E N Z Y M ES Cholesterol Oxidase (Recombinant) · C A T # R E C O-7 0-1 2 2 1 E C # 1. 1. 3. 6 E N Z Y M E S SUBSTRATE SPECIFICITY SUBSTRATE Cholesterol 100% Pregnenolone 116% β-Cholestanol

O R I G I N Original gene expressed in Streptomyces lividans

� S P E C I F I C A T I O N SAppearance Bright yellow to brown free flowing powderActivity >25 U/mg powder at 25°CSpecific Activity >25 U/mg protein at 25°CContaminants Catalase <1%

Glucose Oxidase <0.01%Uricase <0.01%

� U N I T D E F I N I T I O NOne unit of activity is defined as the amount of enzyme that will catalyse the oxidation of 1.0micromole of cholesterol per minute at 25°C under the standard assay method conditions.Refer to Table 1 for guidance on factors to adjust units according to temperature of assay.

Cholesterol Oxidase(Recombinant)

E N Z Y M E S

CAT# RECO-70-1221

EC# 1.1.3.6

C A T # R E C O - 7 0 - 1 2 2 1E C # 1 . 1 . 3 . 6

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ASSAY TEMPERATURE

25°C 1.00

30°C 1.12

37°C 1.24

45°C 1.28

FACTOR RELATIVE TO 25° RESULT

TABLE 1 : T EMPERATURE FACTORS FOR UN I T CONVERS ION

Note: Temperature can influence the level of available oxygen in the reaction mixture.

� A S S AY P R I N C I P L ECholesterol Oxidase (CO) catalyses the following reaction:

Cholesterol + O2 Cholestenone + H2O2CO

CAT# RECO-70-1221

EC# 1.1.3.6

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SUBS TRATE SPEC I F IC I TY

SUBSTRATE

Cholesterol 100%

Pregnenolone 116%

β-Cholestanol 86%

Stigmasterol 55%

Ergosterol 35%

% OF CHOLESTEROL ACTIVITY

SUBSTRATE

Cholesteryl Linoleate 0.9%

Estradiol 0.2%

Cholecalciferol 0.1%

Androsterone <0.1%

Sodium Cholate <0.1%

% OF CHOLESTEROL ACTIVITY

Molecular Weight (by SDS Page): 58kD

Kmvalue (using Eadie-Hofstee): 7 x 10-6 M (Cholesterol)

Optimum pH (Fig. 1): pH 6.5 to 7.5

Optimum Temperature (Fig. 2): 45°C

Stable pH Range (Fig. 3): pH 4.5 to 9.0 (25°C for 20 hrs)

Thermal Stability (Fig. 4): Stable at 60°C and below (pH 7.0 for 15 mins)

Substrate specificity was tested in-house by replacing cholesterol with alternativesubstrates in the CO assay i.e. at 0.2mM concentration.

� C H A R A C T E R I S T I C SThis recombinant grade of CO is highly purified and formulated without additives. Itsmain characteristics are as follows:

� A P P L I C A T I O NCholesterol Oxidase can be used in combination with Cholesterol Esterase (in test stripsor other clinical chemistry formats) to determine the level of cholesterol in blood.

F IGURE 3 : pH S TAB I L I TY(25 °C FOR 20 HOURS )

CAT# RECO-70-1221

EC# 1.1.3.6

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[email protected]

THE AMERICASSekisui Diagnostics, LLC4 Hartwell Place Lexington, MA 02421 Phone: 800 332 1042Fax: 800 762 6311

INTERNATIONALSekisui Diagnostics (UK) LimitedLiphook Way, AllingtonMaidstone, Kent, ME16 0LQ, UKPhone: +44 1622 607800Fax: +44 1622 607801

AP-00-0021.1.1 03/17© 2017 Sekisui Diagnostics, LLC. All rights reserved. Because every result matters™ is a trademark of Sekisui Diagnostics, LLC.

RELATIVE ACTIVITY (%)

pH

120

100

80

60

40

20

0

3 4 5 6 7 8 9 10 11

F IGURE 1 : OP T IMUM pH

F IGURE 4 : THERMAL S TAB I L I TY(pH 7 . 0 FOR 15 M INUTES )

F IGURE 2 : OP T IMUM T EMPERATURE

pH

3 4 5 6 7 8 9 10 11

RELATIVE ACTIVITY (%)

TEMPERATURE (°C)

TEMPERATURE (°C)

120

100

80

60

40

20

0

25 30 35 40 45 50

RELATIVE ACTIVITY (%)

120

100

80

60

40

20

0

RELATIVE ACTIVITY (%)

120

100

80

60

40

20

0

30 35 40 45 50 55 60 65 70

SODIUM ACETATE

POTASSIUM PHOSPHATE

MES

TRIS

MOPS

BICINE

CHES

SODIUM ACETATE

MOPS

SODIUM BORATE

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CHES

POTASSIUM PHOSPHATE

BICINE

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