APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1978, p. 969-971 0099-2240/78/0036-0969$02.00/0 Copyright © 1978 American Society for Microbiology
Vol. 36, No. 6
Printed in U.S.A.
D-Lyxose as a Substrate for Streptomyces D-Xylose Isomerase J. H. PARKERt Miles Laboratories, Inc., Industrial Products Group, Elkhart, Indiana 46515
Received for publication 26 September 1978
Results are presented which show that the D-xylose isomerases present in Streptomyces olivaceus and Streptomyces phaeochromogenes NRRL B-3559 are incapable of utilizing D-lyxose as a substrate. The implications of these findings as related to the use of D-lyxose in the selection of constitutive mutants are discussed. The use of species of Streptomyces for production of D-xylose keto isomerase (EC 5.3.1.5) was first reported by Tsumura and Sato (9). The properties of this enzyme have been well documented (1, 5,6,8,9), and its use in the conversion of D-glucose to D-fructose is finding broad commercial application. The isomerase of Streptomyces species is inducible and requires the presence of xylose in the fermentation medium for production (6, 8, 9). Because of the relatively high cost of xylose, a number of laboratories have sought mutants which are constitutive for enzyme production, and several methods for selecting such mutants have been reported (5; F. C. Armbruster, R. E. Heady, and R. P. Cory, U.S. Patent RE 29,152, March 1977). Sanchez and Quinto (5) reported that D-lyxose (2-epimer of D-xylose) could be employed to isolate mutants constitutive for D-xylose keto isomerase production in Streptomyces phaeochromogenes NRRL B-3559. It was reported that such mutants were obtained by using a selective medium containing D-lyxose as the sole carbon source. Attempts to use the same procedure in a strain of Steptomyces olivaceus are reported in this paper. The basic rationale for this approach is also discussed. The objective of these studies was to isolate glucose isomerase-constitutive mutants of S. olivaceus by using the method reported by Sanchez and Quinto (5). It was confirmed that spores of S. olivaceus 6A-KO are unable to germinate in minimal salts medium containing D-lyxose as the sole carbon source but that both D-xylose and D-glucose support profuse growth. However, in contrast to the previous report (5), preincubation of spores in the presence of D-xylose plus D-glucose and transfer to D-lyxose did not produce growth, (Table 1). To determine whether there are species differences, a culture of S. phaeochromogenes National Regional Research t Present address: Rochester Institute of Technology, 1 Lomb Memorial Drive, Rochester, NY 14623.
Laboratory (NRRL) B-3559 was also tested. The results are compared with the results obtained by Sanchez and Quinto (5) with their strain of S. phaeochromogenes NRRL B-3559. Seven mutants, selected according to their ability to grow in miniimal salts medium containing 1% D-lyxose as the sole carbon source, were obtained after treatment with nitrosoguanidine, and two others resulted from ultraviolet light treatment and selection on minimal salts medium (4) containing 3.3 ,umol of D-lyxose per ml. Whereas all of these mutants grew in the presence of lyxose, there were varying patterns of growth. None of these mutants produced significant amounts of glucose isomerase when grown in the absence of xylose inducer, as determined by the lack of fructose produced in the standard enzyme assay procedure. Enzyme activity was generally measured by determining the rate of conversion of D-glucose, D-xylose, or D-lyxose to their ketose form in the following reaction mixture: 62.5% carbohydrate, 3.08% MgS04.7H20, 0.02975% CoCl2.6H20 in 0.125 M tris(hydroxymethyl)aminomethane-hydrochloride buffer. Ten milliliters of the substrate mixture was incubated with 15-ml enzyme samples (cells plus fermentation medium) at 600C for 2 h, at which time the reaction was terminated by the addition of 1 ml of 2 M perchloric acid. The amount of ketose sugar formed was determined colorimetrically as follows: 20 ,ul of filtered reaction mixture (prepared as described above) was diluted to 10 ml with water. One milliliter of this solution was added to 0.2 ml of 2.4% cysteine-HCl in 6 ml of 75% H2SO4 with immediate mixing. To this was added 0.2 ml of 0.1% carbazole in ethanol, and the mixture was incubated in a 370C water bath for 1 h. Absorbance at 540 nm was determined (2). Mutant 3662.7/12A.2, which produced the most growth on lyxose medium, was further tested to detect possible small increases in the amount of enzyme present in uninduced cells. A
969
970
APPL. ENVIRON. MICROBIOL.
