A new method to screen polysaccharide cleavage enzymes

Enzyme and Microbial Technology 48 (2011) 248–252

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A new method to screen polysaccharide cleavage enzymes S. Badel a,b , C. Laroche a , C. Gardarin a , E. Petit c , T. Bernardi b , P. Michaud a,∗ a

Clermont Université, Université Blaise Pascal, Laboratoire de Génie Chimique et Biochimique, Polytech’ Clermont Ferrand, 24 avenue des Landais BP 206, 63174 Aubière cedex, France BioFilm Control, Biopôle Clermont Limagne 63360 Saint-Beauzire, France c Laboratoire des Polysaccharides Microbiens et Végétaux, IUT d’Amiens, avenue des Facultés, Le Bailly, 80025 Amiens cedex, France b

a r t i c l e

i n f o

Article history: Received 16 June 2010 Received in revised form 9 November 2010 Accepted 11 November 2010 Key words: Polysaccharides Polysaccharide hydrolases and lyases BioFilm Ring Test®

a b s t r a c t The activity of polysaccharide cleavage enzymes has usually been evaluated by qualitative plate screening methods and quantitative colorimetric or chromatographic assays. The recent development of protein engineering has shown the limits of these techniques when applied to high throughput screening. Here we propose a microplate method to measure the activity of polysaccharide cleavage enzymes through small variations in viscosity. Polysaccharide solutions are co-incubated with magnetic particles in enzyme buffers. The cleavage action of polymer-degrading enzymes increases the mobility of the particles in a magnetic field, even at low levels of enzyme activities. This reproducible, sensitive technique was used to evaluate enzymatic specificity towards substrates. BioFilm indices (BFI) determined by associated software were used to follow enzyme kinetics and measure the usual variables. © 2010 Elsevier Inc. All rights reserved.

1. Introduction In IUB-IUPAC nomenclature, a polysaccharide is a glycosidelinked polymer of monosaccharides with a degree of polymerization higher than 10. Polysaccharides can be up to several million daltons in size, and present various structures and different types of linkages. These features lead to a range of specific behaviours in solution [1]. Their texturing properties account for most of their industrial applications. However, uses based on their biological activities have increased significantly [2,3], in particular for oligosaccharides [4,5]. Interest in the enzymes acting on these macromolecules, and especially polysaccharide cleavage enzymes (polysaccharases), has accordingly increased [6–8]. Several modern methods of enzyme modification, e.g. random mutagenesis, sitedirected mutagenesis and directed evolution, have been applied to detect new catalytic properties or modify known ones [9–12]. These methods require efficient tools to screen, measure and characterize the activity of the two classes of polysaccharide-degrading enzymes: the polysaccharide lyases (EC 4.2.2.-) and hydrolases (EC 3.2.1.-). Many methods have been used to detect and (or) quantify the activities of polysaccharide hydrolases (PH) and polysaccharide lyases (PL). Plate screening methods and rheological measurements are often used to detect polysaccharide cleavage enzymes. Several dyes (Congo Red, Ruthenium Red, Calcofluor White, iodine, Direct Green, Mikacion and Ostazin Brilliant Red and Remazol Brilliant

∗ Corresponding author. Tel.: +33 4 73 40 74 25; fax: +33 4 73 40 74 02. E-mail address: [email protected] (P. Michaud). 0141-0229/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2010.11.003

