using these methods in the analysis of a distributed enzyme sample were com- .... Summary of participants' own methods for assay of xylanase activity; the ...
Journal of Biotechnology, 23 (1992) 257-270 © 1992 Elsevier Science Publishers B.V. All rights reserved 0168-1656/92/$05.00
257
BIOTEC 00735
Interlaboratory testing of methods for assay of xylanase activity Michael J. Bailey 1, Peter Biely 2 and Kaisa P o u t a n e n 3 I VTT, Biotechnical Laboratory, Espoo, Finland; 2 Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Czechoslovakia; 3 I/TT, Food Research Laboratory, Espoo, Finland (Received 18 October 1991; revision accepted 10 November 1991)
Summary
Twenty laboratories participated in a collaborative investigation of assays for endo-l,4-fl-xylanase activity based on production of reducing sugars from polymeric 4-O-methyl glucuronoxylan. The substrates and methods already in use in the different laboratories were first recorded and the apparent activities obtained using these methods in the analysis of a distributed enzyme sample were compared. The standard deviation of the results reported in this analysis was 108% of the mean. Significant reduction in interlaboratory variation was obtained when all the participants used the same substrate for activity determination, each with their own assay procedure. The level of agreement was further improved when both the substrate and the method procedure were standardized. In a round robin testing of a single substrate and method, including precise instructions for enzyme dilution, the standard deviation between the results after the rejection of two outliers was 17% of the mean. This figure probably reflects the inherently poor reproducibility of results when using only partially soluble, poorly defined and rather impure polymeric substrates. The final level of variation was however low enough to allow meaningful comparison of results obtained in different laboratories when using the standardized assay substrate and method procedure. Fifteen laboratories also participated in preliminary testing of an assay based on the release of dyed fragments from 4-O-methyl glucuronoxylan dyed with Remazol Brilliant Blue dye. High values of the coefficients of correlation indicated good linearity between the amount of dyed fragments released and enzyme concentraCorrespondence to: M.J. Bailey, VTT, Biotechnical Laboratory, PO Box 202, 02151 Espoo, Finland.
258 tion. The relative standard deviations of the results obtained by fifteen laboratories were about 30% for an optimum range of xylanase activity in the reaction mixture. Xylanase; Assays; Testing; Round robin
Introduction
Xylanases (endo-l,4-/J-D-xylan xylanohydrolase, EC 3.2.1.8) have attracted increasing attention in biotechnical research during the past decade, largely because of their potential applications in cellulose pulp bleaching (e.g. Viikari et al., 1988, 1990; Jurasek and Paice, 1988; Kantelinen et al., 1991). An important criterion in this as in many other applications of enzymes is dosage of activity. Too high a dosage may lead to undesired effects (yield losses in biobleaching) and certainly to poor economic feasibility, whereas insufficient dosage of course decreases the effectiveness of the application. Comparison of the enzyme dosages used in different applications is hindered by the lack of a standardized method for measurement of xylanase activity (Khan et al., 1986; Royer and Nakas, 1989). Although the methods used for the assay of e.g. endoxylanase activity are in most cases reported, realistic comparison between the methods is in practice extremely difficult. Most workers report xylanase activities based on the release of reducing sugars from partially soluble xylan substrates (e.g. Tan et al., 1985), although other methods have also been published based on reduction in turbidity of a suspension of insoluble xylan (Nummi et al., 1985; Bailey and Poutanen, 1989) or release of soluble dyed moieties from a dyed xylan substrate (Biely et al., 1985). Even when the procedural details of different assays are similar, the use of different xylan substrates with different types and degrees of substitution (arabinose, acetic acid, 4-O-methyl glucuronic acid) leads to poor comparability of reported activities. This is particularly true when purified xylanases are used in pulp treatment experiments, because different xylanases have different specific activities against different xylan substrates (e.g. Puls and Poutanen, 1989; Bailey et al., 1991). Finally, it is the experience of these authors that