NOTES
TABLE 1. Effect ofpreincubation in xylosecontaining medium on growth of strains of S. phaeochromogenes and S. olivaceus on lyxose and other carbon sources Growth Strain
S. olivaceus
Carbohydrate in of preinspot platesa
cubated sporesb
D-GlucoSe
+C
D-Xylose D-Lyxose
+
None
S. phaeochromogenes (Sanchez and Quinto)d
D-Glucose D-Xylose D-Lyxose None
+ +
S. phaeochromogenes (Miles)
D-Glucose
+ +
D-Xylose D-Lyxose
+
None
Carbohydrates were added to solid inorganic salts medium (4) to produce the following final concentrations: 26 ,umol of D-glucose, 11 ,umol of D-xylose, and 3.3 ltmol of D-lyxose per ml. b Spores were preincubated at 300C with shaking for 20 h in inorganic salts medium containing 16 ,umol of D-glucose and 46 l.mol of D-xylose per ml. Cells were washed twice with sterile distilled water before plating. Spores were suspended in sterile water, and a drop of suspension was spotted on each type of medium. 'Plates were observed at 24 and 48 h of incubation at 300C. +, Formation of a confluent colony possessing ariel mycelium. d Data from Table 1 of Sanchez and Quinto (5).
Sanchez and Quinto, synthesis of this enzyme is inducible by xylose but not lyxose so that the parent strain is incapable of producing the enzyme when lyxose is the sole carbon source. However, lyxose may serve as a substrate for the enzyme, and, thus, constitutive mutants will grow on lyxose medium, whereas the parent will not. This model is similar to the one proposed by Mortlock et al. (3) for utilization by Aerobacter aerogenes of various "unnatural" pentoses. It was suggested that the use of rare pentoses occurs via enzymes specific for other more common pentoses. The enzyme levels normally present, according to this model, are insufficient to allow growth on the rare pentoses. When mutation resulting in derepression and constitutive TABLE 2. Production of enzyme by two S. olivaceus strains determined by using D-glucose and D-xylose as assay substrates Enzyme activity
a
sample of culture medium plus cells was used, and the incubation period of the assay was extended to 24 h instead of the usual 2 h. The enzyme activity of cells grown in the presence and absence of xylose are indicated in Table 2, using glucose and xylose as enzyme substrates. Note that enzyme samples from mutant cells grown in the presence of lyxose did show an increased conversion of D-xylose to the ketose form, compared to mutant cells grown in the absence of inducer, but no increase in the conversion of D-glucose to D-fructose when D-lyxose was present as an inducer. A similar comparison between S. olivaceus 6A-KO and S. phaeochromogenes was conducted except that incubations for the enzyme assay were carried out for 4 rather than 24 h (Table 3). Lyxose showed no activity as a substrate for the enzyme under any of the conditions tested. The rationale for selecting mutants possessing the ability to grow on D-lyxose, as described by Sanchez and Quinto (5), is that such mutants are able to grow because they constitutively produce D-xylose keto isomerase. According to
(AA/24 h)b
Strain
Parent (6A-KO)
Inducer pres-
en'
None
D-Xylose D-Lyxose None
Glucose
Xylose yo
sub-
substrate
strate
0.058 1.029 0.044
0.196 0.727 0.240
0.084 0.648 0.274 D-Lyxose aStandard production medium containing 3.4% corn steep liquor, 0.3% Buffalo pearl starch, and either 0.44% xylose, lyxose, or no inducer added, at 300C for 30 h. bThe enzyme activity is expressed as the change in absorbance (AA) at 540 nm after 24 h of incubation of enzyme in either glucose or xylose substrate compared to the substrate blank. TABLE 3. Comparison of D-xylose (D-glucose) keto isomerase activity in S. olivaceus 6A-KO and S. phaeochromogenes B-83 by using glucose and xylose as substrates Enzyme activity (AA/4 h)a
3662.7/12A.2
Strain
S. olivaceus
D-Xylose
Inducer
None
Xylose
0.054 0.735 0.052
Glucose substrate
Xylose
Lyxose substrate
0.064 0.477
0.199 0.983
0b
substrate
0
0.041 0.041 0 S. phaeochromo- None 0 Xylose 0.365 0.757 genes a Enzyme activity expressed in the same manner as in Table 2. Incubation time for the assay was 4 h. b The AA, using lyxose as a substrate, was zero or slightly negative in all cases, indicating no conversion of D-lyxose to xylulose.
