Journal of Biochemical and Biophysical Methods, 23 (1991 ) 265-273 © 1991 Elsevier Science Publishers B.V. All rights reserved 0165-022X/91/$03.50
265
JBBM 00909
Estimation of cellulase activity using a glucose-oxidase-Cu(II) reducing assay for glucose Warren L. Baker and Adrian Panow Chemistry Department, Swinburne Institute of Technology, Hawthorn, Victoria, Australia (Received 20 December 1990) (Revision received 7 May 1991) (Accepted 18 June 1991)
Summary In the presence of the Cu(l)-chelating agent neocuproine (2,10-dimethyl-l,9-phenanthroline) hydrogen peroxide acts as a reductant of Cu(II). The reaction does not proceed in the absence of neocuproine and the addition of EDTA to the reaction mixture prior to addition of Cu(II) also inhibits the reduction. Colour development can be arrested and stabilized by addition of EDTA. The reaction can be used to estimate hydrogen peroxide concentrations in the range 0.68-6.8 /zg/ml and glucose concentrations in the range 3.6-36/zg/ml (20-200 ttM). Horseradish peroxidase is not required for the peroxide assay but glucose oxidase must be used for glucose estimations. Thermostable cellulase activity has been estimated at 60°C against cellobiose, carboxymethylcellulose and cellulose substrates by estimation of the glucose released from the substrates. Key words: Glucose; Glucose oxidase; Neocuproine-Cu(1); Cellulase
Introduction Cellulases are a group of protozoal, bacterial or fungal enzymes which rupture t h e e x o - a n d endo-/3 (1 ~ 4) b o n d s o f c e l l u l o s e to r e l e a s e g l u c o s e . A c t i v i t y is usually measured through production of glucose using either the non-specific S o m o g y i - N e l s o n c o p p e r - r e d u c i n g m e t h o d [1], 3 , 5 - d i n i t r o s a l i c y l i c a c i d [2] o r t h e h i g h l y s p e c i f i c g l u c o s e o x i d a s e p r o c e d u r e w i t h o - d i a n i s i d i n e [3] as t h e t e r m i n a l e l e c t r o n d o n o r . T h i s l a t t e r p r o c e d u r e is m o r e s e n s i t i v e b u t has t h e d i s a d v a n t a g e t h a t t h e p e r o x i d a s e e n z y m e is n o t p a r t i c u l a r l y s t a b l e o n p r o l o n g e d s t o r a g e a n d t h e
Correspondence to: Warren L. Baker, Chemistry Dept., Swinburne Institute of Technology, John Street, Hawthorn, Vic 3122, Australia.
266 dye is of questionable safety. An even more sensitive glucose oxidase assay involves use of the electron donor 2,2'-azino-di(3-ethyl-benzthiazoline)-6-sulphonate (ABTS) [4] but also involves use of peroxidase. In experiments in this laboratory it has been observed that hydrogen peroxide acts as a reducing agent for Cu(I1) in the presence of the Cu(I)-chelating agent neocuproine. This reaction has been used as the basis of an enzymic assay for glucose where horseradish peroxidase was not required to transfer electrons from hydrogen peroxide. The assay has been used to estimate cellulase activity. It has a sensitivity range similar to the system using ABTS [4] and only a small volume of sample is required for analysis.
Materials and Methods
Instruments Absorbance readings were made on a Varian-Techtron 635 recording spectrophotometer and fluorescence readings were made with a Shimadzu SP-500 spectrophotofluorimeter. A Metrohm 620 meter was used to measure pH. Chemicals Neocuproine was obtained from Fluka A.G., Buchs, Switzerland. EDTA, glucose and potassium sodium tartrate were purchased from May and Baker, Dagenham, Essex, U.K. and copper sulphate and hydrogen peroxide from Ajax Chemicals, Homebush, N.S.W., Australia. Glucose oxidase ( T / I I ) purified from Aspergillus niger, cellobiose and cellulose "Sigmacel T50" came from Sigma Chemicals, St. Louis, MO, U.S.A. The cellulose was mixed with water and the fines decanted. It was then filtered under vacuum through Whatman No. 1 paper, washed again with water, filtered and air dried. Carboxymethylcellulose was from Whatman (No. 23) and was treated in the same manner as Sigmacel T50. Cellulases The enzymes which were used were Novo Novozyme (/3-glucosidase) and celluclast (cellulase mixture) and were donated by Dr R.F. Dekker of CSIRO Wood Products Division, Clayton, Victoria. These preparations were dialysed for two days at 4°C against distilled water (two changes per day). This procedure removed glucose, small oligosaccharides and much colouring matter. Some Cu(II)reducing substances were not completely removed by this procedure but the remaining concentrations were small. The Novozyme and celluclast were used individually or mixed in the proportion of 1 : 10, respectively. Colour reagent Colour reagent consisted of a mixture of 3 vol. of neocuproine solution (4 m g / m l ) in a 50% solution of ethanol in 0.05 M phosphate buffer, pH 7, 5 vol. of 2% potassium sodium tartrate in buffer and 11 vol. of the same buffer. This solution was stored in a glass container. One volume of 0.5% copper sulphate in
267 water was added immediately before use. The potassium sodium tartrate ensured that the copper and phosphate did not interact and precipitate.
