa glucoamylasefrom S . diastaticus with an extremely high carbohydratecontent (8O0/,) was recently characterized by Modena et al.

A discrepancy was observed between the values for theapparent relative molecular mass determined by SDS-PAGEand gel filtration (Table 2). When using agarose or dextran-based media (Sephadex, Ultrogel, Superose) M , estimateswere significantly lower than for SDS-PAGE. However, M ,

values obtained with a polyacrylamide gel (Bio-Gel P), oran agarose-containing medium (Superose) in 6 M guanidine

~461.


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Intrinsically glucoamylase is more thermally stable thana-amylase, which is also the case for the Schwanniomycesenzymes [45, 55, 561. The much lower energies previouslyreported for the amylases from L . kononenkoae ( < 100 kJmol-') [36] and Schwanniomyces alluvius(< 170 kJ m ol- l)1571 indicate the C . antarctica enzymes to be less susceptibleto heat inactivation. As demonstrated by the parallel shiftsin the Arrhenius plots (Fig. 3 B), the enzymes, especially aamylase, were stabilized by added substrate. This phenome-non has also been described for glucoamylases fromAspergillus spp. [58, 591.

Under standard assay conditions the temperature optimawere 62C for a-amylase and 57C for glucoamylase, withglucoamylase activity being much more temperature-depen-dent (Fig. 3 C). The optimum for glucoamylase is within theusual range for yeast glucoamylases (SO 0C), but the valuefor a-amylase is considerably higher than for most other yeasts(not higher than 50C) [3, 4,27]. However, an a-amylase withoptimum a t 70C is secreted by L. starkeyi [60].

Substrate specificity

The purified a-amylase and glucoamylase from C. antarc-ticu were characterized by their substrate specificity (Table 3).In addition, kinetic constants K,,,,k,) were determined withseveral soluble substrates (Table 3).

In general, high-molecular-mass substrates containinga-1,4 linkages were the better substrates for both enzymes.The enhanced activity with increasing degree of polymeriza-tion was also evident within the homologous series of maltoseup to short -chain amylose, with decreasing K,,,and increasingk , values (Table 3). Similar kinetic results have been obtainedwith Aspergillus and Rhizopus glucoamylases [42, 61 31.The relative activity of purified S. diastaticus glucoamylasesalso increased with increasing degree of polymerization ofmaltooligosaccharides but, as compared to the Candidaenzyme, better activity on small substrates was shown [35,461. As compared to cr-amylase, K , values with polysac-charides were lowest for glucoamylase, resembling the proper-ties of the Succharomycopsis,fibuligeraenzymes [64] but quitedifferent from the L. kononenkoae [36] and theSchwanniomyces[45, 56, 571 amylases.

Only the glucoamylase of C . antarctica slowly hydrolyzeda-1,6 linkages in panose, dextran and pullulan (Table 3). Fur-thermore, glycogen with its relatively high number of a-1,6branch points was a good substrate for glucoamylase, but notfor a-amylase. As specific extracellular debranching enzymes,such as pullulanase or isoamylase, are usually not produced

by moulds or yeasts [2], the glucoamylase debranching activityis essential to enable extensive starch hydrolysis by thesemicroorganisms. Indeed, the most active amylolytic yeastsdisplay such activity [3, 11, 27, 44, 45, 56, 651. However,the debranching activity of S. diustaticus glucoamylases isextremely low, if detectable [33, 35, 461. The C. antarcticaglucoamylase also released, albeit very slowly, glucose fromcross-linked dextran (Sephadex), without affecting its chro-matographic properties, however. This affinity may accountfor the observed, pronounced retardation of the enzymeduring Sephadex G-75 sf gel filtration. A low exodextranaseactivity has equally been assigned to Rhizopusand Aspergillusglucoamylases [66, 671 but it has not yet been reported foranother yeast glucoamylase. The negligible activity of the

Candida glucoamylase on a-glucosides further indicates it tobe a true glucoamylase rather than a starch-degrading

a-glucosidase [60,68]. Glucose was the sole hydrolysis productof the glucoamylase from starch or pullulan as indicated byTLC analysis (results not shown). Further evidence for the exotype of hydrolysis was obtained with endo-specific, artificialsubstrates, namely Amylase B-AR [12],Alphachrome [69] andPhadebas [70], which were not susceptible to the enzyme.

