Purification and characterization of a xylanase and an arabinofuranosidase from Bacillus polymyxa

Purification and characterization of a xylanase and an arabinofuranosidase from Bacillus polymyxa Pilar Morales, Alejo Madarro, Agusti Flors, Jose M. Sendra and JosC A. Ptkez-GonzBlez Departamento de Biotecnologia, Institute de Agroqui’mica y Tecnologia Superior de Investigaciones Cientljcicas, Valencia, Spain

de 10s Alimentos,

Consejo

Two hemicellulases from Bacillus polymyxa were purified and characterized: a xylanase with a molecular mass of 61 kD and pI of 4.7 and an arabinofuranosidase with a molecular mass of I66 kD and pl of 4.7. The xylanase. which showed increased from several

sources.

thermostability

on linear (1+5)-a-L-arabinan, from arabinoxylan

Keywords:

in the presence

The arabinofuranosidase arabinogalactan,

when an active endoxylanase

Bacillus polymyxa; xylanase;

of M&l,,

showed a @pica1 endo-action

mode on xylans

was only active on (145)~a+arabinooligosaccharides and arabinoxylan. was also present

arabinofuranosidase:

Introduction Many microorganisms exhibit a complex system of enzymes involved in the degradation of hemicellulose. The heterogeneous structure of xylan, the major component of hemicellulose, is responsible for the observed diversity of these enzymes. The xylan backbone, constituted of P-Dxylose residues linked by (1+4) bonds, is degraded by xylanases (1,4-P-D-xylan xylanohydrolase, EC 3.2.1.8) and P-xylosidases (1,4-P-D-xylan xylohydrolase, EC 3.2.1.37). Arabinofuranosidases (a-L-arabinohydrolase, EC 3.2.1.55) split the a(l,3) bonds between the xylose chain and r_-arabinofuranose residues of some xylans. Other less well-characterized enzymes are involved in the cleavage of different xylan substituents. ’ The xylanase systems of some fungi and bacteria have been exhaustively studied [for a review, see Coghlan and Hazlewood’]. The large number of potential applications of these enzymes in several fields (food and feed industries, paper pulp treatment, and removal of residues from agricultural and forestry activities) is one of the main reasons for this, but an understanding of how several enzymes with

purification;

However,

it was able to release

but not arabinose

in hydrolysis assays.

characterization

different catalytic and physical properties and substrate specificities could contribute to the degradation of heterogeneous polymers such as the native xylans is also important. Species of the genus Bacillus secrete a large number of industrially relevant enzymes. However, few Bacillus hemicellulolytic enzymes have been studied in comparison to those of other genera. We have focused our attention on the xylanase system of Bacillus polymyxa, which comprises a broad range of enzymes involved in xylan degradation, some of which have not been described in other Bacillus species. Three alkaline xylanases have already been purified and characterized from this microorganism.3 The cloning and sequencing of an arabinofuranosidase gene with two peptide forms has also been reported by our group,4 and the two peptides are now being characterized. Other author? have reported the cloning of an acidic xylanase (PI 4.9) with a molecular mass of 48 kD. We present here the purification and characterization of a second acidic xylanase and an acidic arabinofuranosidase from B. polymyxa.

Materials and methods Bacterial

culture

Bacillus polymyxa CECT 153 was obtained from the Spanish Type Address reprint requests to Jose A. PCrez-Gonzllez. Biotecnologia, Instituto de Agroquimica y Tecnologia CSIC, Jaime Roig 11, 46010 Valencia, Spain Received 16 April 1994; accepted 3 October 1994

Departamento de de 10s Alimentos,

Enzyme and Microbial Technology 17:424-429,1995 0 1995 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

Culture Collection. For production of the extracellular xylanolytic complex, B. polymyxa was grown at 30°C in the Bacillus medium described by Robson and Chambliss.6 Whole medium (3 1) containing 7.5 g I-’ xylan from oat spelts (Sigma) as a carbon source

0141-02291951$10.00 SSDI 0141-0229(94)00062-V

Xylanase was adjusted to pH 7.2 with NaOH and sterilized at 120°C for 30 min, cooled to room temperature, and inoculated with 1% bacterium culture in late logarithmic growth phase. Growth was carried out in an orbital incubator (Gallenkamp) with agitation (200 ‘pm) for 40 h.