considerable variation of results may occur between - or even within - laboratories using the same enzymes, substrates and methods. In the work of the International Energy Agency (IEA) network collaboration programme, entitled Biotechnology for the Conversion of Lignocellulosics, major emphasis was placed on the standardization and characterization of methods for assaying enzymes involved in lignocellulose degradation. A group of laboratories participating in the IEA network agreed in 1989 to undertake collaborative testing of an assay for xylanase activity. The results of this undertaking, coordinated by the authors, are presented here. The initial aims of the work were: 1. To record the methods and substrates for the assay of xylanase activity in use in twenty laboratories around the world. All the methods tested employed detection of reducing sugars (as xylose) as the
259 TABLE 1 Participants in the interlaboratory collaboration Name
Affiliation
Michael Bailey Peter Biely Michael Coughlan Hermann Esterbauer Kurt Fisher Karel Grohman B~rbel Hahn-H~igerdal Dieter Kluepfel Tarja Lahtinen Anna-Maria Marzetti Aneli de Melo Barbarosa Murray Moo-Young Jacques Pourquie Jiirgen Puls Jack Saddler WolfgangSchwald David Senior Walter Steiner Jouko Treuthardt WolfgangZimmermann
VTT, Espoo, Finland Slovak Academyof Sciences, Bratislava, Czechoslovakia University College, Galway,Ireland University of Graz, Austria Technical University, Wien, Austria SERI, Golden, U.S.A. University of Lund, Sweden University of Quebec, Canada Alko, Rajam~iki, Finland Cellulose Research Station, Milano, Italy University of Londrina, Brazil University of Waterloo, Ontario, Canada Institute of Petroleum, Rueil-Malmaison, France BHF, Hamburg, Germany University of British Columbia, Vancouver, Canada Biochemie Ges. m.b.H., Kundl, Austria ICI BiologicalProducts, Ontario, Canada BiotechnologyInstitute, Graz, Austria Cultor, Kantvik, Finland ETH, ZiJrich, Switzerland
criterion for activity measurement. 2. To compare the apparent activities obtained with these methods using an enzyme sample distributed to all the laboratories. 3. To investigate the level of interlaboratory variation in results by round robin testing of a single enzyme, substrate and method protocol distributed to the participating laboratories. During the course of the work, some of the laboratories also agreed to undertake preliminary testing of an assay in which release of colour from dyed substrate was used for quantification of activity. This method (Biely et al., 1985) measures substrate cleavage (reduction in the amount of polymeric substrate) rather than product formation (production of reducing sugars). It is therefore independent of the level of/3-xylosidase in the sample being analyzed, whereas the effect of/3-xylosidase on the assay based on production of reducing sugars has not been investigated.
Method selection
Comparison of xylanase assays used by the participants In a preliminary questionnaire, the participants (twenty laboratories, see Table 1) were asked to report their method for assay of xylanase activity. A summary of the methods is presented in Table 2, in which the laboratories were numbered
26O TABLE 2 Summary of participants' own methods for assay of xylanase activity; the participant laboratories were assigned arbitrary numbers; laboratories 18-20 did not have a xylanase assay in routine use Lab no.
Substrate
Conc. a %
pH
T °C
t min
Sugar assay b
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Larchwood Beechwood Larchwood Oat spelt Oat spelt Beechwood Larchwood Oat spelt Beechwood Oat spelt Oat spelt Oat spelt Oat spelt Oat spelt Oat spelt Beechwood Larchwood
0.5 0.7 0.7 0.5 0.9 0.5 0.5 0.7 0.9 0.5 0.5 0.75 0.5 0.5 0.5 0.7 0.9
5.3 5.3 5.3 5.3 4.8 5.4 5.0 5.0 5.3 5.3 5.0 5.3 5.3 5.3 4.8 5.0 5.3
50 50 50 50 50 30, 50 50 50 50 50 50 50 50 50 65 37 50
5 15 15 3 10 5-60 20 30 5 10 30 10 10 30 30 5 5
DNS DNS DNS S-P DNS S-N S-N DNS DNS DNS DNS DNS DNS DNS DNS DNS S-N
a Substrate concentration in the final reaction mixture b DNS = dinitrosalicylic acid method for detection of reducing sugars; S-N = Somogyi-Nelson method; S-P = method of Sinner and Puls (1978).
arbitrarily. Only two of the 17 laboratories with an assay procedure already in regular use employed exactly the same combination of substrate and method. Variation was between substrates and their concentrations in the final reaction mixture (0.5-0.9%), reaction conditions (time, temperature, pH of incubation) and detection method for reducing sugars (as xylose) produced during the reaction.
