Enzyme preparation containing protease

Details Enzyme preparation containing protease Enzyme preparation containing protease

1995-02-06

2002-09-17

Lankford, Jr., Leon B. (Department: 1651)

Chemistry: molecular biology and microbiology

Process of utilizing an enzyme or micro-organism to destroy...

Depilating hides, bating, or hide treating using enzyme or...

C435S219000

Reexamination Certificate

active

06451586

ABSTRACT:

The present invention pertains to solid enzyme preparations, free of surface active agents, containing proteases, obtained by tanning precipitation, as the active enzyme, and to methods for the soaking, bating, and unhairing of hides in the preparation of leather.
State of the Art
The technique of tannin precipitation has been known for a long time as an occasionally used method variant for the isolation of enzymes from solutions, e.g. from plant juices or aqueous culture media. There is a relatively broad state of the art relevant thereto (cf., e.g., GB-C 1,156,900, DE-A 1,642,619). As a rule, the goal is purification of the enzyme. The addition of gelatin, or working in an acid pH region (pH 3-5) serve as measures assisting good precipitation. However, because tannin disrupts further enzyme processing—after all, it forms an insoluble complex with the enzymes, whereupon they precipitate—it must again be removed, which is usually done by treating the precipitate with organic solvents, e.g. with acetone or ethanol, or by the addition of surface active agents, or by an increase in the pH value of the tannin precipitation. These are all measures which generally can be performed only on a laboratory scale without considerable complications.
Technical use of the tannin complex itself has been described in only a few cases. Thus, a tannin complex together with skim milk is said to be worked up by drying into a stable &agr;-amylase preparation (CS 141,233). For medical use, an insoluble pancreas preparation is provided which was prepared by precipitation of pancreas with tannic acid. The preparation is insoluble in acid stomach juice, but develops its effect in the alkaline intestinal regions. (DE 128,419).
Further, DE-A 2,143,945 describes “water-insoluble, dried enzyme adducts which are compatible with the skin”, inter alia in the form of tannin complexes. For practical use, the incorporation directly into a washing powder of an adduct of a bacterial protease obtained by tannin precipitation is proposed, for example. Since the tannin complex is said to be fully effective as a washing agent during use, i.e. in the washing float, it must “break up”.
Problem and Solution
As is evident from the foregoing picture of the state of the art, the method of tannin precipitation of enzymes from an aqueous milieu is, to be sure, a useful precipitation technique, but the relative stability of precipitated enzyme-tannin complexes under the conditions prevailing in potential uses, seems—apart from the exceptions named—to stand in the way of their industrial utilization.
Heretofore, ammonium sulfate or sodium sulfate have preferably been used for precipitation from the juice of pressed pancreas or from bacterial cultures (cf.
Ullmanns Encyclopädie der technischen Chemie
, 4th edition, volume 10, pages 475-561, page 495 ff., Verlag Chemie, 1975; DE-A 2,234,412).
However, this procedure is by no means ideal—particularly from an ecological viewpoint—since as a rule it incurs a quite considerable loading of the waste water. This is evident from the rule of thumb which says that 50 kg of ammonium sulfate are necessary for precipitation for each 100 liters of juice containing enzyme. Such a salt load is strictly contrary to the present tendency of waste water technology to restrict sulfate loading as much as possible. Rather, an efficient method for precipitating enzymes was to be strived for, in which the waste water loading would be considerably less—as well as, also, more cost effective, particularly in view of the quite particular cost pressure under which the leather industry operates. It has now been found that the proteinase preparation according to the invention comes very close to the aforementioned technical conceptions. The invention pertains to a solid, i.e. powdered or granulated, enzyme preparation essentially free of surface active agents and containing a protease—obtained as a tannin complex by precipitation of the active enzyme from an aqueous medium by the addition of tannin—with the proviso that the enzyme preparation containing at least 50 percent by weight, preferably more than 80 percent by weight, and up to 99.9 percent by weight, of one or more salts conventionally used as extenders or diluent in commercial enzyme preparations.
