APPLID MICROBIOLOGY, Feb. 1975, p. 300-302
Vol. 29, No. 2
Copyright 0 1975 American Society for Microbiology
Printed in U.S.A.
Rapid Method for Preparing Cell-Free Extracts of Aspergillus ochraceus G. A. SANSING', N. D. DAVIS, AND U. L. DIENER*
Department of Botany and Microbiology, Auburn University, Agricultural Experiment Station, Auburn, Alabama 36830 Received for publication 30 July 1974
A rapid method for preparing cell-free extracts of Aspergillus ochraceus was developed. Mycelial mats were prefrozen in liquid nitrogen, ground to a fine powder in a cold mortar, and homogenized in an all-glass mechanical homogenizer. This method provided preparations averaging 43.0 mg of protein per g of mycelium (wet weight). The method was fast, efficient, and did not subject the extract to temperatures above 1 C or to heavy metals. The preparation method was suitable for studying a variety of in vitro fungal enzyme systems. Amylase, acid phosphatase, alkaline phosphatase, catalase, fatty acid synthetase, glucose6-phosphate dehydrogenase, beta-glucosidase, beta fructofuranosidase, and trehalase activities were measurable in the preparations.
The relationship of certain fungi and their metabolical products to animal and human health was recognized during the outbreaks of turkey-X disease (11). The causal agents were found to be toxic polyketide metabolites of Aspergillus flavus named aflatoxins (4). Since then a number of fungal toxins have been reported and research involving the biosynthetic capabilities of these toxigenic fungi has resulted in an increase in literature in this area. One general theme noted in the literature was the need for an efficient method for obtaining cell-free extracts of various toxin-producing fungi with which to study certain enzyme systems in vitro. (Portions of this study were submitted to Auburn University by G. A. Sansing in partial fullfillment of the requirements of the Ph.D. degree). Several methods have been used for preparing cell-free extracts of fungi. Stine et al. (15) compared nine different methods for the extraction of soluble protein and of a nicotinamide adenine dinucleotide phosphate-specific glutamic acid dehydrogenase system from Neurospora crassa, and found that the French pressure cell and Raper-Hyatt press were the most satisfactory for disruption of mycelial cells. Because of the interest in studying the biosynthetic and enzymatic capabilities of A. ochraceus in our laboratory (3), this investigation was initiated to develop a method for preparing cell-free extracts containing high con' Present address: Commercial Solvents Corp., Research and Development Div., Terre Haute, Ind. 47808.
300
centrations of soluble protein and active enzyme systems. Activities of several enzyme systems were measured to determine their presence in the organism, to check the efficiency of the preparations, and to determine the presence of enzyme systems potentially involved in polyketide (ochratoxin A) biosynthesis (17, 18). Aspfrgillus ochraceus NRRL 3174, obtained from C. W. Hesseltine, Northern Regional Research Laboratory, Peoria, Ill., was used throughout this investigation. This ochratoxinproducing strain (3) was originally isolated from cereal and legume products by the Microbiology Research Group, Council for Scientific and Industrial Research, Pretoria, South Africa (12). Cultures were maintained at 25 C on Czapek solution agar with 20% sucrose (8) supplemented with 0.7% yeast extract (3). The liquid medium used throughout this investigation consisted of 4% sucrose and 2% yeast extract medium (3). Flasks (125-ml) containing 25 ml of the 4% sucrose and 2% yeast medium were stoppered with foam plugs, autoclaved at 121 C for 15 min, cooled, and inoculated with a conidial suspension from 8-day-old agar slants of A. ochraceus. Stationary cultures of A. ochraceus were incubated for 8 days at 25 C. Mycelial mats were removed from culture flasks and rinsed twice with glass distilled water. Mats were surface dried by placing on paper towels for 5 min and then frozen by submerging them in liquid nitrogen for 5 sec. Frozen mats were ground to a fine powder in a cold (0 C) mortar. The powder (10 g) was transferred to a cold
301
NOTES
VOL. 29, 1975
75-ml Braun mechanical cell homogenizing flask of a Bronwill model MSK homogenizer containing 45 g of 1.0-mm glass beads. A 45-ml portion of cold (0 C) buffer was added to the flask. The buffers varied according to the enzyme system being assayed for and were those used by authors of individual assay methods utilized in this investigation. The flask was homogenized (4,000 rpm) for varying times (30 s. 1, 2, and 4 min) under a cold CO2 stream from a CO, cylinder equipped with a siphon tube to facilitate cooling. The homogenate was transferred to cold centrifuge tubes and centrifuged for 20 min at 8,000 x g. The supernatant was decanted and stored at 0 C until utilized. Extracts of A. ochraceus NRRL 3174 were prepared according to the protocol presented for other fungi by Tanenbaum (16). The amount of mycelium used was adjusted to ensure that an equal amount of preparation (1 ml) corresponded to that obtained by the mechanical bead method given above. Preparations were stored at 0 C until used. The enzyme assays for and the methods adopted for assaying were as follow: amylase (2); acid phosphatase (9, 13); alkaline phosphatase (9, 13); catalase (1); fatty acid synthetase (6); glucose-6-phosphate dehydrogenase (20); beta-glucosidase (19); beta-fructofuranosidase (10); and trehalase (14). Those assays involving the quantitation of glucose end TABE 1. Comparison of total protein extracted from Aspergillus ochraceus mycelial mats by three methods Protein Proteinf Extrd mycelium)