Selection of method and substrate for round robin testing The method chosen for the round robin test (XYL-DNS) is described in detail in Appendix 1. In a preliminary test, 5 differently substituted, partially soluble and one unsubstituted (insoluble) xylan were compared using the substrate preparation procedure and method protocol described in Appendix 1. The results of this comparison are summarized in Table 3. Three factors contributed to the choice of birchwood 4-O-methyl glucuronoxylan (Roth, product 7500) as substrate for use in the round robin testing: low turbidity of the 1.0% substrate solution, extended range of linearity of the reaction using this substrate and commercial availability of the xylan. The high turbidity of the oat spelt and insoluble beechwood xylans (Table 3) decreased during boiling with DNS because of solubilization in the
261 TABLE 3 Comparison of different xylan substrates in the xylanase assay procedure described in Appendix 1 Substrate
Birchwood glucuronoxylan (Sigma X-0502) Birchwood glucuronoxylan (Roth 7500) Oat spelt arabinoxylan (Sigma X-0376) Beechwoodglucuronoxylan (Ebringerov~ et al., 1967) DMSO acetyl glucuronoxylan (H~igglundet al., 1956) Insoluble, unsubstituted beechwood xylan (Lenz and Schurz, 1986) a
Turbidity of 1% suspension/solution at 620 nm
Limit of linearity at 540 nm in the enzyme reaction
Commercial availability
0.08
0.50
+
0.10
0.70
+
3.00
0.45
+
0.60
0.45
-
0.15
?a
_
3.30
0.55
Reaction mixtures turbid after boiling with DNS.
alkaline conditions. By contrast, the long-chain DMSO xylan became turbid as a result of boiling and the extent of linearity of the enzyme reaction could not be established using this assay. The concentration of the substrate solution (1.0%) was limited, as in the case of many polymeric substrates, by solubility. Therefore, in order to maximize substrate availability in the enzyme reaction, an enzyme : substrate volume ratio of 1 : 9 (200 /zl + 1800/zl) was chosen in preference to the more frequently observed ratio of 50:50. Insufficient substrate availability is a probable cause of non-linearity in assay methods for polymer-degrading hydrolytic enzymes (Bailey, 1988). The reaction conditions (300 s, 50°C, pH 5.3, see Appendix 1) were chosen to be suitable for mesophilic fungal xylanases. An incubation temperature of 50°C is usually used in assays of fungal cellulase and hemicellulase activities and this temperature was chosen to facilitate comparison of activities. However, because some xylanases are unstable during prolonged incubation at this temperature (e.g. Bailey et al., 1991), a short incubation time of only 300 s (5 min) was chosen. The reaction was terminated by addition of 3.0 ml dinitrosalicylic acid solution (DNS; Sumner, 1921; Sumner and Somers, 1949; many workers use a modified reagent developed by Miller, 1959) and boiling for 5 min (Miller, 1959 and Miller et al., 1960 recommended a boiling time of 15 min with the modified DNS reagent) along with the tubes containing reagent blanks and xylose standards. The boiled reaction mixtures were measured as such at 540 nm after cooling, without employing the more usual, but inadvisable procedure of prior dilution with 10 or 20 ml of distilled water (Bailey, 1988). The exact composition of the DNS reagent was not specified in the instructions sent to the participants. Sugar detection by the DNS method was chosen rather than by the SomogyiNelson (SN) method (Nelson, 1944; Somogyi, 1952) partly because the majority of
262 laboratories were already using this reagent (see Table 2), partly because the authors were reluctant to recommend adoption of the more toxic SN reagent and partly because the SN reagent is considerably more sensitive to disturbing factors than DNS (Bailey, unpublished results). The SN method is known to give a lower result than DNS (Breull and Saddler, 1985) because the latter effects partial hydrolysis of oligosaccharides, thus increasing the number of free reducing groups in the colour reaction. Xylanase activities in the distributed method protocol were expressed in SI units, katals (1 kat = 1 mol s-1). This unit of enzyme activity was recommended by the Commission on Biochemical Nomenclature almost twenty years ago (Florkin and Stotz, 1973) to replace the earlier, non-SI International Unit (1 IU = 1 ~mol min - 1 = 16.7 nkat). The katal was adopted in an earlier assay for endo-l,4-/3glucanase (cellulase) activity (Bailey and Nevalainen, 1981; IUPAC, 1987) using hydroxyethyl cellulose (HEC) as substrate. Both the method and the unit definition in the distributed xylanase assay procedure were modeled on the HEC assay and therefore the use of these two assays would allow direct comparison of the molar activities of endoglycosidases against cellulosic and hemicellulosic substrates. The XYL-DNS enzymatic reaction was linear within the range of the series of monosaccharide standards given in Appendix 1 (results not shown).
