Continuous production of S-lactoylglutathione by immobilized Hansenula mrakii cells

Process Biochemistry 29 (1994) 27 1-275

ContinuousProductionofS-Lactoylglutathioneby ImmobilizedHansenula mrakii Cells Yoshiharu Inoue, Hiromi Tsuchiyama & Akira Kimura Research (Received

Institute for Food Science, Kyoto University, Uji, Kyoto 6 11, Japan 15 February

1993; revised version received and accepted

3 1 March 1993)

A methylgboxal resistant yeast, Hansenula mrakii IF0 089.5, was immobilized in the matrices of x-carrageenan gel and production of S -lactoylglutathione was performed. Optimal production was achieved under the following conditions: pH 60,3o”C, ratio of glutathione and methylglyoxal = 14. By packing the gel mam’ces into a column, S -1actoylglutathione was produced continuously for over I week with more than 80% of conversion of glutathione to S 4actoylgiutathione at a space velocity 1 h- I.

concanavalin &induced histamine release from leukocytes,9 (iii) anti-inflamatory effects on carrageenan-induced rat edima,‘O and (iv) inhibitory effect of melanization of mouse melanoma cell B16.10 In order to advance the study of the physiological functions of this ester, a large amount of Slactoylglutathione is required. Hence, we have been engaging in the enzymatic production of Slactoylglutathione using microbial glyoxalase I.“-‘3 Previously we reported that the yeast Hcznsenufu mrukii IF0 0895 was highly resistant to methylglyoxal owing to a high activity of glyoxalase 1.14 ln this paper, we describe the continuous production of S-lactoylglutathione by K-carrageenan gel-immobilized H. mrukii.

INTRODUCTION SLactoylglutathione is an intermediate of the glyoxalase system consisting of glyoxalase I [EC 4.4.1.51 and glyoxalase II [EC 3.1.2.61. Glyoxalase I catalyzes the conversion of methylglyoxal to Slactoylglutathione in the presence of glutathione. The ester is then hydrolyzed to glutathione and lactic acid by glyoxalase II (Fig. 1). Methylglyoxal is a typical 2-oxoaldehyde synthesized enzymatitally in living cells, although the aldehyde arrests growth at very low concentrations. The glyoxalase system is, therefore, one of the detoxification route of methylglyoxal.’ In addition to this route, we have found an alternative route to oxidize methylglyoxal to lactic acid via lactaldehyde in some microorganisms.*-” S-Lactoylglutathione has several physiological functions; including (i) stimulation of microtubule assembly in vitro,7,8 (ii) an inhibitory effect on Corresponding author: Dr. Y. Inoue.Tel: 31 11 (Ext. 2728); Fax: ( + 81)-774-33-3004.

MATFBIALS

Microorgati~m and culture H. mrakii IF0 0895 was obtained from the Institute for Fermentation, Osaka, Japan. The yeast

( +81)-774-32271

Process Biochemistry 0032-9592/94/$7.00

AND METHODS

0 1994 Elsevier Science Limited, England.

272

Yoshiharu Inoue, Hiromi Tsuchiyama, Akira Kimura

‘i

;

CHO

GsT Glol

h%thyl&V0Xd

~

Y3

HtjOH c--o

CH3 y”, Glo II

6G s-l.Ilctoylgl~tblone

HiOH LOH Lactic ecid

Fig. 1. Synthesis and degradation of S-lactoylglutathione in the glyoxalase system (Glo I, glyoxalase 1; Glo II, glyoxalase II; GSH, glutathione).

was cultured in a nutrient medium (1.0% glucose, O-5% peptone, @2% yeast extract, 0.03% K,HPO,, 0.03% KH2P04, O-01% MgCl,; pH 5.5) with reciprocal shaking at 30°C until the stationary phase. After cultivation, the cells were collected, and washed once with O+W& NaCl solution. Enzyme

assay

Glyoxalase I activity was assayed in a mixture ( 1.O ml) containing 50 mM methylglyoxal, 50 lTLMglutathione, 100 mu potassium phosphate buffer (pH 6-O) and O-1 g of H. mrukii cells (as wet weight) at 25°C for 30 min. S-Lactoylglutathione was determined by measuring the absorbance at 240 nm and applying a molar extinction coefficient of 3300 cm-‘~-‘.‘~ The reaction product was identified as S-lactoylglutathione by reverse-phase HPLC (column, PBondasphare, 5 ,&-18, 10 A, 19 X 150 mm; elution, methanol : formic acid : water = 5 : 9.5 : 85.5, v/v/v).Glyoxalase II activity was measured in a mixture (1.0 ml) containing 10 mM S-lactoylglutathione, 100 mM potassium phosphate buffer (pH 7.0) and 0.1 g of H. mrukii cells (as wet weight) at 25°C for 60 min, and the decrease in S-lactoylglutathione concentration was determined as described above.

