Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Yamagata, S. Articles by Iwama, T. Search for Related Content PubMed PubMed Citation Articles by Yamagata, S. Articles by Iwama, T. Agricola Articles by Yamagata, S. Articles by Iwama, T.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, June 2004, p. 3766-3768, Vol. 70, No. 6
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.6.3766-3768.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Immobilization of Saccharomyces cerevisiae Cystathionine -Lyase and Application of the Product to Cystathionine Synthesis

Shuzo Yamagata, Tsuyoshi Akamatsu, and Tomonori Iwama^*

Department of Biotechnology, Faculty of Agriculture, Gifu University, Gifu 501-1193, Japan

Received 4 September 2003/ Accepted 23 February 2004

Top Abstract Introduction References

 
Cystathionine -lyase of Saccharomyces cerevisiae was immobilized to aminohexyl-Sepharose through the cofactor pyridoxal 5'-phosphate and was characterized with respect to its cystathionine -synthase activity. The immobilized product was so stable that it repeatedly catalyzed as many as five cycles of the reaction without losing activity.

Top Abstract Introduction References

 
L-Cystathionine (CTT) -lyase (EC 4.4.1.1) of Saccharomyces cerevisiae catalyzes the CTT -synthase reaction as well (2). The enzymatic production of CTT has been extensively investigated (7-9, 11). However, no utilization of a recombinant enzyme for this purpose has been reported. In this paper, we describe the immobilization of S. cerevisiae CTT -lyase, its characterization, and the recyclic utilization of the immobilized enzyme.

CTT (Sigma), L-homoserine (HS) (Nacalai Tesque, Kyoto, Japan), pyridoxal 5'-phosphate (PLP) (Nacalai Tesque), aminohexyl-Sepharose 4B (Pharmacia), and all other chemicals were obtained commercially. S. cerevisiae CTT -lyase (EC 4.4.1.1) was purified from Escherichia coli cells transformed with the CYS3 gene (12). PLP was bound to aminohexyl-Sepharose by a procedure slightly modified from that described by Guatrecasas (5) and Fukui et al. (3, 6). After preparing p-nitrobenzamidohexyl-Sepharose (approximately 16 g, wet weight), diazonium derivative was obtained by treating this with 0.1 M sodium nitrite. The derivative was incubated with 64 mg of PLP at 4°C and pH 8.0 for 8 h and then at 60°C for 30 min to decompose diazonium derivative without PLP. Bound PLP was determined spectrophotometrically at 292 nm after being liberated from the gel. An apoenzyme solution (containing 13 mg of protein) was mixed with 2 g (wet weight) of PLP-bound Sepharose in 0.1 M potassium phosphate (K-P) buffer (pH 7.8), and the suspension was incubated at 37°C for 30 min. The reaction product was reduced with 3 mM sodium borohydride at room temperature, and Sepharose-bound CTT--lyase was obtained. The amount of the enzyme bound to the gel was calculated as the difference between the amount applied to the gel and the amount that was washed out.

The mixture for the CTT -synthase reaction comprised 0.1 M K-P buffer, 0.1 M HS, 10 mM L-cysteine and dithiothreitol, 1 mM PLP, and the enzyme or the enzyme-bound gel. In many experiments, the reaction was done at 30°C for 30 min in a mixture of 0.5 ml, with approximately 0.05 mg of purified CTT -lyase or 20 mg (wet weight) of the enzyme-bound gel (having 0.1 mg of protein). The activity was determined by assaying cysteine consumption (4) and CTT synthesis (10). Confirmation of CTT as a reaction product was done by high-voltage paper electrophoresis (12). When the enzyme-bound gel was employed as the enzyme, the reaction mixture was gently shaken. The (ß) elimination reactions and activity determination were done as described previously (12). One unit of the enzyme was defined as the amount catalyzing production of 1 µmole of a product per min. The protein assay was done by the method described by Bradford (1).

