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A process for the production of fructosyl amino acid oxidase
514
Patent Survey
OH
I B-A--C-R,
where A is either a single bond, C,-C4alkylene or Cz-Cb-alkenylene; B is phenyl, naphtyl, pyridyl, pyrimidyl etc.; R, is COOH, COOCl-C., alkyl, Cn, C1C4-alkyl etc. To 7.13 mmol of a racemic alcohol, 0.92 g (4.63 mmol) di-vinyl adipate, 0.02 ml sodium phosphate buffer (0.1 M, pH 7.0), 4.5 ml O-xylol and 0.3 g Pseudomonas-lipase were added. The reaction mixture was shaken at 40°C. The enantiomer excess of non-converted alcohol was determined as trifluoro acetyl derivative by means of gas chromatography to a cyclodextrin column. The results obtained are shown in Table 1. lslble 1. Alcohol
3-Methyl-2-butanol 1-Octin-3-01 2-Octanol 2-Chloro-l-phenylethanol
ReactionResult time (h) (%)
168 168 23 188
94 9.5 98 95
References J. Am. Chem. Sot., 107 (1985) 7072. J. Am. Chem. Sot., 53 (1988) 5531.
Synthesis 1989, pp. 933-4. 3 European patent publications.
A process for the production of fructosyl amino acid oxidase (European patent application 678 576 corresponding to Japanese patent application 33488194 to Kyoto Daiichi Kagaku Co., Ltd, Kyoto, Japan)
Nobuo Kato and his co-biochemists, all asignors to the above company, have discovered a novel fructosyl amino acid oxidase derived from genus Fusarium or Gibberella. The oxidase is specific to amadori compounds, particularly to glycated protein, and they have found that the enzymes can be obtained by culturing a strain of the organisms in the presence of fructosyl lysine and/or fructosyl N”-Z-lysine. Examples of strains of Fusarium include Fusarium oxysporum (S-lF4 9FERM BP-5010) and examples of strains of Gibberella include Gibberella fujikuroi (IF0 No. 6356 and IF0 No. 6605). The inventors have also developed a process for producing fructosyl Iysine
and/or fructosyI”-Z-Iysine as a substrate of the enzyme, which is useful for screening and/or culturing a microorganism capable of producing a fructosyl Iysine oxidase (FLOD) being specific to fructosyl lysine and/or a fructosyl-zlycine (FZL). As an example of the preparation and purification of FLOD-G derived from G. fujikuzoi (IF0 No. 6356) is described. Gibberlla fijikuroi (IF0 No. 6356) was inoculated into 10 Iitres of medium (pH 6.0) containing 0.5% FZL (N”-ZIysine), 1.0% glucose, 0.1% dipotassium phosphate, 0.1% monosodium phosphate, 0.5% magnesium sulfate, 0.01% calcium chloride and 0.2% yeast extract, and grown at 28°C for 24 h with aeration (2 litre/min) and stirring (400 rpm) using a jar fermentor. The culture was filtered to harvest mycelia. A portion of mycelia (200g) was suspended in 1 litre of 0.1 M Tris-HCI buffer (pH 8.5) containing 2 mM D’lT and ground Dino-Mill. The ground mixture was centrifuged at 10000 rpm for 15 min to give a crude enzyme solution. To the crude enzyme solution was added ammonium sulfate to 40% saturation and the mixture was stirred and centrifuged at 12000 rpm for 10 min. To the supernatant was added ammonium sulfate to 75% saturation and the mixture was stirred and centrifuged at 12000 rpm for 10 min. The precipitates were dissolved in 50 mM Tris-HCI buffer (pH 8.5) containing 2 mM DTT (buffer A) and the solution was dialysed overnight against buffer A. The dialysed substance was absorbed onto a DEAE-Sephacel column equilibrated with buffer A. After washing with buffer A, the column was eluted with a