Immobilization of H. mrakii cell Immobilization of H. mrakii to Ic-carrageenan gel was essentially as described previously.16 H. mrukii (5 g as wet weight) was suspended in 5.0 ml of 0.85% NaCl solution, and warmed at 40°C. KCarrageenan solution (10 ml of 3.1%) was added to the cell suspension, stirred gently, and then cooled in ice water. Ice-chilled 2.0% KC1 solution (20 ml) was added and the mixture kept at 0°C for 1 h. The gel matrix obtained was cut into 2-mm cubes with a scalpel. To cross-link and harden the gel matrix, the cubes were suspended in 200 ml 350 mu potassium phosphate buffer (pH 7.0) containing 2.0% KC1 and 80 mu hexamethylenediamine. After stirring for 10 min, 8-3 ml of 25%

glutaraldehyde was added and the mixture was stirred gently for 1 h at 0°C. The resulting gel cubes were washed thoroughly with 5.0 rn~ TrisHCl buffer (pH 7-O), and were kept at 4C until use in the same buffer. Production system

of S-lactoylghxtathione

Continuous immobilized

production

in a batch

The H. mrakii-immobilized K-carrageenan gel (0.28 g, corresponding to O-08 g wet weight of H. mrukii) was transferred into a test tube containing 50 rn~ methylglyoxal, 50 mu glutathione and 100 mu potassium phosphate buffer (pH 6-O). The mixture was incubated at 3O”C, and a portion of the mixture was withdrawn periodically. The Slactoylglutathione synthesized was determined as described above. of S-lactoylglutathione

by

H. mrakii The H. mrakii-immobilized K-carrageenan gel ( 10 g) was packed into a column (3-2 x 6.1 cm, gel volume approximately 20 ml). For production of S-lactoylglutathione, the column was eluted with substrate (50 mM methylglyoxal and 50 ITLMglutathione) in 100 rn~ potassium phosphate buffer (pH 6-O) at various space velocities (h-l) at 30°C. After 40 ml of the substrate had been eluted, a portion of the eluate was collected and the concentration of S-lactoylglutathione synthesized was determined. Chemicals

Methylglyoxal, S-lactoylglutathione and Kcarrageenan were purchased from Sigma Chemical Co., Ltd, St Louis, MO, USA. Other chemicals are all analytical grade reagents. RESULTS

AND DISCUSSION

S-Lactoylglutathione is synthesized and degraded by glyoxalase I and glyoxalase II, respectively (Fig. 1). In order to synthesize Slactoylglutathione efficiently with immobilized H. mrakii cells, a treatment which increases the permeability of the cell and simultaneously decreases glyoxalase II activity is preferable. As shown in Fig. 2, the treatement of H. mrakii with hexamethylenediamine increased the permeability of the cell and decreased the glyoxalase II activity was (i.e. degradation of S-lactoylglutathione reduced). It was thought that the glyoxalase II was

273

Production of S -LG by immobilized yeast cell

dominantly inactivated by hexamethylenediamine. Therefore, H. mrakii was immobilized in a Kcarrageenan gel matrix followed by treatment with hexamethylenediamine. The concentration of hexamethylenediamine was varied in combination with the treatment time and the conditions described (80 mM hexamethylencdiamine, 10 min) were those which most efficiently inactivated glyoxalase II without inactivating glyoxalase I (data not shown). Some properties of immobilized H. mrukii were investigated with respect to the stabiity of glyoxalase I. Figure 3 shows the comparison of the thermal stability of glyoxalase I in the immobilized cells with that of purified enzyme. Approximately 40% of the enzyme activity was lost by

incubating the purified glyoxalase I at 50°C for 30 min in potassium phosphate buffer (pH 7.0), although the glyoxalase I activity in the immobilized cell was completely maintained under the same conditions. Some (50 %) of the enzyme activity was lost on incubating the immobilized cell at 65°C for 30 min. The immobilized H. mrukii did not lose the glyoxalase I activity after 1 month storage at 4°C (data not shown). S-Lactoylglutathione was synthesized using the immobilized H. mrukii preparation. The effects of pH, temperature, and ratio of methylglyoxal and glutathione were examined (Fig. 4). The optimal pH for the production of Slactoylglutathione was

ri

100 F-OfSLG (mM/h/O.l 20

40202010

of sl.0

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(mMlhlO.1

gall) 0

0

1

2

2

g-all)

3

4

5

E H t % d

50

0 I

I

I

I

Fig. 2. Effect of hexamethylenediamine on permeability of H. mrakii.Cells (01 g as wet weight) of H. mrakiiwere treated with 80 mM hexamethylenediamine for 10 min as described in the text. The concentrations of KC1 and glutaraldehyde were 2.0% and 25%, respectively. Glutaraldehyde treatment was carried out for 60 min after the treatment with hexamethylenediamine (HMDA, hexamethylenediamine; GA, glutaraldehyde).