VOL. 36, 1978
synthesis occurs, growth on the rare pentoses can take place (3). This model requires that the enzyme (in this case D-xylose isomerase) utilizes the rare pentose (D-lyxose) to a small extent. The present work demonstrates that lyxose does not serve as substrate for the enzyme produced by S. olivaceus 6A-KO. Furthermore, the enzyme produced by S. phaeochromogenes NRRL B-3559 also shows no conversion of lyxose to the keto form, xylulose. Others have reported the inability of D-lyxose to serve as a substrate for D-xylose isomerase, specifically in the organisms Streptomyces albus (6) and Lactobacillus brevis (10). In the latter case, it was shown that D-lyxose serves as a competitive inhibitor of the enzyme (11). Thus, it seems unlikely that constitutive production of enzyme would be sufficient to explain growth when lyxose is the sole carbon source. Rather, it seems necessary that some other enzyme such as a lyxose isomerase be present for growth to occur, as in the case of Aerobacter (3). Sanchez and Quinto (5) reported that preincubation of S. phaeochromogenes spores in Dxylose-containing medium leads to profuse growth upon transfer of the spores to medium containing D-lyxose as the sole carbon source. Our results failed to confirm this finding for either S. olivaceus or S. phaeochromogenes NRRL B-3559. Indeed, it would be expected that enzyme induced in the preincubation medium would be rapidly diluted out upon transfer to medium lacking an inducer. If, as suggested (5), lyxose is not an inducer, the growth would cease after a few generations. That preincubation in xylose medium, under our conditions, did not lead to growth on miniimal medium containing D-lyxose suggests that perhaps the strain of S. phaeochromogenes NRRL B-3559 reported by Sanchez and Quinto (5) may be different from our culture, although both were apparently obtained from the same source (NRRL). Perhaps their strain possesses an inducible D-lyxose isomerase which can be induced by D-xylose or Dlyxose and which is under similar control to the D-xylose keto isomerase. The mutants isolated might then be constitutive for both enzymes. It has been reported that Aerobacter possess a specific D-lyxose isomerase which is inducible only by D-lyxose (3). In Escherichia coli K-12, on the other hand, the ability of mutants to grow on D-lyxose apparently results from constitutive synthesis of a novel D-mannose isomerase (7). The results of our experiment in which D-lyxose was added to the enzyme production medium do not indicate the presence of an inducible Dlyxose isomerase in our strains of S. olivaceus. It is also possible that the D-lyxose used by
NOTES
971
Sanchez and Quinto (5) may have contained sugar impurities which could account for their results. D-Lyxose, as marketed by Sigma Chemical Co., St. Louis, Mo., does not include a statement of purity. Presence of any impurities which could act as a substrate or inducer for D-xylose keto isomerase or D-lyxose keto isomerase would greatly alter the interpretations of their data. In conclusion, it is apparent that the D-lyxose selection method does not operate according to the hypothesized mechanism (5) in the strains tested. In addition, mutants of S. olivaceus which grow on D-lyxose as sole carbon source are not universally constitutive for D-xylose keto is6merase production. It is suggested, therefore, that another explanation is necessary for the success of this method in the case of S. phaeochromogenes. It is my opinion that the D-lyxose selection procedure will not be generally useful for obtaining mutants constitutive for the synthesis of D-xylose keto isomerase. I thank R. J. Erickson, for his critical reading of the manuscript and for his suggestions, and also E. D. Anderson and M. L. Chupp, for their excellent technical assistance.
