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Automated rapid method for microbioassay of amino acids
ANALYTICAL
BIOCHEMISTRY
Automated HIROSHI
60, 573-580 (1974)
Rapid Method for Microbioassay of Amino Acids ITOH, TOMOAKI MORIMOTO, ICHIRO CHIBATA
Department of Biochemistry, Tanabe Seiyaku Co. Ltd.,
Research Kashima-cho,
Received February
Laboratory of Applied Higashiyodogawa-ku,
1, 1974; accepted March
AND
Biochemistl,y, Osaka, Japan
1, 1974
iln automated rapid method for microbioassay of amino acids was investigated by taking advantages of the rapid manual method. The Pye Unicam automatic analytical apparatus was adopted for the automation of assay culture in the rapid microbioassay. The advantages of the present method are that amino acids can be automatically determined on 3.5-hr assay culture, and that an aseptic technique can be omitted. These advantages were confirmed in several amino acid assays. The assay values were the same as those obtained by the conventional method. Application of the automated rapid method to the serine assay showed a linear standard curve without a lag section, leading to more expanded assay range and smaller drift in valves.
The microbiological method is widely used for the assay of amino acids, where the contents of one or several amino acids in a number of samples, or the optical configuration of the amino acid is problem, because of its specificity, sensitivity, and ability to yield many replicate results at the same time. However, the conventional microbiological method requires complicated multiple-step manual operations and a conlparatively longer period of time for assay culture. The use of an automated rapid microbiological method is, therefore, expected to make
significant cont’ributions to routine works. Of automated methods of microbioassay for several biologically active substances, a continuous-flow system has been successfully adopted for antibiotic assays because the sasay can be performed in an extremely short period by measurement of respiratory carbon dioxide (l-3). A semiautomated method for vitamin assays also has been reported by Kuzel and Kavanagh (4) and Berg and Behagel (5). The syst~emconsists of two independently automated modules. One module dilutes samples and dispensesthem into culture tubes. Other module measures the turbidity of the cultures after incubation. However, transfer of multiple tube arrays for incubation is not automated. ,Moreover, the semiautomated 573 Copyright @ 1974 by Academic Press, Inc. 911 rights of reproduction in any form reserved.
574
ITOH,
MORIMOTO
AND
CHIBATA
method requires tedious aseptic technique since the method cannot reduce the period of time for the growth of microorganism in assay culture. For the microbioassay of amino acids, we recently reported the rapid method (6,7). The method is characterized by carrying out intermediate culture between inoculum and assay cultures in order to inoculate large amounts of cells of logarithm phase to assay culture. By this technique the assay can be performed by 2.5-3.5 hr of assay culture. A further advantage of the rapid method is that no aseptic technique is required for the intermediate and assay cultures because of heavy inoculum and short incubation period. This paper describes successful automatic adaptation of t,he rapid method by using a commercially available apparatus based on discrete-sample system. METHODS
Test organism and stock culture. Leuconostoc mesenteroides P-60 was used as a test organism and its stab culture was prepared by the conventional method previously described (6). Culture medium. The composition of semisynthetic complete medium for inoculum and intermediate cultures is shown in Table 1. The composition of synthetic basal medium for assay culture is shown in Table 2. This composition was essentially the same as that described by Henderson and Snell (8), except that pyridoxine and pyridoxamine were supplied in addition to pyridoxal as vitamin B, compounds, and L-isomers of amino acids were used with exception of alanine. The double strength basal medium was prepared at the twice concentration shown in Table 2.
Composition
of the Semisynthetic
Component
TABLE 1 Medium for Inoculum Quantity
Glucose Proteose peptone (Difco) Yeast extract (Difco) KHJ’Or KeHPOb MgSO,.7H,O FeS017HzO MnSO,.nH20 N&l = The medium was adjusted to pH 6.0 before use.
and Intermediate
per 100 ml
g 1
1 0.5 w 25 25 10 0.5 0.5 0.5
Culturesa
AUTOMATED
MICROBIOASSAY
Composition Quantity Component m-Alanine n-Arginine.HCl n-Aspartic acid LCystine n-Glutamic acid Glycine r.-Histidine.HCl Llsoleucine LLeucine L-Lysine.HCl LMethionine r.-Phenylalanine n-Proline n-Serine LThreonine n-Tryptophan n-Tyrosine n-Valine
100 20 50 10 100 10 10 10 10 20 10 10 10
10 10 10 10 10
a The adjusted
2 0.1 2 amino acid to be determined to pH 6.0 before use.