linear gradient of O-O.5 M potassium chloride. The active fractions were collected and fractionated with ammonium sulfate ranging from 55 to 75%, followed by overnight dialysis against buffer A. Ammonium sulfate to 25% saturation was added to the dialysed substance which was then absorbed onto a phenyl-Toyopearl column equilibrated with buffer A containing 25% ammonium sulfate. After washing with the same buffer, the column was eluted with a linear gradient of 25-O% saturation of ammonium sulfate. The active fractions were collected. After addition of ammonium sulfate to 40% saturation, the solution was absorbed onto a butyl-Toyopearl column equilibrated with buffer A containing 40% saturated ammonium sulfate. After washing with the same buffer, the column was eluted with a linear gradient of 40-O% saturation of ammonium sulfate. The active fractions were collected and ammonium
sulfate to 80% saturation was added. The mixture was stirred and centrifuged at 12000 rpm for 10 min. The precipitates were dissolved in 0.1 M buffer A to give an enzyme solution. The enzyme solution was subjected to gel filtration chromatography using Sephacryl S-200 equilibrated with buffer A containing 0.1 M potassium chloride. The active fractions were collected and concentrated by means of ultrafiltration. The concentrate, when treated with Pharmacia FPLC system using a Mono Q column eluting with a linear gradient of O-O.5 M potassium chloride in buffer A, gave the objective purified enzyme of 30-60 units. The molecular weight of the resulting purified enzyme preparation was determined by SDS-PAGE using several standards of known proteins such as phosphorylase B, bovine serum albumin (BSA), ovalbumin, carbonic anhydrase and soybean trypsin inhibitor. SpecificSDS-PAGE was conducted ally, according to a Davis’ method (40 mA, 3 h, 10% gel) and protein was stained with Coumassi brilliant blue G-250. The molecular weight of a subunit referring to a calibration curve, was about 52 kDa. When estimated by gel filtration on Superdex 2OOpg, the molecular weight was about 47 kDa, as is apparent from the calibration curve shown in Fig. 1.
Fig. 1. References Chromatogx Sci., 10 (1979) 659. Clin. Chem., 28 (1982) 2088. Clin. Chem., 26 (1980) 1598. Clin. Chem. Acta, 127 (1982) 87-95. Clin. Chem. Acta, 112 (1981) 179-204. J. Clin. Lab. Inst. Reag., 16 (1993) 33-7.
11 Japanese publications. Bioremediation of hydrocarbon contaminated soil (US patent 5 436 160 to Exxon Research & Engineering Co., NJ, USA)
The
biochemists
Ramesh
Varadaraj,
Patent Survey
OH
I B-A--C-R,
where A is either a single bond, C,-C4alkylene or Cz-Cb-alkenylene; B is phenyl, naphtyl, pyridyl, pyrimidyl etc.; R, is COOH, COOCl-C., alkyl, Cn, C1C4-alkyl etc. To 7.13 mmol of a racemic alcohol, 0.92 g (4.63 mmol) di-vinyl adipate, 0.02 ml sodium phosphate buffer (0.1 M, pH 7.0), 4.5 ml O-xylol and 0.3 g Pseudomonas-lipase were added. The reaction mixture was shaken at 40°C. The enantiomer excess of non-converted alcohol was determined as trifluoro acetyl derivative by means of gas chromatography to a cyclodextrin column. The results obtained are shown in Table 1. lslble 1. Alcohol
3-Methyl-2-butanol 1-Octin-3-01 2-Octanol 2-Chloro-l-phenylethanol
ReactionResult time (h) (%)
168 168 23 188
94 9.5 98 95
References J. Am. Chem. Sot., 107 (1985) 7072. J. Am. Chem. Sot., 53 (1988) 5531.
Synthesis 1989, pp. 933-4. 3 European patent publications.