PLP (0.24 µmol) was bound to 1 ml of the gel to give a concentration of 240 µM. An apoenzyme solution (13 mg of protein, 8.4 U of the enzyme [as HS -lyase]) was subjected to binding to 2 ml (approximately 2 g, wet weight) of the gel, followed by washing of the gel with 0.1 M K-P buffer (pH 7.8). The washing contained 2.7 mg of protein (1.1 U of the enzyme). Therefore, 10.3 mg of protein and 7.3 U of the enzyme were calculated to be bound to the gel, the efficiency being 79 and 87%, respectively. The concentration of the enzyme in the gel was 28 µM. On an assumption that the apoenzyme was bound through one PLP, approximately 12% of bound PLP was calculated to be employed to make the immobilized enzyme.

In the CTT synthase reaction, the native enzyme and the immobilized enzyme showed common optimal temperature and pH, i.e., 30°C and 8.0 (K-P buffer), respectively (data not shown). To compare the heat stabilities of the two enzymes, the native enzyme (0.06 mg) and the enzyme-bound gel (12 mg) were added to 0.088 ml of 0.1 M K-P buffer (pH 7.8)-0.2 mM PLP-1 mM EDTA, and the suspensions were incubated for 10 min at various temperatures. The whole mixtures treated were employed as the enzymes in the HS elimination reaction. The activities obtained after heat treatment are shown in Fig. 1. The two enzyme preparations showed very similar behaviors in response to heat. Sensitivities of the two enzyme preparations to pH were also very similar (data not shown).

View larger version (16K): [in this window] [in a new window]

FIG. 1. Heat stability of the native and the immobilized CTT -lyase. HS -lyase activity was measured at 30°C after the two enzyme preparations were treated for 10 min at the indicated temperatures in 0.1 M K-P buffer (pH 7.8) containing 0.2 mM PLP and 1 mM EDTA. Symbols: , immobilized enzyme; , native enzyme.

Recyclic use of the immobilized enzyme was investigated. At the end of the first reaction of CTT synthesis, the gel was recovered by filtering the whole mixture and was employed as the enzyme in the second reaction. This cycle was repeated five times, and the amounts of CTT synthesized were compared. Results are summarized in Table 1. The immobilized enzyme retained its activity without significant loss up to the fifth use, but it began to lose activity after that. It is also evident that the reaction was catalyzed by the gel-bound enzyme and not by free enzyme liberated from the gel during handling, because soluble materials were removed after each cycle of the reaction.

View this table: [in this window] [in a new window]

TABLE 1. Stability of the immobilized enzyme after repeated usea

In (ß) elimination reactions with CTT, HS, O-acetyl-HS, O-acetyl-L-serine, and O-succinyl-HS as substrates, the two enzyme preparations showed very similar reactivities to these substrates, suggesting that no denaturation of the protein occurred after immobilization (data not shown). The effect of repeated addition of cysteine (10 mM) on maintenance of activity was investigated, as it has been shown for the Streptomyces enzyme (9, 11). The synthase reaction was carried out in a 2-ml mixture containing 80 mg of the enzyme-bound gel at 30°C for up to 6 h. The CTT concentration was determined for an aliquot of the mixture after a certain time. The same reaction, but with cysteine added (at 10 mM) after 2 and 4 h of incubation, was carried out. The results obtained are presented in Fig. 2, in which the effect of repeated addition of cysteine is evident.

View larger version (15K): [in this window] [in a new window]

FIG. 2. Effects of repeated addition of L-cysteine on the CTT synthesis catalyzed by the immobilized enzyme. Two reaction mixtures (2 ml each) of the same composition were incubated with the immobilized enzyme. After 2 and 4 h of incubation (arrows), 11 µmol of L-cysteine and dithiothreitol was added to one mixture (), but another mixture () was incubated for 6 h without the reagents added during the reaction. The CTT concentration for 0.1 ml of the reaction mixture was determined.