575
ACIDS
TABLE 2 of Basal Medium”
per 100 ml (mg)
g
Glucose NaAcetate Na.Citrate
OF AMINO
Quantity
-
Component
-
NH&l KzHPOa MgS01.7HzO FeSOa.7HeO MnSOcnH20 NaCl Adenine.HzSOc2HsO Guanine.HCl2H20 Uracil Xanthine
Thiamine.HCl Riboflavin Pyridoxine.HCl Pyridoxamine.2HCl Pyridoxal.HCl CaPantothenate Niacin p-Aminobenzoic Biotin Folic acid
was excluded
from
per 100 ml bd 300 500 80 4 16 4 1 1 1 1 rg 100 100 100
30 30 100 100
acid
20 1 1
the mixture.
The
medium
was
Preparation of inoculum for assag culture. Inoculum culture was carried out by transfering inoculum from the stock culture and incubated for 17 hr at 37°C in 10 ml of the semisynthetic medium. The whole culture was added to 140 ml of the semisynthetic medium and incubated for 2 hr (Intermediate culture). The cells of logarithm phase were centrifuged and resuspended in 150 ml of the synthetic basal medium. The suspension was used as inoculum for assay culture. Assay procedure. Assay culture was made by mechanizing the individual steps of manual method on Pye Unicam AC 60 Chemical Processing Unit. The Unit functioned in conjunction with a spectrophotometer and recording systems. The assay systems programmed for microbioassay of amino acids are presented in Fig. 1. The AC 60 Chemical Processing Unit incorporates a continuous thermostatted track which consists of 120 carriers. Each carrier holding a sample cup and a 5 ml reaction tube is driven every 30 set by a synchronous
576
ITOH,
0: E,F:
MORIMOTO
Spectrophotometer Vibration
AND
Transfer
CHIBATA
Station
Station
FIG. 1. Flow diagram for automated microbioassay. Chemical Processing Unit was modified in such a way station (C), vibration station (F), and spectrophotometer set to act after all the reaction tubes passed through the
The Pye Unicam AC 60 that the culture dilution transfer station (D) were stations three times.
motor. When a sample cup, in which sample or standard solution was preloaded, arrived at position A, 0.1 ml of the content was withdrawn and then washed into the reaction tube with 0.9 ml of water. After 1 ml of inoculum suspension was added to the tube at position B, the mixture was stirred at position E. Incubation was carried out at 37°C for 3.5 hr by circulating three and a half times on the track. At the termination of incubation, the culture was diluted with 2 ml of water at position C, mixed at position F, and transferred to a flow cell in the spectrophotometer for recording the turbidity. Optical density at 660 nm was recorded on the chart with expansion in such a rate as 0.5 absorbance level was expanded to full scale. RESULTS
Standard curves of amino acids. The typical standard curves of amino acids on 3.5-hr assay culture are shown in Figs. 2 and 3. The assay ranges of isoleucine, lysine, phenylalanine, and aspartic acid were the same as those with the conventional method cultured for 18 hr. As shown in Fig. 3, the response curve of serine on 13-hr incubation with the conventional method was characterized by a lag section at lower concentrations. Although the lag section was diminished by increasing the incubation period to 18 hr, the slope of the curve became steeper, resulting in the narrow assay range only up to a concentration of 10 ,ag/ml. On the other hand, the lag was not observed in the response curve obtained by the automated rapid method in which large amounts of cells of logarithm phase were inoculated. The growth increased linearly with increase of serine concentration to 40 pg/ml. Moreover, as shown in Fig. 3, the coefficient of variations in optical density were 1.3-1.70/O with the automated rapid method and 2.0-2.6s with the conventional ‘method.