Extraction
(mg/gliof
efficiency (% of highest value)
Bead, no prefreeze Bead, prefreeze
8.0 43.0
19 100
French pressure cell
40.0
93
Extraction method
products were conducted using the method of Nelson (7). Spectrophotometric enzyme assays and colorimetric assays were conducted with a Coleman 139 spectrophotometer. Protein was determined by the method of Lowry et al. (5). Mycelial dry weights of A. ochraceus mycelia were determined by filtering mats on Whatman no. 1 filter paper and then drying at 70 C for 12 h. The relationship between concentrations of protein and mycelial disruption times was determined. Soluble protein concentrations, determined at the four homogenization times using the glass bead system, were 30 mg/g of mycelium (wet weight) at 30 s, 43 mg/g at 1 min, 46 mg/g at 2 min, and 47 mg/g at 4 min. Microscope examination of homogenates revealed that most cells were ruptured at the 1-min time and, thus corresponds to the time at which maximum protein concentration is approached. This method of cell disruption was compared with two others and the results are presented in Table 1. The bead-grinding method, without a liquid nitrogen prefreeze, was only 19% as efficient as with the prefreeze (efficiency based on mg/g of mycelium). The French pressure cell method, using the mycelial prefreeze step, was 93% as efficient as the bead method with a mycelial prefreeze. However, preparations are subjected to potential heavy metal contamination with the pressure cell method; an exposure that is avoided with the bead system. Analyses of cell-free homogenates of A. ochraceus, prepared by disrupting prefrozen mycelial mats with the bead system, demonstrated the presence of the enzyme activities listed in Table 2. The high glucoside hydrolase activities demonstrated that the method used was adequate for assaying these enzymes. Activities obtained for other enzyme systems indicated that the method can be expanded to
TAmz 2. Enzyme activities in cell-free Aspergillus ochraceus preparations containing 410 jig/ml ofprotein per ml Enzyme assayed
Amylase Acid phosphatase Alkaline phosphatase Catalase Fatty acid synthetase
Glucose-6-phosphate dehydrogenase Beta-glucosidase Beta-fructofuranosidase Trehalase
Substrate
Soluble starch Phenyldisodium phosphate Phenyldisodium phosphate
Hydrogen peroxide Acetyl CoA Malonyl CoA Glucose-6-phosphate Salicin Sucrose Trehalose
Activity per milliliter of extract
660 ,ig of maltose/ml/12 hr 46 jig of phenol/ml/3 hr 34kgg of phenol/ml/3 hr 16.2 U/ml 2.5 mU/ml
0.016 U/ml 480 Mg of glucose/ml per 12 h 7,860 Mig of glucose/ml per 12 h 560 Mg of glucose/ml per 12 h
302
NOTES
facilitate assays for a wide variety of enzymes. This report describes a rapid method for obtaining cell-free extracts of the filamentous fungus A. ochraceus. Results demonstrate that a liquid nitrogen prefreeze step greatly increases the efficiency of fungal cell disruption by the glass-bead method. This is probably a result of more contact between the finely ground, frozen mycelial particles and the colliding glass beads. The glass-bead method was found to be comparable (milligrams of protein extracted per gram of fresh weight mycelium) to the pressure cell system, thus it is equal in efficiency to the best method described by Stine et al. (15). From a strictly quantitative aspect, the glass-bead system is desirable because it is a closed system and because complete recovery of cellular components is accomplished. The glass-bead system was found to be faster than the pressure cell because two problems that occur when the pressure cell is used are avoided. These are the formation of an ice sleeve along the side of the bore in the precooled pressure cell and a gradual temperature increase during the time pressure is being applied to the cell. These problems are not encountered with the glass-bead method due to the fine temperature control that can be attained with the CO2 stream. In addition, the use of an all glass system precludes possible heavy metal ion contamination that may occur when other systems are used. Because of the variations in assay methods, extract preparation methods, and culture media, the results of the enzyme assays in this investigation are not comparable to similar enzyme assays reported for A. ochraceus and other Aspergillus spp. Therefore, no attempts will be made to compare these results with the specific activities from other reports. However, this method provides a means to examine the intracellular enzymes and components of filamentous fungi and at the same time maintain a high degree of control over temperature, disruption times, and quantitative recovery of cellular components. This investigation was supported by Public Health Service research grant FD-00081 from the Food and Drug Administration.