The S. cerevisiae enzyme was shown to share many properties with other enzymes. However, the results of the present study provided us with new information regarding recyclic utilization of the recombinant enzyme, which is available in abundance through one-step purification from the extract of transformed E. coli cells (12). In addition, the catalytic activity of the immobilized enzyme with HS as one of substrates was not very low compared with those of other enzymes (7-9, 11). A calculation is possible, on the basis of the results (Fig. 2), that 1 g of CTT can be obtained in 6 h by using approximately 85 mg of the immobilized enzyme. Synthesized CTT can be separated from other substances by subjecting the mixture to stepwise chromatography on a Dowex-X8 (H⁺ form) column and can be crystallized with ethanol (11).

 
* Corresponding author. Mailing address: Department of Applied Life Sciences, Faculty of Applied Biological Sciences, Gifu University, Gifu 501-1193, Japan. Phone: 81-58-293-2932. Fax: 81-58-293-2932. E-mail: iwamatom{at}cc.gifu-u.ac.jp.

Top Abstract Introduction References

 

1
1. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.[CrossRef][Medline]
2
2. Cherest, H., D. Thomas, and Y. Surdin-Kerjan. 1993. Cysteine biosynthesis in Saccharomyces cerevisiae occurs through the transsulfuration pathway which has been built up by enzyme recruitment. J. Bacteriol. 175:5366-5374.[Abstract/Free Full Text]
3
3. Fukui, S., S. Ikeda, and M. Fujimura. 1975. Comparative studies on the properties of tryptophanase and tyrosine phenol-lyase immobilized directly on Sepharose-bound pyridoxal 5'-phosphate. Eur. J. Biochem. 51:155-164.[Medline]
4
4. Gaitonde, M. 1967. A spectrophotometric method for the direct determination of cysteine in the presence of other naturally occurring amino acids. Biochem. J. 104:627-633.[Medline]
5
5. Guatrecasas, P. 1970. Protein purification by affinity chromatography. J. Biol. Chem. 245:3059-3065.[Abstract/Free Full Text]
6
6. Ikeda, S., and S. Fukui. 1973. Preparation of pyridoxal 5'-phosphate-bound Sepharose and its use for immobilization of tryptophanase. Biochem. Biophys. Res. Commun. 52:482-488.[CrossRef][Medline]
7
7. Kanzaki, H., M. Kobayashi, T. Nagasawa, and H. Yamada. 1987. Production of L-cystathionine using bacterial cystathionine -synthase. Appl. Microbiol. Biotechnol. 25:322-326.
8
8. Kanzaki, H., M. Kobayashi, T. Nagasawa, and H. Yamada. 1987. Purification and characterization of cystathionine -synthase type II from Bacillus sphaericus. Eur. J. Biochem. 163:105-112.[Medline]
9
9. Kanzaki, H., T. Nagasawa, and H. Yamada. 1986. Highly efficient production of L-cystathionine from O-succinyl-L-homoserine and L-cysteine by Streptomyces cystathionine -lyase. Appl. Microbiol. Biotechnol. 25:97-100.
10
10. Ravanel, S., B. Gakiere, D. Job, and R. Douce. 1998. Cystathionine -synthase from Arabidopsis thaliana: purification and biochemical characterization of the recombinant enzyme overexpressed in Escherichia coli. Biochem. J. 331:639-648.
11
11. Yamada, H., H. Kanzaki, and T. Nagasawa. 1984. Synthesis of L-cystathionine by the -replacement reaction of cystathionine -lyase from Streptomyces phaeochromogenes. J. Biotechnol. 1:205-217.
12
12. Yamagata, S., R. J. D'Andrea, S. Fujisaki, M. Isaji, and K. Nakamura. 1993. Cloning and bacterial expression of the CYS3 gene encoding cystathionine -lyase of Saccharomyces cerevisiae and the physicochemical and enzymatic properties of the protein. J. Bacteriol. 175:4800-4808.[Abstract/Free Full Text]

---

Applied and Environmental Microbiology, June 2004, p. 3766-3768, Vol. 70, No. 6
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.6.3766-3768.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Yamagata, S. Articles by Iwama, T. Search for Related Content PubMed PubMed Citation Articles by Yamagata, S. Articles by Iwama, T. Agricola Articles by Yamagata, S. Articles by Iwama, T.