A process for the production of fructosyl amino acid oxidase (European patent application 678 576 corresponding to Japanese patent application 33488194 to Kyoto Daiichi Kagaku Co., Ltd, Kyoto, Japan)
Nobuo Kato and his co-biochemists, all asignors to the above company, have discovered a novel fructosyl amino acid oxidase derived from genus Fusarium or Gibberella. The oxidase is specific to amadori compounds, particularly to glycated protein, and they have found that the enzymes can be obtained by culturing a strain of the organisms in the presence of fructosyl lysine and/or fructosyl N”-Z-lysine. Examples of strains of Fusarium include Fusarium oxysporum (S-lF4 9FERM BP-5010) and examples of strains of Gibberella include Gibberella fujikuroi (IF0 No. 6356 and IF0 No. 6605). The inventors have also developed a process for producing fructosyl Iysine
and/or fructosyI”-Z-Iysine as a substrate of the enzyme, which is useful for screening and/or culturing a microorganism capable of producing a fructosyl Iysine oxidase (FLOD) being specific to fructosyl lysine and/or a fructosyl-zlycine (FZL). As an example of the preparation and purification of FLOD-G derived from G. fujikuzoi (IF0 No. 6356) is described. Gibberlla fijikuroi (IF0 No. 6356) was inoculated into 10 Iitres of medium (pH 6.0) containing 0.5% FZL (N”-ZIysine), 1.0% glucose, 0.1% dipotassium phosphate, 0.1% monosodium phosphate, 0.5% magnesium sulfate, 0.01% calcium chloride and 0.2% yeast extract, and grown at 28°C for 24 h with aeration (2 litre/min) and stirring (400 rpm) using a jar fermentor. The culture was filtered to harvest mycelia. A portion of mycelia (200g) was suspended in 1 litre of 0.1 M Tris-HCI buffer (pH 8.5) containing 2 mM D’lT and ground Dino-Mill. The ground mixture was centrifuged at 10000 rpm for 15 min to give a crude enzyme solution. To the crude enzyme solution was added ammonium sulfate to 40% saturation and the mixture was stirred and centrifuged at 12000 rpm for 10 min. To the supernatant was added ammonium sulfate to 75% saturation and the mixture was stirred and centrifuged at 12000 rpm for 10 min. The precipitates were dissolved in 50 mM Tris-HCI buffer (pH 8.5) containing 2 mM DTT (buffer A) and the solution was dialysed overnight against buffer A. The dialysed substance was absorbed onto a DEAE-Sephacel column equilibrated with buffer A. After washing with buffer A, the column was eluted with a linear gradient of O-O.5 M potassium chloride. The active fractions were collected and fractionated with ammonium sulfate ranging from 55 to 75%, followed by overnight dialysis against buffer A. Ammonium sulfate to 25% saturation was added to the dialysed substance which was then absorbed onto a phenyl-Toyopearl column equilibrated with buffer A containing 25% ammonium sulfate. After washing with the same buffer, the column was eluted with a linear gradient of 25-O% saturation of ammonium sulfate. The active fractions were collected. After addition of ammonium sulfate to 40% saturation, the solution was absorbed onto a butyl-Toyopearl column equilibrated with buffer A containing 40% saturated ammonium sulfate. After washing with the same buffer, the column was eluted with a linear gradient of 40-O% saturation of ammonium sulfate. The active fractions were collected and ammonium
sulfate to 80% saturation was added. The mixture was stirred and centrifuged at 12000 rpm for 10 min. The precipitates were dissolved in 0.1 M buffer A to give an enzyme solution. The enzyme solution was subjected to gel filtration chromatography using Sephacryl S-200 equilibrated with buffer A containing 0.1 M potassium chloride. The active fractions were collected and concentrated by means of ultrafiltration. The concentrate, when treated with Pharmacia FPLC system using a Mono Q column eluting with a linear gradient of O-O.5 M potassium chloride in buffer A, gave the objective purified enzyme of 30-60 units. The molecular weight of the resulting purified enzyme preparation was determined by SDS-PAGE using several standards of known proteins such as phosphorylase B, bovine serum albumin (BSA), ovalbumin, carbonic anhydrase and soybean trypsin inhibitor. SpecificSDS-PAGE was conducted ally, according to a Davis’ method (40 mA, 3 h, 10% gel) and protein was stained with Coumassi brilliant blue G-250. The molecular weight of a subunit referring to a calibration curve, was about 52 kDa. When estimated by gel filtration on Superdex 2OOpg, the molecular weight was about 47 kDa, as is apparent from the calibration curve shown in Fig. 1.
Fig. 1. References Chromatogx Sci., 10 (1979) 659. Clin. Chem., 28 (1982) 2088. Clin. Chem., 26 (1980) 1598. Clin. Chem. Acta, 127 (1982) 87-95. Clin. Chem. Acta, 112 (1981) 179-204. J. Clin. Lab. Inst. Reag., 16 (1993) 33-7.
11 Japanese publications. Bioremediation of hydrocarbon contaminated soil (US patent 5 436 160 to Exxon Research & Engineering Co., NJ, USA)
The
biochemists
Ramesh
Varadaraj,