Anomeric form of D -glucose produced during enzymolysis

Cmbohydmfe Research Elsevier PublishingCompany, Amsterdam

424

Rimed in Belgium

ANOMERIC F. W. PARRISH

FORM OF D-GLUCOSE PRODUCED AND

DURING

ENZYMOLYSIS

E. T. REEB

Pioneering Research Division, U. S. Amy

Natick Luboratories, Natick, Mass. 01760 (IY. S. A. j

(Received August z6th, 1966)

INTRODUCTION

The initial configuration of sugars resulting from enzymic hydrolysis of polysaccharides has always been determined by means of optical rotation. In a classic example, maltose produced by the action of /3-amylase on starch was found to have a low optical rotation, i.e., it was in the /&form’. The products from c+amylase had higher rotations, i.e., were of the a-configuration’. In a similar way &glucosidases were considered enzymes which released&D-glucose from glucosides, whereas c+glucosidases releaseda+-glucose. Indeed, the anomericnature ofthe product was considered to imply that the same configuration occurred in the glycoside. This, of course, preceded the discovery by Kuhn1 of inversion of configuration by /%amylase. Application of gas-liquid chromatography (g.1.c.) to sugar analysis has shown that not only can simple sugars be determined, but that their anomeric forms are separable2. This technique appeared useful for the determination of anomericconfiguration of sugars released by the action of carbohydrases. We now report application of g.1.c. to systems producing glucose, i.e., glucosidases and exo-glucanases, enzymes whose characteristics we are currently examining. MEl-HODS

Enzymes A. Exo-p-I,3-gkzna.ses [EC 3.2.1-3 I. Sporotrichum pruinosum QM 826 enzyme was produced by growth on cellulose in shake culture3. Solutions of the enzymes, precipitated with acetone from the culture filtrates, were passed through diethylaminoethyl Sephadex (DE&-Sephadex) at pH 7.0, the desired fraction coming through without any hold-back. Over 98% of the cellulase and all of the j?-1,6-glucanase were removed. A small amount of amylase remained, but this was of no importance in the present tests. 2. Basidiomycete sp. QM 806 enzyme was produced by growth on starch in shake culture3, and precipitated with acetone. This ppt. was nearly free of &glucanases, other than the exo-j?-x,3-glucanase, but contained a little amylase. It was used without further treatment. Carbohydrate Res., 3 (1967) 424429

425

t%NOhtERIC FORM OF D-GLUCOSE

B. Exo-a-g~ucmu3ses-g~ucamykzse-a-I,#-gk&xzn-g~ucohydro~ase [EC 3.2.1.3] I. Aspergiiks niger “ Diazyme “, 455DU/gram, Miles Chemical Company, Elkhart, Indiana. 2. Endomyces sp. “Mat&se”, Matsutani Chemical Company (Japan). These enzymes were used without further treatment. C. /3-Glucosidases [EC 3.2.1.211 I. Almond emulsin (gift of Dr. N. K. Richtmyer, National Institutes of Health). 2. Aspergillus niger QM 877 enzyme was produced by growth on dextran hydrolyzate. The &glucosidase was freed from a-glucosidase by fractionation on DEAE-Sephadex. 3. Aspergillus Iuctiuensis QM 873 enzymes were obtained by growth on starch in shake cuItures3. No attempt was made to separate the fi-glucosidase from exo-ar,3-glucanase, amylase, and a-glucosidase. D. a-Glucosidases [3.2.1.20] I. AspergilIus niger QM 877 as above. The a-glucosidase fraction was used. 2. Paecilomyces varioti QM roa was grown on starch. The maltase was freed of a-amylase and of glucamylase by separation on DEAE-Sephadex. 3. Penicillium parvum QM I 878 was grown on dextran hydrolyzate. The maltase was freed of nearly all amylase by passage through a DEAE-Sephadex column. The activity of this enzyme is slightly greater on isomaltose than on maltose. E. Invertase-#?--fnrctofuranosidose [EC 3.2.1.26] A dry preparation, free of melibiase (Nutritional Biochem. Co.) was used. PROCEDURES

A. Enzymolysis. - A concentrated enzyme solution (0.1 ml) was added to a 5 0Asolution of substrate in 0.025M citrate buffer at pH 4.5. (0.4 ml). During incubation at 4o”, samples (0.2 ml) were removed, cooled in dry ice, and freeze dried. Alternatively the samples were dried quickly by blowing air over them (usually about 5 min). B. Analysis. - Dry (CaH,) pyridine (0.1 ml), hexamethyldisilazane (0.2 ml), and trimethylchlorosilane (0.1 ml) were added to each dry sample. After 30 min the solvent was removed in vacua, and the residue was treated with hexane (0.2 ml). A portion (5 ,uI) of the extract was applied to a column (6 ft x r/4 in) of SE 30 (3”& on Chromosorb W at 150°, in conjunction with a hydrogen flame detector. Alter-

natively, a temperature gradient was used. The stream splitter at the column exit deflected most of the solvent from the detector, after which the splitter was rendered inoperative by closing one exit. This procedure gave higher sensitivity and increased resolution. The ratio of &D-glucose to a-D-glucose (R p/a) was determined from the relative peak areas. Carbohydrate Res., 3 (1967) 424-429