Blue) can interact specifically or non-specifically with polysaccharides. Thus when the polysaccharide substrate is embedded in a gelled matrix, its enzymatic degradation can be visualized by a clear halo in a coloured gel. The size of the halo is proportional to the enzyme activity within the enzyme diffusion limits [13]. Similarly, the insolubility of polysaccharides in numerous solvents such as ethanol, acetone or isopropanol can be exploited. When the gel matrix containing the polysaccharide substrate is immersed in the solvent, opacity shows non-degradation while a transparent halo reveals the activity of a polysaccharase [13]. The rheology of a polysaccharide solution can also be followed after addition of an enzyme. Its viscosity decreases as molecules with lower average molecular weights are generated [14]. The kinetics of this process provides information on the cleavage mechanism (endo- or exoenzyme). Products from polysaccharide degradations are rarely coloured, and so their occurrence cannot be followed directly by spectrophotometry except for some dyed polysaccharides [15,16]. However, such polymers, obtained by grafting dyes chemically, are often not commercially available and may not be recognised by enzymes as their natural substrates. Indirect colorimetric assays measuring reducing sugars are therefore the most common ways to quantify polysaccharase activities. Non-hydrolytic methods such as DNS [17,18] or 2,2 -bicinchoninate (BCA) assays [19] are generally chosen by researchers. They are difficult to handle, in particular in microplates for high throughput screening. Other more specific quantitative methods are sometimes used to detect monoor oligosaccharide products. Among these, high performance liquid and thin layer chromatography [20–23] have been successfully developed. The only case in which the detection of enzyme activity is easy and to the same for all enzymes using the same catalytic

S. Badel et al. / Enzyme and Microbial Technology 48 (2011) 248–252

mechanism is the spectophotometric measurement of A235 . This wavelength is specific to unsaturated non-reducing terminal units of PL products [24]. Rigouin et al. recently compared several methods, and concluded that it was difficult to adapt them for high throughput screening as they are generally time-consuming and require special equipment [25]. The BioFilm Ring Test® is a tool to explore biofilms and their constitutive macromolecules [26,27]. Magnetic particles are added to a culture medium containing microorganisms in the microwells of a microplate. Under the effet of a magnetic field, free particles aggregate at the centre of the well, forming a spot. If a biofilm is formed, the particles are immobilized by the extracellular matrix and no spot is seen. Here we describe the use of this method to detect polysaccharide cleavage enzymes.

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inserted in the instrument and the data analysed by the Rheology Advance software. The Cross equation (1) is a suitable rheological model classically used for entangled polymer solutions [30]: ( − ∞ ) 1 = m (0 − ∞ ) (1 + (K ) ˙ )

(1)

where 0 is the Newtonian viscosity at low shear rates, ∞ the Newtonian viscosity at high shear rates, K the consistency (s) and m is the rate index. The parameter taken into account here was 0 , the viscosity at low shear rates.

