Chemistry: molecular biology and microbiology – Process of utilizing an enzyme or micro-organism to destroy... – Cleaning using a micro-organism or enzyme
Reexamination Certificate
1996-12-18
2001-02-20
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Process of utilizing an enzyme or micro-organism to destroy...
Cleaning using a micro-organism or enzyme
C435S069100, C435S221000, C435S265000, C435S471000, C536S023200, C510S392000
Reexamination Certificate
active
06190904
ABSTRACT:
The subject matter of this invention concerns a new high-alkaline protease, its use in the industrial and domestic field, and compositions for the applications mentioned which contain this protease.
The use of protease-containing compositions in industrial applications or processes is well known. For example, in commercial laundry establishments, proteases have long been used, for example, to clean blood-stained hospital linen as well as protective clothes worn in meat-processing plants. For the production of leather, it is still normal practice in the leather treatment industry, for example, to remove the hair from skins and hides in an alkaline processing stage in the so-called beamhouse, which creates the prerequisites for hair removal and which causes the necessary skin digestion, often still using questionable and unsafe chemicals (e.g., inorganic sulfides). Although recently enzymatically supported liming processes (hair removal processes) have been proposed—especially using tryptic enzymes or fungal or bacterial proteases and sometimes even carbohydrolases—in practice, these enzymatic processes for the removal of hair from hides and skins have been applied almost exclusively to hides and skins of small animals and even in this area, the use has been rather restricted. For the removal of hair of large animals, on the other hand, the enzymatic hair removal process has so far not gained any acceptance at all, mainly because in many cases, hair removal is incomplete and because the collagen grain membrane is damaged or because too much of the skin substance is destroyed. In addition, the use of a certain percentage of alkaline proteases during liming with a reduced quantity of chemicals (e.g., sulfides) has been investigated. Although the use of enzymes makes it possible to markedly reduce the quantity of chemicals (e.g., sulfide) and although excellent surface yields with little cicatricial contraction are obtained, leather produced in this manner tends toward grain pipeyness, toward a loose flame scarring structure, and toward a coarse appearance which sometimes resembles the grain of nubuck leather. To the extent that proteases are presently used in the leather manufacturing industry (main soaking cycle and liming), these prior-art proteases, while having high pH values (pH=11 to 13), are not sufficiently effective and have a relatively low activity at the treatment temperatures (28° C. to 30° C.) normally encountered in the limeyard.
Due to the fact that the conditions in industrial processes are more drastic than those in domestic applications (e.g., as a household detergent), the proteases used must meet especially stringent requirements with respect to stability, acceptance of the prevailing environment, and performance. In addition to a satisfactory stability and activity at high alkaline pH values, the proteases should, on the one hand, have an excellent temperature resistance so as to yield good results at a low concentration over the longest possible time at a temperature that for a given industrial application can be very high and, on the other hand, they should be sufficiently active in certain applications (e.g., leather manufacture) even at relatively low temperatures (approximately 30° C.). Furthermore, the alkaline proteases used should be as resistant as possible to the chemicals and ingredients that are conventionally used in industrial processes (e.g., surfactants, bleaching agents, or disinfecting agents, chemicals, and other constituents).
Thus, the need for other alkaline proteases that are suitable for industrial applications, e.g., proteases for industrial textile laundering processes, industrial surface cleaning, or leather treatments and leather manufacture, is undiminished.
Therefore, the problem to be solved by this invention was to create a new alkaline protease which is suitable especially for use in industrial processes and which, in addition, can also be used to advantage for domestic applications.
It was discovered that the alkaline bacillus protease described below can be used highly effectively in a number of industrial processes. Thus, one of the subject matters of this invention concerns an alkaline protease and its use especially in compositions for industrial applications as well as in the domestic field, such as proposed in the claims and described in greater detail below.
Therefore, the subject matter of this invention concerns a high-alkaline protease which is characterized by the fact that it contains an underlying amino acid sequence with a minimum of a 95%, preferably with a minimum of a 98%, homology with respect to the amino acid sequence shown in FIG.
1
and that it is distinguished from this sequence by a triple mutation in the positions 42/114/115 of
FIG. 1
or that is distinguished in the three positions homologous thereto by the fact that arginine has been substituted for the amino acids in the relevant positions.
The alkaline bacillus protease mentioned has a molecular weight of approximately 26,000 to 28,000 g/mol, measured by means of SDS polyacrylamide gel electrophoresis against references proteins with a known molecular weight. The optimum pH value which was determined with soluble model substrates in an analytical test is approximately pH 10.5, with the optimum pH value being defined as that pH range in which the protease has a maximum proteolytic activity. The pH activity is higher than in the original protease (according to FIG.
1
); the optimum effect extends further into the more alkaline range and is pH 10.5 to 11.5. In addition, the mentioned alkaline bacillus protease according to this invention has an excellent pH stability and temperature resistance. Thus, this protease is an extremely high-alkaline protease which is effective in a pH range so far not reached by prior-art proteases.
In this context, homology with respect to the amino acid sequence shown in
FIG. 1
is defined as the structural relationship between the relevant amino acid sequences and the amino acid sequence shown in FIG.
1
. To determine the homology, the segments of the amino acid sequence of
FIG. 1
which structurally correspond to one another and of the amino acid sequence with which they are to be compared are made to coincide in such a way that a maximum structural agreement between the amino acid sequences exists, and differences caused by the deletion or insertion of individual amino acids are taken into consideration and are compensated for by appropriate rearrangements of sequence segments. The number of amino acids which now match one another in the sequences (“homologous positions”), relative to the total number of the amino acids that are contained in the sequence of
FIG. 1
, is the homology in %. Differences in the sequences can be caused by variation and insertion and by deletion of amino acids. It is therefore obvious that, if alkaline proteases are used which are at least 95% homologous with respect to
FIG. 1
, the amino acid positions named with respect to
FIG. 1
refer to the positions of the protease used which are homologous thereto. Deletions or insertions in the amino acid sequences of the proteases that are homologous with respect to
FIG. 1
can lead to a relative rearrangement of the amino acid positions so that the numerical notations of the amino acid positions that correspond to one another need not be identical in homologous fragments of amino acid sequences that are homologous with respect to one another, i.e., it is possible for slight numerical rearrangements to develop relative to the individual numbering of the amino acid positions.
In a preferred modification of this invention, the high-alkaline protease is characterized by an underlying amino acid sequence which is substantially identical to the amino acid sequence shown in FIG.
1
and which differs from this amino acid sequence by a triple mutation in positions 42/114/115 of
FIG. 1
in that the amino acids in the relevant positions have been replaced by arginine. The term “substantially identical” amino acid sequence is here defined t
Amory Antoine
Börner Karsten
Clippe Andre
Herrmann Hubert
Konieczny-Janda Gerhard
Achutamurthy Ponnathapu
Genecor International, Inc.
Moore William W.
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