US 3645851 A
Process for the recovery of glucose oxidase in highly purified form comprising adjusting the pH of an aqueous solution containing impure glucose oxidase to a value of from 6.5 to 7.5, while maintaining the temperature of the solution at from 15 DEG to 25 DEG C., adding methanol up to a concentration of 62 volume percent and separating off the precipitate thereby formed.
Description (OCR text may contain errors)
United States Patent Bergmeyer et al.
[ 1 Feb. 29, 1972  RECOVERY OF GLUCOSE OXIDASE  Inventors: Hans Ulrich Bergmeyer, Tutzing; Wollgang Gruber, Garathshausen; Hans Moel- 1 lering; Waldemar Thum, both of Tutzing, all of Germany  Assignee: Boehringer Mannheim GmbH, Mannheim- Waldhof, Germany  Filed: Nov. 14, 1968  App1.No.: 863,374
 Foreign Application Priority Data Nov. 20, 1967 Germany ..P 16 42 592.6
 US. Cl. ..l95/66 R, 195/62, l95/103.5 C  Int. (1 ..C07g 7/02  Field otSearch ..195l3l,62, 66, 68
 References Cited UNITED STATES PATENTS 2,926,122 2/1960 Goldsmith et a1 ..l95/62 X Dixon, et al., Enzymes, Academic Press 1nc., N.Y., 1964 2nd ed., (pp. 37- 39).
Primary Examiner-Lionel M. Shapiro Assistant Examiner-D. M. Natl' Attorney-Burgess, Dinklage & Sprung  ABSTRACT Process for the recovery of glucose oxidase in highly purified form comprising adjusting the pH of an aqueous solution containing impure glucose oxidase to a value of from 6.5 to 7.5, while maintaining the temperature of the solution at from 15 to 25 C., adding methanol up to a concentration of 62 volume percent and separating off the precipitate thereby formed.
11 Claims, No Drawings RECOVERY OF GLUCOSE OXIDASE The present invention relates to a process for the recovery of glucose oxidase in a highly purified form. More particularly the invention relates to a process for the recovery of glucose oxidase which is free from disturbing amine oxidases.
Several processes are already known for the recovery of glucose oxidase. According to one such process, the glucose oxidase is precipitated from an aqueous extract containing the same by means of I a suspension of sodium gluconate in ethanol. This process, however, results in a product which has too high a content of amine oxidases for use in glucose determinations. Other methods which canbe carried out on a laboratory scale are known which admittedly provide, follow ing a large number of purification steps, a comparatively highly enriched preparation which, nevertheless, still has an undesirably high content of amine oxidases.
Thus, there exists a need for a process for the recovery of glucose oxidase free from amine oxidases. The action of various precipitation agents on the precipitation of the enzyme glucose oxidase has already been investigated. In addition to many other precipitation agents, methanol has been investigated for its usefulness. However, the results which were thereby obtained established only that methanol is hardly suitable for this purpose, and, in the case of an addition of 50 volume-percent, only resulted in a yield of about 11 percent and even when substantially higher concentrations were used, yields of 50 percent were hardly realized. In contradistinction thereto, other precipitating agents, particularly other alcohols, result in substantially better yields.
In accordance with the invention it has now surprisingly been found that when very definite temperature and pH conditions are in effect and with the maintenance of sharply defined precipitation agent concentrations, methanol behaves in a manner which is completely different from that which was to have been expected from previous investigations and results in the recovery of a glucose oxidase product which is substantially free of amine oxidases and that in excellent yields.
Thus, according to the present invention, there is provided a process for the recovery of glucose oxidase, wherein an aqueous extract of glucose oxidase is mixed with methanol up to 62 volume-percent, preferably up to 58 volume-percent, at a pH value of 6.5-7.5, while maintaining the temperature at l5-25 C., preferably at l92l C., followed by separation of the precipitate formed.
As starting material for the process according to the present invention, therecan be used not only freshly obtained glucose oxidase extracts but also solutions of commercially available preparations which contain considerable quantities of amin oxidases.
Glucose oxidase is to be found in numerous tissues but particularly suitable starting materials for the recovery of this enzyme are strains of Penicillium and Aspergillus, particularly Aspergillus niger and Penicillium notatum. By the simple aqueous extraction of these micro-organisms and separation of insoluble solid materials, there can be obtained solutions of glucose oxidase which can be used for the process according to the present invention.
The process according to the present invention can also be used to equal advantage for the purification of already enriched, commercially available glucose oxidase preparations. in this case, not only are the disturbing amine oxidases eliminated but a further considerable enrichment of the glucose oxidase also occurs.