Interlaboratory collaboration Preliminary testing of methods and substrates Identical packages were sent to each participating laboratory, containing: a sample of Trichoderma reesei enzyme preparation kindly provided by Dr. Niels Krebs Lange, Novo Nordisk, Denmark; a sample of birchwood glucuronoxylan (Roth 7500); the method protocol described in Appendix 1. The participants were requested to assay the xylanase activity in a 1.0% (w/v) solution of the distributed T. reesei enzyme preparation using: (A), the method and substrate in use in the recipient laboratory; (B), the method of the recipient laboratory but with the distributed xylan substrate; (C), the distributed substrate and the distributed method. The laboratories were allowed to choose their own dilution(s) of the enzyme preparation after first preparing a 1.0% stock solution in 0.05 M citrate buffer, pH 5.3. However, it was stressed that only dilutions yielding results within the range of the xylose standards should be used for activity calculations in method C (see Appendix 1, Note 2). The results of this collaboration are presented in Fig 1. By far the greatest interlaboratory variation, between about 1000 and 28000 nkat ml-1, was recorded when the participants all used their own method and substrate (Fig. 1A). The standard deviation of the results was 108% of the mean. This variation was considerably decreased when all the participants used the same substrate, still using their own methods (Fig. 1B). Further reduction in the standard deviation of
263
0
m m m m
A
t
~SD(%) 6390 108
m
0
A
m
I ,
,
10000
20000
m m m m
,
~
i 4000 D(%)i
0
m m m m
0
m m
51
m m
,
20000
10000
o o
3
m m m m m
m m m
C
m m
~
m
5630
SD(%) ~ 2 4
m m m m m m m m m m mmmmm m m m m m
10000
20000
Assay results (nkat m1-1) Fig. 1. Assay results reported by the participating laboratories in the collaborative method comparison and testing of xylanase methods. (A), own method/own substrate; (B), own method/distributed substrate; (C), distributed method/distributed substrate.
the results was obtained when both the substrate and the method were standardized (Fig. 1C). When using own method and own substrate, the lowest results were obtained by those participants using Somogyi-Nelson reagent for detection of reducing sugars, as predicted (laboratories 6, 7 and 17). However, no common factor could be identified to explain the very high values obtained by laboratories 12, 13 and 15 (Fig. 1A). The explanation was apparently at least partly in the methods used by these laboratories, because the same three participants also reported the highest apparent activities in the test using own method with the distributed substrate, although in this case the difference was less pronounced (Fig. 1B).