3. Results and discussion 3.1. Determination of polysaccharide concentrations

2. Materials and methods 2.1. Enzymes and polysaccharides Glucuronan lyase (EC 4.2.2.14) was extracted from Trichoderma sp. GL2 culture as described by Delattre et al.et al. [28]. It was dissolved in potassium acetate buffer 50 mM, pH 5.5. The glucuronan was obtained from Sinorhizobium meliloti M5N1CS and deacetylated by alkaline treatment [29]. Pullulanase (EC 3.2.1.41) from Sigma (P2986) was used in 50 mM sodium acetate buffer, pH 4.5. Its substrate, pullulan, was obtained from Hayashibara Co. Ltd. (Japan). A cellulolytic preparation from Trichoderma reesei (celluclast) (EC 3.2.1.4) was obtained from Sigma (C2730). Buffer was 100 mM sodium acetate pH 5.6. Carboxymethylcellulose (CMC) was from Fluka (21904). A 200 mM Tris–HCl buffer supplemented by NaCl (2 M), pH 7.5 was used to evaluate the activity of alginate lyase (4.2.2.3) from Sigma (A6973). Its substrate was sodium alginate (RARESEA) from Degussa Texturant Systems (France). 2.2. Enzymatic activity Enzymatic starter samples were 5.6 U/mL for glucuronan lyase, 1.6 U/mL for pullulanase, 0.03 U/mL for alginate lyase and 1.2 U/mL for cellulase in specific buffers. One unit was defined as the amount of enzyme needed to release 1 ␮mol of reducing sugar per minute. Reducing sugars were evaluated in glucose equivalent by BCA assay according to the method described by Waffenschmidt and Jaenicke [19]. This method is based on the reduction of Cu2+ to Cu+ by reducing the terminal unit in the presence of l-serine. Cu+ ions then complex with BCA to produce a purple colour. The colour intensity was quantified by measuring absorbance at 540 nm. To prepare inactivated enzymes, solutions were heated for 5 min at 95 ◦ C except for the cellulase solution, which was autoclaved at 121 ◦ C for 20 min. 2.3. The BioFilm Ring Test® technique This assay is based on quantifying the mobility of insoluble magnetic microparticles in a solution under the effect of a magnetic field. Polysaccharide solutions were prepared in specific buffers according to the degrading enzymes. Tween 20 was added at 0.002% (w/v) and magnetic particles (toner) were then added at 10 ␮L/mL. After adding a defined volume of enzymatic starter sample, each solution was homogenized and distributed in microwells (a microplate contains 12 strips of 96 microwells (200 ␮L)). Two concentrations of polysaccharide cleavage enzymes were tested for each polysaccharide. Wells were covered with Contrast Liquid (inert oil) and immediately scanned with a dedicated plate reader to record an image I0 . The strips were then placed for 1 min on a magnetizer and re-scanned to record an image I1 . The magnetizer consisted of 96 magnets each centred under the bottom of a well. In the magnetic field, free particles were attracted to the centre of the bottom of the wells, forming a spot, while those held in the polysaccharide remained stationary. Images of each well before (I0 ) and after (I1 ) magnetic treatment were compared by the Biofilm Elements® software. An algorithm estimates the discrepancy between the two images of the same well, and assigns a value to it, termed the BioFilm Index (BFI), ranging from 0 to 20. High BFI values correspond to a high mobility of particles under magnetic action (e.g. control wells) while low values correspond to major immobilization of particles (no difference for a well between images I0 and I1 shows full immobilization). This operation was repeated for every sample taken from the reaction volume. 2.4. Rheological measurements Rheological measurements were made with a double concentric cylinder arrangement on a stress-controlled Rheometer AR-G2 (TA Instrument) with a Peltier temperature control system. Temperature was set at 25 ◦ C and the viscosity was evaluated at shear rates over the range 10−4 to 103 s−1 . Samples (13 mL) from polysaccharide degradation runs by specific enzymes were collected at 0, 30 and 60 min and heated for 5 min or 1 h (for cellulase) at 100 ◦ C. Lastly the samples were

The Biofilm Ring Test® works well with bacteria-rich media such as Brain Heart Infusion (BHI), Luria-Bertani (LB) and Trypton Soy Broth with Yeast Extract (TSBYE). The first step in these experiments was to test the enzyme buffers, which can increase electrostatic forces that interact with the charged magnetic particles. In bacteria-rich media, organic particles such as proteins and peptides counteract these interactions,preventing the accretion of toner on the sides of the well (so that magnetic particles are immobilized in the bottom of wells and cannot gather in the centre). Magnetization of toner suspended in the four buffer types was tested at T0 (initial time) and T24 (24 h of incubation) (data not shown). No clearly-formed spot was revealed, BFI <2 indicating a total immobilization of toner, meaning that electrostatic forces were present. Accordingly, we used Tween 20 in the buffers at 0.002% (w/v). This concentration, which had no impact on enzyme activities (data not shown), maintained BFI >12. This value was necessary to obtain a clearly defined spot at all experiment times for the control (buffer + toner) incubation. To observe enzymatic degradation of polysaccharides with the BioFilm Ring Test® , the substrates had to be concentrated enough to immobilize particles of toner before enzyme addition. These “blocking concentrations” were evaluated by dissolving each polysaccharide at several concentrations in appropriate buffers. Four microwells per sample were filled (200 ␮L) and the microplate immediately read. Polysaccharide solutions inducing BFI <2 were 6 g/L for glucuronan, 2 g/L for CMC, 30 g/L for pullulan and 4 g/L for alginate. Enzymatic degradations were then performed with these concentrations.