APPL. MICROBIOL. LITERATURE CITED
1. Beers, R. F., and I. W. Sizer. 1952. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. Biol. Chem. 195:133-140. 2. Bernfeld, P. 1951. Enzymes of starch degradation and synthesis. Adv. Enzymol. 12:379-428. 3. Davis, N. D., J. W. Searcy, and U. L. Diener. 1969. Production of ochratoxin A by Aspergillus ochraceus in a semisynthetic medium. Appl. Microbiol. 17:742-744. 4. Goldblatt, L. A. 1969. Aflatoxin. Academic Press Inc., New York. 5. Lowry, 0. H., N. J. Rosenbrough, A. L. Farr, and R. J. Randall. 1951. Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193:265-268. 6. Lynen, F. 1962. Fatty acid synthesis from malonyl coA, p. 443-451. In S. P. Colowick and N. 0. Kaplan (ed.), Methods in enzymology, vol. 5. Academic Press Inc., New York. 7. Nelson, N. N. 1944. Photometric adaption of the Somogyi method for determination of glucose. J. Biol. Chem. 153:375-380. 8. Raper, K. B., and D. I. Fennell. 1965. The genus Aspergillus, p. 36-37. The Williams and Wilkins Co., Baltimore. 9. Rodriguez-Kabana, R. 1969. Enzymatic interactions of Sclerotium rolfsii and Trichoderma viride in mixed soil culture. Phytopathology 59:910-921. 10. Rodriguez-Kabana, R., and E. A. Curl. 1968. Saccharase activity of Sclerotium rolfsii in soil and the mechanism of antagonistic action by Trichoderma viride. Phytopathology 58:985-992. 11. Sargeant, K., R. Allcroft, and R. B. A. Carnaghan. 1961. Groundnut toxicity. Vet. Rec. 73:865. 12. Scott, de B. 1965. Toxigenic fungi isolated from cereal and legume products. Mycopathol. Mycol. Appl. 14:213-222. 13. Skujins, J. J. 1967. Enzymes in soil, p. 371-414. In A. D. McLaren and G. H. Peterson (ed.), Soil biochemistry. Marcel Dekker, New York. 14. Smith, R E., and R. Rodriguez-Kabana. 1971. Trehalase activity in natural and fungus-colonized soil. Phytopathology 61:912. 15. Stine, G. J., W. N. Strickland, and R. W. Barratt. 1964. Methods of protein extraction from Neurospora crassa. Can. J. Microbiol. 10:29-35. 16. Tanenbaum, S. W. 1967. Biosynthesis of aromatic compounds and their congeners in cell-free extracts from fungi imperfecti. Dev. Ind. Microbiol. 8:88-95. 17. Turner, W. B. 1971. Fungal metabolites. Academic Press Inc., New York. 18. van der Merwe, K. J., P. S. Steyn, L. Fourie, de B. Scott, and J. J. Theron. 1965. Ochratoxin A, a toxic metabolite produced by Aspergillus ochraceus Wilh. Nature (London) 205:1112-1113. 19. Veibel, S. 1950. beta-Glucosidase. In J. B. Summer and K. Myrback (ed.), The enzymes. Academic Press, Inc., New York. 20. Worthington Biochemicals Corporation. 1972. Worthington enzyme manual, p. 13. Freehold, N.J.