Separation

and concentration

of culture liquid

Culture medium was clarified by centrifugation. The supematant was then concentrated 35-fold (final volume, 80 ml) and dialyzed against 20 mM Bis-Tris buffer (pH 6.3) in an Amicon unit cartridge with a 50,000 mol wt cut-off. This concentrate was used as the source to carry out the purification of enzymes reported in this study.

Purification

of enzymes

The concentrated dialyzed culture fluid was applied to a DEAEBiogel A column (2 . I5 cm) previously equilibrated with 20 mM Bis-Tris buffer (pH 6.3). Elution was carried out at a flow rate of 42 ml h ’ with equilibration buffer. Once a stable basal A280 was established, an NaCl gradient of 0 to 1 M in the same buffer was applied over a 2-h period. Fraction volumes of 4.2 ml were collected. Fractions with xylanase and arabinofuranosidase activities were pooled. concentrated. and dialyzed against 20 mM l-methylpiperazine buffer (pH 5.7) in an ultrafiltration unit (Filtron) with a 10.000 mol wt cut-off membrane. Fast protein liquid chromatography (FPLC) was carried out with a biocompatible high-pressure liquid chromatography system from Kontron. The first step was chromatofocusing on a prepacked Mono P HR 515 column (Pharmacia). The eluents used were: buffer A (25 mM 1-methylpiperazine. pH 5.7) and buffer B (a IO-fold dilution of Polybuffer 74; Pharmacia). A flow rate of 0.5 ml min _ ’ was used. Stepwise elution was carried out using buffer A for the initial 5 min. switching to 100% buffer B for the duration of the chromatography. The peak containing both xylanase and arabinofuranosidase activities was pooled, concentrated, and dialyzed against 20 mM 1-methylpiperazine buffer (pH 5.7 I as described in the previous step. The second FPLC step employed a Mono Q HR 515 column (Pharmacia). The eluents used were: buffer C (20 mM l-methylpiperazine. pH 5.7) and buffer D ( 1 M NaCl in buffer C). A flow rate of 0.5 ml mini ’ was used. Elution was carried out with buffer C for the first 2 min after injection, a linear gradient from 0 to 45% buffer D for the following 21 min, and 100% buffer D for the last 5 min. Xylanase and arabinofuranosidase activities were detected in different peaks. Fractions were pooled separately. concentrated, and dialyzed against 20 mM I -methylpiperazine buffer (pH 5.7 as described previously. The pool containing X6 I was rechromatographed under different conditions. The eluents used were: buffer E (IO mM l-methylpiperazine. pH 5.7) and buffer F ( I M NaCl in buffer E). Elution was carried out with buffer E for the first 2 min after injection, a linear gradient from 0 to 30% buffer F for the following 14 min. and a wash of 100% buffer F for the last 5 min.

Gel electrophoresis focusing (IEF)

and analytical isoelectric

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) was performed as described previously.’ Native electrophoresis was performed by the method of Davis.8 IEF was carried out on precast gels with a pH gradient from 3.5 to 9.3 (PharmaciaLKB) following the instructions of the manufacturer. To detect xylanase activities, SDS-PAGE of the samples was carried out on gels incorporating 0.2% oat spelt xylan. Immediately after electrophoresis. SDS was removed by soaking the gel in

characterization:

P. Morales

et al.

50 mM phosphate buffer (pH 6.5) containing 2.5% Triton X-100 for 30 min. The gel was then incubated for 15 min at 50°C and stained with Congo Red by the method of Begum.’ To detect arabinofuranosidase activities, the polyacrylamide gels (after SDS removal) were overlaid on 1.25% agarose detection gels that contained 3 mu p-nitrophenyl-cx+arabinofuranoside (pNPA) in 50 mM phosphate buffer (pH 6.5). The sandwich was incubated for 15 min at 50°C. Arabinofuranosidase activity was visualized as a yellow band in a colorless gel.