Glucose oxidase preparations having minimum activity of about 1 U/mg are particularly suited for the process according to the present invention. in the case of the use of crude extracts, it may be advantageous to carry out an enrichment thereof according to the previously-known processes if the activity values are substantially below the above-mentioned range of about 1 U/mg For this purpose, there can be used, for example, the process described by Colowick and Kaplan in METHODS OF ENZYMOLOGY, Volume I, pages 340 345.
Glucose oxidase preparations having substantially higher activity values can, of course, also be used for the process according to the present invention.
An exact maintenance of the temperature and pH conditions is of critical importance for the process according to the present invention. For achieving the best results, the temperature should be maintained at 2011? C. Satisfactory results can, however, be obtained at temperatures of between 15 and 25 C. The pH should be maintained at a value of between 6.5 and 7.5, the preferred pH value being 7.0. As, during the methanol precipitation, the pH value is shifted towards the alkaline range, it is usually necessary to add some acid in order to maintain the above-defined pH range. While, when the pH value is displaced to the acid side of the above-defined pH range, the filterability of the precipitate which is formed is admittedly improved, the separation of the undesired foreign oxidases is impaired. The best results are obtained at a pH value of 7.0. i
The addition of methanol takes place in an amount to provide a concentration of up to, at the most, 62 volume-percent and preferably of up to 58 volume-percent. In order to ensure a complete separation of the amine oxidases, about 5-10 percent of the glucose oxidase activity should remain in the supernatant liquid. In the case of the above-mentioned preferred temperature and pH value conditions and with the addition of methanol in an amount of 58 volume-percent, there is obtained a yield of about 90-95 percent of a very pure product.
A direct relationship exists between the temperature at which the methanol precipitation is carried out and the volume percentage addition of methanol insofar as in the case of the higher temperatures in the given range, there can also be used an addition of methanol falling at the upper limit, of the methanol concentration range, i.e., at a temperature of 24-25 C, up to 62 volume-percent of methanol can be added.
The precipitation of the glucose oxidase under the abovedescribed conditions takes place over a certain period of time. It is, therefore, advantageous to allow the methanol-containing solution to stand for some time, preferably for at least 1 hour, with the exact maintenance of the stated conditions. The precipitate is thereafter separated off, while the temperature is maintained constant.
For the separation of the precipitate, it is advantageous to add filter adjuvants. For thispurpose, there can be used, for example, the known, commercially available filter adjuvants. However, care has to be taken to avoid selecting a filter adjuvant which has a deleterious effect on the activity of the en'- zyme.
In the case where filter adjuvants are employed, the precipitate is very easily filtered off or separated on a filter press. If a filter adjuvant is not to be used, the product can be centrifuged off directly and, following drying in air, used in the form thereby obtained.
Where a filter adjuvant is used, following the precipitation, the separation of the enzyme is carried out by dissolving the precipitate in water having a neutral to weakly acid pH value, separating off any insoluble matter, concentrating the filtrate and lyophilization. The dissolving or slurrying of the filter-adjuvant-containing preparation is preferably effected using water having a pH value of 5-6. However, it is also possible to use water having a pH value falling outside of this range, but in this case, the known limits of the pH value stability of the enzyme must, of course, be maintained. These limits range from about pH 3.5 to 8.0. If, before the lyophilization, the glucose oxidase solution is concentrated, then the concentration is preferably carried out in a vacuum at only moderately increased temperatures. It is advantageous not to exceed a temperature of about 45 C. Particularly good results are obtained at a temperature of about 37 C.
The process according to the present invention results in a product which is substantially free of foreign oxidases. Within the meaning of the present invention, a product which is-free of disturbing foreign oxidases and particularly amine oxidases, is regarded as being one which, at a temperature of 25 C. and
an activity of 10 U/ml., results in an extinction difference in a photometer of less than 0.010 per hour at 436 nm. and l-cm. path length.
The photometric determination of the purity of the enzyme product is carried out using a reagent which is composed of 100 ml. 0.1M phosphate buffer having a pl-i of 7.0, 4 mg. peroxidase and 6.6 mg. o-dianisidine (in the form of a solution of 6.6 mg./ml.).
in addition to the major advantage of the removal of foreign oxidases which cause considerable disturbances particularly in connection with the use of the glucose oxidase in the determination of glucose and the presence of which hitherto rendered impossible the preparation of test reagents which remained usable in solution for an entire day, the process according to the present invention also leads to a further enrichment of the enzyme of up to about 300 percent. When the commercially available glucose oxidase having an activity of 20 U/ mg. is utilized as starting material in the process according to the present invention, there is obtained an enzyme product having an activity of about 60 U/mg.