Round robin testing of the selected DNS method In a second series of collaborative analyses, the participants were sent samples of a second T. reesei enzyme concentrate (supplied by N. Krebs Lange, Novo Nordisk) and of the birchwood glucuronoxylan (Roth 7500) for preparation of substrate. They were requested to perform a series of three specified dilutions, chosen by the authors to be within the range of the standard line constructed as
264 First d uton
(1:2oooo)
A v
4 5 6 7
B
'=Seconddilution
D
Third dilution (1:6650)
',0:1oooo)
9
~~" lo11 ~ 12 J~ 13
~t 1~ 15 lg 17 18 19 217
100000
2OOOOO
3OOOOO
4OO0O0
Assay results (nkat m1-1) Fig. 2. Assayresults reported in the round robin testing of a single enzyme,substrate and xylanase assay method. Each participant performed the method according to the protocol in Appendix 1 using three specified dilutions. The mean of the whole data was 180000 nkat ml-1 and the standard deviation as a percentage of the mean was 36%, but with the two outliers removed (laboratories 15 and 17, see text) the correspondingfigures were 170000 nkat ml-1 and 17%. described in Appendix 1. The dilutions were to be performed using glass pipettes and graduated flasks of specified volumes, to reduce the risk of inaccuracies due to incorrect calibration of automatic pipettes (sometimes a source of apparent nonlinearity of enzyme reactions). The results of the round robin test are presented in Fig. 2. One laboratory (no. 1) did not respond in this round of testing and two sets of results were clearly erroneous: laboratories 15 and 17 reported that some or all of the absorbance results (A540) obtained from the enzyme reactions were well above the range of the standards. The reasons for these outlying results remained unclear, despite queries sent out by the authors. When the outliers were rejected, the standard deviation of the results from the other 17 laboratories as a percentage of the mean was 17%. This level of variation is probably due to the inherent inaccuracy involved in the use of only partially soluble, heterogeneous and viscous substrates. Another source of variation may have arisen from the fact that the participants were allowed to use their own recipe for preparation of the DNS reagent - although as they also prepared their own standard lines this factor should not in theory cause significant variation.
Round robin testing of dyed xylan substrate Fifteen laboratories also agreed to participate in the testing of a xylanase assay based on the release of dyed fragments from 4-O-methyl glucuronoxylan covalently dyed with Remazol Brilliant Blue dye (RBB xylan; Biely et al., 1985). The substrate (0.555% in 0.05 M sodium acetate buffer, pH 5.3) was dissolved by heating to 70-80°C (with stirring) in 80% of final volume, cooling and making up to volume.
265
Procedural details were: 0.1 ml enzyme (dilutions in acetate buffer, pH 5.3); 0.9 ml RBB xylan solution; incubation 15 min, 30°C; termination by addition of 2.0 ml 96% ethanol, mixing, standing for 20 min, mixing again and centrifuging at 2500 × g for 3 min; measurement of colour released at 595 nm (1 cm light path) against a substrate blank prepared by incubating with 0.1 ml buffer in place of enzyme.
A m
0.15
m
+
i
1
2
3
5
7
6
8
9
10
11
12
13
14
15
i i i 16 17
18
Laboratory (no,)
f::: 8o ~LI~
~
~
I~ (.1 ~.gl
~,~
N ~ ~ m n,II I
I
I
I
II
2
4
6
8
~1
'
36
Xylanase in reaction mixture (nkat) Fig. 3. (A) Slopes of the functions y = kx + q, corresponding to the relationship between dyed fragments released from RBB-dyed xylan substrate (y, A595) and XYL-DNS activity in the 1.0 ml reaction mixture (x, nkat ml-1, calculated from a mean of 180000 nkat ml-1 in the undiluted enzyme, see Fig. 2) for a series of 9 specified enzyme dilutions in 15 participant laboratories. Numbers in the columns represent the intralaboratory coefficients of correlation between the values of x and y for the 9 dilutions used. (B) Interlaboratory relative standard deviation (RSD%) of the values of A595 obtained for 9 different enzyme dilutions by 15 participant laboratories in the assay based on release of coloured oligosaccharides from RBB-dyed xylan. The xylanase activities in the reaction mixture were calculated from the mean XYL-DNS activity of 180000 nkat ml-~ in the undiluted enzyme.