4

(0

(a)

W

8 8 10

10203040

TOP. (W PB Fig. 4. Optimum conditions for the production of S-lactoylglutathione by immobilized H. mrakiicells. Conditions for production are described in the text. (A) Buffers used were: 0, sodium acetate buffer (pH 40-6.0); 0, potassium phosphate buffer (pH 5.5-9.0); A, Tris-HCl buffer (pH 5-5-8.0); A, glycine-KOH buffer (pH 9.0-l 1). The activity with potassium phosphate buffer (pH 6.0) was relatively taken as 100%. (B) The activity at 30°C was taken as 100%. (C) The activity at GSH/MG = 1.0 was taken as 100% (MG, methylglyoxal; GSH, glutathione).

lYY!iL 60

t

40

30

20

10

0 E

20408080

Temperature (“c) Fig. 3. Effect of temperature on stability of glyoxalase I. Furitied glyoxalase I from H. tnrukii(0) and K-carrageenanimmobilized H. mrakii cells (0) were incubated in 10 mM potassium phosphate buffer (pH 70) at various temperatures for 30 min, and the remaining activity was assayed as described in the text.

0

2

1

3

mn-00

Fig. 5. Production of S-lactoylglutathione by immobilized H. mmkii in a batch system. The reaction conditions are described in the text. Symbols are: 0, 10 mu methylglyoxal + 10 rnMglutathione; 0, 20 mu methylglyoxal + 20 mu glutathione; q,50 mM methylglyoxal+ 50 rnMglutathione; n , 100 mk4methylglyoxal + 100 mhi glutathione.

274

Yoshiham Inoue, Hiromi Tsuchiyama,Akira Kimura

Fig. 6. Continuous production of S-lactoylglutathione. (A) Concentration of substrate, 50 rnM methylglyoxal and 50 mM glutathione; column size, 3.2 x 6.1 cm (SV, space velocity). (B) Operational stability of immobilized H. mm&iiin continuous oroduction of S-lactovlalutathione. Concentration of substrate, 50 mM methylglyoxal and 50 mM glutathione; space velocity, 1.0 h- I; column size, 3.2 & 5.1 cm.

pH 6.0, and a high production yield was obtained between pH 5.5 and pH 7.0. Production of Slactoylglutathione using immobilized H. mrakii increased with the temperature up to 30°C but above 35°C the productivity decreased. The concentration of glutathione was varied in the reaction mixture containing a fixed concentration of methylglyoxal(50 n-m). As shown in Fig. 4(C), the optimum ratio of methylglyoxal and glutathione was 1.0. Under the optimum conditions obtained above (pH 6.0, 3O”C, methylglyoxal/glutathione = l*O), S-lactoylglutathione was produced in the batch system. As shown in Fig. 5, S-lactoylglutathione increased with incubation time, and approximately 40% of glutathione was converted to Slactoylglutathione after 2-h incubation. We have previously produced S-lactoylglutathione using the cell extracts of glycerol-adapted Saccharomyces cerevisiae cells or genetically engineered Escherichia coli carrying the glyoxalase I gene from Pseudomonas putida.” In these cases the Slactoylglutathione formed was degraded during incubation because of the remaining activity of glyoxalase II. As shown in Fig. 2, however, the glyoxalase II activity was diminished in the immobilized H. mrukii because of the treatment with hexamethylenediamine. Therefore, S-lactoylglutathione once formed in the reaction mixture was not degraded. Immobilized H. mrakii was then packed into a column and the production of S-lactoylglutathione was carried out by supplying the substrate continuously. Production was monitored when the space velocity was varied. As shown in Fig. 6(A), approximately 80% of glutathione was converted to S-lactoylglutathione at a space velocity of 1 h-l and continuous production of S-lactoylgluta-

thione was performed under these conditions (Fig. 6(B)). S-Lactoylglutathione was produced continuously for almost 1 week with 80% yield. Recently we demonstrated the continuous production of S-lactoylglutathione using immobilized glyoxalase I which was partially purified from H. mrakii. However, the enzyme was inactivated by exposure to substrate for a long period and 50% of the activity was lost within 12 h.13 This was presumably due to methylglyoxal in the reaction mixture, because methylglyoxal is known to bind to arginyl residues of the protein and it has been reported to cause the inactivation of several enzymes.2-5~17By using immobilized H. mrakii, this problem could be overcome and S-lactoylglutathione was produced at least for 1 week without decreasing the initial product yield.