Analytical methods Protein concentrations were measured against a standard of crystalline lysozyme using the Coomassie blue G250 dye binding procedure [5]. Hydrogen peroxide was standardized against freshly prepared potassium permanganate solution [6] and its disappearance estimated against standards using the fluorescent dimer formed from p-hydroxyphenylacetic acid in the presence of horseradish peroxidase [7]. Glucose estimations were performed by incubating 1 ml of glucose solution (20-200/xM) in 0.05 M phosphate buffer, pH 7, with 0.1 ml of glucose oxidase (1 m g / m l ) for 30 min at 37°C. Colour reagent (2 ml) was then added and the solution was incubated for a further 30 min when 0.1 ml of 0.2 M E D T A was added. Absorbance readings were made at 450 nm. When hydrogen peroxide was estimated by this procedure the glucose oxidase incubation was not performed. Cu(II) concentrations in the neocuproine complex were estimated against standard copper solutions reduced by excess hydroxylamine in the presence of neocuproine [8].
Cellulase estimations Ten ml of a 1.2% solution or suspension of substrate in buffer (usually 0.05 M acetate buffer, pH 5) containing either 0.5 ml of cellulase mixture, 0.05 ml of /3-glucosidase plus 0.5 ml of water or 0.55 ml of enzyme mixture as described above was continuously mixed at 60°C. At specified times 1-ml samples were taken, treated with an equal volume of 8% TCA and filtered. Duplicate samples (0.05, 0.1 or 0.2 ml) were diluted to 1 ml with 0.2 M phosphate buffer, pH 7, and 0.1 ml of glucose oxidase solution added. Further protocol was as described above for glucose. Substrate blanks containing all reagents except enzyme and enzyme blanks containing all reagents except substrate were used with each enzyme analysis. Corrections were made for reduction of Cu(II) by the enzyme solution. Substrate blanks gave absorbance values in the range 0.035-0.080 while blanks containing enzyme varied with absorbance values as high as 0.16. Cellulase activity is reported as mg (or/xg) glucose f o r m e d / m l after reference to a glucose standard curve.
Results
Reduction of Cu (II) by hydrogen peroxide The Cu(I)-chelating agent neocuproine was essential for the reduction reaction of Cu(II) (Fig. 1) since the hydrogen peroxide acted as a reductant and disappeared only in its presence (Fig. 2). Pretreatment of the peroxide with catalase inhibited Cu(II) reduction. E D T A arrested colour development at any stage and while it was not strictly necessary at equilibrium it was added routinely to stabilize the colour.
268
0.8! 0.7 0,6
#
,
f
~.__q
~0.5 c
~0.4 o .< 0.3 0.2 ¸
0.1 0 0
t
I
10 20 Time (min)
30
Fig. 1. Development of colour of 200 nmol/ml of hydrogen peroxide treated with 2 ml of colour reagent as described under Materials and Methods. Arrow indicates addition of 20/xmol EDTA.
G l u c o s e s t a n d a r d curt,e The calibration curve for glucose was linear within the concentration
range
2 0 - 8 0 0 ~ M b u t t h e m o s t c o n v e n i e n t r a n g e w a s 2 0 - 2 0 0 j z M ( 3 . 6 - 3 6 ~ g ) (Fig. 3) and this range was used routinely. Solutions showing glucose concentrations above
2 2oo
c_. QJ
a_ 10C
30
Time
(rain)
Fig. 2. Disappearance of hydrogen peroxide in the presence of neocuproine-copper colour reagent. Incubated mixture ([3) contained 9 ml of 400 ~M H202, 8.4 ml of 0.1 M phosphate buffer, pH 7, containing 56 /zmol (12 mg) of neocuproine and 0.6 ml of 4 mM CuSO 4. Other solutions in the same volume lacked neocuproine but contained (©) hydrogen peroxide alone, (o) hydrogen peroxide plus copper and (A) hydrogen peroxide, copper and 60 ttmol EDTA. At relevant times duplicate 1-ml samples were taken and mixed with 2 ml of phosphate buffer containing 100 ttg horseradish peroxidase, 400 /zg hydroxyphenylacetic acid and 5 tzmol EDTA and incubated at 37 °C for ½h before treatment with 0.2 ml of 1 M NaOH. The fluorescence was then read at 415 nm after exciting at 325 nm. The amount of peroxide was determined with reference to a standard curve treated similarly.