AUTOMATED
MICROBIOASSAY
OF AMINO
0.6
t
E
0.4
0.2 E
L-lie
z ‘n e (II h .z "7 r
d 2 .” c1 ,"
L-Phe
E
0.6
577
ACIDS
10
20
30
40
20
40
60
80
10
20
30
40
20
40
60
80
t
0.4
0.2
ugtml
of
Sample
FIG. 2. Growth response curve of amino acids. Incubation was carried out for 3.5 hr (-O-) with the automated rapid method and for 18 hr (+) with the conventional method. In the automated method optical density was recorded with expansion in such a rate as 0.5 absorbance level was expanded to full scale. In the conventional method optical densiB was determined on Hitachi model EPO-B electric photometer.
Application of automated rapid method. The assay of isoleucine, lysine, phenylalanine, aspartic acid, and serine in several samples were carried out by the automated rapid method, and the assay values were compared with those obtained by the conventional method. As shown in Table 3, there appears no difference between the assay values obtained by two methods. The reproducibility of assay values for isoleucine, lysine, phenylalanine, and aspartic acid with the automated rapid method was similar to that with the conventional method. In the serine assay, however, the reproducibility observed was slightly higher in the automated rapid method than that in the conventional method. DISCUSSION
An automated method for the rapid microbioassay has been accomplished by mechanization of individual steps in assay culture of the manual rapid method previously reported (7). The automated method
578
ITOH,
MORIMOTO
AND
20
10 pglml
of
CHIRATA
30
40
L-Serine
FIG. 3. Growth response curve of n-se&e. Incubation was carried out for 3.5 hr (-O-) with the automated rapid method and for 13 hr (--@--) and 18 hr C-e) with the conventional method. In both methods optical density was determined as described in Fig. 2. The figures in parenthesis indicate coefficient of varintions for six determinations,
TABLE Comparative
Amino acid Isoleucine Lysine Phenylalanine Aspartic acid Serine
Method Automated Conventional Automated Conventional Automated Conventional Automated Conventional Automated Conventional
Assays
3 for Amino Acids
Shiitakea extract (mid100 .d 4.Vd 4.67 33.0 33.3 2.19 2.41 10.4 10.3 18.5 18.2
(1.42)e (1.66) (1.4) (1.5) (1.40) (1.36) (1.5) (1.3) (1.8) (2.8)
Casein* hydrolyzate k/l~ d 5.15d 5.09 8.73 8.83 4.95 5.08 6.56 6.30 5.72 5.58
(1.35)e (1.29) (1.31) (1.26) (1.25) (1.35) (1.62) (1.43) (1.75) (2.55)
Fermentedc broth (w/ml) 10.6d (1.5)” 10.4 (1.3)
a The fresh Shiitake (Lentinus edodes) was extracted with 50% ethanol. * The casein (Hammarsten) was hydrolyzed with 6 N HCl for 24 hr at 110°C. c The fermented broth of Serrutiu marcescens (9). d Mean of six determinations. 0 Coefficient of variations.
AUTOMATED
MICROBIOASSAY
OF AllIN
ACIDS
579
is generally classified into discrete-sample and continuous-flow systems. Although the period of time required for the growth of microorganism is reduced extremely in the proposed rapid method, it is still considerably longer than the time required for usual enzymatic or chemical assays by automated methods. With the continuous-flow system, incubation should not be extended beyond half an hour. If longer incubation is forced to perform by using a longer incubation coil, the great resistance to flow causes high pressures which result in erratic flow rates and frequent ruptures of joints in the tubing. The continuous-flow system, therefore, is unsuitable for the automation of microbioassay of amino acids. On the other hand, incubat.ion period on the discrete-sample system can be set at the desired time by varying circulation times on the track and moving the positions of sampling and inoculating probes. In this paper, the Pye Unicam automatic analytical apparatus was found to fit the requirements for the automation of rapid microbioassay. This apparatus consists of 120 reaction tubes and the assay of one amino acid in 25 samples can be run in duplicate at. two levels in a time and results are obtained successively after 3.5hr assay culture. The automated rapid method, in addition, requires no aseptic technique, making the method applicable to the assay of amino acids in samples which becomes tubid by heat sterilizat,ion. The method offers another advantage in the assay of amino acids which show a sigmoidal standard curve with the conventional method. The serine standard curve with L. mesenteroides has a lag section at lower concentrations due to the inhibition of serine utilization by threonine. The similar sigmoidal standard curve is also observed in glutamic acid assay with Lactobacillus arabinosw. In such cases, the variation in assay values was reported to follow the extent of the lag (10,ll). Kirimura (12) reported that the lag disappeared from the standard serine curve when cells of L. mesenteroides grown in the medium containing small amounts of serine were used as inoculum for assay culture, On the improved procedures for glutamic acid assay, Hat et al. (13‘) used a heavy inoculum and Ishikura et al. (14) inoculated the cells obtained by the culture of shorter period. Our automated rapid method, using large amounts of cells of logarithm phase as inoculum, gave the satisfactory standard curlre and highly reproducible values in serine assay (Fig. 3). AS the advantages mentioned above are not obtained by the conventional method, the automated rapid method will be widely acceptable as one of the convenient assay procedures for amino acids. The automated rapid method is also expected to he applied to the assay for other substances that can be performed by the conventional microbiological method.