The above substrates were readily hydrolyzed by theCandida a-amylase. The hydrolysis products from starch weremaltose (major product), small maltooligosaccharides and aminor amount of glucose (results not shown). Like a fewother a-amylases [3, 11,71- 731, the C . antarctica enzyme wascapable of hydrolyzing cyclic, non-reducing dextrins(Table 3), which further confirms its endo mode of action.TLC analysis revealed that P-cyclodextrin wdS first linearizedto maltoheptaose, which was further hydrolyzed to maltoseand glucose (results not shown). The effect of the degree ofpolymerization of the cyclic dextrins on the a-amylase activity,also evident from the kinetic parameters (Table 3), was muchmore pronounced than with the linear maltooligosaccharides.In fact, only y-cyclodextrin was hydrolyzed at a comparablerate. This typical kinetic pattern, also described for theAspergillus [71, 721 and the pancreas and saliva [73]

a-amylases, is quite different from the one observed for truecyclodextrinases [74]. As far as yeasts are concerned, such aspecific enzyme has only been reported for L . kononenkoae

The observation that the C. antarctica a-amylase andglucoamylase, respectively, hydrolyzed P-cyclodextrin andpullulan slowly and selectively was exploited routinely toestimate their activities in column effluents during purifica-tion, employing the simple 3,s-dinitrosalicylate method. Thus,extensive dilutions and the use of the more laboriousAlphachrome and peroxidase/glucose oxidase/2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) ethods was avoided.

Raw starches, especially those from wheat and potato,were hydrolyzed by the C. antarctica a-amylase and gluco-amylase, the lat ter enzyme displaying the highest activity. Theability of some amylases, especially from filamentous fungi,to degrade raw starch has been established for some time [75,761. However, apart from the glucoamylases of Saccharomy-copsis f ibdige ra [44] and C . antarctica (this paper), this phe-nomenon has not yet been reported for yeast amylases. Thepotential of yeast species for digesting raw starch merits fur-ther investigation as it has been suggested that this propertycould be advantageously used for the alcoholic fermentationof starch without precooking [76].

~ 9 1 .

Inhihition

The effect of several potential amylase inhibitors on theC . antarctica enzymes was characterized as shown in Table 3.Glucoamylase was competitively inhibited by cyclic

dextrins and a-D-glucosides (Ki between 15mM and 60 mM)and, somewhat more strongly, by glucose, amino alcohols(Tris, 2-amino-2-ethyl-l,3-propanediol nd maltitol (Ki 513 mM). However, a-amylase was only very weakly affectedby maltitol and no t at all by the amino alcohols. The inhibitionof a-amylase by a and P-cyclodextrin was similar to theireffects on glucoamylase. Maltose was a non-competitive in-hibitor (Ki = 8.8 mM) of a-amylase. The inhibitory effect ofmaltose and glucose on a-amylase and glucoamylase, respec-tively, has previously been observed for the L . kononenkoaeenzymes [36], but further kinetic da ta on yeast amylase inhibi-

tion are scarcely available, if at all. On the other hand, themould enzymes have been intensively studied. Inhibition of