266 Each participant performed a series of 9 dilutions of the second enzyme sample obtained from Novo Nordisk. Absorbances obtained against the substrate blank were reported (results not shown). The slopes of the lines corresponding to the relationship between dyed fragments released from the substrate (measured as A595 after removal of unhydrolyzed substrate by ethanol precipitation) and the XYL-DNS activity in the 1 ml reaction mixture (calculated from the mean activity of 180000 nkat m1-1 obtained for the undiluted enzyme in the round robin test, see Fig. 2) are presented in Fig. 3A. Although the interlaboratory variation in the range of the slopes obtained was rather high, the dependences obtained in each laboratory were apparently linear: coefficients of correlation between colour release from dyed xylan and the calculated XYL-DNS activities in the dilutions used exceeded 0.95 in all cases (Fig. 3A). The relative standard deviation of the results from the 15 participating laboratories was calculated for each enzyme dilution and plotted as a function of the calculated amount of XYL-DNS activity in the reaction mixture. The curve obtained is presented in Fig. 3B. High relative deviation was observed when very low or high enzyme activities were used in the reaction. However, with xylanase activities (XYL-DNS) in the reaction mixture between about 3 and 10 nkatals the interlaboratory deviation between the results was reduced to about 30%, which can be considered as a reasonable figure for a preliminary testing in which many of the participants probably performed this type of assay for the first time.
Discussion
According to the results of the interlaboratory method comparison, the major source of variation between apparent xylanase activities obtained for a single distributed xylanase sample was the choice of substrate (Fig. 1). The variation in results due to differences in the method protocols was smaller but still significant. When both the method and the substrate were standardized and the dilution procedure was precisely specified, the interlaboratory variation of the results was further decreased. The standard deviation of the results in the round robin testing after exclusion of 2 incorrectly performed outliers was 17% of the mean (Fig. 2), compared with a value of 108% in the first comparison in which the participants used their own methods and substrates. The level of deviation between results obtained using a poorly defined, viscous polymeric substrate cannot in practice decrease significantly below the 17% obtained in this interlaboratory collaborative testing. It is the experience of these authors that the variation in assay results obtained within a single laboratory by the same operator may be almost as high when using such substrates. However, the final level of variation was low enough to allow meaningful comparison of results obtained in different laboratories when using the standardized assay substrate and method procedure (Appendix 1). The method and substrate described therein are recommended for use when laboratories wish to compare endo-/3-1,4-xylanase activities of different enzyme samples. The method as specified is suitable for most
267 mesophilic fungal xylanases. In the case of thermophilic a n d / o r bacterial enzymes, the reaction conditions (temperature, p H / b u f f e r ) should be adjusted as necessary. The use of dyed xylan substrate may be an attractive alternative to assays based on the release of reducing sugars from xylan. According to the results of collaborative testing by fifteen laboratories, colour release during the reaction is linear as a function of enzyme dosage and interlaboratory variation is acceptably low when the xylanase activity in the reaction mixture is optimized. Thus the method using RBB-dyed xylan substrate could provide a useful alternative assay for endo-fl-l,4xylanase. The biochemical correlation between release of dyed fragments and production of reducing oligosaccharides requires further investigation. Product analysis from these and other xylan substrates using purified xylanases would provide important information about the hydrolysis mechanisms of different xylanolytic proteins, with and without the assistance of accessory enzymes.
Acknowledgements The authors thank Dr Jiirgen Puls (BHF, Hamburg) for providing samples of non-commercial xylans for testing, Dr J. Caplovic and Dr J. Svec for providing RBB xylan and Dr J. Strecko for help in the statistical analysis of the results of the RBB xylan assay. The technical assistance of Tarja Hakkarainen in Espoo and Mr M. Cziszfirov~ in Bratislava is gratefully acknowledged.
Appendix 1 Xylanase assay (DNS-stopping) Substrate 1.0% birchwood 4-O-methyl glucuronoxylan (Roth 7500) in 0.05 M Na-citrate buffer, pH 5.3. Homogenize 1.0 g of xylan in ca. 80 ml buffer at 60°C (e.g. using a kitchen blender) and heat to boiling point, preferably on a heating magnetic stirrer. Cool with continued stirring, cover and stir slowly overnight. Make up to 100 ml with buffer. Store at 4°C for a maximum of 1 week or freeze aliquots of e.g. 25 ml at -20°C~ Mix well after thawing! Procedure 1. Add 1.8 ml substrate solution to a 15 ml test tube, preferably using an automatic pipette. Temperate to 50°C. 2. Add 200 tzl enzyme diluted appropriately in citrate buffer, mix. 3. Incubate 300 s (5 min), 50°C. 4. Add 3.0 ml DNS, mix and remove the tube from the water bath. Boil for 5 min, cool in cold water. Read note 1 below! 5. Measure the colour produced at 540 nm against the reagent blank. 6. Correct the absorbance (5) for background coiour in the enzyme blank (see below) if necessary.