580
ITOH,
MORIMOTO
AND
CHIBATA
ACKNOWLEDGMENTS We are grateful to Mr. Takayanagi, managing director helpful advice and encouragement in this study.
of this company
for his
REFERENCEiS 1. GERKE, J. R., HANEY, T. A., AND PAGANO, J. F. (1962) Ann. N. Y. Acad. Sci. 87, 872. 2. HANEY, T. A., GERKE, J. R., MADIGAN, M. E., AND PAGANO, J. F. (1962) Ann. N. Y. Acad. Sci. 93, 627. 3. SHAW, W. H. C., AND DUNCOMBE, R.. E. (1963) Analyst 88, 694. 4. KUZEL, N. R., AND KAVANAGH, F. W. (1971) J. Pharm. Sci. 60, 767. 5. BERG, T. M., AND BEHAGEL, H. A. (1972) Appl. Microbial. 23, 531. 6. ITOH, H., KAWASHIMA, K., AND CHIBATA, I. (1973) Agr. Biol. Chem. 37, 2227. 7. ITOH, H., MORIMOTO, T., KAWASHIMA, K., AND CHIBATA, I. (1974) Agr. Biol. Chem. 38, 869. 8. HENDERSON, L. M., AND SNELL, E. E. (1948) J. Biol. Chem. 172, 15. 9. KISTJMI, M., KATO, J., KOMATSUBARA, S., AND CHIBATA, I. (1970) Appl. Microbial. 21, 569. 10. LYMAN, C. M., KUIKEN, K. A., BLOTTER, L., AND HALE, F. (1945) J. Biol. Chem. 157, 395. 11. MEINKE, W. W., AND HOLLAND, B. R. (1948) J. Biol. Chem. 173, 535. 12. KIRIMURA, J. (1961) J. Agr. Chem. Sot. Jap. 35, 644. 13. HAC, L., SNELL, E. E., AND WILLIAMS, R. J. (1945) J. Biol. Chem. 159, 273. 14. ISHIKURA, T.. SAKAMOM, T., KAWASAKI, I., TSUNODA, T., AND NARUI, K. (1964) Agr. BioZ. Chem. 28, 700.
BIOCHEMISTRY
Automated HIROSHI
60, 573-580 (1974)
Rapid Method for Microbioassay of Amino Acids ITOH, TOMOAKI MORIMOTO, ICHIRO CHIBATA
Department of Biochemistry, Tanabe Seiyaku Co. Ltd.,
Research Kashima-cho,
Received February
Laboratory of Applied Higashiyodogawa-ku,
1, 1974; accepted March
AND
Biochemistl,y, Osaka, Japan
1, 1974
iln automated rapid method for microbioassay of amino acids was investigated by taking advantages of the rapid manual method. The Pye Unicam automatic analytical apparatus was adopted for the automation of assay culture in the rapid microbioassay. The advantages of the present method are that amino acids can be automatically determined on 3.5-hr assay culture, and that an aseptic technique can be omitted. These advantages were confirmed in several amino acid assays. The assay values were the same as those obtained by the conventional method. Application of the automated rapid method to the serine assay showed a linear standard curve without a lag section, leading to more expanded assay range and smaller drift in valves.