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Table 3. Substrate specificity andkinetic constants of the C. antarctica amylasesRelative activities 1/13 were determined under standard assay conditions (3,5-dinitrosalicylate method) with oligosaccharides, a-glucosides(I 0 mM) and polysdccharides (I /.). N o activity of n-amylase and glucoamylase was found with melibiose, trehalose, saccharose, methyla-D-glucoside or phenyl a-wglucoside (n.d. = not detectable). Values between { } correspond to activities on boiled starches (previouslyheated for 45 min at 100C in substrate buffer). Michaelis constants (K,) and reaction rates at infinite substrate concentrations ( V ) wereobtained 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 fromDixon plots. N o inhibitory effect on a-amylase was observed for Tris, 2-amino-2-ethyl-l,3-propanediol nd 1-deoxynojirimycin. A n averageM , of 3750 was assumed for the homologous inhibitor mixture Bay e 4609 [84]. Activities were determined with the 3,5-dinitrosalicylatemethod (40C; pH 4.2). Concentrations causing 50 inhibition of glucoamylase adsorption onto raw starch (Zs0) were determined at 4 C i n0.1 M sodium citrate buffer pH 5 according to Takahashi et al. [26]

Substrate or inhibitor a-Amylase Glucoamylase~~

Km k, A , Ki K , k , A , K , I5

Glucosep-Nitrophenyl a-D-glucosidePanoseMaltoseMaltotrioseMaltopentaoseMaltoheptaose

Short-chain aniylosea-C yclodextrinj-Cyclodextriny-Cyclodex rinMaldex 15Soluble starch (Difco)Soluble starch (UCB)Soluble starch (Merck)Soluble starch (Zulkowsky type)Amylose (potato)Amylopectin ( potato)Glycogen (rat liver)Glycogen (oyster)PullulanDextran

Wheat starchCorn starchWaxy corn starchRice starchPotato starchAcarboseTrestatin ATrestatin CBay e 46091-DeoxynojirimycinMaltitolTris2-Amino-2-ethyl-I ,3-propanediolMethyl a-D-glucosidePhenyl a-D-glucoside

YO

n.d.n. d.

0.020.37

23.034.2

43.80.081.9

32.255.860.197.194.098.084.586.2

4.57.1

n. d.n. d.43.9 {100:

0.22 (86.3)0.22 i73.2)1.8 {83.8)

44.6 (98.7)

mM S - %

0.270.182.64.8

10.913.814.8n.d.n.d.n.d.62.360.892.294.581.150.790.679.470.6

0.400.05

92.7 {IOO)6.0 (96.3)

23.4 (99.8)1.2 C97.3)

43.8 {99.5}

mM

13.3(C) 2140

40259.642.227.0

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

~

0.087h (C) 63.10.066' (C) 32.20.037 (C) 24.78 S b C) 21.80.45(C) 5325.3 (C) 5009.4 (C)8.5 (C)

56.2 (C)34.8 (C)

the glucoamylases from Aspergillus and Rhizopus species byglucose and a-glucosides has been demonstrated [61, 771,whereas maltose is known to inhibit the Aspergillus awamoria-amylase [37]. Recently Iwama et al . [78] noticed that severalamino alcohols, including Tris and 2-amino-2-ethyl-l,3-pro-panediol and maltitol affect the Aspergillus and Rhizopusglucoamylases, reporting K i alues close to the one we foundfor the C. antarctica glucoamylase. Although immobilized a

and P-cyclodextrin have been adopted to purify, from varioussources, different types of amylolytic enzymes, which were

inhibited by these cyclic oligosaccharides (a-amylase, P-amy-lase, pullulanase) [79 21, the relatively high Ki values( > 10 mM) for both the C. untarctica glucoamylase and a-amylase indicate these ligands not to be suitable for effectiveaffinity chromatography of these yeast amylases. During thelast decade several amylase inhibitors of microbial origin havebeen isolated, including acarbose (Bay g 5421) and Bay e4609 [83, 841, 1-deoxynojirimycin [85] , amylostatins [86] and

trestatins [87], which are all characterized by a mono-saccharide or oligosaccharide composition. A limited amount


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of literature data, almost exclusively dealing with enzymesother than yeast amylases, are available on the characteriza-tion of these substances. Therefore, several of thesecompounds were included in our study (Table3). 1-Deoxynojirimycin selectively inhibited glucoamylase (Ki =0.45 mM). Bay e 4609 displayed considerably lower Ki values(< 10 pM) for both amylases, with a-amylase being slightlymore affected. An even stronger effect on a-amylase (Ki - ' . T C S ~ P ~ ~ I Y F ~ ~ .S i i p r i o s r I ? c v l i i r n n (HIS i O I 3 0 1 * a s

o p c r , i t c d a I d f l n n r d t r o i 70 m 1 . h " i i i a P h a r m a c i a F P L C

5 Y S t F r n .

F o r g e l f i l t r a t i o n i n t h e p r e s e n c e o f g u d n i d i n e h y d r o -

c h l o r i d ~ , 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

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 M a g ? I Z I } .