A further advantage of the process according to the present invention is that it can also be carried out on a technical scale and does not require the use of expensive adjuvants.
The following examples are given for the purpose of illustrating the present invention and are in nowise to be construed as limiting the same.
EXAMPLE 1 Ten kilograms of a commercially available glucose oxidase having a specific activity of 20 U/mg. were dissolved in 195 liters of completely desalted water. The pH value of the solution was adjusted to 7.0 and the temperature to 20 C. Thereafter, under continuous correction of the pH to maintain a value of 7.0 by addition of 2N hydrochloric acid, the solution was mixed with 270 liters methanol to a concentration of 58 volume-percent. The methanol-containing solution was then allowed to stand for 1 hour, with the temperature being precisely maintained at 20 C. Thereafter, 5 kg. of l-lyflo-Supercel (filter adjuvant manufactured by Mansfield, U.S.A.) were added and the resulting precipitate separated off in a filter press.
The filter cake thusly obtained was homogenized, free of foam, with 50 liters of cold, completely desalted water for IS minutes. The pH value of the suspension was then adjusted to 6.0 using 2N hydrochloric acid and the suspension which was obtained filtered.
The recovered filtrate was concentrated to about 20 liters at a temperature of 35-40 C. in a glass circulation evaporator and then lyophilized.
There were obtained about 3 kg. of glucose oxidase having an activity of about 60 U/mg. This corresponded to a yield of 90 percent of the initial activity.
The product which was obtained had, at 436 nm. using a 1- cm. cuvette and at a temperature of 25 C., an extinction difference of less than 0.010 per hour. Under the same conditions, the commercially available preparation used as starting material had an extinction difference of about 0.08 per hour.
EXAMPLE 2 Five grams of a crude oxidase obtained from mycelia by the direct extraction thereof with salt-containing water and precipitation of the proteins with alcohol, were dissolved in 100 ml. water and adjusted to a pH value of 6.9. The salt content of the resulting solution amounted to 00] and the activity to 1.23 U/mg.
Ninety-seven milliliters of this solution, which contained 4.85 g. of the crude oxidase starting material, was adjusted to 1 a methanol concentration of 61 percent at a temperature of 20 C. The pH value was adjusted to 6.0 using a small quantity of hydrochloric acid and the solution then centrifuged. The precipitate which was obtained contained the glucose oxidase. Following the determination of the glucose oxidase and amine oxidase contents, the product was concentrated and the residue dried.
The degree of enrichment and purification realized by the foregoing procedure are Ethe following table:
1. Process for the recovery of substantially pure glucose oxidase which comprises adjusting the pH of an aqueous solution containing impure glucose oxidase to a pH value of 6.5 to 7.5, adding methanol thereto to a minimum concentration of about 5 8 volume-percent and up to a concentration of 62 volume-percent while maintaining the temperature of the solution at between 15 and 25 C. and recovering the precipitate which is thereby formed.
2. Process according to claim 1 which comprises adding the methanol while maintaining the temperature of the solution at between 19 and 21 C.
3. Process according to claim 1 which comprises maintaining the pH of the solution at a value of 7.0 during the addition of the methanol.
4. Process according to claim l which comprises adding the methanol to a concentration of about 58 volume-percent.
5. Process according to claim l which comprises allowing the precipitate formed by the addition of the methanol to stand for at least 1 hour before recovering the same.
6. Process according to claim 1 which comprises recovering the precipitate by centrifugation.
7. Process according to claim 6 which comprises air-drying the recovered precipitate.
8. Process according to claim 1 which comprises admixing the methanol-precipitate mixture with a filter adjuvant, filtering the resultant admixture, dispersing the resultant filter cake in water having a pH of 3.5 to 8.0, separating off the insoluble material contained in said dispersion by filtering, concentrating the remaining filtrate and lyophilizing the concentrate.
9. Process according to claim 8 wherein said filter cake is dispersed in water having a pH of 5 to 6.0.
10. Process according to claim 8 wherein said filtrate is concentrated under vacuum at a temperature not exceeding 45 C.
11. Process according to claim 8 wherein said filtrate is concentrated under vacuum at a temperature not exceeding 37 C.
UNITED s'm'ms PATENT OFFICE CERTEHQATE 0F CGRREC'HQN Pateht No. 3,645,851 Dated February 29, 1972 InVentOI-(S) Hans Ulrich Bergmeyer et a1 It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:
Column 1 Cancel lines 64 to 72.
Signed and sealed this ZLLth day of October 1972.
EDWARD M.FLJE'IPCHEIR,JRo ROBERT GOTTSCHALK Attesting Officer Commissioner of Patents