3.2. Determination of initial rates in classical enzyme degradation Polysaccharide solutions were prepared in buffers supplemented with Tween 20 (0.002%) at the previously determined concentrations. The levels of enzyme activities used for each polymer were set according to the supplier’s recommendations. The BCA method allowed the detection of reducing terminal units released by the degradation of glycoside bonds. Initial rates against enzyme concentrations were measured during the first minutes (Table 1). In the usual manner, the amount of reducing sugars increased linearly to reach a stationary phase when no significant evolution was detected. This final phase appeared at about 120 min, 60 min, 120 min and 60 min for pullulan, CMC, alginate and glucuronan degradations, respectively (data not shown). Alginate cleavage by 3.8 × 10−5 U/mL of alginate lyase and degradation of CMC by 1.2 × 10−4 U/mL of cellulase were weakly detectable by BCA assay (values were comparable to the control without enzyme). However, the viscosity had visibly decreased during the first hour of reaction, showing enzymatic activities. Another method was therefore tried.

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S. Badel et al. / Enzyme and Microbial Technology 48 (2011) 248–252

Table 1 Initial rate determination by BRT® and BCA methods. Substrate

Enzyme activities (U/mL)

Initial rate (␮mol/min/L)a

Pullulan

0.016 0.12 1.2 × 10−4 1.2 × 10−2 3.8 × 10−5 1.9 × 10−3 6.7 × 10−3 0.034

17.8 102.2 0.9 23.9 0.2 2.3 6.9 32.2

CMC Alginate Glucuronan a b

Glucose equivalent concentration (␮mol/L) at 60 min

Initial rate (BFI/min)b

Without enzyme

With enzyme

2777.78 2777.78 5.56 5.56 55.56 55.56 277.80 277.80

4186.6 9270.3 46.3 598.1 56.90 100.00 669.59 2012.88

0.04 0.13 0.16 1.19 0.01 0.58 0.22 1.32

Determined by BCA method. Determined by BRT® method.

3.3. Degradation of polysaccharides with the BioFilm Ring Test® Earlier experiments with the BioFilm Ring Test® had been conducted to study the role of biopolymers (nucleic acids, proteins and polysaccharides) in the formation of a biofilm matrix. They showed that enzymatic degradation helped to evaluate their function in biofilms [27,31]. These studies also revealed the influence of biopolymer viscosities in the BFI variations [31]. To apply this approach to measurements of PL and PH activities, a qualitative experiment was conducted at the blocking concentration of each polysaccharide, with two concentrations of the specific enzymatic activity (Tables 2 and 3). BFI ≤2 indicated a total immobilization of magnetic particles, while BFI >2 showed a decrease in viscosity, releasing some particles. Glucuronan lyase, pullulanase and alginate lyase specifically depolymerized their substrates (Table 3). As expected, the BFI values obtained were inversely proportional to the enzyme activities. A cellulolytic extract from Trichoderma (Celluclast) was detected that degraded CMC and glucuronan with significant BFI values of 10.9 and 11.4, respectively. This was not surprising as commercial enzymatic preparations contain not only purified enzyme but often other contaminating activities [32]. All the enzymes were deactivated by heating for 5 min at 95 ◦ C except for cellulase, which was treated for 20 min at 121 ◦ C. Enzymes were then tested for 120 min on polysaccharide solutions. As expected, we found all BFI <2 throughout the incubation (data not shown). To quantify the activity of enzymes, results from BRT® and BCA had to be compared. Fig. 1 gives the example of CMC, a neutral polysaccharide, while Fig. 2 represents an example of anionic