A S i i p e r o s e 1 2 F P L C c o l u m n ( H H 1 0 1 3 0 ) w a s e q u i l i b r a t e d i n

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

pH 7 a n d c l u t e d w i t h t h i s s o i v r n t a t 20 r n1 .h .' . G u a n i d i n e

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 F o h l r n an e t a 1 . 1 ? ? 1 .

E f f e c t s o f t e m p e r a t u r ? a n d pH

T h e ~ n i t i a i 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 m p ~ r a t u r e s l u ~ s 25 'C-75 OC) b y t h e O N 5

m e t h o d ~ m p l o y i n q u l k o w s k y s n l < i h l s s t d r c h ( 2 i n i 4 c i l v a i n e

b u f i e r p H 4 . 2 ) a s s u b s t r a t e . T h c 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 y d r o l y s i s w e r c o b t a i n e d f r o m A r r h e n i u s

p l o t s .

Fn/vrn? s a m p l e s W C F P 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 l e m p r r a t u P ~v a l u e s ( 4 5 * C - 7 0 ' r l f u r 5 t a 60 m i " ,

t h r n r a p i d l y c o n l ~ l i n i r e , a n d a s s a y e d f o r r e s i d u a l a c t i -

v i t y ( D N S m c t h o d l . F o r s o m e e x p e r i m e n t s 1 . 6 7 % L u i k o a s k y

~ o l u b l r 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 .

T i b e r m d l i n a c t i v a t i o n r a t e s w ~ r c a l c u l a t e d f r o m p l o t s o f

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 . F r o m R r r h e n i u s

p l o t s t h e d p p d r e n t a c t i v a t i o n e n e r g i e s f o r t h e r m a l i n a c -

1 4 i 0 n we r e c* I c I 1 t e .

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

u n d e r s t a n d a r d a s s a y c o n d i t i o n s w a s d e t e rm i n e d b y m ~ a s u r i i i q

d c t i v i t i v s i n t h e r a n q e 35 O C - 9 0 C u s ~ n y h e D N S m c t h o d

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 h n M c I l v a i n e b u f f c r pH

4 . ) a s I U h S t r a t i . .

T h r s o m e s ~ b s t r a t c a s d i s s o l v e d i n 5 0 m M g i y c i n ? / H C i

( p H < 3 . 8 1 , M c i l v d i n e b u f f e r ( p H 3 . 4 - 1 . 6 o r C l a r k I L u b b s

b u f f r r 1 2 3 1 ( p H > 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

55 O C w i t h t h ? DNS m e th o d i n t h e r a n g e pH 2 - 9 . T h e s e b u f -

f C r s W P ~ P l r o u s e d 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 a t

d 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 he n i n c u b a t e d

f o ? 1 4 h d t 3 5 'C

H a w s t a r c h . i d s o r p t i o n

T h r p r o c e d u r e 1used by l l r d a a n d c o - w o r k e r s i 2 4 . 2 5 1 w a s

m o d i f i e d d 5 f o l i a w s . C i u c u a m y l d s e s . ir n p ie s ( u p t o 8000 U N S

u n i t s 1 s ~ r r i x r ~ dw i t h 200 m y v f r a w ~ t d r r h n 4 m l o f O . i M

s o d i u m < : i t r d t e b u f f ? = ( p t l 3 - 6 . 2 ) a n d i n c u b d t e d a t 4 O C f o r

? O r n i n w i t h r r c l ~ l d r m i x i n q . S t a r c h w a s t h e n p r e c i p i t a t e d

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