DE3044455C1 - Production of pectin esterase - Google Patents

Description

(It should be emphasized, however, that both the problem of the accompanying enzymes and also the sensitivity to pH values in the alkaline range not for all Pectin esterases applies. So seem z. B. pectin esterases of vegetable origin, like those made from tomatoes, citrus etc. obtained essentially freely of PG and PTE activities) It is also known to the person skilled in the art that the conditions to be complied with for the selective inactivation of the disruptive accompanying enzymes compromise between inactivating undesirable depolymerase activity and the impairment must also represent the desired esterase activity.

Expediently, when using the method according to the invention the three parameters pH, temperature and time are matched to one another in such a way that inactivation the disruptive depolymerase activity without impairing the esterase effect is achieved.

The following information can serve as a guide: Incubation in pH range 8-9: incubation time 0.1-12 hours, preferably 1-5 hours, temperature range 35-55 "C.

In the pH range> 9-10: incubation time 0.2-5 hours, preferably 0.5-4 hours, in the temperature range from room temperatures to 45 "C, preferably up to 400 C.

In the pH range> 10-12: incubation time 0.1-5 hours, preferably 0.2 to 4 hours, in the temperature range from room temperature to 400 ° C., preferably up to 35 ° C.

Another help in choosing the one for the method according to the invention The data on enzyme activities shown in Table 1 provide optimal conditions depending on the process parameters mentioned. The setting of the required: Chen pH values can be measured in the manner known in enzymology, e.g. B. using of alkali lye, of buffers etc., whereby it should be noted that the used Cations after neutralization in the form of salts of those used for neutralization Acid are present in the approach.

In general, one is used to set the desired pH Alkali, ammonia and / or alkali carbonate or bicarbonate, alkali or ammonium salts other weak acids, for example acetates, dialkali hydrogen phosphates as well Use buffers such as Tris buffer, borate buffer, disodium phosphate buffer, etc.

The neutralization at the end of the incubation period can, for example by adding hydrochloric acid, phosphoric acid, acetic acid, etc. -The pectin esterase, which one assumes can be in solution or in a solid state. So suitable z. B. the known commercial forms of pectinases of fungal origin as starting products for the method according to the invention. If present in dissolved form, the above specified manner, d. H. by adding alkalis, ammonia, etc. the desired pH value adjusted and, if necessary, maintained (pH stat).

The work-up of the process products from the inventive Process can be carried out in the preparations take place in the usual manner, for example by freeze drying, spray drying, precipitation operations, chromatography, etc. (see Ullmann's Encyclopedia loccit.).

However, the enzyme can also be dissolved directly as a process product Shape, d. H. after neutralization or

Adjustment of what is desired for the use or storage stability pH value use.

The determination of the pectin esterase activity can be known per se Way, e.g. B. According to Methods in Enzymology, Academic Press Inc, N.Y., 1955, I.

159 - 160 (incubation at pH 4.5). The control According to R. Albersheim, possibly remaining activity of the PTE can possibly be

Killias, Arch. Biochem. Biophys., 97, 107 (1962). The activity of the PG can include

viscometric according to H. Suzuki et al., Journal of Ferment. Technol., 45.73 45, 73 (1967).

The method according to the invention generally results in inactivation at least 90% of the disruptive accompanying enzymes, especially of the PG and PTE types, usually> 90% to mostly> 99% achieved, without any significant Impairment of the desired esterase activity.

The following examples serve to illustrate the invention.

The protein content of the solutions can vary within wide ranges. Concentrations of 5-15% protein are appropriate.

Table 1 Enzyme activities as a function of the process parameters (Incubation temperature, time, pH).

Note o s are guidelines.

Incubation residual activities PE PG PTE pH 8.5 400 4h 90% <1% <1% pH 8.5 45 "2 h 88% <1% <1% pH 8.5 500 0.2 h 90% <1% <1% pH 10 400 ih 87% <1% <1% pH 12 350 0.5 h 85% <1% <1% example 10 1 pectinase solution (10% F.S) from the production of PECTINOL C is adjusted to pH 8.5 with 1N NaOH and heated to 40 °. The solution is kept at 400 for 4 h (with pH correction), then cooled to approx. 20 ° C. and adjusted to pH 5 with 1N hydrochloric acid.

A soluble pectin esterase product is obtained in a yield of 90% PE and less than 1% ° / Q of the initial activities PG and PTE, which stabilizes and can be standardized.

