of a-amylase from thermomonospora

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1977, P. 391-397Copyright X) 1977 American Society for Microbiology

Vol. 34, No. 4Printed in U.S.A.

Production, Purification, and Characterization of a-Amylasefrom Thermomonospora curvata

J. L. GLYMPH AND F. J. STUTZENBERGER*Department of Microbiology, Clemson University, Clemson, South Carolina 29631

Received for publication 9 May 1977

Thermomonospora curvata produces an extracellular a-amylase. Maximalamylase production by cultures in a starch-mineral salts medium occurred atpH 7.5 and 53°C. The crude enzyme was unstable to heating (65°C) at pH 4 to 6,and was activated when heated at pH 8. The enzyme was purified 66-fold witha 9% yield and appeared homogeneous on discontinuous gel electrophoresis.The pH and temperature optima for activity of the purified enzyme were 5.5 to6.0 and 65°C. The molecular weight was calculated to be 62,000. The Km f6rstarch was 0.39 mg/ml. The amylolytic pattern consisted of a mixture ofmaltotetraose and maltopentaose.

A U.S. Public Health Service study (19) hasdemonstrated the feasibility of open-windrowcomposting for the conversion of biodegradablemunicipal solid waste to a usable product. Thefinished compost is useful as a soil supplementin strip mine reclamation and as an agricul-tural mulch (7). In the evaluation of finishedcompost quality, starch utilization has beenadvocated as a rate indicator in the compostingprocess and as one criterion in determining thebiological stability of the product (13, 26). Al-though many studies (10, 20-23) have beenpublished on some of the thermophilic cellulo-lytic flora isolated from the composting process,no report has characterized the amylase pro-duced by any ofthe isolates. Thermomonosporacurvata, a cellulolytic, thermophilic actinomy-cete frequently isolated from composting mate-rial, produces an extracellular amylase. Thepurification and characterization of this amy-lase was undertaken for two reasons: (i) theliterature contains no reports on the character-ization of highly purified amylases from ther-mophilic actinomycetes, and (ii) the study ofthe amylase would help to evaluate the role ofT. curvata in the starch degradation whichmust occur before the compost can be consid-ered a finished, marketable product.

MATERIALS AND METHODS

Culture maintenance. Cultures of T. curvatawere maintained at 4°C on a 1% agar mediumcontaining the constituents listed in the next sec-tion.Minimal medium for amylase production. The

minimal medium for amylase production contained:1% soluble starch (Fisher Scientific Co.) or 1%maltose, 0.2% (NH4)2SO4, 0.02% MgCl2 6H20), 0.1-

,ug/ml concentrations of biotin and thiamine, 10-iM CaCl2, and 10-1 M potassium phosphate (finalpH, 7.8). Appropriate amounts of dry carbohydrate,mineral salts, and vitamins were autoclaved sepa-rately and combined aseptically after cooling.Amylase production. Erlenmeyer flasks (500 ml)

containing 200 ml of medium were inoculated withapproximately 8 mg (dry weight) of washed inocu-lum. Cultures were routinely incubated at 50 to53°C in a New Brunswick G-76 rotary water bathshaker at 160 rpm for 3 days.Amylase assay. Amylase activity was measured

in magnetically stirred reaction mixtures contain-ing 2 ml of 4% soluble starch (Fisher), 1 ml of 1.0 Msodium acetate buffer (pH 6.0), and 0.6 ml of water.After equilibration in a water chamber maintainedat 6500 by a Haake FK2 constant-temperature cir-culator, the reaction was initiated by addition of 0.4ml of appropriately diluted enzyme. One-millilitersamples were drawn at zero time and after 10 minof incubation. Accumulation of reducing sugar wasestimated by the dinitrosalicylic acid method ofBernfeld (2). An amylase activity unit was definedas the release of 1 j,mol of reducing sugar (as glu-cose) per min.Amylase purification. After clarification by cen-

trifugation (17,500 x g at 4°C for 15 min), culturefluids were concentrated on an Amicon model TCF10ultrafiltration unit by using a membrane filter (PM-10; molecular weight exclusion of >104) under anatmosphere of N2 with a pressure of 25 lb/in2 at 60C.Approximately 3 liters of culture fluid could beconcentrated to 50 ml in 25 h. The concentratedculture filtrate was adjusted to pH 8 and stirredslowly at 20C as precooled ethanol (95%, -10°C)was added dropwise to a final concentration of 75%.The precipitate from this step was redissolved in 4ml of 0.01 M phosphate buffer, pH 8, and applied toa Sephadex G-150 column (1.5 by 60 cm) equilibratedat 60C with a 0.01 M phosphate-0.1 mM calciumacetate buffer, pH 8, containing 0.02% sodium azide.Fractions (3 ml) were collected at a flow rate of 15