The microbiological method is widely used for the assay of amino acids, where the contents of one or several amino acids in a number of samples, or the optical configuration of the amino acid is problem, because of its specificity, sensitivity, and ability to yield many replicate results at the same time. However, the conventional microbiological method requires complicated multiple-step manual operations and a conlparatively longer period of time for assay culture. The use of an automated rapid microbiological method is, therefore, expected to make
significant cont’ributions to routine works. Of automated methods of microbioassay for several biologically active substances, a continuous-flow system has been successfully adopted for antibiotic assays because the sasay can be performed in an extremely short period by measurement of respiratory carbon dioxide (l-3). A semiautomated method for vitamin assays also has been reported by Kuzel and Kavanagh (4) and Berg and Behagel (5). The syst~emconsists of two independently automated modules. One module dilutes samples and dispensesthem into culture tubes. Other module measures the turbidity of the cultures after incubation. However, transfer of multiple tube arrays for incubation is not automated. ,Moreover, the semiautomated 573 Copyright @ 1974 by Academic Press, Inc. 911 rights of reproduction in any form reserved.
574
ITOH,
MORIMOTO
AND
CHIBATA
method requires tedious aseptic technique since the method cannot reduce the period of time for the growth of microorganism in assay culture. For the microbioassay of amino acids, we recently reported the rapid method (6,7). The method is characterized by carrying out intermediate culture between inoculum and assay cultures in order to inoculate large amounts of cells of logarithm phase to assay culture. By this technique the assay can be performed by 2.5-3.5 hr of assay culture. A further advantage of the rapid method is that no aseptic technique is required for the intermediate and assay cultures because of heavy inoculum and short incubation period. This paper describes successful automatic adaptation of t,he rapid method by using a commercially available apparatus based on discrete-sample system. METHODS
Test organism and stock culture. Leuconostoc mesenteroides P-60 was used as a test organism and its stab culture was prepared by the conventional method previously described (6). Culture medium. The composition of semisynthetic complete medium for inoculum and intermediate cultures is shown in Table 1. The composition of synthetic basal medium for assay culture is shown in Table 2. This composition was essentially the same as that described by Henderson and Snell (8), except that pyridoxine and pyridoxamine were supplied in addition to pyridoxal as vitamin B, compounds, and L-isomers of amino acids were used with exception of alanine. The double strength basal medium was prepared at the twice concentration shown in Table 2.
Composition
of the Semisynthetic
Component
TABLE 1 Medium for Inoculum Quantity
Glucose Proteose peptone (Difco) Yeast extract (Difco) KHJ’Or KeHPOb MgSO,.7H,O FeS017HzO MnSO,.nH20 N&l = The medium was adjusted to pH 6.0 before use.
and Intermediate
per 100 ml
g 1
1 0.5 w 25 25 10 0.5 0.5 0.5
Culturesa
AUTOMATED
MICROBIOASSAY
Composition Quantity Component m-Alanine n-Arginine.HCl n-Aspartic acid LCystine n-Glutamic acid Glycine r.-Histidine.HCl Llsoleucine LLeucine L-Lysine.HCl LMethionine r.-Phenylalanine n-Proline n-Serine LThreonine n-Tryptophan n-Tyrosine n-Valine
100 20 50 10 100 10 10 10 10 20 10 10 10
10 10 10 10 10
a The adjusted
2 0.1 2 amino acid to be determined to pH 6.0 before use.
575
ACIDS
TABLE 2 of Basal Medium”
per 100 ml (mg)
g
Glucose NaAcetate Na.Citrate
OF AMINO
Quantity
-
Component
-
NH&l KzHPOa MgS01.7HzO FeSOa.7HeO MnSOcnH20 NaCl Adenine.HzSOc2HsO Guanine.HCl2H20 Uracil Xanthine
Thiamine.HCl Riboflavin Pyridoxine.HCl Pyridoxamine.2HCl Pyridoxal.HCl CaPantothenate Niacin p-Aminobenzoic Biotin Folic acid
was excluded
from
per 100 ml bd 300 500 80 4 16 4 1 1 1 1 rg 100 100 100
30 30 100 100
acid
20 1 1
the mixture.