269
c°0"6~ ~0. o4 0,8--
265
JBBM 00909
Estimation of cellulase activity using a glucose-oxidase-Cu(II) reducing assay for glucose Warren L. Baker and Adrian Panow Chemistry Department, Swinburne Institute of Technology, Hawthorn, Victoria, Australia (Received 20 December 1990) (Revision received 7 May 1991) (Accepted 18 June 1991)
Summary In the presence of the Cu(l)-chelating agent neocuproine (2,10-dimethyl-l,9-phenanthroline) hydrogen peroxide acts as a reductant of Cu(II). The reaction does not proceed in the absence of neocuproine and the addition of EDTA to the reaction mixture prior to addition of Cu(II) also inhibits the reduction. Colour development can be arrested and stabilized by addition of EDTA. The reaction can be used to estimate hydrogen peroxide concentrations in the range 0.68-6.8 /zg/ml and glucose concentrations in the range 3.6-36/zg/ml (20-200 ttM). Horseradish peroxidase is not required for the peroxide assay but glucose oxidase must be used for glucose estimations. Thermostable cellulase activity has been estimated at 60°C against cellobiose, carboxymethylcellulose and cellulose substrates by estimation of the glucose released from the substrates. Key words: Glucose; Glucose oxidase; Neocuproine-Cu(1); Cellulase
Introduction Cellulases are a group of protozoal, bacterial or fungal enzymes which rupture t h e e x o - a n d endo-/3 (1 ~ 4) b o n d s o f c e l l u l o s e to r e l e a s e g l u c o s e . A c t i v i t y is usually measured through production of glucose using either the non-specific S o m o g y i - N e l s o n c o p p e r - r e d u c i n g m e t h o d [1], 3 , 5 - d i n i t r o s a l i c y l i c a c i d [2] o r t h e h i g h l y s p e c i f i c g l u c o s e o x i d a s e p r o c e d u r e w i t h o - d i a n i s i d i n e [3] as t h e t e r m i n a l e l e c t r o n d o n o r . T h i s l a t t e r p r o c e d u r e is m o r e s e n s i t i v e b u t has t h e d i s a d v a n t a g e t h a t t h e p e r o x i d a s e e n z y m e is n o t p a r t i c u l a r l y s t a b l e o n p r o l o n g e d s t o r a g e a n d t h e
Correspondence to: Warren L. Baker, Chemistry Dept., Swinburne Institute of Technology, John Street, Hawthorn, Vic 3122, Australia.
266 dye is of questionable safety. An even more sensitive glucose oxidase assay involves use of the electron donor 2,2'-azino-di(3-ethyl-benzthiazoline)-6-sulphonate (ABTS) [4] but also involves use of peroxidase. In experiments in this laboratory it has been observed that hydrogen peroxide acts as a reducing agent for Cu(I1) in the presence of the Cu(I)-chelating agent neocuproine. This reaction has been used as the basis of an enzymic assay for glucose where horseradish peroxidase was not required to transfer electrons from hydrogen peroxide. The assay has been used to estimate cellulase activity. It has a sensitivity range similar to the system using ABTS [4] and only a small volume of sample is required for analysis.
Materials and Methods
Instruments Absorbance readings were made on a Varian-Techtron 635 recording spectrophotometer and fluorescence readings were made with a Shimadzu SP-500 spectrophotofluorimeter. A Metrohm 620 meter was used to measure pH. Chemicals Neocuproine was obtained from Fluka A.G., Buchs, Switzerland. EDTA, glucose and potassium sodium tartrate were purchased from May and Baker, Dagenham, Essex, U.K. and copper sulphate and hydrogen peroxide from Ajax Chemicals, Homebush, N.S.W., Australia. Glucose oxidase ( T / I I ) purified from Aspergillus niger, cellobiose and cellulose "Sigmacel T50" came from Sigma Chemicals, St. Louis, MO, U.S.A. The cellulose was mixed with water and the fines decanted. It was then filtered under vacuum through Whatman No. 1 paper, washed again with water, filtered and air dried. Carboxymethylcellulose was from Whatman (No. 23) and was treated in the same manner as Sigmacel T50. Cellulases The enzymes which were used were Novo Novozyme (/3-glucosidase) and celluclast (cellulase mixture) and were donated by Dr R.F. Dekker of CSIRO Wood Products Division, Clayton, Victoria. These preparations were dialysed for two days at 4°C against distilled water (two changes per day). This procedure removed glucose, small oligosaccharides and much colouring matter. Some Cu(II)reducing substances were not completely removed by this procedure but the remaining concentrations were small. The Novozyme and celluclast were used individually or mixed in the proportion of 1 : 10, respectively. Colour reagent Colour reagent consisted of a mixture of 3 vol. of neocuproine solution (4 m g / m l ) in a 50% solution of ethanol in 0.05 M phosphate buffer, pH 7, 5 vol. of 2% potassium sodium tartrate in buffer and 11 vol. of the same buffer. This solution was stored in a glass container. One volume of 0.5% copper sulphate in
267 water was added immediately before use. The potassium sodium tartrate ensured that the copper and phosphate did not interact and precipitate.