By “tannins” are meant according to the present invention the polyphenols, as a rule of natural origin, encompassed under this name, particularly the tannic acids. (More exact data are found in Ullmann,
Encyclopädie der Tech. Chemie,
3rd edition, volume 11, pages 593-594; Kirk-Othmer,
Encyclopedia of Chemical Technology,
2nd edition, volume 12, pages 319-325, J. Wiley 1967;
Fortschritte der Chemie organischer Naturstoffe
, Zechmeister, editor, volume 41, 1-46, Springer Verlag).
By “proteases” are to be understood the enzymes included under E.C.3.4. (Cf. Kirk-Othmer,
Encyclopedia of Chemical Technology,
3rd edition, volume 9, pages 173-223, J. Wiley 1980; E. Pfleiderer and R. Reiner in H. J. Rehm & G. Reed,
Biotechnology
, volume 6
b
, pages 729-742, VCH 1988; K. Aunstrup in
Industrial Aspects of Biochemistry
, B. Spencer, editor, volume 30(I), pages 23-46, North Holland 1974).
Various sub-criteria are applied, among them differentiation according to origin:
a) animal origin, such as
&agr;) rennin (E.C.3.4.23.4)
&bgr;) pancreas proteases
pancreatin, particularly trypsin; chymotrypsin (pH region of efficacy ca. 7-10); pepsin (E.C.3.4.23.1) (pH region of efficacy ca. 1.5-4.0; cathepsin (E.C.3.4.23.5) (region of efficacy ca. 4.0-5.0)
b) plant origin
&agr;) papain (E.C.3.4.22.1) pH region of efficacy ca. 5.0-8.0
&bgr;) ficin (E.C.3.4.22.3) pH region of efficacy ca. 4.0-9.0
&ggr;) bromelin (E.C.3.4.22.4 and 3.4.22.5) pH region of efficacy ca. 5.0-7.0
c) microbial origin (cf. L. Keay in
Process Biochemistry
1971, pages 17-21)
&agr;) from Bacillus species
such as
B.subtilis, B.licheniformis, B.alkalophilus, B.cereus, B.natto, B.vulgatus, B.mycoides
&bgr;) from Streptococcus species
&ggr;) from Streptomyces species
such as
Streptomyces fradiae, S.qriseus, S.rectus
&dgr;) from Aspergillus species
such as
Aspergillus flavus-oryzae, A.niger, A.saitoi, A.usamii
&egr;) from Mucor and Rhizopus species
such as
Mucor pusillus, M.miehei
&zgr;) Endothia species
such as
Endothia parasitica
&eegr;) from Trametes species
such as
Trametes sanquinea.
Besides this differentiation according to origin, differentiation according to the kind of attack (exo- versus endo-enzymes) and on the basis of the “active site” of the proteases (serine proteases, which are inhibited by diisopropylfluorophosphate, sulfhydryl enzymes) is also used.
Further, the pH dependence of the enzyme activity is of considerable practical significance. Here, differentiation is, above all, according to practical criteria.
i) alkaline proteases, having an activity optimum about in the region from pH 7.5 to 13,
particularly alkaline bacterial proteases (E.C.3.4.21.) (which mostly belong to the serine type) and alkaline fungal proteases
ii) neutral proteases, having an activity optimum in the range from pH 6.0-9.0,
particularly neutral bacterial proteases (E.C.3.4.24) (which belong to the metalloenzymes) and fungal proteases, for example Bacillus proteases, Pseudomonas proteases, Streptomyces proteases, Aspergillus proteases
iii) acid proteases, having an activity maximum in the range from pH 2.0-5.0 (E.C.3.4.23)
particularly acid fungal proteases, e.g. from Rhizopus species, Aspergillus species, Penicillium species, Mucor species, and
Impex lacteus
and
Endothia parasitica.
Subtilisins, alkaline bacterial proteases of the serine type which are stable in the pH region 9-10 and are to some degree insensitive to perborate, are especially mentioned as alkaline proteases.
The use of proteolytic enzymes is an established part of leather manufacture, particularly in the beamhouse, since the introduction of enzymatic bating (tryptic digestive enzymes of the pancreatic gland) in the “OROPON” bate by Dr. Otto Röhm (DE-PS 200,519) about 80 years ago. In addition to use in the bate (DP-PS 927,464; DE

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