LITERATURE CITED 1. Chou, C. C., M. R. Ladisch, and G. T. Tsao. 1976. Studies on glucose isomerase for Streptomyces species. Appl. Environ. Microbiol.:32:489-493. 2. Dische, Z. 1962. Color reaction of ketoses with carbazole and sulfuric acid, p. 481. In R. L. Whistler et al. (ed.), Methods in carbohydrate chemistry. Academic Press, Inc., New York. 3. Mortlock, R. P., D. D. Fossitt, and W. A. Wood. 1965. A basis for utilization of unnatural pentoses and pentitols by Aerobacter aerogenes. Proc. Natl. Acad. Sci. U.S.A. 54:572-579. 4. Pridham, T. G., P. Anderson, C. Foley, L. A. Lindenfelser, C. W. Hesseltine, and R. G. Benedict. 1957. A selection of media for maintenance and taxonomic study of Streptomyces. Antibiot. Annu. 57:947-953. 5. Sanchez, S., and C. M. Quinto. 1975. D-Glucose isomerase: constitutive and catabolite repression-resistant mutants of Streptomyces phaeochromogenes. Appl. Microbiol. 30:750-754. 6. Sanchez, S., and K. L. Smiley. 1975. Properties of Dxylose isomerase from Streptomyces albus. Appl. Microbiol. 29:745-750. 7. Stevens, F. J., and T. T. Wu. 1976. Growth on D-lyXose of the mutant strain of Escherichia coli K12 using a novel isomerase and enzymes related to D-xylose metabolism. J. Gen. Microbiol. 97:257-265. 8. Takasaki, Y., Y. Kosugi, and A. Kanbayashi. 1969. Streptomyces glucose isomerase, p. 561-570 In D. Perlman (ed.), Fernentation advances. Academic Press, Inc., New York. 9. Tsumura, N., and T. Sato. 1965. Enzymatic conversion of D-glucose to D-fructose. VI. Properties of the enzyme from Streptomyces phaeochromogenes. Agric. Biol. Chem. 29:1129-1134. 10. Yamanaka, K. 1968. Purification, crystallization and properties of the D-Xylose isomerase from Lactobacillus brevis. Biochim. Biophys. Acta 151:670-680. 11. Yamanaka, K. 1975. D-Xylose isomerase from Lactobacillus brevis. Methods Enzymol. 51:466-471.
Vol. 36, No. 6
Printed in U.S.A.
D-Lyxose as a Substrate for Streptomyces D-Xylose Isomerase J. H. PARKERt Miles Laboratories, Inc., Industrial Products Group, Elkhart, Indiana 46515
Received for publication 26 September 1978
Results are presented which show that the D-xylose isomerases present in Streptomyces olivaceus and Streptomyces phaeochromogenes NRRL B-3559 are incapable of utilizing D-lyxose as a substrate. The implications of these findings as related to the use of D-lyxose in the selection of constitutive mutants are discussed. The use of species of Streptomyces for production of D-xylose keto isomerase (EC 5.3.1.5) was first reported by Tsumura and Sato (9). The properties of this enzyme have been well documented (1, 5,6,8,9), and its use in the conversion of D-glucose to D-fructose is finding broad commercial application. The isomerase of Streptomyces species is inducible and requires the presence of xylose in the fermentation medium for production (6, 8, 9). Because of the relatively high cost of xylose, a number of laboratories have sought mutants which are constitutive for enzyme production, and several methods for selecting such mutants have been reported (5; F. C. Armbruster, R. E. Heady, and R. P. Cory, U.S. Patent RE 29,152, March 1977). Sanchez and Quinto (5) reported that D-lyxose (2-epimer of D-xylose) could be employed to isolate mutants constitutive for D-xylose keto isomerase production in Streptomyces phaeochromogenes NRRL B-3559. It was reported that such mutants were obtained by using a selective medium containing D-lyxose as the sole carbon source. Attempts to use the same procedure in a strain of Steptomyces olivaceus are reported in this paper. The basic rationale for this approach is also discussed. The objective of these studies was to isolate glucose isomerase-constitutive mutants of S. olivaceus by using the method reported by Sanchez and Quinto (5). It was confirmed that spores of S. olivaceus 6A-KO are unable to germinate in minimal salts medium containing D-lyxose as the sole carbon source but that both D-xylose and D-glucose support profuse growth. However, in contrast to the previous report (5), preincubation of spores in the presence of D-xylose plus D-glucose and transfer to D-lyxose did not produce growth, (Table 1). To determine whether there are species differences, a culture of S. phaeochromogenes National Regional Research t Present address: Rochester Institute of Technology, 1 Lomb Memorial Drive, Rochester, NY 14623.