391

392 GLYMPH AND STUTZENBERGER

ml/h; those containing amylase activity were com-bined and applied to diethylaminoethyl (DEAE)-cellulose (Sigma, medium mesh) equilibrated withthe same buffer in a column (1.5 by 30 cm) at 6°C.Fractions (2 ml) were collected during elution witha linear NaCl gradient (0 to 0.6 M) in buffer at aflow rate of 12 ml/h. Fractions with the highestspecific activity were combined and concentrated onan ultrafiltration unit (model 12, Amicon) with amembrane filter (UM-10) under the same conditionsas previously described.

Electrophoresis. Polyacrylamide gels, preparedaccording to the procedure of Davis (4), were usedin discontinuous electrophoresis equipment (model12, Canalco). Standard 7% gels were prepared intubes (0.5 by 7.5 cm) and run at pH 8.3. Samples(0.2 ml) containing about 100 ,ug of protein weremixed with a 40% sucrose solution and pipetteddirectly onto the stacking gels. A 1-mA current wasapplied to each tube until the tracking dye enteredthe separating gel. The current was then raised to 3mA per tube for the remainder of the run (approxi-mately 3 h). Gels were stained with either amidoblack or Coomassie brilliant blue R250 and de-stained electrophoretically (amido black), with 7%acetic acid as the electrolyte, or by diffusion (Coo-massie brilliant blue) at 25°C in the same solvent.Unstained gels were sliced into sections correspond-ing to the electrophoretic pattern of stained gels,macerated in tubes containing 2 ml of buffer, storedovernight at 2°C, and assayed for enzyme activity.The eluate from active slices was pooled and concen-trated. Gels were stained for carbohydrate by theprocedure of Zacharius et al. (30).Chromatography of hydrolysis products. The en-

zymatic action on starch was followed by a methodsimilar to the procedure of Welker and Campbell(29). Samples (0.5 ml) from standard amylase assaymixtures were removed at 5-min intervals up to 30min and placed in precooled tubes in an ethanolbath at -20°C. Samples were thawed and heated ina boiling water bath for 7 min, deionized with 2 g ofa mixture (1:1) of Amberlite 1R-4B and IRC-50chemically pure resin, and placed at 2°C overnight.Resin was removed by centrifugation at 14,500 x gat 4°C, and unknown samples (10 1I), along withthe reference sugars (glucose, maltose, and malto-triose at 1% concentrations) were spotted 4 cm aparton Whatman no. 3 chromatography paper (24 by 30cm). Chromatograms were run for 4 h with a solventconsisting of n-butanol-pyridine-water (6:4:3) andair dried. Chromatograms were redeveloped for atotal of five ascents. Reducing sugars were detectedby the silver-dip method of Mayer and Larner (16),modified as follows. Solution A was prepared bydiluting 2 ml of saturated AgNO3 to 12 ml withdistilled water and then to 400 ml with acetone.Solution B contained 100 ml of 10% aqueous solutionof 0.05 M sodium thiosulfate. Solutions were pouredinto separate pans, and the paper was dipped rap-idly in solution A and allowed to dry. The paperwas next dipped into solution B until the character-istic black spots appeared (about five dips). Afterwashing in tap water for 1 h with continuous flow,the chromatogram was dipped into solution C until

the background coloration disappeared (about 5min). A 60-min washing in tap water with continu-ous flow yielded a stable chromatogram.