268 7. Using the standard line, convert the corrected absorbance (6) to enzyme activity units (nkat ml-1). Read Note 2 below! 8. Calculate the activity in the original (undiluted) sample by multiplying activity units (7) by the dilution factor.
Reagent blank 1.8 ml substrate solution 5 min, 50°C 3.0 ml DNS 0.2 ml buffer Boil, cool. Use this solution to zero the spectrophotometer.
Enzyme blank 1.8 ml substrate solution 5 min, 50°C 3.0 ml DNS 0.2 ml enzyme dilution (Note pipetting order!) Boil, cool. Enzyme blanks are required if the dilution is rather small a n d / o r if the sample contains a high level of reducing sugars. Xylose standard
The standard is pure xylose (e.g. Merck 8692), stock solution 0.01 M ( = 0.15 g per 100 ml buffer). Stock solution can be stored in aliquots at -20°C. After thawing the tubes must be mixed particularly well, because the solution becomes "layered" on freezing. A poorly mixed solution will result in an unacceptable standard. Read note 3 below! The stock solution is diluted (in buffer) as follows: 1 : 1 ( = undiluted) = 10.0/zmol ml 1--,33.3 nkat m1-1. 1:2 = 5.0 16.7 1:3 = 3.33 11.1 1 :5 = 2.0 6.7 * Example: If colour produced in the enzymatic reaction corresponds to the colour in the strongest standard point (10/xmol ml-1), xylose production (reducing sugars as xylose) is: 10/zmol ml-1 300 s
0.033/zmol m1-1 s -1 = 33.3 nmol m1-1 s -1 (nkat m1-1)
Standard solutions are treated as e.g. the enzyme blanks: 1.8 ml substrate solution 5 min, 50°C 3.0 ml DNS 0.2 ml standard solution Boil, cool and measure against the reagent blank.
269 The standard line is constructed using the four standard pbints, with absorbances on the ordinate and xylose concentrations converted to nkat m l - 1 , as in the example above, on the abscissa.
Notes 1. W h e n using the modified D N S recipe described e.g. by Miller et al. (1960) a boiling time of 15 min is required, not 5 min as with the original reagent of Sumner (1921). 2. The standard line is based on a purely chemical reaction and is linear to a very high absorbance. The linearity of the enzymatic reaction must be checked using a representative batch of enzyme produced by the microorganism being investigated. W h e n carrying out this collaborative assay, use dilution(s) of the 1% xylanase solution which give absorbances within the range of the four standard points. 3. It is advisable to construct a standard line for every series of assays, in order to minimize variation due to e.g. different batches of substrate and DNS, boiling conditions, automatic pipette calibration, etc.
References Bailey, M.J. (1988) A note on the use of dinitrosalicylic acid for determining the products of enzymatic reactions. Appl. Microbiol. Biotechnol. 29, 494-496. Bailey, M.J. and Nevalainen, K.M.H. (1981) Induction, isolation and testing of stable Trichoderma reesei mutants with improved production of solubilizing cellulase. Enzyme Microb. Technol. 3, 153-157. Bailey, M,J. and Poutanen, K. (1989) Production of xylanolytic enzymes by strains of Aspergillus. Appl. Microbiol. Biotechnol. 30, 5-10. Bailey, M.J., Puls, J. and Poutanen, K. (1991) Purification and properties of two xylanases from Aspergillus oryzae. Biotechnol. Appl. Biochem. 13, 380-389. Biely, P., Mislovicov~i, D. and Toman, R. (1985) Soluble chromatogenic substrates for the assay of endo-l,4-/3-xylanases and endo-l,4-/3-glucanases. Anal. Biochem. 144, 142-146. Breull, C. and Saddler, J.N. (1985) Comparison of the 3,5-dinitrosalicylic acid and Nelson-Somogyi methods of assaying for reducing sugars and cellulase activity. Enzyme Microb. Technol. 