Vol. 29, No. 2
Copyright 0 1975 American Society for Microbiology
Printed in U.S.A.
Rapid Method for Preparing Cell-Free Extracts of Aspergillus ochraceus G. A. SANSING', N. D. DAVIS, AND U. L. DIENER*
Department of Botany and Microbiology, Auburn University, Agricultural Experiment Station, Auburn, Alabama 36830 Received for publication 30 July 1974
A rapid method for preparing cell-free extracts of Aspergillus ochraceus was developed. Mycelial mats were prefrozen in liquid nitrogen, ground to a fine powder in a cold mortar, and homogenized in an all-glass mechanical homogenizer. This method provided preparations averaging 43.0 mg of protein per g of mycelium (wet weight). The method was fast, efficient, and did not subject the extract to temperatures above 1 C or to heavy metals. The preparation method was suitable for studying a variety of in vitro fungal enzyme systems. Amylase, acid phosphatase, alkaline phosphatase, catalase, fatty acid synthetase, glucose6-phosphate dehydrogenase, beta-glucosidase, beta fructofuranosidase, and trehalase activities were measurable in the preparations.
The relationship of certain fungi and their metabolical products to animal and human health was recognized during the outbreaks of turkey-X disease (11). The causal agents were found to be toxic polyketide metabolites of Aspergillus flavus named aflatoxins (4). Since then a number of fungal toxins have been reported and research involving the biosynthetic capabilities of these toxigenic fungi has resulted in an increase in literature in this area. One general theme noted in the literature was the need for an efficient method for obtaining cell-free extracts of various toxin-producing fungi with which to study certain enzyme systems in vitro. (Portions of this study were submitted to Auburn University by G. A. Sansing in partial fullfillment of the requirements of the Ph.D. degree). Several methods have been used for preparing cell-free extracts of fungi. Stine et al. (15) compared nine different methods for the extraction of soluble protein and of a nicotinamide adenine dinucleotide phosphate-specific glutamic acid dehydrogenase system from Neurospora crassa, and found that the French pressure cell and Raper-Hyatt press were the most satisfactory for disruption of mycelial cells. Because of the interest in studying the biosynthetic and enzymatic capabilities of A. ochraceus in our laboratory (3), this investigation was initiated to develop a method for preparing cell-free extracts containing high con' Present address: Commercial Solvents Corp., Research and Development Div., Terre Haute, Ind. 47808.
300
centrations of soluble protein and active enzyme systems. Activities of several enzyme systems were measured to determine their presence in the organism, to check the efficiency of the preparations, and to determine the presence of enzyme systems potentially involved in polyketide (ochratoxin A) biosynthesis (17, 18). Aspfrgillus ochraceus NRRL 3174, obtained from C. W. Hesseltine, Northern Regional Research Laboratory, Peoria, Ill., was used throughout this investigation. This ochratoxinproducing strain (3) was originally isolated from cereal and legume products by the Microbiology Research Group, Council for Scientific and Industrial Research, Pretoria, South Africa (12). Cultures were maintained at 25 C on Czapek solution agar with 20% sucrose (8) supplemented with 0.7% yeast extract (3). The liquid medium used throughout this investigation consisted of 4% sucrose and 2% yeast extract medium (3). Flasks (125-ml) containing 25 ml of the 4% sucrose and 2% yeast medium were stoppered with foam plugs, autoclaved at 121 C for 15 min, cooled, and inoculated with a conidial suspension from 8-day-old agar slants of A. ochraceus. Stationary cultures of A. ochraceus were incubated for 8 days at 25 C. Mycelial mats were removed from culture flasks and rinsed twice with glass distilled water. Mats were surface dried by placing on paper towels for 5 min and then frozen by submerging them in liquid nitrogen for 5 sec. Frozen mats were ground to a fine powder in a cold (0 C) mortar. The powder (10 g) was transferred to a cold
301
NOTES
VOL. 29, 1975
75-ml Braun mechanical cell homogenizing flask of a Bronwill model MSK homogenizer containing 45 g of 1.0-mm glass beads. A 45-ml portion of cold (0 C) buffer was added to the flask. The buffers varied according to the enzyme system being assayed for and were those used by authors of individual assay methods utilized in this investigation. The flask was homogenized (4,000 rpm) for varying times (30 s. 1, 2, and 4 min) under a cold CO2 stream from a CO, cylinder equipped with a siphon tube to facilitate cooling. The homogenate was transferred to cold centrifuge tubes and centrifuged for 20 min at 8,000 x g. The supernatant was decanted and stored at 0 C until utilized. Extracts of A. ochraceus NRRL 3174 were prepared according to the protocol presented for other fungi by Tanenbaum (16). The amount of mycelium used was adjusted to ensure that an equal amount of preparation (1 ml) corresponded to that obtained by the mechanical bead method given above. Preparations were stored at 0 C until used. The enzyme assays for and the methods adopted for assaying were as follow: amylase (2); acid phosphatase (9, 13); alkaline phosphatase (9, 13); catalase (1); fatty acid synthetase (6); glucose-6-phosphate dehydrogenase (20); beta-glucosidase (19); beta-fructofuranosidase (10); and trehalase (14). Those assays involving the quantitation of glucose end TABE 1. Comparison of total protein extracted from Aspergillus ochraceus mycelial mats by three methods Protein Proteinf Extrd mycelium)