Determination of molecular weight. The sodiumdodecyl sulfate-polyacrylamide gel method of Weberand Osborn (27) was used. Gels (10%) were preparedin tubes (0.75 by 10.2 cm). Protein samples wereprepared according to method 1 in the presence ofsodium dodecyl sulfate at 100°C for 3 min. A 1-mAcurrent was applied per tube until the tracking dyeentered the sample gel (1.5 h), at which time thecurrent was raised to 3 mA per tube. The averagetime for all runs was 4.5 h. Gels were stained withCoomassie brilliant blue R250 (Bio-Rad) and de-stained by diffusion. Proteins of known molecularweight, bovine serum albumin (0.5 mg/ml), ovalbu-min (1 mg/ml), pepsin (1 mg/ml), and chymotrypsin(1 mg/ml), served as reference markers.

RESULTSConditions for maximal amylase produc-

tion. The pH and temperature requirementsfor amylase production in maltose-mineralsalts-vitamin medium are illustrated in Fig. 1and 2, respectively. During variation of bothparameters, the accumulation ofamylase activ-ity in culture fluids was proportional to growth,the optima being pH 7.5 and 53°C.Heat stability of crude amylase. During

testing of the heat stability of the crude amy-lase composting temperatures (60 to 70°C), theenzyme was rapidly inactivated at pH 4 to 5,relatively stable at pH 6 to 7, and activated atpH 7 to 8 (Fig. 3). When incubated at 65°C and

6 7 8p H

FIG. 1. Effect ofpH on amylase production, accu-mulation ofextracellular protein, and dry cell weightafter 3 days of incubation in shaken culture, 50°C,with 1 % starch as carbohydrate source.

APPL. ENVIRON. MICROBIOL.

AMYLASE OF T. CURVATA 393

TEM PERATURE(C)

FIG. 2. Effect of temperature on amylase produc-tion, extracellular protein concentration, and drycell weight after 3 days of incubation in stationaryculture with 1% starch as carbohydrate source.

o=ACETATE *= PHOSPHATEro I IT

pH 4.0 pH 6.0

1.0C __0 -

o 2=a In I

pH 7.0

*--

pH 6.0 pH &0

_ _. _ _ A Ae_!1-0-0 /0-0---.

I[ IL IL..I ...

h 1.CA0

1.

0510 30 0510

TIME(MINUTES)

FIG. 3. Effect ofpH on the heat stability of crudeamylase. AhIAo is the ratio of heated (65°C) sampleactivity to that ofan unheated control.

pH 5.0

S.°'. ~

pH 8 for 5 to 10 min before mixing with sub-strate, an increase in activity of about 60%was observed when later measured in the stan-dard reaction mixture.

Purification of the amylase. A summary ofresults from the purification procedure is givenin Table 1. A 66-fold purification was achieved,with a yield of 9% of the original activity. Thepurified preparation appeared to be homogene-ous on discontinuous gel electrophoresis (Fig.4), and all detectable amylase activity in thegel coincided with a single protein bond. Theamylase from this section of the gel could bequantitatively eluted (>85%) by maceration inhydroxymethylaminomethane-glycine buffer,pH 8.3, and allowing the elution to proceedovernight at 20C.Temperature and pH optima. Under the

conditions of the routine assay system, theoptimal temperature for the purified amylasewas 650C (Fig. 5). The Q,o values (calculatedfrom triplicate experiments) for the range 45 to650C varied from 1.75 to 1.82. The enzymeexhibited a rather broad pH optimum in therange ofpH 5.5 to 6.5, with half-maxima at pH4.8 and 7.5 (Fig. 6). The purified enzyme wasrapidly inactivated at pH values below 7.0when held at 650C. At pH 7 to 8, the enzymewas stable, but did not show the heat activationobserved with the crude preparations.

In stability studies on microbial thermosta-ble amylases (1, 3), the presence of bovineserum albumin (BSA) afforded substantial pro-tection against heat denaturation. However,the presence of BSA at 650C caused no changein the heat stability of the purified T. curvataamylase, and at 900C it caused more rapidinactivation. This deleterious effect is shownin Fig. 7. The enzyme in the presence of BSAwas completely inactivated within 5 min at900C, whereas enzyme alone or in the presenceof substrate lost only about 40% of its activity.The presence ofthe substrate had no significantprotective effect.