polysaccharide degradation. Scales were adapted, but as the molar extinction coefficient was different for each type of released sugar, it was difficult to evaluate the maximum quantity degraded by the enzymes. We chose the maximum value on the BCA scale corresponding to the theoretical maximum degraded concentration: the initial polymer concentration expressed in molarity of glucose equivalent. On the BFI scale, the maximum corresponded to the value when all particles were free to move in the microwell, i.e. the BFI recorded when no polysaccharide was dissolved (only buffer + Tween). The differences between the two concentrations were greater with the BRT® results (Figs. 1 and 2). The end of cleavage was recorded sooner with this method. The enzymes are endoenzymes and cleave the polysaccharide inside its chain. Molecular weight decreased faster and as particles were trapped by crowding, viscosity fell. The BRT® measured microviscosities indirectly with a high precision. With this method, degradation by lower enzyme activities (1 × 10−4 or 1 × 10−5 U/mL) were visualized, at variance with results obtained with the BCA assay, showing that BRT® was the more sensitive method. In the literature, work on polysaccharide degradation by enzymes such as carboxymethyl cellulose cleavage by endoglucanase have been carried out with viscosimetry measurements [33,34]. The authors state that a single viscosity evaluation took 40 min, not counting sample work-up time (homogenizing, centrifuging or filtering). Also, the precision depended on physical parameters such as capillary diameter and type of system employed (automated or manual). Concerning colorimetric assays, to increase the number of treated samples per measure for the DNS method, King et al.et al. developed this method in microplates [35]. During enzymatic hydrolysis, the plate needed to be sealed with an

11 x 103

12

10 x 103

10

33 x 103

12

32 x 103

10

600 400

2 0

200 0

0

10

20

30

40

50

60

Time (min) Fig. 1. Correlation of values for CMC degradation evaluated by both BRT® and BCA methods: () cellulase 1.2 × 10−4 U/mL, BRT® ; () cellulase 0.012 U/mL, BRT® ; () BRT® Control; () cellulase 1.2 × 10−4 U/mL, BCA; (䊉) cellulase 0.012 U/mL, BCA; (*) BCA control.

BFI

BFI

800

4

8 6 3000

4

[glc] µmol/

6

[glc] µmol/L

8

2000 2 0

1000 0

10

20

30

40

50

60

0

Time (min) Fig. 2. Correlation of values for glucuronan degradation evaluated by both BRT® and BCA methods: () glucuronan lyase 6.7 × 10−3 U/mL, BRT® ; () glucuronan lyase 0.034 U/mL, BRT® ; () BRT® Control; () glucuronan lyase 6.7 × 10−3 U/mL, BCA; (䊉) glucuronan lyase 0.034 U/mL, (*) BCA control.

S. Badel et al. / Enzyme and Microbial Technology 48 (2011) 248–252

251

Table 2 Comparison between the BRT® method and measure of viscosity (0 ). Enzyme/substrate

Enzyme activities (U/mL)

Initial

Glucuronane/glucuronan lyase

1.7

3.8 × 10−5

0.016

1.5

0.12

CMC/cellulase

60 min

Initial

2.1

4.0

5.7

9.3

1.9

5.2

4.8

7.5

2.1

3.8

4.6

10.4

4.0

4.1

10.7

10.9

32.30

0.50

30 min

60 min

1.30

0.48

0.46

0.60

0.02

0.05

0.01

0.04

nd

nd

nd

nd

0.28

0.34

0.53

0.12

1.6

1.9 × 10−3

Pullulan/pullulanase

30 min

6.7 × 10−3

0.034

Alginate/alginate lyase

Viscosity 0 (Pa s)

BFI

1.2 × 10−4

1.9

1.2 × 10−2

nd

13.20

nd = not determined.