426

F.-W. -PARRISH, E. Ti REESE

Optical rotation, coupled to concurrent measurements of extent of hydrolysis, followed the principles of Weill et d4. and was recorded on a Bendix Automatic Polarimeter Type 143A at 27”. FEsuLTs

Any method for dete rmining the anomeric form in which glucose is released requires a highly active enzyme and a highly susceptible substrate to minimize the effect of mutarotation. The enzymes that yield &D-glucose caused most difbculty, in part because the equilibrium mixture j?/a ishigh (1.76 from optical rotation data’; 1.51 from g.l.c2; our value based on g.1.c. peak areas is 1.35). The result varies with different detectors, and standard mixtures were used for calibration. This, however, should be compensated somewhat by the lower rate of mutarotation of p-form to a-for&. A list of substrates that we have found suitable for our work is included in Table I. For exo-a-glucanases, starch was unsatisfactory, maltotriose of doubtful value, but maltodextrin mixture (maltotetraose and higher mol. wt. oligosaccharides) gave good results. For exo-/I-r,3-glucanases, the tetrasaccharide obtained from laminaran was better than the trisaccharide, but laminaran also gave satisfactory results. For &glucosidase, no substrate was very good. For a-glucosidases, maltotriose gave the best results. Minimal reaction time is important, since longer incubation periods give R B/a values approaching the equilibrium value, and it is then diflicult to determine whether inversion has occurred (Table I). Because of mutarotation, it is necessary to stop the reaction as soon as possible by freeze drying. Although requiring several h, this is done under conditions rchGmSng mu&rotation. The small sample, when dried quickly with a stream of air or by freeze-drying, gave satisfactory results (Table I). The purity of the enzymes is not of great importance. It is necessary that exo+r,3glucanase be free of /I-glucosidase, and vice versa, and similarly, that exo-a-glucanase and a-glucosidase be free of one another. That is, there should be but one enzyme present capable of acting on the substrate used. The enzymes used passed this test (except perhaps A. Zuchuensis where no purification was attempted). Six preparations examined for mutarotase activity all proved to be negative. The trimethylsilyl ethers of fructose and of the glucose anomers were well separated by isothermal g.1.c. the retention times, relative to that of a-n-glucose, for /I-D-fructose and &D-glucose being 0.73 and 1.58, respectively. With a constant temperature (150”) during elution, pyridine, trimethylsilylating reagents, and citrate formed early peaks which interfered with the a-peak in some ‘chromatographic systems. These were eliminated by replacing the pyridine with hexane or by the streamsplitting technique. In the g.1.c. of the hydrolyzate of sucrose, Qructose appears before a-Dglucose; and sometimes interferes. This interference was eliminated by temperature-pro gramming, a technique which also eliminated the pyridine and citrate problems. Both exo-p-r,3-glucanases gave glucose as the a-n-anomer, and both exo-a-Carboilydrare Res., 3 (1967) 424-429

427

ANGMJZRICFORM OF D-GLUCOSE

TABLE I OF CONFIGURATION BY CARBOHYDRASES

RJ=ENTION AND INVERSION Enzyme

Time

SubsImIe

(m in)

a-D-glucose,dry

None None None None Exe-j?-I,g-glucanases Sporotrichum

pruinosum

Basidiomycete

QM

826

sp. QM 806

&Glucosidases AspergiIIus niger QM 877 Aspergillus Zuchuensis QM 873 Almond emulsin (Richtmyer) Fxo-a-glucanases (glucamylase) AspergiIIns niger. (Diazyme 455) Endomyces sp. (Matulase) a-Glucosidases AspergilIus niger QM 877 Paecifomyces varioti QM IOO PeniciIIium parvum QM 1878

/SFructofuranosidase Invertase, powder NBC (melibiose-free)

5

0.05 0.48

IO 4000

1.05 I-35

0

cc-D-glucose in solution at room temperature a-o-glucose, in solution at 40” cc-D-glucose, at equilibrium

laminarane tetrasaccharide laminarane tetrasaccharide lanlinaran laqinarane tetrasaccharide laminaran

Iaminarane trisaccharide salicin

salicin

maltodextrius mahodextrins maltodextrins maltodextrins

R I% D- GIucose

10

o-39

30

o-93

IO

O-75

10 IO

o-59

6 5 4

I.59 I-55 1.60

0.46

:::

4-5 3.8 4-I 4.0

maltotriose maltotriose maltotriose

IO 6 IO

o-57 o-54 0.0

sucrose

I IO

0.44 0.50

,‘I

a Freeze-dried. @ Blown dry.