EnLyme assays The xylanase activity of X61 was measured by mixing a volume of an appropriate dilution of the enzyme ( 1.35 pg ml- ’ final concentration) with a suspension of oat spelt xylan (60 mg ml- ’ final concentration) in 50 mu MES (2-N-morpholino-ethanesulfonic acid) buffer (pH 6.5) containing 5 mM MgC12. The mixture was incubated for IO min at 50°C. The reducing sugars formed were measured, as xylose equivalents, by the Somogyi method.“’ Blanks were prepared by incubating enzyme and substrate solutions separately. One unit of activity was defined as the amount of enzyme releasing I pmol of reducing sugar equivalent min _ ’ In some cases, remazol brilliant-blue xylan was used as substrate at a final concentration of 16 mg ml ’ in the same buffer. For the latter, 2 vol of ethanol was added after incubation. and precipitated xylan was removed by 5 min centrifugation in a microcentrifuge. Dye release was measured at 590 nm. The arabinofuranosidase activity of AF166 was measured by mixing a volume of an appropriate dilution of the enzyme (90 ng ml ’ final concentration) with a solution of a p-nitrophenyl or methylumbelliferyl derivative (0.8 mg ml - ’ final concentration) in 30 mM MES buffer (pH 6.5). The mixture was incubated for 8 min at 50°C. One volume of 1 M Na,CO, was added to the sample. The release of p-nitrophenol was measured at 420 nm and the release of methylumbelliferone at 365 nm. Blanks were prepared by incubating enzyme and substrate solutions separately. One unit of activity was defined as the amount of enzyme releasing I p.mol of p-nitrophenol or methylumbelliferone min - ’

Measurement

of enzyme properties

The effect of temperature on reaction rates was assessed by incubating reaction mixtures at different temperatures in the range from 30 to 65°C. For the corresponding study of pH effects, several buffers adjusted to different pHs in the range 5.5-8.5 were used. The influence of metallic ions on activity was determined. The thermostability of enzymes was monitored by incubating the reaction mixtures at a fixed temperature and removing aliquots at intervals to test the activity. In the case of X61, the effect of MgClz on thermostability was also studied. The effect of substrate concentration on X61 was studied with I .35 p,g ml- ’ of enzyme in 50 mM MES buffer (pH 6.5) containing 5 mM MgCl? and oat spelt xylan at a final concentration rangingfromOto80mgml-‘. Samples were incubated for 10 min at 50°C. The effect of substrate concentration on AF166 was studied with 90 ng ml-’ of enzyme in 50 mM MES buffer (pH 6.5) and pNPA at a final concentration ranging from 0 to 1.8 mg ml _ ’ Samples were incubated for 8 min at 5O’C.

Enzymatic hydrof_vsis of xvlans, xylooligosaccharides, and arabinooligosaccharides The analysis of products from the hydrolysis of xylans from different sources was performed by HPLC on a Sugar-Pack 1 column (Waters) placed in an oven. The temperature of the column was adjusted to 85°C. and that of the refraction index detector (Waters) to 40°C. The eluent used was water at a flow rate of 0.5 ml min _ ’

Enzyme

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1995, vol. 17, May

425

Papers Twenty-microliter samples from reaction mixtures were injected. Xylose, arabinose (both from Sigma), and (1+4)-p-~xylooligosaccharides of 2 to 4 xylose units in length and (l+S)a-L-arabinooligosaccharides of 2 to 8 arabinose units in length (all from Megazyme) were used as standards. Oat spelt xylan, 4-O-D-methyl-D-glucurono-D-xylan from larchwood, birchwood xylan (all from Sigma), wheat flour arabinoxylan (from Megazyme), or sugar beet linear (l-+5)-cu-L-arabinan (from Megazyme) were used as substrates. The reaction mixtures contained 5.5 p,g tn-10fAF166and/or4.2~gml~‘ofX61, 16mgml-‘ofone of the mentioned xylans, or 3 mg ml - ’ of arabinan, 50 mM MES buffer (pH 6.5), and 5 mu MgCl, in the case of X61. Reaction mixtures were incubated for 20 h at 50°C. Similar mixtures and hydrolysis conditions were performed to determine the products of hydrolysis of (l-+4)-B-D-xylooligosaccharides and (1+5)-o-~arabinooligosaccharides using a substrate concentration of 2 mg ml - ’ in these cases.

Analytical

methods

Protein concentration was measured either by the method of Bradford” or by A280 nm using albumin as standard.