TABLE 1. Purification summary of amylase from T. curvata

Step Vol (mI) Total activity Total pro- Sp act Putrifica Yield (Step Vol (ml) ~~(U) tein (mg) tuiona Yed %

(1) Crude culture fil- 2,733 12,300 752 16.35 1 100trate

(2) Retentate from 50 12,500 60 208 12.7 100PM-10 filter

(3) Ethanol precipi- 4 6,480 13 498 30.5 53tate at 75% satu-ration

(4) Eluted from 9 2,313 3.8 608 37 19Sephadex G-150

(5) Eluted from 14 1,086 1.0 1,086 66 9DEAE-cellulose

VOL. 34, 1977

2


i yielded a Km of 0.39 mg/ml. Reaction rates forthe purified enzyme with either starch or amy-lose were initially linear under standard assayconditions (Fig. 9). The enzyme caused no de-tectable hydrolysis of maltose or maltotriose.

Molecular weight determination. Electro-phoretic mobility profiles of the known molecu-lar weight proteins, together with the purifiedamylase in SDS-gel electrophoresis, are shownin Fig. 10. From these results, the molecularweight of the amylase was calculated to be62,000 at pH 8.0.Determination of the amylolytic pattern.

Chromatography of products of the amylaseacting on starch for 10, 20, and 30 min underthe routine assay conditions yielded only twospots with identical Rf values (0.29 and 0.39)for all three reaction times. In various studies(8, 24, 28) on the correlation of carbohydrate

100-

80~~~~~_,~~~~~~~~~~~ 0

FIG. 4. Protein patterns from discontinuous gelelectrophoresis studies on (a) concentrated crude <60culture fluids, (b) precipitate from ethanol treatment,(c) combined fractions from gel filtration, (d) com- <40-bined fractions from DEAE-cellulose.

2 0

100- 04 5 6 7 6

_ pH

FIG. 6. EffectofppH on amylase activity. Reactions5~~~~~cteuids, were buffered with0e25 M acetate or0t25 M phos-(600 phate and incubated for 10 min at65Ci at each pHbined0ractionsfromDEAE-cellulose./value.

0~~~~~

20-~~ 20 °

20 100 O~~~~~~~~~*AMYLASE0 AMYLASE+ STARCH(1O%)

40 50 60 70 0TEMPERATURE (C) >- 80

FIG. 5. Effect of temperature on amylase activity. >Reaction mixtures were buffered with 025 M acetate or0uat pH 6 and incubated for 10minat each tempera- <pHture. 0v

Kinetic parameters. Although the Line- < 40weaver-Burk linear transformation of the Mi-chaelis-Menten equation is most commonlyused for the calculation of Km and Vm, the 20Hofstee transformation (9) has been shown tobe the most reliable and discerning method forthe calculation of the kinetic parameters (6). ~ 102 306

40~~~~~~~~~~~500 2070 80

When reaction rates for the purified amylase TIME MINUTES)were measured over a 100-fold range of starch FIG. 7. Thermal inactivation of amylase at 90'Cconcentrations, under standard assay condi- in 0.01 M phosphate-0.01 M calcium acetate buffer,tions, a Hofstee plot of V versus V/S (Fig. 8) pH 8.0.



YS

FIG. 8. Hofstee plot for the determination of theKm value for starch with the purified amylase. TheV values are expressed as micromoles of reducingsugar (as glucose) released per minute under stan-dard assay conditions. The S values are expressedas percent starch. The line was fitted to the points bythe least-squares method for unweighted data.

10TIME (MINUTES)

FIG. 9. Time course of reducing sugar liberationby amylase from starch (29%) and amylose (1.29%)under standard assay conditions of65°C andpH 6.0.

structure with paper chromatogram mobility,a plot of log(Rf /1 - Rf) against the degree ofpolymerization of oligosaccharides was astraight line. When Rf values of glucose, mal-tose, maltriose, and the two unknown spotswere subjected to this treatment, a straightline was formed (Fig. 11). The two unknownspots appear to be maltotetraose and maltopen-taose when estimated in this manner.