Laboratory (NRRL) B-3559 was also tested. The results are compared with the results obtained by Sanchez and Quinto (5) with their strain of S. phaeochromogenes NRRL B-3559. Seven mutants, selected according to their ability to grow in miniimal salts medium containing 1% D-lyxose as the sole carbon source, were obtained after treatment with nitrosoguanidine, and two others resulted from ultraviolet light treatment and selection on minimal salts medium (4) containing 3.3 ,umol of D-lyxose per ml. Whereas all of these mutants grew in the presence of lyxose, there were varying patterns of growth. None of these mutants produced significant amounts of glucose isomerase when grown in the absence of xylose inducer, as determined by the lack of fructose produced in the standard enzyme assay procedure. Enzyme activity was generally measured by determining the rate of conversion of D-glucose, D-xylose, or D-lyxose to their ketose form in the following reaction mixture: 62.5% carbohydrate, 3.08% MgS04.7H20, 0.02975% CoCl2.6H20 in 0.125 M tris(hydroxymethyl)aminomethane-hydrochloride buffer. Ten milliliters of the substrate mixture was incubated with 15-ml enzyme samples (cells plus fermentation medium) at 600C for 2 h, at which time the reaction was terminated by the addition of 1 ml of 2 M perchloric acid. The amount of ketose sugar formed was determined colorimetrically as follows: 20 ,ul of filtered reaction mixture (prepared as described above) was diluted to 10 ml with water. One milliliter of this solution was added to 0.2 ml of 2.4% cysteine-HCl in 6 ml of 75% H2SO4 with immediate mixing. To this was added 0.2 ml of 0.1% carbazole in ethanol, and the mixture was incubated in a 370C water bath for 1 h. Absorbance at 540 nm was determined (2). Mutant 3662.7/12A.2, which produced the most growth on lyxose medium, was further tested to detect possible small increases in the amount of enzyme present in uninduced cells. A
969
970
APPL. ENVIRON. MICROBIOL.
NOTES
TABLE 1. Effect ofpreincubation in xylosecontaining medium on growth of strains of S. phaeochromogenes and S. olivaceus on lyxose and other carbon sources Growth Strain
S. olivaceus
Carbohydrate in of preinspot platesa
cubated sporesb
D-GlucoSe
+C
D-Xylose D-Lyxose
+
None
S. phaeochromogenes (Sanchez and Quinto)d
D-Glucose D-Xylose D-Lyxose None
+ +
S. phaeochromogenes (Miles)
D-Glucose
+ +
D-Xylose D-Lyxose
+
None
Carbohydrates were added to solid inorganic salts medium (4) to produce the following final concentrations: 26 ,umol of D-glucose, 11 ,umol of D-xylose, and 3.3 ltmol of D-lyxose per ml. b Spores were preincubated at 300C with shaking for 20 h in inorganic salts medium containing 16 ,umol of D-glucose and 46 l.mol of D-xylose per ml. Cells were washed twice with sterile distilled water before plating. Spores were suspended in sterile water, and a drop of suspension was spotted on each type of medium. 'Plates were observed at 24 and 48 h of incubation at 300C. +, Formation of a confluent colony possessing ariel mycelium. d Data from Table 1 of Sanchez and Quinto (5).