A

205 116 97.4 66

45

Figure 1 SDS-PAGE analysis of purified enzymes. (A) Lane 1, xylanase X61; lane 2, molecular mass marker proteins. (B) Lane 1, nonboiled arabinofuranosidase AF166; lane 2, boiled arabinofuranosidase AF166; lane 3, molecular mass marker proteins. The numbers to the right are the values in kilodaltons of the molecular mass marker proteins

Results and discussion Production

of activities by B . polymyxa

Zymogram analysis of cell-free culture samples showed that B. polymyxu produces at least five xylanase activities of molecular masses 22, 34, 48, 61, and 120 kD under the conditions used for the culture of this bacterium.12 The 61 kD xylanase enzyme, names X61, seemed to have a high activity, and has not been studied previously. Zymogram analysis of a-L-arabinofuranosidase activity using nonboiled cell-free culture samples showed two bands corresponding to proteins of 166 and 140 kD (not shown). Neither of the a-r_-arabinofuranosidases, herein named AF166 and AF140, has been previously described.

centrated culture supematant, although the final activity recovered was low (0.5%). This may be due to the presence of multiple xylan-reducing-sugar-releasing activities in the concentrate, although most of the low-molecular-weight enzymes were removed during the concentration step itself. AF166 was purified 145fold over the specific activity determined in supematant. Although little arabinofuranosidase activity was lost during supematant concentration, as can be deduced from Table 1, a much higher loss of activity was observed after the chromatofocusing step. The multimerit nature of the enzyme could be responsible for both phenomena.

Enzyme purification

Characterization

X61 and AF166 enzymes were purified from B. polymyxa extracellular culture fluid by anionic exchange chromatography on DEAE-Biogel A, chromatofocusing and anionic FPLC on a Mono Q column, as described in Materials and methods. Both enzymes coeluted in the first two steps. They were detected in the same adsorbed and NaCl-eluted fractions from DEAE-Biogel A chromatography. A pH gradient from 5 to 4 applied to a Mono-P chromatofocusing column was unable to separate the activities, both of which were detected in the same protein peak. This confirmed the similarity of the pIs of both proteins previously observed in preliminary IEF experiments. However, the chromatofocusing step was very efficient in removing contaminating proteins. AF166 was subsequently purified in a single step on a Mono-Q column from which the two activities were detected in separate protein peaks. It was necessary to carry out an extra chromatographic step in the same column using different conditions (see Materials and methods) to remove contaminants from the peak containing X61. Both enzymes seemed to be purified to homogeneity, as deduced from SDS-PAGE analysis (Figure I). A summary of the purification of both activities is presented in Table I. X61 was purified 30-fold over the specific activity detected in con-

X61 showed a single protein band by SDS-PAGE analysis corresponding to a molecular mass of 61 kD (Figure IA). Gel filtration chromatography indicated that the enzyme may be in a dimeric form. Zymogram analysis performed with boiled and nonboiled samples suggested that both the dimeric and monomeric forms were active (not shown). With regard to the arabinofuranosidases, some of those reviously described are composed of several subunits. *i: A single protein band corresponding to a molecular mass of 166 kD was seen in an SDS-polyacrylamide gel when a nonboiled sample of AF166 protein was loaded. However, two protein bands corresponding to molecular masses of 65 (more intense band) and 33 kD could be seen when the AF166 sample was boiled prior to loading (Figure 1B). Nondenaturing PAGE analysis also showed that a single band and gel filtration chromatography estimation of AF166 molecular mass was about 180 kD. A single positive signal corresponding to a protein of similar size could be detected by zymogram analysis (not shown). All of these data suggest that the native enzyme could be constituted by two polypeptides, one of 65 kD and another of 33 kD, giving a total mass of 163 kD, in good agreement with the 166~kD value determined by SDS-PAGE of the nonboiled sample.