DISCUSSIONThe requirements for amylase production by

T. curvata are markedly different from those

ofthe closely related Thermomonospora viridis(25) and Thermoactinomyces vulgaris (11).Both T. viridis and T. vulgaris required acomplex nitrogen source such as peptone foramylase production, whereas T. curvata pro-duced high levels of the enzyme in a chemicallydefined medium. The crude amylase of T. cur-vata is unusual in its heat stability as relatedto pH. It was inactivated by heating at 650C atpH 4 to 6 and significantly activated in the pHrange of 7 to 8. The amylase of T. vulgaris hasopposite requirements for heat stability, beingmore stable at pH 5 to 6.5 and rapidly inacti-

87- BSA

'06 RAmylase

Ov

(D ~~~~~~PE(4

-J CTD2

-j0

O Q2 0.4 0.6 0,8 1.0MOBILITY

FIG. 10. Standard curve for proteins of knownmolecular weight determined by SDS-gel electropho-resis (BSA, bovine serum albumin; OV, ovalbumin;PE, pepsin; CT, chymotrypsin) used in calculationofamylase molecular weight.

GLUCOSE UNITS

FIG. 11. Correlation of amylase reaction products(UKI and UK2) with known reference sugars (Gl,glucose; G2, maltose; G3, maltotriose).

VOL. 34, 1977


vated at pH 7 to 8. The temperature optimumfor amylase production by T. curvata is 530C;during the first 7 to 10 days of the compostingprocess, the average temperatures of municipalsolid waste reach 50 to 600C to provide thatoptimum (20). By that time, the average pH ofthe refuse has reached the range of 7 to 8 (19),which also encompasses the optimum for amy-lase production by T. curvata. Therefore, itwould appear that in these respects compostingconditions during the early stages ofthe processwould be the most favorable for starch degra-dation by T. curvata. During analysis of carbo-hydrate content in composting (19), the solublesugar content (initially about 0.8%) was rapidlydepleted, followed by starch (from 4% initiallydown to less than 1.0% by 28 days), and finallyby cellulose (from 50% initially down to 30% atthe end of the 49-day process). T. curvata,which can utilize all three classes of carbohy-drates and which could probably establish siza-ble population densities early in the process,has the potential of playing a major role in therate of conversion to a finished product.The purified amylase described here has a

pH optimum of 5.5 to 6.0 and a temperatureoptimum of 650C. In this regard, it appearssimilar to the amylases from other therno-philes (1, 18, 12). However, the Km for starch,0.39 mg/ml, is considerably lower than thatreported for thermostable amylases from Ther-momonospora vulgaris, 1.4 mg/ml (1), Bacillusmacerans, 3.3 mg/ml (5), -or Bacillus stearo-thermophilus, 1.0 mg/ml (15). A molecularweight of 62,000 was calculated for the amylaseby SDS-gel electrophoresis. We could find noreport of a molecular weight for a purifiedamylase of another thennophilic actinomycete.However, the amylase of Aspergillus niger(which is also unstable at acidic pH, like theT. curvata amylase) has a molecular weight of61,000 (17), and most bacterial amylases havemolecular weights in the range of 50,000 to60,000.One unusual feature of the amylase from

T. curvata is its amylolytic pattern, consistingof oligosaccharides that appear to be malto-tetraose and maltopentaose. In contrast, thepartially purified amylase from T. vulgaris (1)produced only glucose and maltose from solublestarch. This apparent difference between amy-lases from closely related species may be dueto the presence of an associated a-glucosidasethat hydrolyzes the larger oligosaccharides toglucose and maltose in the T. vulgaris amylasepreparations. A comparison of the action pat-terns of pure amylase preparations from thetwo species on a variety of malto-oligosaccha-rides is needed to resolve this question.

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16. Mayer, F. C., and J. Larner. 1959. Substrate cleavagepoint of the alpha and beta amylases. J. Am. Chem.Soc. 81:188-193.

17. Minoda, Y., M. Arai, Y. Torigoe, and K. Yamada.1968. Acid-stable a-amylase of black Aspergilli. PartIII. Separation of acid-stable a-amylase and acid-unstable a-amylase from the same mold amylasepreparation. Agr. Biol. Chem. 32:110-116.

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of a-amylase from thermomonospora

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