Sanchez and Quinto, synthesis of this enzyme is inducible by xylose but not lyxose so that the parent strain is incapable of producing the enzyme when lyxose is the sole carbon source. However, lyxose may serve as a substrate for the enzyme, and, thus, constitutive mutants will grow on lyxose medium, whereas the parent will not. This model is similar to the one proposed by Mortlock et al. (3) for utilization by Aerobacter aerogenes of various "unnatural" pentoses. It was suggested that the use of rare pentoses occurs via enzymes specific for other more common pentoses. The enzyme levels normally present, according to this model, are insufficient to allow growth on the rare pentoses. When mutation resulting in derepression and constitutive TABLE 2. Production of enzyme by two S. olivaceus strains determined by using D-glucose and D-xylose as assay substrates Enzyme activity
a
sample of culture medium plus cells was used, and the incubation period of the assay was extended to 24 h instead of the usual 2 h. The enzyme activity of cells grown in the presence and absence of xylose are indicated in Table 2, using glucose and xylose as enzyme substrates. Note that enzyme samples from mutant cells grown in the presence of lyxose did show an increased conversion of D-xylose to the ketose form, compared to mutant cells grown in the absence of inducer, but no increase in the conversion of D-glucose to D-fructose when D-lyxose was present as an inducer. A similar comparison between S. olivaceus 6A-KO and S. phaeochromogenes was conducted except that incubations for the enzyme assay were carried out for 4 rather than 24 h (Table 3). Lyxose showed no activity as a substrate for the enzyme under any of the conditions tested. The rationale for selecting mutants possessing the ability to grow on D-lyxose, as described by Sanchez and Quinto (5), is that such mutants are able to grow because they constitutively produce D-xylose keto isomerase. According to
(AA/24 h)b
Strain
Parent (6A-KO)
Inducer pres-
en'
None
D-Xylose D-Lyxose None
Glucose
Xylose yo
sub-
substrate
strate
0.058 1.029 0.044
0.196 0.727 0.240
0.084 0.648 0.274 D-Lyxose aStandard production medium containing 3.4% corn steep liquor, 0.3% Buffalo pearl starch, and either 0.44% xylose, lyxose, or no inducer added, at 300C for 30 h. bThe enzyme activity is expressed as the change in absorbance (AA) at 540 nm after 24 h of incubation of enzyme in either glucose or xylose substrate compared to the substrate blank. TABLE 3. Comparison of D-xylose (D-glucose) keto isomerase activity in S. olivaceus 6A-KO and S. phaeochromogenes B-83 by using glucose and xylose as substrates Enzyme activity (AA/4 h)a
3662.7/12A.2
Strain
S. olivaceus
D-Xylose
Inducer
None
Xylose
0.054 0.735 0.052
Glucose substrate
Xylose
Lyxose substrate
0.064 0.477
0.199 0.983
0b
substrate
0
0.041 0.041 0 S. phaeochromo- None 0 Xylose 0.365 0.757 genes a Enzyme activity expressed in the same manner as in Table 2. Incubation time for the assay was 4 h. b The AA, using lyxose as a substrate, was zero or slightly negative in all cases, indicating no conversion of D-lyxose to xylulose.
VOL. 36, 1978
synthesis occurs, growth on the rare pentoses can take place (3). This model requires that the enzyme (in this case D-xylose isomerase) utilizes the rare pentose (D-lyxose) to a small extent. The present work demonstrates that lyxose does not serve as substrate for the enzyme produced by S. olivaceus 6A-KO. Furthermore, the enzyme produced by S. phaeochromogenes NRRL B-3559 also shows no conversion of lyxose to the keto form, xylulose. Others have reported the inability of D-lyxose to serve as a substrate for D-xylose isomerase, specifically in the organisms Streptomyces albus (6) and Lactobacillus brevis (10). In the latter case, it was shown that D-lyxose serves as a competitive inhibitor of the enzyme (11). Thus, it seems unlikely that constitutive production of enzyme would be sufficient to explain growth when lyxose is the sole carbon source. Rather, it seems necessary that some other enzyme such as a lyxose isomerase be present for growth to occur, as in the case of Aerobacter (3). Sanchez and Quinto (5) reported that preincubation of S. phaeochromogenes spores in Dxylose-containing medium leads to profuse growth upon transfer of the spores to medium containing D-lyxose as the sole carbon source. Our results failed to confirm this finding