ACKNOWLEDGEMENTS We thank Mr Shigeaki Ikemoto, Industrial Technology Center of Wakayama Prefecture, for his technical advice on the immobilization of H. mrakii cell.

REFERENCES Murata, K., Inoue, Y., Rhee, H.-I. & Kimura, A., 2-Oxoaldehyde metabolism in microorganisms. Can. J.

MicrobioI.,35 (1989) 423-31. Murata, K., Fukuda, Y., Shimosaka, M., Watanabe, K., Saikusa, T. 8~ Kimura, A., Metabolism of 2-oxoaldehvdes in veasts. Purification and characterization of GADPH-dependent methylglyoxal reducing enzyme from Saccharomwes cetetiiae. Eur. J. B&hem.. 151 (1985) 631-6. Inoue, Y., Rhee, H.-I., Watanabe, K., Murata, K. & Kimura, A., Metabolism of 2-oxoaldehydes in mold.

Production

4.

5.

6.

7.

8.

9.

10.

of S -LG by immobilized

Purification and characterization of two methylglyoxal reductase from Aspergillus niger. Eur. J. Biochem., 17 1 (1988) 213-18. Saikusa, T., Rhee, H.-I., Watanabe, K., Murata, K. & Kimura, A., Metabolism of 2-oxoaldehydes in bacteria. Purification and characterization of methylglyoxal reductase from Esherichia coli. Agric. Biol. Chem., 51 (1988) 1893-9. Inoue, Y., Tran, L.-T., Yoshikawa, K., Murata, K. & Kimura, A., Purification and characterization of methylglyoxal reductase from Hansenula mrakii. J. Ferment. Bioengng., 71(1991) 134-6. Inoue, Y., Watanabe, K., Shimosaka, M., Saikusa, T., Murata, K. & Kimura, A., Metabolism of 2-oxoaldehydes in yeasts. Purification and characterization of lactaldehyde dehydrogenase from Saccharomyces cerevisiae. Eur. J. Biochem., 153 (1985) 243-7. Gillespie, E., Cell-free microtubule assemblv: evidence for control by glyoxalase. Fed. Proc. Am. So& Exp. Biol., 34 (1975) 541. Thornalley, P. J., Della Bianca, V., Bellavite, P. & Rossi, F., S-n-Lactovlahnathione in resting and activated human neutrophils. Biochem. Biophysy Res. Commun., 145 (1987) 769-74. Gillespie, E., Effect of S-lactoylglutathione and inhibitors of glyoxalase I on histamine release from human leukocytes. Nature, 227 (1979) 135-6. Kojima, H., Kosugi, N., Kawai, Y., Konishi, H. & Kimura, A., Production of S-lactoylglutathione (SLG)

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yeast cell

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and its physiological activity. J. Jpn. Sot. Cutan. Hlth, 26 (1991) 52-6. Kosugi, N., Inoue, Y., Rhee, H.-I., Murata, K. & Kimura, A., Production of S-lactoylglutathione by glyceroladapted Saccharomyces cerevisiae and genetically engineered Escherichia coli cells. Appl. Microbial. Biotechnol,, 28 (1988) 263-7. Inoue, Y., Tsuchiyama, H., Kosugi, N. & Kimura, A., Production of S-lactovlalutathione by organic-solventextracted glyoxalase I &om Hansenula mrakii. Appl. Microbial. Biotechnok, 36 (1992) 469-72. Inoue, Y., Tsuchiyama, H:, T&t, L.-T., Kosugi, N. & Kimura, A., Continuous production of S-lactoylglutathione by immobilized glyoxalase I from Hansen&a mrakii. J. Ferment. Bioengng., 73 (1992) 116-20. lnoue, Y., Tran, L.-T., Yoshikawa, K., Murata, K. & Kimura, A., Purification and characterization of glyoxalase I from Hansenula mrakii. J. Ferment. Bioengng., 71 -(1991) 131-3. Racker, E., The mechanism of action of glyoxalase. J. Biol. Chem., 190 (1951) 685-93. Chibata, I., Tosa, T., Sato, T. & Takata, I., Immobilization of cells in carrageenan. In MethodF in Enzymology, ed. K. Mosbach. Academic Press, New York, 1987, pp.189-98. Riordan, J. F., McElvany, K. D. & Bordes, C. L., Jr, Arglnyl residues: anion recognition sites in enzymes. Science, 195 (1977) 884-6.