aluminium film and for the colorimetric reaction a PCR thermocycler allowed heating and cooling. To read the absorbance, transfer into new microwells was needed. BRT® thus showed itself to be a time-saving, efficient tool, as no transfer phase or time-consuming steps were required. BFI values obtained by the BFC Elements® software also allowed enzyme kinetic parameters to be determined. A

new variable, expressed in BFI/min, reflects the quantity of particles being released in the course of degradation and so indirectly gives a measure of degraded polysaccharide (Table 1). As the recording required the acquisition of I0 (image of well before plate magnetization), magnetization and acquisition of I1 (image of well after 1 min of plate magnetization), the first real points were recorded at

Table 3 BFI determination according to substrate/enzymes specificity, with the BRT® . Enzyme/substrate

Glucuronan

Pullulan

CMC

Alginate

Glucuronane lyase

9.3

1.7

2.3

1.6

Pullulanase

2.5

10.4

1.4

1.7

11.4

1.6

10.9

1.6

1.3

1.7

2.4

7.5

Cellulase

Alginate lyase

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S. Badel et al. / Enzyme and Microbial Technology 48 (2011) 248–252

about 5 min. The BFI at T0 was the BFI value of controls measured at the first reading (polysaccharide solution without addition of enzymes). To quantify the initial rate, curves for pullulan and glucuronan were taken into account from 5 min. The stationary phase observed at the start of the run should correspond to this BFI control value measured at T0 . Values obtained by the two methods (BRT® and BCA) were not comparable owing to marked differences in method. The BCA assay quantitatively evaluates the cleavage of a glycoside bond, whereas BRT® measures the mobility of magnetic particles determined by medium viscosity. Thus when an endo-polysaccharide cleavage enzyme acts on its substrate the impact on viscosity is initially greater than the impact on the rate of reduction of the terminal units. This is easily explained by the influence of polymer molecular weight on viscosity. However, even though BRT® measures viscosity indirectly, its sensitivity and its application in microplates are advantageous compared with classical rheological measurements. 3.4. Viscosity of the polysaccharide solutions Viscosities of polysaccharide solutions with and without enzymes were determined to correlate them with BFI. Measurements were made on samples before introducing the enzyme, and after 30 min and 60 min of enzyme reaction (Table 2). The pullulan solution, even before addition of pullulanase, was not viscous enough to obtain correct values. The viscosities attained at low shear rates were too low to permit reproducibility. For the other three polysaccharides, the viscosity decreased significantly during the first 30 min. When small values of 0 were obtained, the inter-measure variability was too great. This observation highlights the lack of precision of this technique for the degradation of non-viscous polysaccharides. Here, BRT® was evaluated for screening degrading enzymes on different substrates, qualitatively and quantitatively. It offers a new way to characterize polysaccharide degradation, rather than an improvement of existing techniques. Results show that this new technique is a powerful tool to detect polysaccharase activities with a high specificity and sensitivity. Compared with other current assays based on spectrophotometric and rheological analysis, results were more precise, but most importantly they took less time to obtain. Also, the BRT® does not require reagents that may interfere with enzyme activities, or need high acidic or alkaline conditions or heating. The development of BRT® in microplate assay and the rapidity of the measurement underline its suitability for high throughput screening. Hence this method could help to assess the efficiency of enzyme cocktails on a broad range of substrates in the same microplate. Finding and screening new native or modified cleavage enzymes also emerges as a possible application. References [1] Rinaudo M. Role of substituents on the properties of some polysaccharides. Biomacromolecules 2004;5:1155–65. [2] Duboc P, Mollet B. Applications of exopolysaccharides in the dairy industry. Int Dairy J 2001;11:759–68. [3] Ruas-Madiedo P, Salazar N, de los Reyes-Gavilan CG. Exopolysaccharides produced by lactic acid bacteria in food and probiotic applications. In: Moran A, Holst O, Brennan PJ, von Itzstein M, editors. Microbial glycobiology – structures, relevance and applications. Elsevier Inc; 2010. p. 885–902. [4] Wichienchot S, Jatuporapipat M, Rastall RA. Oligosaccharides of Pitaya (dragon fruit) flesh and their prebiotic properties. Food Chem 2010;120:850–7.

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