Extraction
(mg/gliof
efficiency (% of highest value)
Bead, no prefreeze Bead, prefreeze
8.0 43.0
19 100
French pressure cell
40.0
93
Extraction method
products were conducted using the method of Nelson (7). Spectrophotometric enzyme assays and colorimetric assays were conducted with a Coleman 139 spectrophotometer. Protein was determined by the method of Lowry et al. (5). Mycelial dry weights of A. ochraceus mycelia were determined by filtering mats on Whatman no. 1 filter paper and then drying at 70 C for 12 h. The relationship between concentrations of protein and mycelial disruption times was determined. Soluble protein concentrations, determined at the four homogenization times using the glass bead system, were 30 mg/g of mycelium (wet weight) at 30 s, 43 mg/g at 1 min, 46 mg/g at 2 min, and 47 mg/g at 4 min. Microscope examination of homogenates revealed that most cells were ruptured at the 1-min time and, thus corresponds to the time at which maximum protein concentration is approached. This method of cell disruption was compared with two others and the results are presented in Table 1. The bead-grinding method, without a liquid nitrogen prefreeze, was only 19% as efficient as with the prefreeze (efficiency based on mg/g of mycelium). The French pressure cell method, using the mycelial prefreeze step, was 93% as efficient as the bead method with a mycelial prefreeze. However, preparations are subjected to potential heavy metal contamination with the pressure cell method; an exposure that is avoided with the bead system. Analyses of cell-free homogenates of A. ochraceus, prepared by disrupting prefrozen mycelial mats with the bead system, demonstrated the presence of the enzyme activities listed in Table 2. The high glucoside hydrolase activities demonstrated that the method used was adequate for assaying these enzymes. Activities obtained for other enzyme systems indicated that the method can be expanded to
TAmz 2. Enzyme activities in cell-free Aspergillus ochraceus preparations containing 410 jig/ml ofprotein per ml Enzyme assayed
Amylase Acid phosphatase Alkaline phosphatase Catalase Fatty acid synthetase
Glucose-6-phosphate dehydrogenase Beta-glucosidase Beta-fructofuranosidase Trehalase
Substrate
Soluble starch Phenyldisodium phosphate Phenyldisodium phosphate
Hydrogen peroxide Acetyl CoA Malonyl CoA Glucose-6-phosphate Salicin Sucrose Trehalose
Activity per milliliter of extract
660 ,ig of maltose/ml/12 hr 46 jig of phenol/ml/3 hr 34kgg of phenol/ml/3 hr 16.2 U/ml 2.5 mU/ml
0.016 U/ml 480 Mg of glucose/ml per 12 h 7,860 Mig of glucose/ml per 12 h 560 Mg of glucose/ml per 12 h
302
NOTES
facilitate assays for a wide variety of enzymes. This report describes a rapid method for obtaining cell-free extracts of the filamentous fungus A. ochraceus. Results demonstrate that a liquid nitrogen prefreeze step greatly increases the efficiency of fungal cell disruption by the glass-bead method. This is probably a result of more contact between the finely ground, frozen mycelial particles and the colliding glass beads. The glass-bead method was found to be comparable (milligrams of protein extracted per gram of fresh weight mycelium) to the pressure cell system, thus it is equal in efficiency to the best method described by Stine et al. (15). From a strictly quantitative aspect, the glass-bead system is desirable because it is a closed system and because complete recovery of cellular components is accomplished. The glass-bead system was found to be faster than the pressure cell because two problems that occur when the pressure cell is used are avoided. These are the formation of an ice sleeve along the side of the bore in the precooled pressure cell and a gradual temperature increase during the time pressure is being applied to the cell. These problems are not encountered with the glass-bead method due to the fine temperature control that can be attained with the CO2 stream. In addition, the use of an all glass system precludes possible heavy metal ion contamination that may occur when other systems are used. Because of the variations in assay methods, extract preparation methods, and culture media, the results of the enzyme assays in this investigation are not comparable to similar enzyme assays reported for A. ochraceus and other Aspergillus spp. Therefore, no attempts will be made to compare these results with the specific activities from other reports. However, this method provides a means to examine the intracellular enzymes and components of filamentous fungi and at the same time maintain a high degree of control over temperature, disruption times, and quantitative recovery of cellular components. This investigation was supported by Public Health Service research grant FD-00081 from the Food and Drug Administration.