glucanases produced glucose as the B-D anomer (Table I, II). Thus, dl exo-glucanases brought about inversion in cotiguration. Three $-glucosidases, when used on susceptible substrates, seem to liberate B-D-glucose, but the values R B/a are not much above the equilibrium value (1.35). We believe that values within 0.2 of the equilibrium value (i.e., 1.35 + 0.2) are of questionable significance, and further attention is being given to the j?-glucosidases. The three a-glucosidases clearly produced cr-D-glucose from maltotriose. This substrate is more susceptible to these enzymes than is maltose, and has, in addition, the advantage that the aglycon is maltose not D-glucose. Invertase released D-glucose in the a-D form. . curbohydrute

Res., 3 (1967) 424-429

F. W.

428 TABLE INVJZRSION

PARRISH,

E. T. REESE

II OF CONFKiURATION

(OPTICAL-ROTATION

BY

MO-GLIJCANASES

rmmo0)

Enzyme

Substrare

F.x0+0,3hhmm3se

Iaminaran

Basidiomycete sp. QM 806 Sparotrichutn pruinosum QM 826

EXO-CU-gIUCanaSeS

Diazyme 455 Mat&se CakuIated values, for: al1 glucose is a-D-&cose glucose is an equilibrium mixture all glucose is /l-n-glucose

maltodextdo

ardue to

D-&COSp

fO.042 +0.039 -to.017 fO.023 to.056 to.026 +o.oog

aOptical rotation at 50% hydrolysis of substrate. The time required was less than 15 min at room temperature. DISCUS!XON

Determination of conjiguration. - G.1.c. has been found satisfactory for the measurement of a-D-glucose and fl-D-glucose released by enzyme action. The two forms are well separated, and interference by the buffer and by pyridine can be eliminated. The high equilibrium ratio generally makes it easier to detect the a than the jI form. Two optical-rotation methods have been used for determining configuration. One depends on the abrupt change in rotation (to the equilibrium value) brought about by addition of ammonia to the hydrolyzate. Whereas others516 have used successfully this method, we have observed changes of such small magnitude that we hesitate to draw conclusions from the data. The other method4 is based on simultaneous determination of the glucose produced and optical-rotation. The rotation observed is, of course, made up of contributions from all components present. In g.1.c. each specific component is determined separately. Glucosidases versus exe-gl~canases. - The present work is part of a study to emphasize the differences between glucosidases and exo-glucanases. As they have in common the ability to hydrolyze dimers and trimers of D-glucose, they have both been considered “glucosidases”. Yet they are clearly different in their action on long-chain po1ymer-s. The present work uses g.1.c. to conSrm earlier results (using optical rotation) showing inversion of configuration by exo-a-glucanases (glucamylases) and retention by a-glucosidase and by &glucosidase (the results are less satisfactory with j?-glucosidases.) We show for the first time that inversion accompanies the action of exo-/31,3&canases, i.e. that the glucan having only &D-linkages yields a-D-glucose. Li, Flora, and King’ have obtained inversion by an exo+glucanase of broad speciCarbohydrate Res., 3 (1967) 424429

ANOMERIC

429

FORM OF D-GLUCOSE

ficity, an enzyme associated with the hydrolysis of /3-I,4-ghrcans. It seems fair to generalize that glucosidases differ from exo-glucanases in that the former act to retain configuration, whereas the latter act in such a way as to invert it. In line with this generalization, pancreatic maltase is a true a-glucosidase, whereas the Takadiastase “maltase” (which produces inversion) appears to be an exo-a-glucanase6. Yeast invertase hydrolyzes5 sucrose by transferring the fructosyl moiety to water’. This leaves the ghrcosyl unit attached to the bridge oxygen atom in the a-Dform. The present data substantiate this explanation. Throughout all the work on configurational changes brought about by enzymes, the assumption is made that the resulting product is entirely of one form, i.e., all a-D- or all P-D. Certainly in transfer reactions (Le., glucosidases), all the products retain configuration, so although the assumption is probably correct, the data do not exclude the possibility that a very small percentage of product is of the unexpected form. ACKNOWLEDGMENT

Dr. George Dateo was of great assistance with the g.1.c. SUMMARY

G.1.c. offers a satisfactory method for determining the configuration of the D-glucose formed by enzymic hydrolysis of glucosides and of glucans. Exo-glucanases both a and j3,give inversion; glucosidases, both a and /3, give retention ofconfiguration. REFERENCES I z 3 4 5 6 7

R. C. E. C. L. E. D.

KUHN, Ber., 57 (1924) 1965. C. SWEELEY, R. B ENTLEY, M. MAKITA, AND W. W. WELLS, J. Am. Chem. Soc.,8g REESEAND M. MANDELS. Can. J. Microbial., 5 (rgsg) 173. E. WEILL, R. J. BURCH, AND J. W. VANKYK, Cereal Chem., 31 (1964) ISO. H. LI, R. M. FLORA, AND K. W. KING, Arch. Biochem. Biophys., II (1965) 439. BEN-GERSHOM AND J. LIEBOWITZ, Enzymologia, 20 (1958) x48. E. KOSHLAND AND SYLVIA STEIN, J. Biol. Chem., 208 (1954) 139.

Carbohydrate

(x963) 2497.

Rex, 3 (1967) 424-429