Analytical methods Protein concentrations were measured against a standard of crystalline lysozyme using the Coomassie blue G250 dye binding procedure [5]. Hydrogen peroxide was standardized against freshly prepared potassium permanganate solution [6] and its disappearance estimated against standards using the fluorescent dimer formed from p-hydroxyphenylacetic acid in the presence of horseradish peroxidase [7]. Glucose estimations were performed by incubating 1 ml of glucose solution (20-200/xM) in 0.05 M phosphate buffer, pH 7, with 0.1 ml of glucose oxidase (1 m g / m l ) for 30 min at 37°C. Colour reagent (2 ml) was then added and the solution was incubated for a further 30 min when 0.1 ml of 0.2 M E D T A was added. Absorbance readings were made at 450 nm. When hydrogen peroxide was estimated by this procedure the glucose oxidase incubation was not performed. Cu(II) concentrations in the neocuproine complex were estimated against standard copper solutions reduced by excess hydroxylamine in the presence of neocuproine [8].
Cellulase estimations Ten ml of a 1.2% solution or suspension of substrate in buffer (usually 0.05 M acetate buffer, pH 5) containing either 0.5 ml of cellulase mixture, 0.05 ml of /3-glucosidase plus 0.5 ml of water or 0.55 ml of enzyme mixture as described above was continuously mixed at 60°C. At specified times 1-ml samples were taken, treated with an equal volume of 8% TCA and filtered. Duplicate samples (0.05, 0.1 or 0.2 ml) were diluted to 1 ml with 0.2 M phosphate buffer, pH 7, and 0.1 ml of glucose oxidase solution added. Further protocol was as described above for glucose. Substrate blanks containing all reagents except enzyme and enzyme blanks containing all reagents except substrate were used with each enzyme analysis. Corrections were made for reduction of Cu(II) by the enzyme solution. Substrate blanks gave absorbance values in the range 0.035-0.080 while blanks containing enzyme varied with absorbance values as high as 0.16. Cellulase activity is reported as mg (or/xg) glucose f o r m e d / m l after reference to a glucose standard curve.
Results
Reduction of Cu (II) by hydrogen peroxide The Cu(I)-chelating agent neocuproine was essential for the reduction reaction of Cu(II) (Fig. 1) since the hydrogen peroxide acted as a reductant and disappeared only in its presence (Fig. 2). Pretreatment of the peroxide with catalase inhibited Cu(II) reduction. E D T A arrested colour development at any stage and while it was not strictly necessary at equilibrium it was added routinely to stabilize the colour.
268
0.8! 0.7 0,6
#
,
f
~.__q
~0.5 c
~0.4 o .< 0.3 0.2 ¸
0.1 0 0
t
I
10 20 Time (min)
30
Fig. 1. Development of colour of 200 nmol/ml of hydrogen peroxide treated with 2 ml of colour reagent as described under Materials and Methods. Arrow indicates addition of 20/xmol EDTA.
G l u c o s e s t a n d a r d curt,e The calibration curve for glucose was linear within the concentration
range
2 0 - 8 0 0 ~ M b u t t h e m o s t c o n v e n i e n t r a n g e w a s 2 0 - 2 0 0 j z M ( 3 . 6 - 3 6 ~ g ) (Fig. 3) and this range was used routinely. Solutions showing glucose concentrations above
2 2oo
c_. QJ
a_ 10C
30
Time
(rain)
Fig. 2. Disappearance of hydrogen peroxide in the presence of neocuproine-copper colour reagent. Incubated mixture ([3) contained 9 ml of 400 ~M H202, 8.4 ml of 0.1 M phosphate buffer, pH 7, containing 56 /zmol (12 mg) of neocuproine and 0.6 ml of 4 mM CuSO 4. Other solutions in the same volume lacked neocuproine but contained (©) hydrogen peroxide alone, (o) hydrogen peroxide plus copper and (A) hydrogen peroxide, copper and 60 ttmol EDTA. At relevant times duplicate 1-ml samples were taken and mixed with 2 ml of phosphate buffer containing 100 ttg horseradish peroxidase, 400 /zg hydroxyphenylacetic acid and 5 tzmol EDTA and incubated at 37 °C for ½h before treatment with 0.2 ml of 1 M NaOH. The fluorescence was then read at 415 nm after exciting at 325 nm. The amount of peroxide was determined with reference to a standard curve treated similarly.
269
c°0"6~ ~0. o4 0,8--
