Applied and Environmental Microbiology, June 2004, p . 3766-3768, Vol . 70, No . 6

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

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) .

 
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 .

 
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 .

 
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) .

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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.
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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.
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. 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. 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. 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.

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