426

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of X61 and AF166

Xylanase Table 1

Purification

P. Morales

et al.

balance of X61 and AF166 Xylanase

Protein

Supernatant Concentrate DEAE-Biogel A Chromatofocusing 1 Mono Q-AF166 1 Mono Q-X61 2 Mono Q-X61 PF, Purification

characterization:

Arabinofuranosidase

activity

Total (mg)

%

U rng-’

Recovery (%I

PF

1,173 552 118 5.7 0.088 0.68 0.092

100 47 IO 0.5 0.007 0.06 0.008

4.5 1.1 0.76 IO

100 14.8 9.4

1 0.69 9.1

19.6 33

2.2 0.5

U rng-’ 0.98 1.78 4.7 9.7 142

activity

Recovery (%)

PF

100 85.5 48.3 4.8 1.1

1 1.82 4.8 9.9 145

17.8 30

fold

IEF analysis of both X61 and AF166 enzymes revealed an identical p1 of 4.7. The temperature optima determined for both enzymes were 5O”C, and the pH optima were also identical for both activities, 6.5. In comparison, the enzymes differed in their thermostability. AF166 was more thermostable than X61 (Figure 2), although the xylanase showed an increase in its thermostability in the presence of MgCl,. From an analysis of the effects of some metal ions on the activities of both enzymes it was concluded that AF166 was unaffected by most of the metals tested, except for Cu*+ , which partially inhibited this enzyme. X61 activity was enhanced in the presence of Mg*+ ions, with 5 mM being the most effective concentration. The increase in xylanase activity in the presence of this metal ion was not high (122% over the control without Mg*+), but 5 mM MgCl, was included in all experiments mainly because of the thermostability conferred to the enzyme. When the effect of substrate concentration on the enzyme activity was studied, a decrease in reaction rates was observed for X61 at substrate concentrations >32 mg ml _-‘, yielding a curve that suggested classical substrate inhibition. The apparent kinetic constants K,,, and V,,, of X6 1 for the hydrolysis of oat spelt xylan were 17.7 mg ml - 1 and 112.0 U of xylanase activity mg- ’ of protein, respectively,

0

10

20 30 40 50 60 time (min)

Figure 2 Thermostability of X61 (0, V), X61 in the presence of 5 mM MgCI, (0) and AF166 (0, v) enzymes. The enzymes were incubated at 45°C (0,O) and 55°C (V, ‘I, 0) for the times indicated prior to activity assays

and a Ki of 33.7 mg ml-‘, as calculated from the fitted equations for this type of kinetics, by parametric regression analysis. Substrate inhibition has also been suggested for the kinetics observed for xylanase 2 of Fibrobacter succinogenes. l4 The kinetic constants K, and V,,, of AF166, 0.324 mg ml- ’ and 214.1 U of arabinofuranosidase activity mg-’ of protein, respectively, were calculated according to the Michaelis-Menten kinetic equations using pNPA as substrate. The effects of xylose and xylobiose on the xylanase activity of X61 was also studied using Remazol brilliant-blue modified xylan as substrate. Whereas xylobiose had no effect on enzyme activity. xylose showed a stimulatory effect on the enzyme, with an increase of 50% in the rate of xylan hydrolysis when xylose concentrations ranging from 100 to 200 pg ml _ ’ were used. The substrate specificity of both enzymes is summarized in Table 2. X61, which was only able to hydrolyze xylan, showed similar specificity for xylans from different sources and of different chemical structures. AF166 acted on arti-

Table 2 Specific activity (U mg -‘) of purified enzymes on different substrates Substrate=

X61

AF166b

Oat spelts xylan Birchwood xylan 4-O-Methyl-glucuronoxylan Arabinoxylan Arabinan Arabinogalactan p-Glucan Carboxymethylcellulose p-NP-u-L-Arabinofuranoside p-NP-P-o-Xyloside p-NP-P-o-Mannopyranoside p-NP-a-L-Mannopyranoside MU-c*-L-Arabinopyranoside MU-p-o-Xyloside MU-p-o-Glucopyranoside

33.0 42.7 43.0 38.0 0 0 0 0 0 0 ND ND 0 0 ND

0 0 0 0 0 N”I, ND 89.0 0 0 0 45.0 0 0

ND, Not determined ‘The concentration of polymers in assays was 16 mg ml -’ and the concentration of artificial substrates, 0.8 mg ml-’ bThe incubation time of reaction mixtures containing AF166 and one of the polymers was extended for 4 h. The other assays were made as described in Materials and methods.