Activities of the initial activity after treatment solution (101) (approx. 111 solution) PE 80 U PE / mg (100%) 64 U PE / ml (90%) PG 940 U / ml (100%) 5 U / ml (<1%) PTE 3440 U / ml (100%) 10 U / ml (<1%)

Claims (3)

Claims: 1. Process for the production of pectin esterase, which is essentially free of accompanying depolymerase activities, such as G e k e n n n -z e n e t that one pectin esterase preparation of fungal origin in a solution in the pH range from 8 to 12 at least 10 minutes and at most 12 Incubated hours at temperatures between 10 and 55 "C until the accompanying depolymerase activity no longer interferes, and then acidified on the for the application or Storage stability brings suitable pH. 2. The method according to claim 1, characterized in that the Incubation in the pH range from 8 to 9 for 0.1 to 12 hours, preferably 1 to 5 hours, and in the temperature range from 35 to 55 "C, or that one the incubation in the pH range from> 9 to 10 for 0.2 to 5 hours, preferably 0.5 to 4 hours, and in the temperature range from room temperature to 45 ° C., preferably to 400 C, or that the incubation is carried out in the pH range from> 10 to 12 for 0.2 to 4 hours in the temperature range from room temperature to 40 ° C., preferably up to 35 "C. The subject of the invention is the method specified in claim 1; claim 2 names embodiments of this process. As is well known, pectins consist of high molecular weight polyuronides, the a-1,4-glycosidically composed of D-galacturonic acid. The carboxyl groups are partially methyl esterified. In their native state, however, pectin substances usually come chemically not uniformly before, but they also contain other carbohydrates, such as arabans and galactans as impurities. Generally soluble pectins fill the fruits with high water-binding capacity from the intereellular space. The enzymatic one The breakdown of pectin substances is a complex process in which, in addition to an esterase (pectin methylesterase) enzymes that are still depolymerizing and cleave within the polymer chain (endo-polymerhylgalacturonases) and exoenzymes which split off galacturonic acid units are involved in the end (see Ullmanns Encyclopadie der Technischen Chemie, 4th edition, Vol. 10, pages 514 and 515, Verlag Chemie, 1975). Technically, pectinases are used to detach pectin coatings from plant-based ones Fibers, for removing cloudiness in fruit juices and in beer, for production used by fruit juices and purees and the like. Commercial preparations are often a mixture of different depolymerases and esterases and usually also have secondary activities. The pectin methylesterase, the pectin in pectic acid and methanol divides, appears very specific. Gels as a result of the saponification of the ester groups the dissolved pectin in the presence of Ca + 2 ions. (The activity can be determined by titration the released carboxyl groups or by determining the methanol formed are measured.) Pectins with a low degree of esterification or pectic acids because of their property in the presence of polyvalent cations, especially of Ca + 2, to act as a gelling agent, of technical interest. Through the action of the Pectin esterase, which reduces the degree of esterification of pectins, is thus the The ability of pectins to form gels of the type mentioned above is increased. The naturally occurring pectin depolymerases, how polygalacturonases (PG) and pectin transeliminases (PTE) disturb, even destroy on the other hand, gel formation as a result of the decrease in the degree of polymerization that occurs. The previously known preparations containing pectin esterase from mold cultures (Aspergillus and Rhizopus species) all contain the disturbing accompanying enzymes of the type of PG and PTE. The task was therefore to provide pectin esterases, which are free from the accompanying enzymes PG and PTE. The solution to this problem offered itself a selective inactivation of the disruptive accompanying enzymes under as far as possible Preservation of pectin esterase activity. From US-PS 25 99 531 the inactivation of polygalacturonase is in aqueous solution by treatment with highly concentrated solutions of urea or thiourea. The pectin esterases from mushrooms have, as from theirs Activity and its origin was to be expected, consistently an acidic pH optimum Area. It has now been found that preparations of pectinesterase can be obtained from fungi let win whose accompanying enzymes of the type of polygalacturonases (PG) and pectin transeliminases (PTE) have been inactivated to such an extent that they have no interfering activity can develop more if you contain the disruptive accompanying enzymes of the type mentioned Pectin esterase according to the invention in a solution in the pH range from 8 to 12 at least Incubated for 10 minutes and a maximum of 12 hours at temperatures between 10 and 55 "C. and then by acidification to the one suitable for the application or storage stability pH brings. The pectin esterases to be used according to the invention are are those, the pectin depolymerases, especially endo-enzymes of the type of PG and the PTE included, d. H. Pectin esterases of fungal origin, in particular from Aspergillaceen and Mucoraceae. Particularly mentioned are the Aspergillus species A.niger, Aflavus, A.oryzae, A.fumigatus, A.parasitans, A.fumari, A.wentii, as well as Rhizopus species, such as Rh.tritici, Rh.nigricans and Penicillium species such as P.glaucum and others. According to the current classification, they are from disruptive accompanying enzymes Enzymes to be liberated are generally of the pectin methylesterase type [E.C. 3.1.1.11] to be assigned, while the enzymes to be inactivated generally belong to the classes E.E. 3.21.41 or E.C. 3.2.1.15 belong. The method according to the invention is quite surprising for the person skilled in the art, since pH ranges above the neutral point are harmful to fungal pectinases, in are considered completely unsuitable in any case. had to.