The
medium
was
Preparation of inoculum for assag culture. Inoculum culture was carried out by transfering inoculum from the stock culture and incubated for 17 hr at 37°C in 10 ml of the semisynthetic medium. The whole culture was added to 140 ml of the semisynthetic medium and incubated for 2 hr (Intermediate culture). The cells of logarithm phase were centrifuged and resuspended in 150 ml of the synthetic basal medium. The suspension was used as inoculum for assay culture. Assay procedure. Assay culture was made by mechanizing the individual steps of manual method on Pye Unicam AC 60 Chemical Processing Unit. The Unit functioned in conjunction with a spectrophotometer and recording systems. The assay systems programmed for microbioassay of amino acids are presented in Fig. 1. The AC 60 Chemical Processing Unit incorporates a continuous thermostatted track which consists of 120 carriers. Each carrier holding a sample cup and a 5 ml reaction tube is driven every 30 set by a synchronous
576
ITOH,
0: E,F:
MORIMOTO
Spectrophotometer Vibration
AND
Transfer
CHIBATA
Station
Station
FIG. 1. Flow diagram for automated microbioassay. Chemical Processing Unit was modified in such a way station (C), vibration station (F), and spectrophotometer set to act after all the reaction tubes passed through the
The Pye Unicam AC 60 that the culture dilution transfer station (D) were stations three times.
motor. When a sample cup, in which sample or standard solution was preloaded, arrived at position A, 0.1 ml of the content was withdrawn and then washed into the reaction tube with 0.9 ml of water. After 1 ml of inoculum suspension was added to the tube at position B, the mixture was stirred at position E. Incubation was carried out at 37°C for 3.5 hr by circulating three and a half times on the track. At the termination of incubation, the culture was diluted with 2 ml of water at position C, mixed at position F, and transferred to a flow cell in the spectrophotometer for recording the turbidity. Optical density at 660 nm was recorded on the chart with expansion in such a rate as 0.5 absorbance level was expanded to full scale. RESULTS
Standard curves of amino acids. The typical standard curves of amino acids on 3.5-hr assay culture are shown in Figs. 2 and 3. The assay ranges of isoleucine, lysine, phenylalanine, and aspartic acid were the same as those with the conventional method cultured for 18 hr. As shown in Fig. 3, the response curve of serine on 13-hr incubation with the conventional method was characterized by a lag section at lower concentrations. Although the lag section was diminished by increasing the incubation period to 18 hr, the slope of the curve became steeper, resulting in the narrow assay range only up to a concentration of 10 ,ag/ml. On the other hand, the lag was not observed in the response curve obtained by the automated rapid method in which large amounts of cells of logarithm phase were inoculated. The growth increased linearly with increase of serine concentration to 40 pg/ml. Moreover, as shown in Fig. 3, the coefficient of variations in optical density were 1.3-1.70/O with the automated rapid method and 2.0-2.6s with the conventional ‘method.
AUTOMATED
MICROBIOASSAY
OF AMINO
0.6
t
E
0.4
0.2 E
L-lie
z ‘n e (II h .z "7 r
d 2 .” c1 ,"
L-Phe
E
0.6
577
ACIDS
10
20
30
40
20
40
60
80
10
20
30
40
20
40
60
80
t
0.4
0.2
ugtml
of
Sample
FIG. 2. Growth response curve of amino acids. Incubation was carried out for 3.5 hr (-O-) with the automated rapid method and for 18 hr (+) with the conventional method. In the automated method optical density was recorded with expansion in such a rate as 0.5 absorbance level was expanded to full scale. In the conventional method optical densiB was determined on Hitachi model EPO-B electric photometer.