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Papers ficial arabinose derivatives such as pNPA and methylumbelliferyl-a-t_-arabinopyranoside (MUA), but was unable to hydrolyze arabinose containing polymers such as arabinoxylans, arabinans, or arabinogalactans. Hydrolysis products

analysis

*1

Products of the hydrolysis of different substrates (reaction time, 20 h) were analyzed by HPLC. Similar hydrolysis profiles were observed when different xylans were degraded by X61. Neither xylose nor arabinose was detected in the chromatograms, indicating a typical endoxylanase mode of action for the enzyme without arabinose releasing activity (Figure 3). The effect of the enzyme on several (l-+4)+D-xylooligosaccharides was also studied. X61 was able to hydrolyze xylooligosaccharides of three or more xylose residues, but not xylobiose. The other highly active endoxylanase (X22) from B. polymyxa3 requires longer xylooligosaccharides for efficient hydrolysis. As mentioned before, AF166 only showed activity on pNP or MU arabinose derivatives, but not on natural arabinose-containing polymers. However, (1+.5)-o-~arabinooligosaccharides were completely degraded to arabinose after 20 h incubation in the presence of this enzyme. Shorter reaction experiments using (1--+5)-o-~arabinooctaose as substrate suggested that this enzyme has an exo-mode of action, removing arabinose residues from the oligomer (Figure 4). This enzyme was also able to split (x(l-3) in addition to a( 1+5) bonds, as deduced from the production of arabinose when arabinoxylan was incubated with both X61 and AF166 (see Figure 3). According to the arabinofuranosidase classification proposed by Kaji,” this enzyme could be classified into the Streptomyces purpurascens type. Two enzymes from this group, an arab-

P

u q

c

2

l3 I

3

4

ti

Figure 3 HPLC analysis of xylan hydrolysis products. Oat spelt xylan (A), birchwood xylan (B), methylglucuronoxylan (C) and wheat flour arabinoxylan (D-F) were the substrates degraded by xylanase X61 (A-D), arabinofuranosidase AF166 (E), or by both X61 and AF166 enzymes (F). Controls of nonhydrolyzed substrates are identical to that seen in E. Changes in refractive index were detected. 1, xylose; 2, xylobiose; 3, xylotriose; 4, xylotetraose; A, arabinose

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0l-l

3h

I

6n

20

h

hydrolysis Figure 4 HPLC analysis of (l-+5)-a-L-arabinooctaose products by arabinofuranosidase AF166. Incubation times of the oligomer with the enzyme are indicated. Al, arabinose; A3, arabinotriose; A8, arabinooctaose; An, (1+5)-a-~arabinooligosaccharides of four to eight residues

inofuranosidase from S. purpurascens’5 and the arabinofuranosidase A from Aspergillus niger, l6 are also active on pNPA and ( l-5)-o-t_-arabinooligosaccharides and inactive on arabinan and arabinoxylan, although it is not known whether these enzymes are able to release arabinose from endoxylanase-treated arabinoxylan. However, arabinofuranosidase B from the latter microorganism is active against all of these substrates,16 and would be classified in the other group of enzymes, with a broader range of substrates, proposed by Kaji. Another species from this fungal genus, Aspergillus awamori, has a very specific enzyme named arabinoxylan arabinofuranohydrolase, which releases arabinose only from the singly substituted xyloxyl residues in arabinoxylan.‘7,‘8 Clostridium acetobutylicum has an arabinofuranosidase that is inactive against arabinoxylan or arabinogalactan, but it is active against arabinan and endoxylanase-treated arabinoxylan. l9 There are few arabinofuranosidase activities described in Bacillus species. For one of them from B. subtilis, on an exo-mode of action on arabinan has been reported.”

Acknowledgments This work was supported by Grant ALI91-0336 from the Comision Interministerial de Ciencia y Tecnologia of the Spanish Government. P. Morales was the recipient of a fellowship from Bancaja. Thanks are due to A. P. McCabe for a critical reading of the manuscript.

Xylanase

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Laemmli. U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227, 680-685 Davis. B. J. Disc electrophoresis-method and application to human serum proteins. Ann. NY Acad. Sci. 1964, 121, 404-427 Beguin. P. Detection of cellulase activity in polyacrylamide gels using Congo red-stained replicas. Anal. Biochem. 1983. 131, 333-

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