7, 327-332. Ebringerov~, A., Kramar, A., Rendos, F. and Domansky, R. (1967) Die Stufenextraktion der Hemicellulosen aus dem Holz der Hagebuche (Carpinus betulus L.). Holzforschung 21, 74-77. Florkin, M. and Stotz, E.H., Eds. (1973) Recommendations (1972) of the Commission on Biochemical Nomenclature on the nomenclature and classification of enzymes together with their units and the symbols of enzyme kinetics. Comprehensive Biochemistry, Vol. 13, 3rd ed., pp. 26-27. H~igglund, E., Lindberg, B. and McPherson, J. (1956) Dimethylsulphoxide, a solvent for hemicelluloses. Acta Chem. Scand. 10, 1160-1164. IUPAC (International Union of Pure and Applied Chemistry) (1987) Measurement of cellulase activities. Pure Appl. Chem. 59, 257-268. Jurasek, L. and Paice, M.G. (1988) Biological bleaching of pulp. Int. Pulp Bleaching Conference, Orlando, FL, USA, June 5-9, 1988. Tappi proceedings, pp. 11-13. Kantelinen, A., Sundqvist, J., Linko, M. and Viikari, L. (1991) The role of reprecipitated xylan in the enzymatic bleaching of Kraft pulp. 6 th Int. Symp. on Wood and Pulping Chem., Melbourne, Australia. Proceedings, Vol. 1, pp. 493-500. Khan, A.W., Tremblay, D. and LeDuy, A. (1986) Assay of xylanase and xylosidase activities in bacterial and fungal cultures. Enzyme Microb. Technol. 8, 373-377. Lenz, J. and Schurz, J. (1986) Neue Wege zur Verwertung von Hemicellulosen. (~. Chem. 1986, 318-321. Miller, G.L. (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31, 426-428.
270 Miller, G.L., Blum, R., Glennon, W.E. and Burton, A.L. (1960) Measurement of carboxymethylcellulase activity. Anal. Biochem. 2, 127-132. Nelson, N. (1944) A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem. 153, 375-380. Nummi, M., Perrin, J-M., Niku-Paavola, M-L. and Enari, T-M. (1985) Measurement of xylanase activity with insoluble xylan substrate. Biochem. J. 226, 617-620. Puls, J. and Poutanen, K. (1989) Mechanisms of enzymatic hydrolysis of hemicelluloses (xylans) and procedures for determination of the enzyme activities involved. In: Coughlan, M.P., Ed. Enzyme Systems for Lignoceilulose Degradation. Elsevier, London and New York, pp. 151-165. Royer, J.C. and Nakas, J.P. (1989) Xylanase production by Trichoderma longibrachiatum. Enzyme Microb. Technol. 11, 405-410. Sinner, M. and Puls, J. (1978) Non-corrosive dye reagent for detection of reducing sugars in borate complex ion-exchange chromatography. J. Chromatogr. 156, 197-204. Somogyi, M. (1952) Notes on sugar determination. J. Biol. Chem. 195, 19-23. Sumner, J.B. (1921) Dinitrosalicylic acid: a reagent for the determination of sugar in normal and diabetic urine. J. Biol. Chem. 47, 5-9. Sumner, J.B. and Somers, G.F. (1949) Dinitrosalicylic acid for glucose. In: Laboratory Experiments in Biological Chemistry, 2nd ed., Academic Press, New York, pp. 38-39. Tan, L.U.L., Wong, K.K.Y., Yu, E.K.C. and Saddler, J.N. (1985) Purification and characterization of two D-xylanases from Trichoderrna harzianum. Enzyme Microb. Technol. 7, 425-430. Viikari, L., Ranua, M., Kantelinen, A., Linko, M. and Sundqvist, J. (1988) Application of enzymes in bleaching. 4th Int. Symp. on Wood and Pulping Chem., Paris 27-30 April, 1987. Proceedings, Vol. 1, pp. 151-154. Viikari, L., Kantelinen, A., Poutanen, K. and Ranua, M. (1990) Characterization of pulps treated with hemicellulolytic enzymes prior to bleaching. In: Biotechnology in Pulp and Paper Manufacture. Applications and Fundamental investigations. Kirk, T.K. and Chang, H.M. (Eds.), ButterworthHeinemann, Boston, pp. 145-151.