for either S. olivaceus or S. phaeochromogenes NRRL B-3559. Indeed, it would be expected that enzyme induced in the preincubation medium would be rapidly diluted out upon transfer to medium lacking an inducer. If, as suggested (5), lyxose is not an inducer, the growth would cease after a few generations. That preincubation in xylose medium, under our conditions, did not lead to growth on miniimal medium containing D-lyxose suggests that perhaps the strain of S. phaeochromogenes NRRL B-3559 reported by Sanchez and Quinto (5) may be different from our culture, although both were apparently obtained from the same source (NRRL). Perhaps their strain possesses an inducible D-lyxose isomerase which can be induced by D-xylose or Dlyxose and which is under similar control to the D-xylose keto isomerase. The mutants isolated might then be constitutive for both enzymes. It has been reported that Aerobacter possess a specific D-lyxose isomerase which is inducible only by D-lyxose (3). In Escherichia coli K-12, on the other hand, the ability of mutants to grow on D-lyxose apparently results from constitutive synthesis of a novel D-mannose isomerase (7). The results of our experiment in which D-lyxose was added to the enzyme production medium do not indicate the presence of an inducible Dlyxose isomerase in our strains of S. olivaceus. It is also possible that the D-lyxose used by
NOTES
971
Sanchez and Quinto (5) may have contained sugar impurities which could account for their results. D-Lyxose, as marketed by Sigma Chemical Co., St. Louis, Mo., does not include a statement of purity. Presence of any impurities which could act as a substrate or inducer for D-xylose keto isomerase or D-lyxose keto isomerase would greatly alter the interpretations of their data. In conclusion, it is apparent that the D-lyxose selection method does not operate according to the hypothesized mechanism (5) in the strains tested. In addition, mutants of S. olivaceus which grow on D-lyxose as sole carbon source are not universally constitutive for D-xylose keto is6merase production. It is suggested, therefore, that another explanation is necessary for the success of this method in the case of S. phaeochromogenes. It is my opinion that the D-lyxose selection procedure will not be generally useful for obtaining mutants constitutive for the synthesis of D-xylose keto isomerase. I thank R. J. Erickson, for his critical reading of the manuscript and for his suggestions, and also E. D. Anderson and M. L. Chupp, for their excellent technical assistance.
LITERATURE CITED 1. Chou, C. C., M. R. Ladisch, and G. T. Tsao. 1976. Studies on glucose isomerase for Streptomyces species. Appl. Environ. Microbiol.:32:489-493. 2. Dische, Z. 1962. Color reaction of ketoses with carbazole and sulfuric acid, p. 481. In R. L. Whistler et al. (ed.), Methods in carbohydrate chemistry. Academic Press, Inc., New York. 3. Mortlock, R. P., D. D. Fossitt, and W. A. Wood. 1965. A basis for utilization of unnatural pentoses and pentitols by Aerobacter aerogenes. Proc. Natl. Acad. Sci. U.S.A. 54:572-579. 4. Pridham, T. G., P. Anderson, C. Foley, L. A. Lindenfelser, C. W. Hesseltine, and R. G. Benedict. 1957. A selection of media for maintenance and taxonomic study of Streptomyces. Antibiot. Annu. 57:947-953. 5. Sanchez, S., and C. M. Quinto. 1975. D-Glucose isomerase: constitutive and catabolite repression-resistant mutants of Streptomyces phaeochromogenes. Appl. Microbiol. 30:750-754. 6. Sanchez, S., and K. L. Smiley. 1975. Properties of Dxylose isomerase from Streptomyces albus. Appl. Microbiol. 29:745-750. 7. Stevens, F. J., and T. T. Wu. 1976. Growth on D-lyXose of the mutant strain of Escherichia coli K12 using a novel isomerase and enzymes related to D-xylose metabolism. J. Gen. Microbiol. 97:257-265. 8. Takasaki, Y., Y. Kosugi, and A. Kanbayashi. 1969. Streptomyces glucose isomerase, p. 561-570 In D. Perlman (ed.), Fernentation advances. Academic Press, Inc., New York. 9. Tsumura, N., and T. Sato. 1965. Enzymatic conversion of D-glucose to D-fructose. VI. Properties of the enzyme from Streptomyces phaeochromogenes. Agric. Biol. Chem. 29:1129-1134. 10. Yamanaka, K. 1968. Purification, crystallization and properties of the D-Xylose isomerase from Lactobacillus brevis. Biochim. Biophys. Acta 151:670-680. 11. Yamanaka, K. 1975. D-Xylose isomerase from Lactobacillus brevis. Methods Enzymol. 51:466-471.