Application of automated rapid method. The assay of isoleucine, lysine, phenylalanine, aspartic acid, and serine in several samples were carried out by the automated rapid method, and the assay values were compared with those obtained by the conventional method. As shown in Table 3, there appears no difference between the assay values obtained by two methods. The reproducibility of assay values for isoleucine, lysine, phenylalanine, and aspartic acid with the automated rapid method was similar to that with the conventional method. In the serine assay, however, the reproducibility observed was slightly higher in the automated rapid method than that in the conventional method. DISCUSSION
An automated method for the rapid microbioassay has been accomplished by mechanization of individual steps in assay culture of the manual rapid method previously reported (7). The automated method
578
ITOH,
MORIMOTO
AND
20
10 pglml
of
CHIRATA
30
40
L-Serine
FIG. 3. Growth response curve of n-se&e. Incubation was carried out for 3.5 hr (-O-) with the automated rapid method and for 13 hr (--@--) and 18 hr C-e) with the conventional method. In both methods optical density was determined as described in Fig. 2. The figures in parenthesis indicate coefficient of varintions for six determinations,
TABLE Comparative
Amino acid Isoleucine Lysine Phenylalanine Aspartic acid Serine
Method Automated Conventional Automated Conventional Automated Conventional Automated Conventional Automated Conventional
Assays
3 for Amino Acids
Shiitakea extract (mid100 .d 4.Vd 4.67 33.0 33.3 2.19 2.41 10.4 10.3 18.5 18.2
(1.42)e (1.66) (1.4) (1.5) (1.40) (1.36) (1.5) (1.3) (1.8) (2.8)
Casein* hydrolyzate k/l~ d 5.15d 5.09 8.73 8.83 4.95 5.08 6.56 6.30 5.72 5.58
(1.35)e (1.29) (1.31) (1.26) (1.25) (1.35) (1.62) (1.43) (1.75) (2.55)
Fermentedc broth (w/ml) 10.6d (1.5)” 10.4 (1.3)
a The fresh Shiitake (Lentinus edodes) was extracted with 50% ethanol. * The casein (Hammarsten) was hydrolyzed with 6 N HCl for 24 hr at 110°C. c The fermented broth of Serrutiu marcescens (9). d Mean of six determinations. 0 Coefficient of variations.
AUTOMATED
MICROBIOASSAY
OF AllIN
ACIDS
579
is generally classified into discrete-sample and continuous-flow systems. Although the period of time required for the growth of microorganism is reduced extremely in the proposed rapid method, it is still considerably longer than the time required for usual enzymatic or chemical assays by automated methods. With the continuous-flow system, incubation should not be extended beyond half an hour. If longer incubation is forced to perform by using a longer incubation coil, the great resistance to flow causes high pressures which result in erratic flow rates and frequent ruptures of joints in the tubing. The continuous-flow system, therefore, is unsuitable for the automation of microbioassay of amino acids. On the other hand, incubat.ion period on the discrete-sample system can be set at the desired time by varying circulation times on the track and moving the positions of sampling and inoculating probes. In this paper, the Pye Unicam automatic analytical apparatus was found to fit the requirements for the automation of rapid microbioassay. This apparatus consists of 120 reaction tubes and the assay of one amino acid in 25 samples can be run in duplicate at. two levels in a time and results are obtained successively after 3.5hr assay culture. The automated rapid method, in addition, requires no aseptic technique, making the method applicable to the assay of amino acids in samples which becomes tubid by heat sterilizat,ion. The method offers another advantage in the assay of amino acids which show a sigmoidal standard curve with the conventional method. The serine standard curve with L. mesenteroides has a lag section at lower concentrations due to the inhibition of serine utilization by threonine. The similar sigmoidal standard curve is also observed in glutamic acid assay with Lactobacillus arabinosw. In such cases, the variation in assay values was reported to follow the extent of the lag (10,ll). Kirimura (12) reported that the lag disappeared from the standard serine curve when cells of L. mesenteroides grown in the medium containing small amounts of serine were used as inoculum for assay culture, On the improved procedures for glutamic acid assay, Hat et al. (13‘) used a heavy inoculum and Ishikura et al. (14) inoculated the cells obtained by the culture of shorter period. Our automated rapid method, using large amounts of cells of logarithm phase as inoculum, gave the satisfactory standard curlre and highly reproducible values in serine assay (Fig. 3). AS the advantages mentioned above are not obtained by the conventional method, the automated rapid method will be widely acceptable as one of the convenient assay procedures for amino acids. The automated rapid method is also expected to he applied to the assay for other substances that can be performed by the conventional microbiological method.
580
ITOH,
MORIMOTO
AND
CHIBATA
ACKNOWLEDGMENTS We are grateful to Mr. Takayanagi, managing director helpful advice and encouragement in this study.
of this company
for his
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