APPL. MICROBIOL. LITERATURE CITED
1. Beers, R. F., and I. W. Sizer. 1952. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. Biol. Chem. 195:133-140. 2. Bernfeld, P. 1951. Enzymes of starch degradation and synthesis. Adv. Enzymol. 12:379-428. 3. Davis, N. D., J. W. Searcy, and U. L. Diener. 1969. Production of ochratoxin A by Aspergillus ochraceus in a semisynthetic medium. Appl. Microbiol. 17:742-744. 4. Goldblatt, L. A. 1969. Aflatoxin. Academic Press Inc., New York. 5. Lowry, 0. H., N. J. Rosenbrough, A. L. Farr, and R. J. Randall. 1951. Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193:265-268. 6. Lynen, F. 1962. Fatty acid synthesis from malonyl coA, p. 443-451. In S. P. Colowick and N. 0. Kaplan (ed.), Methods in enzymology, vol. 5. Academic Press Inc., New York. 7. Nelson, N. N. 1944. Photometric adaption of the Somogyi method for determination of glucose. J. Biol. Chem. 153:375-380. 8. Raper, K. B., and D. I. Fennell. 1965. The genus Aspergillus, p. 36-37. The Williams and Wilkins Co., Baltimore. 9. Rodriguez-Kabana, R. 1969. Enzymatic interactions of Sclerotium rolfsii and Trichoderma viride in mixed soil culture. Phytopathology 59:910-921. 10. Rodriguez-Kabana, R., and E. A. Curl. 1968. Saccharase activity of Sclerotium rolfsii in soil and the mechanism of antagonistic action by Trichoderma viride. Phytopathology 58:985-992. 11. Sargeant, K., R. Allcroft, and R. B. A. Carnaghan. 1961. Groundnut toxicity. Vet. Rec. 73:865. 12. Scott, de B. 1965. Toxigenic fungi isolated from cereal and legume products. Mycopathol. Mycol. Appl. 14:213-222. 13. Skujins, J. J. 1967. Enzymes in soil, p. 371-414. In A. D. McLaren and G. H. Peterson (ed.), Soil biochemistry. Marcel Dekker, New York. 14. Smith, R E., and R. Rodriguez-Kabana. 1971. Trehalase activity in natural and fungus-colonized soil. Phytopathology 61:912. 15. Stine, G. J., W. N. Strickland, and R. W. Barratt. 1964. Methods of protein extraction from Neurospora crassa. Can. J. Microbiol. 10:29-35. 16. Tanenbaum, S. W. 1967. Biosynthesis of aromatic compounds and their congeners in cell-free extracts from fungi imperfecti. Dev. Ind. Microbiol. 8:88-95. 17. Turner, W. B. 1971. Fungal metabolites. Academic Press Inc., New York. 18. van der Merwe, K. J., P. S. Steyn, L. Fourie, de B. Scott, and J. J. Theron. 1965. Ochratoxin A, a toxic metabolite produced by Aspergillus ochraceus Wilh. Nature (London) 205:1112-1113. 19. Veibel, S. 1950. beta-Glucosidase. In J. B. Summer and K. Myrback (ed.), The enzymes. Academic Press, Inc., New York. 20. Worthington Biochemicals Corporation. 1972. Worthington enzyme manual, p. 13. Freehold, N.J.
